HUCMSCs capable of efficiently repairing ischemic injury as well as preparation method and application of hUCMSCs

Abstract

The invention relates to hUCMSCs for efficient ischemic injury repair and a preparation method and application thereof, and relates to the technical field of biology, the preparation method comprises the following steps: obtaining P2-generation hUCMSCs cells; under the conditions that the oxygen concentration volume fraction range is 1%-10% and an inflammatory factor pre-activation composition is added, subculture multiplication culture is conducted on the P2-generation hUCMSCs cells, and the hUCMSCs for efficient ischemic injury repair are obtained, wherein the inflammatory factor pre-activation composition is prepared from the following components: 10 to 50 ng / ml of TNF (Tumor Necrosis Factor)-a, 10 to 50 ng / ml of IL-1beta and the like. According to the invention, the problems of low content of paracrine cytokines for promoting angiogenesis and inhibiting inflammatory response, poor in-vivo microenvironment adaptability and the like of the existing hUCMSCs are solved, the hUCMSCs can play a better role in repairing tissue ischemic injury, and the defects of the prior art are overcome.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to hUCMSCs for highly efficient ischemic damage repair and a preparation method and application thereof. Background Art

[0002] Limb artery ischemic disease includes a variety of diseases such as lower extremity arteriosclerosis obliterans (ASO), thromboangiitis obliterans (TAO) and diabetic limb artery occlusive disease (DLAO). Its clinical manifestations are diverse, including weakness of the affected limb, intermittent claudication, rest pain, muscle atrophy, and even ulcers, necrosis and even the risk of amputation in severe cases. This type of disease not only brings great pain to patients, but also has a long and complicated treatment process. It is also accompanied by high disability and mortality rates, which seriously reduces the quality of life of patients and causes heavy economic and social burdens. At present, clinical treatment mainly relies on arterial bypass grafting and endovascular technology, but these methods are limited by strict indications, and the prognosis is often unsatisfactory, especially for the elderly and frail, who cannot tolerate surgery or have poor endovascular treatment effects, and are at risk of amputation. There is a lack of effective treatment methods. Therefore, exploring new treatment methods and drugs to deal with peripheral arterial occlusive disease has become an urgent clinical need.

[0003] In recent years, a large number of studies have shown that the paracrine mechanism of mesenchymal stem cells (UCMSCs) plays a vital role in improving lower limb ischemia. These stem cells secrete a series of bioactive molecules, such as epithelial growth factor, vascular endothelial growth factor, basic fibroblast growth factor, fibroblast growth factor, blood cell growth factor, adhesion protein and angioprotein-1, which act on specific signal regulation pathways, activate and promote the migration, proliferation and differentiation of fibroblasts and endothelial cells, and thus accelerate angiogenesis. This process can significantly increase the blood supply to the ischemic area, thereby improving the healing ability of ischemic ulcers. In addition, these cytokines also have the function of inhibiting inflammatory response at the wound site, which helps to accelerate the wound healing process. However, conventional mesenchymal stem cells (UCMSCs) face challenges in application, mainly manifested in the low content of their paracrine pro-angiogenic factors and anti-inflammatory response factors, and limited adaptability to the microenvironment in the body, which greatly limits their therapeutic effect.

[0004] On the other hand, oxygen concentration is a key factor affecting the growth of cells and microorganisms, and its regulation is crucial for cell culture. For example, mouse embryonic fibroblasts cultured in a hypoxic environment of 3% showed a healthier growth state, less DNA damage, and a milder stress response compared to a conventional oxygen concentration of 20%. In experiments simulating acute inflammatory environments, TNF-α and IL-1β, as inflammatory cytokines highly expressed in ischemic and damaged tissues, are often used in research. However, conventional mesenchymal stem cells (such as hUCMSCs, human umbilical cord mesenchymal stem cells, etc.) are difficult to fully realize their potential in the repair of tissue ischemic damage due to insufficient paracrine factor content and poor adaptability to the in vivo microenvironment.

