Immortalized adipose-derived mesenchymal stem cells as well as preparation method and application thereof

Abstract

The invention provides immortalized adipose-derived mesenchymal stem cells as well as a preparation method and application thereof, and belongs to the technical field of cells, the preparation method comprises the following steps: separating adipose tissues, constructing cells to be screened by the obtained adipose-derived mesenchymal stem cells, and screening to obtain the immortalized adipose-derived mesenchymal stem cells; the immortalized adipose-derived mesenchymal stem cells are used for treating neurological diseases, respiratory system diseases, cardiovascular diseases, osteoarticular diseases, urinary system diseases, digestive system diseases, skin diseases or autoimmune diseases. According to the invention, by preparing the immortalized canine adipose-derived mesenchymal stem cells and utilizing an intranasal administration mode, the nervous system degenerative diseases can be safely and effectively relieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell screening, and particularly relates to an immortalized adipose-derived mesenchymal stem cell and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) can be isolated from various tissues, such as umbilical cord tissue, adipose tissue and bone marrow tissue. Among them, MSCs isolated from adipose tissue are called adipose-derived mesenchymal stem cells (AD-MSCs). Compared with MSCs from other sources, AD-MSCs have many advantages. On the one hand, the source is abundant, and the adipose tissue covers the inguinal subcutaneous fat and the buttock fat, etc. In the sterilization surgery of dogs, the trimmed adipose tissue is often discarded as medical waste, but it can actually be used as a donor tissue for AD-MSCs. On the other hand, the collection site and method are less invasive, and the probability of complications such as infection during the collection process is lower. Moreover, AD-MSCs also have the same advantages as MSCs from other sources, such as anti-apoptosis, anti-inflammatory, chemotaxis and promotion of angiogenesis.

[0003] MSCs show potential in the treatment of many neurodegenerative diseases, such as Alzheimer's disease, Parkinson's syndrome, Huntington's disease and amyotrophic lateral sclerosis. The principle of the therapeutic effect of MSCs is that when they are delivered into the human or animal body by different routes, MSCs can secrete various cytokines and growth factors, and release nutrients that promote endogenous repair in the hypoxic, apoptotic or inflammatory area by means of paracrine and autocrine. These secreted bioactive substances can inhibit local immune response, promote angiogenesis, reduce free radical content, suppress fibrosis and apoptosis process, and stimulate the recruitment, retention, proliferation and differentiation activities of stem cells in tissues.

[0004] At present, there are many nervous system diseases in animals, but there is no significant and effective treatment method, such as canine cognitive dysfunction, spinal cord injury and brain trauma, and AD-MSCs can make up for this gap. Many studies have shown that MSCs can secrete nutrients (such as brain-derived neurotrophic factor), regulate microglia polarization (inhibit M1 pro-inflammatory phenotype and promote M2 anti-inflammatory phenotype), reduce neuronal apoptosis and other pathways to improve nerve function. In addition, stem cell treatment research for canine diseases is a bridge connecting basic research and human clinical research. Dogs, as an experimental animal model with high similarity to human physiological structure, the research of AD-MSCs can not only provide new therapies for canine nervous system diseases (such as spinal cord injury and traumatic brain injury), but also provide reference for the treatment mechanism and safety evaluation of human diseases. However, the clinical application of mesenchymal stem cells is affected by its heterogeneity, proliferation ability, stability and drug delivery route.

[0005] In summary, the present application transfects mesenchymal stem cells to obtain immortalized mesenchymal stem cells, and explores their anti-aging ability and stability. In addition, the present application will explore the feasibility, safety and effectiveness of intranasal administration of immortalized mesenchymal stem cells, in order to solve the problems of canine neurodegenerative diseases and other related problems. SUMMARY

[0006] In view of the above problems, the present application provides an immortalized adipose-derived mesenchymal stem cell and a preparation method and application thereof.

[0007] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] A preparation method of immortalized adipose-derived mesenchymal stem cells, the preparation method comprising the following steps:

[0009] I. Isolation of adipose-derived mesenchymal stem cells

[0010] After cleaning the surface blood vessels and connective tissue of the adipose tissue, the adipose-derived mesenchymal stem cells are obtained by washing, cutting, digesting, rinsing and culturing in turn;

[0011] II. Establishment of immortalized adipose-derived mesenchymal stem cells

[0012] 1) The adipose-derived mesenchymal stem cells are inoculated into complete culture medium for culture, and then replaced with opti-MEM culture medium for culture, to obtain cells to be transfected;

[0013] 2) The cells to be transfected are taken, a DNA-liposome complex mixture is added, incubated, then replaced with complete culture medium, and then subcultured to obtain cells to be screened;

[0014] III. Screening of immortalized adipose-derived mesenchymal stem cells

[0015] The cells to be screened are sequentially cultured in complete culture medium, complete culture medium containing different gradients of hygromycin, digested, proliferated in complete culture medium, digested again, and expanded in complete culture medium, thereby obtaining immortalized adipose-derived mesenchymal stem cells.

[0016] Further, the preparation method of the DNA-liposome complex mixture in the process of establishing the immortalized adipose-derived mesenchymal stem cells is as follows:

[0017] Take opti-MEM medium and mix with Lipofectamine 3000 to obtain a liposome diluent;

[0018] Take opti-MEM medium, add 1 μg of piggyBac plasmid, PBase plasmid and P3000 reagent, mix well to obtain a DNA premix;

[0019] After mixing the liposome diluent and the DNA premix in equal volumes, stand at room temperature, a DNA-liposome complex mixture is obtained.

[0020] Further, the preparation method further comprises step four: culture and subculture of the immortalized adipose-derived mesenchymal stem cells, which is specifically as follows:

[0021] 1) The immortalized adipose-derived mesenchymal stem cells are inoculated into complete culture medium for culture;

[0022] 2) When the cells are confluent to 85-90%, rinse to remove impurities, cell debris and residual culture medium;

[0023] 3) Add trypsin digestion solution for digestion, then add complete culture medium to terminate digestion, and mix well;

[0024] 4) Centrifuge, discard the supernatant, resuspend the cells with complete culture medium, and culture, thereby obtaining the subcultured immortalized adipose-derived mesenchymal stem cells.

[0025] Further, in the process of culture and subculture of the immortalized adipose-derived mesenchymal stem cells,

[0026] In step 1), the culture process is constant temperature culture, wherein the complete culture medium is replaced every 2-3 days;

[0027] In step 2), the reagent used for washing is sterile PBS buffer;

[0028] In step 3), the digestion temperature is 37℃ and the digestion time is 1-1.5 min;

[0029] In step 4), the culture is constant temperature culture, wherein the complete culture medium is replaced every 2-3 days.

[0030] Further, the specific process of screening the immortalized adipose-derived mesenchymal stem cells is as follows:

[0031] 1) Resuspend the adipose-derived mesenchymal stem cells in complete culture medium, then inoculate into cell culture dishes, and after the cells adhere, replace with complete culture medium containing 60-70 μg / mL hygromycin B;

[0032] 2) Maintain culture for 13-15 days, replace with complete culture medium containing 45-55 wt% screening concentration of hygromycin B for continuous culture, remove all culture medium, rinse with PBS buffer, and place the obtained dome-shaped single cell colony in a closed space, add trypsin digestion solution, digest, obtain cell suspension by gentle blowing, and transfer to a 24-well culture plate containing complete culture medium;

[0033] 3) Culture and proliferate, then digest into a 12-well culture plate containing complete culture medium and culture for 3-5 days, then digest the cells into a culture flask containing complete culture medium for expansion to 80-90% of the cell culture dish, and finally obtain the immortalized adipose-derived mesenchymal stem cells.

[0034] Further, in the screening process of the immortalized adipose-derived mesenchymal stem cells,

[0035] In step 2), the digestion time is 1-1.5 min.

[0036] Further, the specific process of establishing the immortalized adipose-derived mesenchymal stem cells is as follows:

[0037] 1) Take the adipose-derived mesenchymal stem cells and inoculate into complete culture medium for 22.5-23 h, replace with opti-MEM medium for 1-1.5 h, and obtain the cells to be transfected;

[0038] 2) Take the cells to be transfected, add DNA-liposome complex mixture, shake gently to distribute the solution uniformly, then incubate at 5% CO2, 36-38°C for 4-8 h, then replace with complete culture medium, continue to culture stably for 24-48 h, then subculture one generation, and obtain the cells to be screened.