[0005] In summary, for the treatment of limb artery ischemic diseases, it is urgent to develop new mesenchymal stem cell therapies or related drugs that can effectively promote angiogenesis, inhibit inflammatory responses, and adapt to the complex microenvironment in the body to meet clinical needs and improve patient prognosis. Summary of the invention

[0006] In order to solve the above problems, the present invention provides hUCMSCs with high efficiency in repairing ischemic damage and a preparation method and application thereof.

[0007] In a first aspect, the present invention provides a method for preparing hUCMSCs with high efficiency in repairing ischemic damage, the preparation method comprising the following steps:

[0008] Obtain P2 generation hUCMSCs cells;

[0009] Under the condition of an oxygen concentration volume fraction range of 1%-10% and the addition of an inflammatory factor pre-activation composition, the P2 generation hUCMSCs cells are subcultured and expanded to obtain the hUCMSCs with high efficiency in repairing ischemic damage;

[0010] Wherein, the inflammatory factor pre-activation composition comprises the following components: 10-50ng / ml TNF-a, 10-50ng / ml IL-1β, 0-20umol / LLPA, and 10-100ng / ml HGF.

[0011] Furthermore, the step of obtaining P2 generation hUCMSCs cells includes: isolating from umbilical cord tissue, and obtaining P2 generation hUCMSCs cells by adherent culture method.

[0012] Furthermore, the step of obtaining P2 generation hUCMSCs cells includes the following process:

[0013] Step 1: Cut and clean the gelatin and place it in a culture dish for later use;

[0014] Step 2: Inoculate the treated tissue blocks in a culture bottle with a spacing of 1-2 cm between tissue blocks, and incubate at 37°C and 5.0% CO2;

[0015] Step 3: Add human mesenchymal stem cell serum-free culture medium, inoculate labeled hUCMSCs P0, observe cell growth regularly and change the medium;

[0016] Step 4: hUCMSCs P0 cells are grown to a confluence of 80%-90%, and the P0 cells are digested and collected. The tissue blocks are removed by centrifugation to obtain P0 umbilical cord mesenchymal stem cells hUCMSCs P0;

[0017] Step 5: Inoculate hUCMSCs P1 at 1*10^6-2*10^6 / T75cm2 flask, culture to 80%-90% confluence and then collect again, subculture and collect cells to obtain P2 generation hUCMSCs cells.

[0018] Furthermore, step 4 includes the following process: culturing for 12-15 days, hUCMSCs P0 cells grow to a confluence of 80%-90%, discarding the supernatant culture medium, adding 3-5 ml / bottle of physiological saline to wash 1-2 times, using 0.25% trypsin / EDTA digestion solution, 0.5-1 ml / bottle to digest the crawling cells and tissue blocks, and using 1-3 ml / bottle of serum-free trypsin stop solution to terminate the digestion; using 50 ml to collect tissue blocks and cell suspension, centrifuging at 1000-2000 rpm for 5 minutes, discarding the supernatant, adding an appropriate amount of physiological saline to mix, filtering with a 70-100 μm filter to remove tissue blocks, and obtaining P0 generation umbilical cord mesenchymal stem cells hUCMSCs P0.

[0019] Furthermore, step 5 includes the following process: inoculating hUCMSCsP1 at 1*10^6-2*10^6 / T75cm2 bottle, adding 10-20ml human mesenchymal stem cell serum-free culture medium to each bottle, placing it in a 37°C 5.0% CO2 incubator and culturing it every 2-4 days until the cell fusion rate reaches 80%-90%, repeating step 4 to collect hUCMSCs P1 generation cells, and passing hUCMSCs P2 by cell counting at 1:3. When the cell fusion rate reaches 80%-90%, repeating step 4 to collect hUCMSCs P2 generation cells.