[0039] Further, the specific process of isolating the adipose-derived mesenchymal stem cells is as follows:

[0040] In a sterile environment, the fat tissue is cleaned of surface blood vessels and connective tissue, then washed with HBSS, then cut into small pieces, and HBSS buffer (purchased from Savel Biosciences Co., Ltd.) is added dropwise during the cutting process, then tissue digestion solution is added to the cut fat tissue, shaken to form a creamy thick liquid, then poured into complete medium to terminate digestion, then filtered through a cell sieve, centrifuged, then the supernatant is aspirated, resuspended with red blood cell lysis solution, incubated at room temperature, then terminated with PBS buffer (purchased from Savel Biosciences Co., Ltd.), filtered, centrifuged, aspirated the supernatant, and rinsed with PBS buffer containing 2% penicillin-streptomycin mixture [configured by mixing 2 mL of penicillin-streptomycin mixture (purchased from Beijing Solabio Science and Technology Co., Ltd.) with 98 mL of PBS buffer], centrifuged again, aspirated the PBS buffer, resuspended with complete medium, and cultured to obtain the adipose-derived mesenchymal stem cells.

[0041] An immortalized adipose-derived mesenchymal stem cell prepared by the preparation method, wherein the immortalized adipose-derived mesenchymal stem cell has an anti-aging ability.

[0042] An application of the immortalized adipose-derived mesenchymal stem cell,

[0043] The application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a nervous system disease;

[0044] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a respiratory system disease;

[0045] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a cardiovascular disease;

[0046] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a bone and joint disease;

[0047] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a urinary system disease;

[0048] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a digestive system disease;

[0049] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating a skin disease;

[0050] Or, the application of the immortalized adipose-derived mesenchymal stem cell in the preparation of a drug for treating an autoimmune disease.

[0051] The immortalized adipose-derived mesenchymal stem cell, the preparation method and the application of the immortalized adipose-derived mesenchymal stem cell have the following beneficial effects:

[0052] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0053] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0054] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0055] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0056] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0057] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness.

[0058] The iAD-MSCs prepared in the application have significant anti-aging ability, and can be effectively delivered to the nervous system through the intranasal route and have safety and effectiveness. BRIEF DESCRIPTION OF DRAWINGS

[0059] Figure 1 is the mesenchymal stem cell morphology in Example 1 of the application; the left graph is the cell morphology of AD-MSCs, and the right graph is the cell morphology of iAD-MSCs;

[0060] Figure 2 is the expression result of the immortalized canine adipose-derived mesenchymal stem cell marker in Example 1 of the application; Figure 2In the figure, CD90 represents the identification result of CD90, CD29 represents the identification result of CD29, CD44 represents the identification result of CD44, CD45 represents the identification result of CD45, AD-MSCs represents the identification result of AD-MSCs, and iAD-MSCs represents the identification result of iAD-MSCs;

[0061] Figure 3 is the result of inducing the canine adipose-derived mesenchymal stem cells into chondrogenic differentiation in the embodiment 1 of the present application; Figure 3 In the figure, AD-MSCs represents the result of AD-MSCs, and iAD-MSCs represents the result of iAD-MSCs;

[0062] Figure 4 is the expression of the senescence-related proteins of AD-MSCs and iAD-MSCs in the embodiment 1 of the present application; in the figure, A represents the Western Blot protein band of AD-MSCs, B represents the relative expression of Klotho protein of AD-MSCs, C represents the relative expression of P21 protein of AD-MSCs, D represents the Western Blot protein band of iAD-MSCs, E represents the relative expression of Klotho protein of iAD-MSCs, and F represents the relative expression of P21 protein of iAD-MSCs; in the figure, the data is expressed as mean ± standard deviation (Mean ± SD), n = 3, P > 0.05 is not marked, *P < 0.05, **P < 0.01; in the figures B, C, E and F, 2, 4, 6 and 8 respectively represent the second generation, the fourth generation, the sixth generation and the eighth generation of cells;

[0063] Figure 5 is the relative telomere length of AD-MSCs and iAD-MSCs in the embodiment 1 of the present application; in the figure, A represents the relative length of the telomere of AD-MSCs, and B represents the relative length of the telomere of iAD-MSCs; in the figure, the data is expressed as mean ± standard deviation (Mean ± SD), n = 5, *P < 0.05, **P < 0.01; in the figures A and B, CTRL represents the blank control group, 3, 5, 7 and 9 respectively represent the third generation, the fifth generation, the seventh generation and the ninth generation of cells;

[0064] Figure 6 is the growth curve of AD-MSCs and iAD-MSCs in the embodiment 1 of the present application; in the figure, iAD-MSCs P2 represents the second generation of iAD-MSCs, iAD-MSCs P4 represents the fourth generation of iAD-MSCs, iAD-MSCs P6 represents the sixth generation of iAD-MSCs, AD-MSCs P2 represents the second generation of AD-MSCs, and AD-MSCs P4 represents the fourth generation of AD-MSCs;

[0065] Figure 7is the mCherry fluorescence protein expression of Lm-iAD-MSCs in embodiment 1 of the present application; Figure 7 The serial numbers 1, 2, 3, 4, 7, 8, 12, and 14 are cell line numbers screened, DAPI represents the cell nucleus, mCherry represents spontaneous fluorescence, and MERGE represents the morphology after superimposition of different fluorescence;

[0066] Figure 8 is the fluorescence expression intensity of Lm-iAD-MSCs in embodiment 1 of the present application, wherein **P<0.01; Figure 8 The serial numbers 1, 2, 3, 4, 7, 8, 12, and 14 are cell line numbers screened;

[0067] Figure 9 is the in-vitro bioluminescence efficiency of Lm-iAD-MSCs in embodiment 1 of the present application; wherein, A is the IVIS detection cell line bioluminescence result, and B is the cell line bioluminescence intensity; the data in the figure is expressed by mean ± standard deviation (Mean ± SD), n = 3, *P<0.05; A-A-H in the figure are cell position numbers; the serial numbers 1, 2, 3, 4, 7, 8, 12, and 14 in B are cell line numbers screened;

[0068] Figure 10 is the survival time and migration track of the bioluminescence revealed cells in embodiment 1 of the present application;

[0069] Figure 11 is the fluorescence tracing analysis of the cell migration track in embodiment 1 of the present application;

[0070] Figure 12 is the brain tissue HE staining in embodiment 1 of the present application; wherein, the magnification of all pictures is 20x; Figure 12 CTRL in the figure represents the CTRL group, PBS represents the PBS group, U87 represents the U87 cell group, and iAD-MSCs represents the iAD-MSCs group; Nasal Cavity represents the nasal cavity, Olfactory Bulb represents the olfactory bulb, Hippocampus represents the hippocampus, Cortex represents the cortex, and Cerebellum represents the cerebellum;

[0071] Figure 13 is the inflammatory factor detection result of the mouse brain tissue in embodiment 1 of the present application; wherein, A is the inflammatory factor result after one week of administration, and B is the inflammatory factor result after one month of administration; the data in the figure is expressed by mean ± standard deviation (Mean ± SD), n = 6, nsP>0.05, *P<0.05, **P<0.01; Figure 13CTRL represents the CTRL group, PBS represents the PBS group, U87 represents the U87 cell group, and iAD-MSCs represents the iAD-MSCs group;

[0072] Figure 14 are experimental results of new object recognition in embodiment 1 of the present application; in which, A is the exploration time in the adaptation stage of the new object recognition experiment, and B is the discrimination index in the test stage of the new object recognition experiment; the data in the figure is expressed by mean ± standard deviation (Mean ± SD), n = 6, nsP > 0.05, *P < 0.05, **P < 0.01; Figure 14 WL represents the wild type control group, AD represents the Alzheimer's disease model group, AD-PBS represents the placebo group of the Alzheimer's disease model mice, and AD-Treat represents the Alzheimer's disease treatment group;

[0073] Figure 15 are experimental results of Y maze in embodiment 1 of the present application; the data in the figure is expressed by mean ± standard deviation (Mean ± SD), n = 6, nsP > 0.05, *P < 0.05, **P < 0.01; Figure 15 WL represents the wild type control group, AD represents the Alzheimer's disease model group, AD-PBS represents the placebo group of the Alzheimer's disease model mice, and AD-Treat represents the Alzheimer's disease treatment group;

[0074] Figure 16 are the results of brain tissue hippocampus immunofluorescence in embodiment 1 of the present application; in which, MERGE represents the shape after superposition of different fluorescence, DAPI represents the nucleus, Thios represents Aβ amyloid protein, GFAP represents astrocyte, and DCX represents newborn neuron; WL represents the wild type control group, AD represents the Alzheimer's disease model group, AD-PBS represents the placebo group of the Alzheimer's disease model mice, and AD-Treat represents the Alzheimer's disease treatment group;

[0075] Figure 17 are the results of brain tissue hippocampus immunofluorescence analysis in embodiment 1 of the present application; in which, Thios represents Aβ amyloid protein, GFAP represents astrocyte, and DCX represents newborn neuron; WL represents the wild type control group, AD represents the Alzheimer's disease model group, AD-PBS represents the placebo group of the Alzheimer's disease model mice, and AD-Treat represents the Alzheimer's disease treatment group;

[0076] Figure 18 are the results of brain tissue ELISA in embodiment 1 of the present application; in which, A is the concentration of p-Tau in the brain tissue of mice, and B is the concentration of Aβ in the brain tissue of mice; the data in the figure is expressed by mean ± standard deviation (Mean ± SD), n = 6, nsP > 0.05, *P < 0.05, **P < 0.01;Figure 18 In the figure, WL represents a wild type control group, AD represents an Alzheimer's disease model group, AD-PBS represents an Alzheimer's disease model mouse placebo group, and AD-Treat represents an Alzheimer's disease treatment group. DETAILED DESCRIPTION

[0077] The technical solutions in the embodiments of the present application are described below in a clear and complete manner. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application, but the present application can also be implemented in other manners different from those described herein, and a person skilled in the art can make similar generalizations without departing from the connotation of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0078] Example 1 Preparation method of immortalized canine adipose-derived mesenchymal stem cells (iAD-MSCs)

[0079] Prepare complete culture medium: take DMEM / F12 (Thermo Fisher Scientific, USA) and fetal bovine serum (Biocode, China) in a weight ratio of 9:1 and mix well to obtain complete culture medium.

[0080] Prepare tissue digestion solution: mix an appropriate amount of collagenase type I and Hank's Balanced Salt Solution (HBSS) to prepare a tissue digestion solution containing 0.075wt% collagenase.

[0081] In a sterile environment, collect abdominal fat tissue from a canine sterilization surgery, pour the fat tissue into a culture dish, use forceps to hold the fat tissue, clean the surface blood vessels and connective tissue, and then lift the fat tissue and rinse with HBSS.

[0082] Replace the forceps, scissors and culture dish, use the forceps to hold the fat tissue, use the scissors to cut the fat tissue, cut for about 30 minutes, and drop the 2wt% concentration of HBSS buffer (purchased from Saver Biological Technology Co., Ltd.) containing penicillin and streptomycin in the process to keep the fat tissue moist, but the culture dish should be kept with as little 2wt% concentration of HBSS buffer containing penicillin and streptomycin as possible.

[0083] Put the cut fat tissue into the centrifuge tube, put into 2 times the volume of tissue digestion solution, seal the centrifuge tube with sealing film, and put it into the EP glove, then put it into the water bath, and shake for 45 min, so that it becomes a chylomicron-like thick liquid. Then pour into an equal volume of complete medium to terminate digestion. After termination of digestion, filter with 100 pm cell screen into a 50 mL centrifuge tube, centrifuge at 1400 rpm for 5 min after screening, then use a Pasteur pipette to suck off the upper liquid, then add 1 mL of 10 wt% red blood cell lysing solution per 5 mL of obtained fat cells, incubate at room temperature for 5 min, then add an equal volume of PBS buffer (purchased from Savel Biotech Co., Ltd.) to terminate digestion, and filter through a 40 pm cell filter into a 15 mL centrifuge tube.

[0084] Put the centrifuge tube into the centrifuge, centrifuge at 1200 rpm / min for 5 min, then suck off the upper liquid, and add 3-5 mL of PBS buffer containing 2% gentamicin mixture [configured with 2 mL of gentamicin mixture (purchased from Beijing Solabio Technology Co., Ltd.) and 98 mL of PBS buffer], centrifuge again, suck off the PBS buffer, and add 4-5 mL of complete medium for resuspension.

[0085] Dispense into 6 cm cell culture dishes, place in a 37°C, 5% CO2 cell incubator for 24 h, observe the cell growth state, and replace the complete medium, and the obtained canine adipose-derived mesenchymal stem cells are named AD-MSCs.

[0086] II. Establishment of immortalized canine adipose-derived mesenchymal stem cells [i.e. establishment of canine adipose-derived mesenchymal stem cells (Lm-iAD-MSCs) expressing Luciferase luciferase and mCherry fluorescent protein]

[0087] 1) One day before transfection, AD-MSCs were inoculated into 6-well cell culture plates containing complete medium for culture, so that the cell density was about 60% after 12 h, and the complete medium was replaced with opti-MEM medium (Thermo Fisher Scientific, USA) 1 h before transfection. The cells to be transfected;

[0088] 2) Experimental grouping design:

[0089] One group is a complete blank control group (does not contain transfection reagent and plasmid, only contains cells to be transfected), one group is a control group (only adds transfection reagent and cells to be transfected, does not contain plasmid), and two groups are experimental groups (add transfection reagent, plasmid and cells to be transfected), as follows:

[0090] 21) Prepare 2 centrifuge tubes, add 125 μL of opti-MEM medium respectively, then add 3 μL of Lipofectamine 3000 respectively, and mix gently to obtain a liposome diluent;

[0091] 22) Prepare 2 centrifuge tubes, add 125 μL of opti-MEM medium respectively, and add 1 μg of piggyBac plasmid (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.), 1 μg of PBase plasmid (purchased from Yunzhou Biotechnology (Guangzhou) Co., Ltd.) and 4 μL of P3000 reagent in one of the centrifuge tubes as the experimental group, and add only 4 μL of P3000 reagent in the other centrifuge tube as the control group, and then mix gently to obtain the corresponding DNA premix respectively;

[0092] 23) Mix the liposome diluent and the DNA premix in equal volumes, mix gently, and stand at room temperature for 15 min to obtain the corresponding DNA-liposome complex mixture;

[0093] 24) Add the cells to be transfected in a 6-well plate, and when they grow to 70-80% confluence, add 250 μL of the corresponding DNA-liposome complex mixture dropwise into the 6-well plate, shake the plate gently to distribute the solution evenly, and obtain the complete blank control group, the control group and the experimental group, then place it in a 5% CO2 cell incubator and incubate at 37°C for 4-8 h;

[0094] 25) Replace the above transfection reagent with complete culture medium, and after stable culture for 24-48 h, subculture the cells, and the obtained cells can start the positive screening procedure of the stable transfection cell line.

[0095] III. Screening of immortalized canine adipose-derived mesenchymal stem cells

[0096] 1) Resuspend the cells obtained in step II in complete culture medium at a density of 10%, and inoculate them in a 10 cm cell culture dish at a density of 10%, and after the cells adhere, replace them with complete culture medium containing 65 μg / mL of hygromycin B;

[0097] 2) maintain culture for 14d to negative control group cell death, more than 50% of the screening concentration of hygromycin B-containing complete medium to continue to form single cell clone colony, remove all culture medium, and then use PBS buffer gently washed 2 times, under the microscope to pick the clear boundary dome-shaped single cell clone colony, take the clone ring dipped with a suitable amount of sterilized silicon grease, the clone ring fixed in the target clone area to form a closed space, after the silicon grease dry; add 200 μL trypsin digestion solution in the clone ring, after 1-1.5 min digestion, the cell suspension was obtained by gentle blowing method, and was transferred to a 24-well cell culture plate containing complete medium;

[0098] 3) the 24-well cell culture plate was placed in a cell culture incubator under standard culture conditions (37℃, 5% CO2) for culture and proliferation to 80-90% density, then the cells in each well of the 24-well plate were sequentially digested to 12-well cell culture plates containing complete medium for culture to 80-90% density, then the cells in the 12-well cell culture plates were digested to T25 cell culture bottles containing complete medium for expansion to 80-90% density, and the obtained different cell strains were subjected to functional evaluation test, finally the immortalized canine adipose-derived mesenchymal stem cells were obtained, named iAD-MSCs.