[0020] Furthermore, the step of subculturing the P2 generation hUCMSCs cells under the condition of an oxygen concentration volume fraction range of 1%-10% and the addition of an inflammatory factor pre-activation composition to obtain the hUCMSCs with high efficiency in repairing ischemic damage comprises the following process:

[0021] hUCMSCs-P2 generation cells were subcultured and expanded using a three-gas incubator with an oxygen volume fraction concentration range of 1%-10%, 20-30ml / T175cm2 human mesenchymal stem cell serum-free culture medium, an inflammatory factor pre-activation composition, and hUCMSCs-P3 were subcultured at a ratio of 1:3 according to cell count. The cells were cultured for 2-4 days. When the cell fusion rate reached 80%-90%, step 4 was repeated to collect hUCMSCs P3 generation cells and their cell supernatant.

[0022] In a second aspect, the present invention provides hUCMSCs with high efficiency in repairing ischemic damage, wherein the hUCMSCs with high efficiency in repairing ischemic damage is prepared by any preparation method described in the first aspect.

[0023] Furthermore, the VEGF protein secretion level of the hUCMSCs with high efficiency in repairing ischemic damage was 483.76±92.34 pg / ml.

[0024] In a third aspect, the present invention provides a use of the hUCMSCs with high efficiency in repairing ischemic damage as described in any one of the first aspect and the second aspect in the preparation of a drug for treating limb artery ischemic diseases.

[0025] The above technical solution provided by the embodiment of the present invention has at least the following advantages compared with the prior art:

[0026] The embodiment of the present invention provides hUCMSCs for efficient ischemic injury repair and its preparation method and application. The present invention, under the specific conditions of oxygen concentration volume fraction range of 1%-10% and addition of inflammatory factor pre-activation composition, the P2 generation hUCMSCs cells are cultured for subculture, so that UCMSCs adapt to the pathological microenvironment of hypoxia in vivo during the in vitro induction culture stage, induce UCMSCs to remember the damaged microenvironment, protect UCMSCs against adverse microenvironment, and maintain gene stability. Maintaining the undifferentiated state of UCMSCs, promoting the proliferation and survival of UCMSCs, and promoting the enhancement of its paracrine function, such as the increase in the secretion of angiogenic factors VEGF, HGF and basic fibroblast growth factor bFGF, EGF, etc., so that it exhibits better characteristics and has a better repair effect of lower limb ischemic injury, solves the problems of low content of paracrine angiogenic and inflammatory inhibitory cytokines in existing hUCMSCs, poor ability to adapt to the microenvironment in vivo, and makes up for the shortcomings of the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0029] Figure 1 This is a CCK8 proliferation curve diagram of each group of cells in the present invention.

[0030] Figure 2 The concentration and protein ratio of exosome particles of hUCMSCs in each group in the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0033] The invention solves the problems of conventional mesenchymal stem cells (hUCMSCs) such as low content of paracrine cytokines that promote angiogenesis and inhibit inflammatory response and poor ability to adapt to the microenvironment in the body, so that hUCMSCs can better repair tissue ischemic damage.

[0034] To solve the above problems, the technical solution provided by the present invention is as follows:

[0035] The present invention relates to hUCMSCs for highly efficient ischemic injury repair and a preparation method and application thereof, comprising:

[0036] 1. Isolate from umbilical cord tissue and obtain P2 hUCMSCs cells by adherent culture method.

[0037] ①Umbilical cord tissue with no infectious diseases, no genetic diseases, and normal chromosomes should be selected, and the length of the tissue should be 15-20cm. Use sterile flat scissors to cut the umbilical cord into 2-3cm tissue segments, wash it repeatedly with 0.9% saline for 2-3 times to clean the residual blood in the tissue, separate the 2 arteries and 1 vein of the umbilical cord with sterile tissue forceps, peel off the Walton's jelly, cut the Wharton's jelly into 0.5-2cm tissue blocks with sterile flat scissors, wash it repeatedly with 0.9% saline for 2-3 times, and place it in a 9cm sterile culture dish for attachment and culture.