[0099] After the operating table was alcohol disinfected, the T25 cell culture bottle expanded in step three was gently taken out from the cell culture box and placed under an inverted microscope for observation, and the camera was used to collect images, the results are shown in Figure 1 , iAD-MSCs meet the morphological requirements of mesenchymal stem cells.

[0100] Four, culture and subculture of immortalized canine adipose-derived mesenchymal stem cells

[0101] 1) the immortalized canine adipose-derived mesenchymal stem cells were inoculated into complete medium, and the cells were placed in a constant temperature incubator under standard culture conditions (37℃, 5% CO2) for culture, and the complete medium was replaced every 2-3d;

[0102] 2) when the cells were confluent to 85-90%, the cell culture dish was gently washed with sterile PBS buffer twice to remove impurities, cell debris and residual medium;

[0103] 3) add 1 mL trypsin digestion solution, digest in a 37℃ constant temperature incubator for 1-1.5 min, after the cells are round and fall off, quickly add double volume of complete medium to terminate digestion, gently blow and mix, and put into a 5 mL centrifuge tube;

[0104] 4) Place the centrifuge tubes in a centrifuge and centrifuge at 1000 rpm for 5 minutes. Use a pipette to remove the supernatant, then resuspend the cells in complete culture medium and add them to a new culture dish at a ratio of 1:3. Place the dish in a constant temperature incubator under standard culture conditions (37℃, 5% CO2) and change the complete culture medium every 2-3 days to obtain passaged immortalized canine adipose-derived mesenchymal stem cells.

[0105] V. Identification of canine adipose-derived mesenchymal stem cells

[0106] 1) Expression of biomarkers of immortalized canine adipose-derived mesenchymal stem cells

[0107] Flow cytometry was used to identify the surface markers CD29, CD90, CD44, and CD45 of immortalized canine adipose-derived mesenchymal stem cells (iAD-MSCs). Specifically, iAD-MSCs were seeded into 12-well cell culture plates containing complete culture medium. When the cell density reached 90%, the cells were gently washed three times with pre-chilled PBS buffer. Then, 1 mL of EDTA-free trypsin digestion solution was added, and the cells were incubated at 37°C for 5–10 min. When the cells became rounded and detached, an equal volume of complete culture medium was added to stop the digestion, and the cells were gently pipetted to form a single-cell suspension. The cell suspension was transferred to centrifuge tubes and centrifuged at 1000 rpm for 5 min. The supernatant was discarded, and the cells were blocked with blocking buffer (containing 3%). Resuspend FBS in PBS buffer and block at 4°C for 30 min. After repeated centrifugation, discard the blocking buffer, add 100 μL of blocking buffer to resuspend, add 3–5 μL of antibody, and incubate at 4°C in the dark for 45 min. After centrifugation, discard the supernatant, resuspend in PBS, centrifuge again and discard the supernatant to remove impurities. Resuspend the cells in 200 μL of PBS and filter through a 40 μm cell sieve into a dedicated flow cytometry tube to remove clumped cells. Collect cell fluorescence signals using a BD flow cytometer and perform quantitative analysis of cell subpopulations using the FlowJo 10.0 analysis platform.

[0108] Experimental results are as follows Figure 2 As shown in the figure. The experimental results show that the positive rates of CD29, CD90, and CD44 are above 90%, while the positive rate of CD45 is below 1%.

[0109] 2) In vitro differentiation capacity of canine adipose-derived mesenchymal stem cells

[0110] AD-MSCs and iAD-MSCs were induced to differentiate using adipogenic, osteogenic, and chondrogenic reagents, respectively. The experimental methods are as follows:

[0111] 21) Induction and identification of canine adipose-derived mesenchymal stem cells into adipogenic differentiation

[0112] a, evenly spread 500 μL of 0.1 wt% gelatin solution in water into the 12-well cell culture plate to ensure complete coverage of the well bottom, and place in a 37 °C incubator for 30 min, and then remove the gelatin; b, prepare adipogenic differentiation solution A and B according to the instructions of the adipogenic differentiation kit; c, resuspend the canine adipose-derived mesenchymal stem cells in complete culture medium, and then seed into the 12-well plate at a density of 50%, when the cell density reaches 90% confluence, replace the complete culture medium with adipogenic differentiation solution A, and after 48-72 h of culture, replace with adipogenic differentiation solution B for 24 h, and then replace with adipogenic differentiation solution A again; d, repeat the above alternating induction scheme until lipid droplet structures appear in the cells; e, after termination of differentiation, discard the induction and differentiation medium, and gently wash with PBS for 3 times, and then add 1 mL of 4 wt% paraformaldehyde fixing solution per well, and fix at room temperature for 30 min; f, mix oil red O stock solution and deionized water at a volume ratio of 3:2, vortex well, and then centrifuge at 1000 rpm for 4 min, take the clear working solution as the oil red O working solution for subsequent staining; g, after discarding the fixing solution, gently wash with PBS for 3 times, add 1 mL of oil red O working solution per well, and stain at room temperature for 30 min; h, remove the oil red O working solution, and gently wash with PBS for 3 times to remove residual staining solution, add 1 mL of PBS per well, and observe the morphology of lipid droplets using an inverted microscope.

[0113] 22) Induction and identification of osteogenic differentiation of canine adipose-derived mesenchymal stem cells

[0114] a, prepare osteogenic induction and differentiation medium according to the instructions of the osteogenic differentiation kit; b, seed the canine adipose-derived mesenchymal stem cells into the 12-well plate at a certain density, when the cell density reaches 70% confluence, replace the complete culture medium with the osteogenic differentiation solution, and replace every 48-72 h until obvious calcium nodules appear; c, discard the induction and differentiation medium, and gently wash with PBS for 3 times, add 1 mL of 4 wt% paraformaldehyde fixing solution per well, and fix at room temperature for 30 min; d, discard the fixing solution, and gently wash with PBS for 3 times, add 1 mL of alizarin red staining solution per well, and stain at room temperature for 10 min; e, discard the alizarin red staining solution, gently wash with PBS for 3 times, and retain 1 mL of PBS, and then place the 12-well plate under an inverted microscope to observe the osteogenic staining effect.

[0115] 23) Induction and identification of chondrogenic differentiation of canine adipose-derived mesenchymal stem cells

[0116] a, prepare the chondrogenic induction and differentiation premix solution according to the instructions of the chondrogenic differentiation kit; b, resuspend 3 x 10 5c. After the supernatant is discarded, the cell precipitate is resuspended with 0.5 mL of the premix, centrifuged at 1000 rpm for 5 min; d. Repeat step c to wash the cells again. e. Mix the chondrogenic induction premix and chondrogenic induction additives at a volume ratio of 100:1 to prepare the chondrogenic induction complete medium; f. Resuspend the cells with 0.5 mL of the induction complete medium, centrifuge at 1000 rpm for 5 min, then loosen the centrifuge tube cap to maintain gas exchange, and place the tube vertically in a cell incubator at 37°C and 5% CO2 for induction culture; g. When the cell mass appears to be aggregated, tap the bottom of the centrifuge tube to suspend the chondrosphere in the liquid, replace the induction complete medium every 48-72 h until the chondrosphere is formed to a size of 1.5-2 mm; h. Wash the chondrosphere with PBS and fix it with 4 wt% paraformaldehyde for more than 30 min; i. Perform gradient dehydration of the chondrosphere with 50% ethanol, 70% ethanol, 80% ethanol, 95% ethanol, and anhydrous alcohol, each for 30 min; g. Mix xylene and anhydrous alcohol at a volume ratio of 1:1, immerse the chondrosphere in the mixture for 2 h, then immerse it in pure xylene for 1.5 h, and finally replace the xylene with fresh xylene and continue to immerse the chondrosphere for 1 h; k. To remove the clearing agent from the chondrosphere, perform wax immersion treatment, first mix xylene and paraffin at a volume ratio of 1:1, immerse the chondrosphere in the mixture, and place it in a 40°C oven for 40 min, and finally immerse the chondrosphere in paraffin and place it in a 55°C oven for 30 min; l. Place the sample in a mold, pour in paraffin, trim the paraffin block after cooling and solidification; m. Prepare 3 μm serial sections and dry them in a 35°C oven; n. Immerse the sections in pure xylene for 15 min, then replace the xylene with fresh xylene and immerse the sections again for 10 min, and finally immerse the sections in a xylene-anhydrous alcohol solution (mixed at a volume ratio of 1:1) for 10 min. Finally, immerse the sections in 95% ethanol, 85% ethanol, 70% ethanol, and 50% alcohol, each for 10 min, and dry them; o. Add alizarin red staining solution dropwise to cover the tissue, stain at 37°C for 1 h, rinse with flowing deionized water for 5 min, and dry; p. Observe the staining effect of the sections under a microscope.

[0117] The experimental results are shown in Table 1. Figure 3 As shown in Table 1, both AD-MSCs and iAD-MSCs can be induced to differentiate into adipocytes, osteocytes, and chondrocytes. In summary, iAD-MSCs still maintain the characteristics of MSCs.

[0118] 3) Identification of the immortalization ability of immortalized canine adipose-derived mesenchymal stem cells

[0119] Western Blot, qPCR and CCK-8 kit were used to identify cell aging related proteins, relative length of telomere and growth curve, and the experimental method was as follows:

[0120] 31) Western Blot (WB)

[0121] First step, protein extraction

[0122] a, AD-MSCs and iAD-MSCs were inoculated into 6-well cell culture plates respectively, and when the cells were confluent to 90% density, they were gently washed twice with PBS; b, the RIPA cell lysis buffer was mixed with phosphatase inhibitor and protease inhibitor at a volume ratio of 100:1:1 to prepare cell lysis buffer, and it was pre-cooled in ice bath environment; c, the 6-well cell culture plate was placed in ice bath environment, 100 μL of pre-cooled cell lysis buffer was added to each well, and the cells were scraped thoroughly in one direction using a sterile cell scraper; d, collect the lysis product and transfer it to a 1.5 mL sterile centrifuge tube, centrifuge at 12000 rpm at 4°C for 5 min, and collect the upper clear liquid as the protein sample.

[0123] Second step, protein concentration determination of the sample using Kangweishiji BCA protein quantification kit a, the standard protein solution was diluted by RIPA cell lysis buffer (dilution gradient was 1:2, a total of 5 times dilution), and 20 μL of each of the 6 gradient concentration standards and 1 blank control containing only RIPA cell lysis buffer was taken to 96-well enzyme-labeled plate; b, each sample was made into 2-3 replicate holes, and 20 μL of sample was added to each hole; c, mix BCA-A and BCA-B at a volume ratio of 50:1, vortex to mix thoroughly, and get BCA working solution; d, add 200 μL of BCA working solution to each reaction well, mix thoroughly, and incubate at 37°C for 30 min, then use the enzyme-labeled instrument to measure the absorbance value of each well at 570 nm wavelength; e, draw a standard curve according to the sample data, which is used to calculate the protein concentration of each sample.

[0124] Third step, protein concentration normalization

[0125] a, according to the actual protein concentration and the target protein concentration (ensure that the protein loading amount is about 10-15 μg each time), dilute the protein, and mix the Loading Buffer and the sample at a volume ratio of 1:4; b, place the mixture in a metal bath heating module for denaturation treatment at 90°C for 10 min, and store it in a-80°C refrigerator after aliquoting.

[0126] Fourth step, according to the configuration of SDS-PAGE gel of Plrlie UltraGel broad-spectrum super-clear gel kit

[0127] a, Use special fixture to fix the glass plate, verify the sealing of the assembly by adding deionized water. After the water is completely volatilized, proceed with the subsequent operation; b, Mix the separation glue A liquid, B liquid and coagulant according to the volume ratio of 50:50:1 to prepare the separation glue working solution, slowly inject the separation glue solution along the inner wall of the glass plate to the predetermined height; c, Immediately fill the anhydrous ethanol above the glue surface to eliminate bubbles and form a flat interface; after the separation glue is fully solidified, discard the liquid; d, Prepare the concentrated glue according to the same volume ratio, and inject it above the separation glue, insert the toothed comb to prepare the sample loading hole; e, After the concentrated glue is completely solidified, transfer the gel device to the electrophoresis buffer, and store it in a 4°C refrigerator for standby.

[0128] Fifth step, electrophoresis separation

[0129] a, Place the SDS-PAGE gel and the protein sample to be tested in a room temperature environment, and prepare the rapid electrophoresis liquid according to the instructions of the Savillex rapid electrophoresis liquid powder; b, Fix the gel plate in the electrophoresis tank device, correctly connect the positive and negative electrode wires, inject sufficient electrophoresis buffer between the electrophoresis tank and the gel plate, make the liquid level flush with the top end of the gel, remove the sample loading comb vertically, and ensure the integrity of the hole; c, Use a micro-volume sample injector to add 10-20 μL of the sample to be tested in the sample loading hole, and add 5 μL of protein molecular weight standard as a reference in the two side edge holes; d, Supplement the electrophoresis buffer to the specified liquid level, set a constant voltage of 200V for electrophoresis separation, and run for 30 min until the tracer reaches the bottom of the separation gel.

[0130] Sixth step, membrane transfer

[0131] a, Measure the size of the SDS-PAGE gel and cut the PVDF membrane to the corresponding specification. Prepare the rapid membrane transfer working solution according to the instructions of the Savillex membrane transfer reagent; b, Membrane activation treatment: inject anhydrous ethanol and membrane transfer liquid into different containers, immerse the PVDF membrane in anhydrous ethanol for 30s, and then transfer it to the membrane transfer liquid for equilibration; c, Cut off the unnecessary area, make a beveled mark on the initial loading side, stack the layers in the order of "filter paper-gel-PVDF membrane-filter paper", use a roller to remove air bubbles between the layers, and close the membrane transfer clamp after confirming that the electrode direction is correct; d, Place the assembled transfer device vertically in the membrane transfer tank, and supplement the membrane transfer liquid to completely cover it. Place the membrane transfer tank in an ice bath environment, set a constant voltage of 400V, and run for 60-72 min; e, Soak the PVDF membrane in TBST for 3 times, 5 min each time, to ensure that the residual membrane transfer liquid is washed away.

[0132] Seventh step, antibody incubation

[0133] a, immerse the transferred PVDF membrane into the rapid sealing buffer and place it on the horizontal shaker (low speed mode) for sealing treatment for 30 min; b, use TBST buffer to rinse the PVDF membrane on the horizontal shaker at a high speed for 3 times, 10 min each time; c, according to the molecular weight of the target protein, accurately cut the membrane and immerse the target band into the corresponding primary antibody working solution and incubate it in the refrigerator at 4°C overnight; d, the washing method is the same as above; e, immerse the PVDF membrane into the corresponding secondary antibody solution and incubate it at room temperature for 2 h; f, the washing method is the same as above.

[0134] Step 8, chemiluminescence detection of protein (ECL)

[0135] a, mix Abbkine chemiluminescence substrate A and B in equal volume and then add them to the PVDF membrane; b, use Tian Neng T400 gel imaging system to capture the fluorescent signal and use Image J image analysis software to quantitatively analyze the gray value of the target band.

[0136] 32) Real-time fluorescent quantitative PCR (qPCR)

[0137] Step 1, use Nuoyuan cell DNA extraction kit to extract cell DNA

[0138] a, centrifuge AD-MSCs and iAD-MSCs (1000 rpm, 5 min) respectively to collect the cells, and then add 220 μL of PBS, 10 μL of RNase Solution and 20 μL of Proteinase K successively after sucking off the supernatant, suspend and lyse at room temperature for 15 min; b, add 250 μL of Buffer GB to the lysis system, vortex well and then treat at 65°C water bath for 15-30 min; c, add 180 μL of absolute ethanol, vortex well for 15-20 s to form a homogeneous solution; d, transfer the mixed solution to the adsorption column (preloaded in a 2 mL collection tube) and centrifuge at 12000 rpm for 1 min; e, discard the filtrate, add 500 μL of Washing Buffer A to the adsorption column and then centrifuge at 12000 rpm for 1 min; f, discard the filtrate, add 650 μL of Washing Buffer B to the adsorption column and then centrifuge at 12000 rpm for 1 min; g, repeat the above step again, discard the filtrate, and remove the residual by centrifuging the empty tube at 12000 rpm for 2 min; h, transfer the adsorption column to a sterile 1.5 mL centrifuge tube, add 50 μL of Elution Buffer preheated to 70°C and stand for 3 min, and then centrifuge (12000 rpm, 1 min) to collect the DNA solution; i, use Nano Drop ultramicro spectrophotometer to quantitatively analyze the solution.