[0038] ② Use a cell scraper to inoculate the colloidal tissue blocks into a T75cm2 breathable culture flask. The interval between the tissue blocks is 1-2cm to reserve growth space for hUCMSCs P0 cells. After inoculation, place the cells in a 37℃ 5.0% CO2 incubator for 20-30 minutes to make the tissue blocks adhere more firmly to the wall.

[0039] ③ Add 5-10ml / T75cm2 human mesenchymal stem cell serum-free culture medium (Tianjin Haoyang SC2013-GA 450ml, SC2013-GB 50ml, fully mixed) to inoculate the labeled hUCMSCs P0, observe whether there is contamination on the second day, change 3-5ml of medium every 2-3 days and a half, and observe the cells crawling out (pay attention to the smooth handling of the culture bottle during the process to avoid the movement of the tissue block.

[0040] ④ After culturing for about 12-15 days, hUCMSCs P0 cells grow to a confluency of 80%-90%, discard the supernatant culture medium, add 3-5ml / bottle of physiological saline to wash 1-2 times, use 0.25% trypsin / EDTA digestion solution (Tianjin Haoyang TE2004Y), 0.5-1ml / bottle to digest and crawl out cells and tissue blocks, and use serum-free trypsin stop solution (Tianjin Haoyang SC2013-GE) 1-3ml / bottle to stop digestion; use 50ml to collect tissue blocks and cell suspension, centrifuge at 1000-2000rpm for 5min, discard the supernatant, add appropriate amount of physiological saline to mix, filter with 70-100μm filter to remove tissue blocks, and obtain P0 generation umbilical cord mesenchymal stem cells hUCMSCs P0;

[0041] ⑤ Count the cells and inoculate hUCMSCs P1 at 1*10^6-2*10^6 / T75cm2 flask. Add 10-20 ml of human mesenchymal stem cell serum-free culture medium to each flask and culture in a 37°C 5.0% CO2 incubator. Repeat step ④ to collect hUCMSCs P1 cells every 2-4 days when the cell fusion rate reaches 80%-90%. Count the cells and pass them to hUCMSCs P2 at a ratio of 1:3. Repeat step ④ to collect hUCMSCs P2 cells when the cell fusion rate reaches 80%-90%.

[0042] 2. Comparison of hUCMSCs-P3 and conventional UCMSCs-P3 culture methods for efficient ischemic injury repair

[0043] The hUCMSCs-P2 cells harvested from step 1-⑤ were subcultured and expanded, and the hUCMSCs-P3 cells were subcultured at a ratio of 1:3 according to the cell count. The cells were cultured for 2-4 days. When the cell fusion rate reached 80%-90%, steps 1-④ were repeated to collect hUCMSCs P3 cells and their cell supernatant.

[0044] The culture methods are compared as follows:

[0045] Experimental group: using a three-gas incubator (providing a low oxygen culture environment, oxygen concentration range 1% -10%), 20-30ml / T175cm2 human mesenchymal stem cell serum-free culture medium (Tianjin Haoyang SC2013-GA 450ml, SC2013-GB 50ml fully mixed), inflammatory factor pre-activation composition (10-50ng / ml TNF-a, 10-50ng / ml IL-1β, 0-20umol / LLPA, 10-100ng / ml HGF), and a culture period of 2-4 days.

[0046] It should be noted that the components and raw materials involved in the hUCMSCs for efficient ischemic damage repair and the preparation method and application thereof provided in the embodiments of the present invention, unless otherwise specified or specified, can be directly commercially available products or homemade using existing public preparation methods; at the same time, the steps and parameters involved, unless otherwise specified or specified, can be carried out according to existing processing techniques or directly using existing equipment, and the present invention document will not describe them one by one.

[0047] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually measured according to national standards. If there is no corresponding national standard, then the conditions recommended by the manufacturer are followed.

[0048] Example 1

[0049] This example provides hUCMSCs with high efficiency in repairing ischemic damage. The preparation method of hUCMSCs with high efficiency in repairing ischemic damage comprises the following steps:

[0050] 1. Isolate from umbilical cord tissue and obtain P2 hUCMSCs cells by adherent culture method.