[0139] Step 2, qPCR

[0140] Reference literature of internal reference gene 36B4 and target gene Telomere are shown in Table 1. qPCR system is shown in Table 2. ABI Step One Plus real-time fluorescent quantitative PCR system is used for amplification of samples, and the running program parameters are shown in Table 3. The data is normalized by using the expression level of 36B4, and the relative quantitative analysis is carried out by 2-ΔΔCt method. Three parallel holes are set for each sample, and the independent test is repeated for three times.

[0141] Table 1 qPCR primer sequence

[0142]

[0143] Table 2 qPCR reaction system

[0144]

[0145] Table 3 qPCR reaction program

[0146]

[0147] 33) Cell growth curve

[0148] The cell growth curve is determined by using Solapao cell proliferation and toxicity detection kit (CCK-8), and the method is as follows:

[0149] 331) Inoculate 5×10 3 cells per hole into 7 96-hole cell culture plates, set 3 parallel holes every day, and take out one 96-hole plate every 24 h for detection;

[0150] 332) Aspirate the culture medium, and replace it with 100 μL of fresh complete culture medium, add 10 μL of CCK-8 reagent per hole, and incubate in a 37℃ cell incubator for 1-4 h;

[0151] 333) Use the enzyme-labeled instrument to determine the absorbance value at 450 nm;

[0152] 334) Use GraphPad Prism 9.0 to analyze the data and generate the growth curve of the cells.

[0153] The results are as follows Figure 4 , 5As shown in FIGS. 6A and 6B, the expression of Klotho protein in AD-MSCs gradually decreased with the increase of passages, and the difference was significant (P < 0.05), and the expression of P21 protein gradually increased with the increase of passages, and the difference was significant (P < 0.05). The expression of Klotho protein in iAD-MSCs showed a decreasing trend, but there was no significant difference (P > 0.05), and the expression of P21 protein showed no trend, and the difference was not significant (P > 0.05). This shows that iAD-MSCs do not significantly age with cell passages. The experimental results show that the telomere length of AD-MSCs significantly shortens with passages (P < 0.01), and the shortening degree of P3 to P5 is significantly higher than that of P5 to P9, as shown in FIG. 6C. Figure 5 In addition, Figure 5 The telomere length of iAD-MSCs of different passages significantly increased (P < 0.05) compared with the control group of P2 AD-MSCs in FIG. 6D, and there was no obvious trend and no significant difference (P > 0.05) between different passages. The growth ability of AD-MSCs of the second passage and the eighth passage was different, and the growth ability of P8 was lower than that of P2. The growth ability of iAD-MSCs showed no obvious difference. The growth curves of the two kinds of cells showed an "S" shape, but the growth curve of iAD-MSCs showed a certain degree of decline when the density was too high.

[0154] 4) Feasibility determination

[0155] In order to track the migration process of cells administered nasally, a double-labeled tracing system was constructed based on the stable co-expression cell strain of Luciferase 2 luciferase and mCherry fluorescent protein according to the cell line establishment method in steps two to four. Specifically as follows:

[0156] 41) Screening of canine adipose-derived mesenchymal stem cells expressing Luciferase luciferase and mCherry fluorescent protein (Lm-iAD-MSCs)

[0157] a, the cells obtained in step two were seeded in 10 cm cell culture dishes at a density of 10%, and after the cells adhered, the complete medium containing 0.22 μg / mL of hygromycin B was replaced; b, the culture was maintained for 14 days, and after the cells in the negative control group died, the complete medium containing 0.11 μg / mL of hygromycin B was replaced for continued culture until the round-topped single-cell clone colonies with clear boundaries were formed; c, after the complete medium was removed and the round-topped single-cell clone colonies with clear boundaries were selected under a microscope, a suitable amount of sterilized silicone grease was taken with a cloning ring, and the cloning ring was accurately fixed on the target cloning area to form a closed space, and after the silicone grease was dried; 200 μL of trypsin digestion solution was added into the cloning ring, and after 1-1.5 min of digestion, the cell suspension was obtained by gentle blowing and transferred into a 24-well cell culture plate containing complete medium; d, the cell culture plate was placed in a cell culture incubator under standard culture conditions (37°C, 5% CO2) for culture and proliferation, and the cells in each well of the 24-well plate were sequentially digested into 12-well cell culture plates containing complete medium, and then expanded in T25 cell culture bottles containing complete medium, and the obtained different cell strains were subjected to functional evaluation tests, and the finally obtained immortalized canine adipose-derived mesenchymal stem cells were named as Lm-iAD-MSCs.

[0158] 42)IVIS Lumina detection of cell line luciferase expression

[0159] a, Lm-iAD-MSCs were seeded into a black 96-well cell culture plate at a density of 1 × 10 4 / well, and three parallel holes were set; b, the complete medium containing 1 mM luciferase substrate D-Luciferin sodium was prepared according to the MCE instructions; c, after the cells adhered, the old medium was aspirated and replaced with the above-mentioned medium, and placed in the IVIS Lumina III system for bioluminescence detection; d, the software was used for quantitative analysis and comparison of bioluminescence intensity, and the cell line with the highest luciferase expression was screened for use in the following tests.

[0160] 43)Cell line mCherry fluorescent protein expression

[0161] a, Cells were seeded in 12-well cell culture plates at an appropriate density, and when the cells grew to an appropriate density, the complete culture medium was aspirated; b, PBS was used to rinse 3 times, 5 min each time; c, 1 mL of 4 wt% paraformaldehyde fixing solution was added to each well, and fixed at room temperature for 15 min; d, the fixing solution was aspirated, and the rinsing step was repeated; e, the cells were permeabilized with 0.2% Triton X-100 at room temperature for 5 min; f, the permeabilization solution was aspirated, and the rinsing step was repeated; g, 100 μL of DAPI was added to each well, and incubated in the dark for 5 min; h, the DAPI was aspirated, and the rinsing step was repeated; i, an appropriate amount of anti-fluorescence quenching mounting medium was added, and then the cells were photographed under a fluorescence microscope.

[0162] The bioluminescence and fluorescence expression of Lm-iAD-MSCs are shown in Figure 7 、 Figure 8 and Figure 9 . Then, 5 x 10 5 cells / 12 μL were administered to the mice by intranasal administration, and the Luciferase 2 luciferase was detected by IVIS Lumina small animal live imaging technology at 0, 1, 3, 6, 12, 24, 72 and 120 h after single administration, and then the brain tissue was removed after mouse heart perfusion to detect the mCherry protein by tissue immunofluorescence technology. The experimental method is as follows:

[0163] Muscle injection of 5 mg / kg of Vetsulin 50 induced anesthesia, and multi-time point bioluminescence detection was performed according to the predetermined time sequence (0, 1, 3, 6, 12, 24, 72 and 120 h). After the experiment was terminated, the mouse was fixed on a foam board, and the chest was opened with scissors to expose the heart; a 0.5 mL syringe needle connected to a peristaltic pump was inserted into the left ventricle, and the other end was placed in low-temperature PBS, the right atrium was cut open, and then PBS was perfused at a rate of 10 mL / min; when the liver and limbs turned white, and the outflow was transparent liquid, it indicated that the blood was basically emptied; one end of the peristaltic pump was placed in a low-temperature 4 wt% paraformaldehyde fixing solution, and perfusion was performed at the same rate for 10 min; when the liver texture became hard, and the limbs stiff, the perfusion was stopped, and the brain tissue was collected; after the brain tissue was immersed and fixed in a 4 wt% paraformaldehyde fixing solution for 48 h, the surface liquid of the brain tissue was wiped clean and placed in a 30% sucrose solution for dehydration for 24-48 h; the sample was loaded into an embedding box, and OCT embedding agent was uniformly perfused to cover all the tissues; immediately transferred to pre-cooled anhydrous ethanol, and frozen at a speed of 30 min, then transferred to a-80°C refrigerator for 48 h; the target area was continuously cut at a thickness of 20 μm using a freezing microtome, dried at 37°C for 10-20 min, immersed in fixing solution for 30 min, and gently washed in PBS for 3 times, 5 min each time; after the surface of the slice was slightly dried, a hydrophobic pen was used to draw a staining and sealing circle. DAPI nuclear staining agent was added dropwise, incubated at room temperature for 10 min in the dark, and used to label the cell nucleus; the slice was gently washed in PBS for 4 times, 5 min each time. Covering the anti-fluorescence quenching mounting agent completed the slide mounting treatment; the laser confocal microscope was used to capture the fluorescence signal.

[0164] The results, as shown in Figure 10 and Figure 11 , show that the bioluminescence intensity of the cells gradually decreased over time and disappeared within the first three days. At 12 h, the cells appeared in the abdominal cavity, which may be due to the fact that part of the cells entered the digestive system through the nasopharyngeal pathway during administration. One hour after administration, the mCherry positive signal was mainly located in the Olfactory Nerve Layer (ONL) and Glomerular Layer (GL) of the olfactory bulb, indicating that the cells initially migrated through the olfactory nerve pathway; after 3 h, the signal spread to the External Plexiform Layer (EPL) of the olfactory bulb, showing the progressive infiltration of the cells along the hierarchical structure of the olfactory bulb. At 12 h, the Granule Cell Layer (GCL) appeared signal, and fluorescence signals were detected in the frontal-parietal cortex of the forebrain, hippocampus, lateral ventricle and cerebellum. At 24 h, the signal further infiltrated the hindbrain structures, including the fourth ventricle and brainstem. Notably, Figure 11After 3 days, the signal distribution changed: only the GL and EPL in the olfactory bulb retained signals, the forebrain signals were concentrated around the lateral ventricles, and the hindbrain signals were distributed in the cerebellum, fourth ventricle, and brainstem regions; after 5 days of administration, signals were only detectable in the ONL and GL in the olfactory bulb and the thalamus and cortex of the forebrain. Finally, at 7 days, no fluorescent signals were detected in the brain tissue, indicating that the cells degraded within 5 to 7 days.

[0165] 5) Safety testing

[0166] The safety of iAD-MSCs was then tested. Twenty-four immunodeficient mice (BalB / c-nu) were divided into four groups of six mice each: the CTRL group (no nasal feeding), the PBS group (nasal feeding with PBS), the iAD-MSCs group (nasal feeding with iAD-MSCs), and the U87 cell group (nasal feeding with U87 cells). The cells were administered intranasally twice weekly, with each administration containing approximately 5 × 10⁻⁶ cells. 6 The animals were administered the medication for a total of 8 weeks, followed by a 2-month rearing period. Brain and nasal tissues were collected via cardiac perfusion. The brain tissue was sectioned coronally and stained using hematoxylin and eosin (HE) staining. The experimental results are as follows: Figure 12 As shown, lymphocyte and granulocyte infiltration was observed in the lamina propria of the nasal cavity of mice in the immortalized mesenchymal stem cell group (black arrows), while no obvious abnormalities were observed in the nasal cavities of other mice. A small number of round vacuoles were observed in the olfactory bulb, hippocampus, cortex, and cerebellum of the CTRL, PBS, and iAD-MSCs groups, with indistinct demarcation between neuronal nuclei and cytoplasm (yellow arrows), and dilated blood vessels (red arrows), but no obvious inflammatory cell infiltration, necrosis, or space-occupying lesions were observed. In the U87 group, a well-defined heterogeneous mass was observed near the hippocampus, containing significantly enlarged nuclei with widespread nuclear fragmentation and dissolution, encased in eosinophilic cytoplasm (green arrows). Furthermore, widespread neurofibrillary degeneration was observed in the cortex (blue arrows). These experimental results indicate that intranasal delivery of U87 induces neurological pathological structures, but delivery of iAD-MSCs, apart from inducing a small number of inflammatory cells in the nasal cavity, does not induce any abnormal neurological pathological structures.

[0167] Twenty-four mice (BalB / c) were then divided into four groups of six each: the CTRL group (no nasal feeding), the PBS group (nasal feeding with PBS), the iAD-MSCs group (nasal feeding with iAD-MSCs), and the U87 cell group (nasal feeding with U87 cells). Each mouse received one dose of the drug via intranasal administration, for a total of 5 × 10⁶ doses. 5 Cells. Mice were sacrificed by cervical dislocation one week and one month after drug administration, and brain tissue was collected. The brain tissue was then homogenized, and the levels of IL-1β, IL-2, IL-6, IL-10, and TNF-α in the brain tissue were detected. The experimental results are as follows: Figure 13As shown in the figure, there were no statistically significant differences in the levels of any cytokines between the PBS group and the CTRL group (P>0.05). The levels of all cytokines in the U87 group were significantly higher than those in the control group (P<0.05). In the iAD-MSCs group, IL-6 and TNF-α were significantly higher than those in the control group after 1 week of iAD-MSCs administration (P<0.01), while the levels of IL-1β and TNF-α were significantly lower after 1 month (P<0.05). The other indicators were not statistically significant (P>0.05).

[0168] 6) Effectiveness testing

[0169] Following the feasibility and safety evaluation of intranasal delivery of immortalized canine adipose-derived mesenchymal stem cells, it is necessary to evaluate the effectiveness of intranasal delivery. Therefore, this invention targets 4*FAD Alzheimer's disease model mice, delivering immortalized mesenchymal stem cells multiple times intranasally over one month. The therapeutic effect of the immortalized mesenchymal stem cells is then evaluated through behavioral tests, such as the Y-maze test and novel object recognition test, as well as immunofluorescence and ELISA tests, aiming to lay a solid foundation for the clinical application of immortalized mesenchymal stem cells.

[0170] Mice were divided into four groups of six each: 18 female B6 / JGpt-Tg(Thy-APP / Thy-PSEN1)5 / Gpt mice (4*FAD, 12 weeks old) and 6 wild-type C57BL / 6JGpt mice (12 weeks old). They were further divided into a wild-type control group (WL), an Alzheimer's disease model group (AD), an Alzheimer's disease model mouse placebo group (AD-PBS), and an Alzheimer's disease treatment group (AD-Treat). In the AD-Treat group, iAD-MSCs were delivered intranasally, iAD-MSCs were resuspended in PBS at a concentration of 5 × 10⁻⁶. 5 The AD-PBS group received 12 μL of PBS per dose, while the AD-PBS group received 12 μL of PBS per dose. Each mouse was administered the medication intranasally twice a week for a total of four weeks.

[0171] The mice underwent two phases of the NORT experiment. The results of the first phase exploration time are as follows: Figure 14 As shown in Figure A, no significant differences were observed among the four groups of mice (P>0.05). The results of the discrimination index in the second stage are as follows: Figure 14 As shown in Figure B, the AD group was significantly lower than the WL group (P<0.05), while there was no significant difference between the AD group and the AD-PBS group (P>0.05). The AD-Treat group, however, was significantly higher than both the AD-PBS group and the WL group (P<0.05). These results indicate that intranasal delivery of iAD-MSCs can effectively improve short-term memory in AD mice.

[0172] The results of the Y-maze experiment are as follows:Figure 15 As shown in FIG. 6, the alternation success rate of spontaneous alternation behavior of the WL group mice was significantly higher than that of the AD group (P < 0.01), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the AD-Treat group was significantly higher than the AD-PBS group (P < 0.05), and no significant difference was found between the AD-Treat group and the WL group (P > 0.05). The experimental results showed that intranasal delivery of iAD-MSCs could effectively improve the spatial recognition ability and working memory ability of AD mice.

[0173] The mouse brain tissue amyloid protein (Amyloid-β, Aβ) was labeled using Thioflavin S, the newborn neurons were labeled using DCX, and the astrocytes were labeled using GFAP, and the immunofluorescence results are shown in FIG. 7. Figure 16 Figure 17 As shown in FIG. 7, the number of amyloid plaques of the AD group was significantly higher than that of the WL group (P < 0.01), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the number of amyloid plaques of the AD-Treat group was significantly lower than that of the AD-PBS group (P < 0.01), but significantly higher than that of the WL group (P < 0.01). The number of astrocytes of the AD group was significantly higher than that of the WL group (P < 0.05), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the number of astrocytes of the AD-Treat group was significantly lower than that of the AD-PBS group (P < 0.01), but no significant difference was found between the AD-Treat group and the WL group (P > 0.05). The number of newborn neurons of the AD group was significantly lower than that of the WL group (P < 0.01), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the number of newborn neurons of the AD-Treat group was significantly higher than that of the AD-PBS group (P < 0.01), but no significant difference was found between the AD-Treat group and the WL group (P > 0.05).

[0174] The results of the Aβ concentration and the phosphorylated tubulin associated unit (p-Tau) concentration of the mouse brain tissue are shown in FIG. 8. Figure 16 As shown in FIG. 8, the total Aβ concentration of the WL group was significantly lower than that of the AD group (P < 0.01), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the total Aβ concentration of the AD-PBS group was significantly higher than that of the AD-Treat group (P < 0.01), and the total Aβ concentration of the AD-Treat group was significantly higher than that of the WL group (P < 0.01). The p-Tau concentration of the WL group was significantly lower than that of the AD group (P < 0.01), no significant difference was found between the AD group and the AD-PBS group (P > 0.05), the p-Tau concentration of the AD-PBS group was significantly higher than that of the AD-Treat group (P < 0.05), and the p-Tau concentration of the AD-Treat group was significantly higher than that of the WL group (P < 0.01).

[0175] Preparation method of immortalized adipose-derived mesenchymal stem cells​

[0176] Embodiments 2-5 are each a method for preparing immortalized adipose-derived mesenchymal stem cells, wherein the preparation processes in Embodiments 2-5 are basically the same as those in the experimental group in Steps 1-4 of Embodiment 1, except that some process parameters are different, and the specific details are shown in Table 4:

[0177] Table 4: Formulation list of Embodiments 6-9

[0178]

[0179] The other parts of Embodiments 6-9 are the same as Embodiment 2, and are not repeated here.

[0180] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

Claims

1. A method for preparing immortalized adipose-derived mesenchymal stem cells, characterized by, The preparation method comprises the following steps: I. Isolation of adipose-derived mesenchymal stem cells After cleaning the surface blood vessels and connective tissue of the adipose tissue, the adipose-derived mesenchymal stem cells are obtained by washing, cutting, digestion, rinsing and culturing in sequence; II. Establishment of immortalized adipose-derived mesenchymal stem cells 1) The adipose-derived mesenchymal stem cells are inoculated into complete culture medium for culture, and then are replaced with opti-MEM culture medium for culture, so as to obtain cells to be transfected; 2) The cells to be transfected are taken, and a DNA-liposome complex mixed solution is added for incubation, and then the complete culture medium is replaced for stable culture, and the cells are subcultured for one generation, so as to obtain cells to be screened; III. Screening of immortalized adipose-derived mesenchymal stem cells The cells to be screened are taken and sequentially cultured in complete culture medium, complete culture medium containing different gradients of hygromycin, digestion, proliferation in complete culture medium, re-digestion and expansion in complete culture medium, so as to obtain the immortalized adipose-derived mesenchymal stem cells.

2. The immortalized adipose-derived mesenchymal stem cell preparation method according to claim 1, characterized in that, The preparation method of the DNA-liposome complex mixed solution in the process of establishing the immortalized adipose-derived mesenchymal stem cells is as follows: The opti-MEM culture medium is mixed with Lipofectamine 3000 to obtain a liposome diluent; The opti-MEM culture medium is taken, 1 μg of piggyBac plasmid, PBase plasmid and P3000 reagent are added and mixed to obtain a DNA premix solution; The liposome diluent and the DNA premix solution are mixed in equal volume, and then are left at room temperature for standing, so as to obtain the DNA-liposome complex mixed solution.

3. The immortalized adipose-derived mesenchymal stem cell preparation method of claim 1, wherein the immortalized adipose-derived mesenchymal stem cell preparation method is a method for preparing immortalized adipose-derived mesenchymal stem cells by introducing a telomerase gene into adipose-derived mesenchymal stem cells. The preparation method further comprises step four: culture and subculture of the immortalized adipose-derived mesenchymal stem cells, which are specifically as follows: 1) The immortalized adipose-derived mesenchymal stem cells are inoculated into complete culture medium for culture; 2) When the cells are confluent to 85-90%, the impurities, cell fragments and residual culture medium are removed by washing; 3) The trypsin digestion solution is added for digestion, and then the complete culture medium is added to terminate the digestion, and then the mixture is mixed; 4) Centrifugation is performed, the supernatant is discarded, the cells are resuspended with complete culture medium, and then the cells are cultured, so as to obtain the subcultured immortalized adipose-derived mesenchymal stem cells.

4. The immortalized adipose-derived mesenchymal stem cell preparation method according to claim 3, characterized in that, During the culture and subculture of the immortalized adipose-derived mesenchymal stem cells, In step 1), the culture process is constant-temperature culture, wherein the complete culture medium is replaced every 2-3 days; In step 2), the washing reagent is sterile PBS buffer; In step 3), the digestion temperature is 37℃, and the digestion time is 1-1.5 min; In step 4), the culture is constant-temperature culture, wherein the complete culture medium is replaced every 2-3 days.

5. The method for preparing immortalized adipose-derived mesenchymal stem cells according to any one of claims 1-4, characterized in that, The specific process of screening the immortalized adipose-derived mesenchymal stem cells is as follows: 1) The adipose-derived mesenchymal stem cells are resuspended in complete culture medium, and then are inoculated into a cell culture dish, and after the cells adhere, the complete culture medium containing 60-70 μg / mL of hygromycin B is replaced; 2) The culture is maintained for 13-15 days, the complete culture medium containing 45-55 wt% of the screening concentration of hygromycin B is replaced for continuous culture, all the culture medium is removed, the PBS buffer is used for washing, the obtained round dome-shaped single cell clone colony is placed in a closed space, the trypsin digestion solution is added for digestion, the cell suspension is obtained by using a gentle blowing method, and then is transferred into the complete culture medium; 3) culture proliferation, then digest into complete culture medium and culture for 3-5 days, then digest the cells into complete culture medium and expand to 80-90% of the cell culture dish, and finally obtain the immortalized adipose-derived mesenchymal stem cells.

6. The method for preparing immortalized adipose-derived mesenchymal stem cells according to claim 5, characterized in that, In the screening process of the immortalized adipose-derived mesenchymal stem cells, In step 2), the digestion time is 1-1.5 min.

7. The method for preparing immortalized adipose-derived mesenchymal stem cells according to any one of claims 1-4 and 6, characterized in that, The specific process of establishing the immortalized adipose-derived mesenchymal stem cells is as follows: 1) Take the adipose-derived mesenchymal stem cells and inoculate them into complete culture medium for 22.5-23 h, replace them with opti-MEM medium for 1-1.5 h, and obtain the cells to be transfected; 2) Take the cells to be transfected, add DNA-liposome complex mixed solution, shake gently to distribute the solution uniformly, then incubate at 36-38℃ for 4-8 h, then replace with complete culture medium, continue to stabilize culture for 24-48 h, then pass one generation, and obtain the cells to be screened.

8. The method for preparing immortalized adipose-derived mesenchymal stem cells according to any one of claims 1-4 and 6, characterized in that, The specific process of isolating the adipose-derived mesenchymal stem cells is as follows: In a sterile environment, clean the surface blood vessels and connective tissue of the adipose tissue, then wash it with HBSS, then cut the adipose tissue, drop HBSS buffer solution during cutting, then add tissue digestion solution to the cut adipose tissue, shake to make it a creamy thick liquid, then pour it into complete culture medium to stop digestion, then filter, centrifuge, discard the upper liquid, resuspend with red blood cell lysis solution, incubate at room temperature, then add PBS buffer solution to stop digestion, filter, centrifuge, discard the upper liquid, and add PBS buffer solution containing an amikacin mixture for rinsing, centrifuge again, discard the PBS buffer solution, resuspend with complete culture medium, and then culture to obtain the adipose-derived mesenchymal stem cells.

9. The immortalized fat-derived mesenchymal stem cell produced by the production method according to any one of claims 1 to 8, characterized in that, The immortalized adipose-derived mesenchymal stem cells have anti-aging ability.

10. Use of the immortalized fat-derived mesenchymal stem cells according to claim 9, characterized in that, The application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating neurological diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating respiratory system diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating cardiovascular diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating bone and joint diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating urinary system diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating digestive system diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating skin diseases; Or, the application of the immortalized adipose-derived mesenchymal stem cells in the preparation of a drug for treating autoimmune diseases.

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