[0051] ①Umbilical cord tissue with no infectious diseases, no genetic diseases, and normal chromosomes should be selected, and the length of 20 cm should be collected under sterile operation. The umbilical cord should be cut into 2 cm tissue segments with sterile flat scissors, and the residual blood in the tissue should be washed repeatedly with 0.9% saline for 3 times. Two arteries and one vein of the umbilical cord should be separated with sterile tissue forceps, and Walton's jelly should be peeled off. The Wharton's jelly should be cut into 0.5 cm tissue blocks with sterile flat scissors, and washed repeatedly with 0.9% saline for 2 times, and placed in a 9 cm sterile culture dish for attachment and culture.

[0052] ② Use a cell scraper to inoculate the colloidal tissue blocks into a T75cm2 breathable culture flask. The interval between tissue blocks is 1cm to reserve growth space for hUCMSCs P0 cells. After inoculation, place the cells in a 37℃ 5.0% CO2 incubator for 30 minutes to make the tissue blocks adhere more firmly.

[0053] ③ Add 10 ml / T75 cm2 human mesenchymal stem cell serum-free medium (Tianjin Haoyang SC2013-GA 450 ml, SC2013-GB 50 ml fully mixed) to inoculate the labeled hUCMSCs P0, observe whether there is contamination on the second day, replace 5 ml of medium every 3.5 days, and observe the cell crawling out (pay attention to the smooth handling of the culture bottle during the process to avoid the movement of the tissue block.

[0054] ④ After culturing for about 15 days, hUCMSCs P0 cells grow to a confluence of more than 85%, discard the supernatant culture medium, add 5 ml / bottle of physiological saline to wash twice, use 0.25% trypsin, 0.8 ml / bottle to digest the crawling cells and tissue blocks, and use 3 ml / bottle of stop solution to stop digestion; use a 50 ml centrifuge tube to collect tissue blocks and cell suspension, centrifuge at 1200 rpm for 5 min, discard the supernatant, add an appropriate amount of physiological saline to mix, and filter with a 70 μm filter to remove tissue blocks, thus obtaining P0 generation umbilical cord mesenchymal stem cells hUCMSCsP0;

[0055] ⑤ Count the cells and inoculate hUCMSCs P1 at 2*10^6 cells / T75cm2 flask. Add 15 ml of human mesenchymal stem cell serum-free medium to each flask and culture in a 37°C 5.0% CO2 incubator. Repeat step ④ every 3 days when the cell fusion rate reaches more than 85%, collect hUCMSCs P1 cells, count the cells and pass them to hUCMSCs P2 at 1:3. When the cell fusion rate reaches 85%, repeat step ④ to collect hUCMSCs P2 cells.

[0056] 2. Different effects of different oxygen concentrations and different inflammatory factor dosages on hUCMSCs-P3

[0057] The hUCMSCs-P2 cells harvested in step 1-⑤ were passaged and expanded, and the hUCMSCs-P3 was passaged at a ratio of 1:3 according to the cell count. The cells were cultured for 3 days in a three-gas incubator with a hypoxic environment of 1%, 3%, 5%, and 10%, respectively. Pre-activated inflammatory factors were added under different dosage conditions of TNF-a (10ng / ml, 25ng / ml, 50ng / ml), IL-1β (10ng / ml, 25ng / ml, 50ng / ml), LPA (5umol / L, 10umol / L, 20umol / L), and HGF (5ng / ml, 10ng / ml, 20ng / ml) for pre-activation culture. When the cell fusion rate reached 85%, steps 1-④ were repeated to collect hUCMSCs P3 cells and their cell supernatant. The ELISA method was used to detect the differences in the secretion of VEGF protein levels in the cell supernatant; the CCK8 method was used to detect the differences in the proliferation activity of UCMSCs-P3 cells cultured under different oxygen concentrations and inflammatory factor dosages. The results showed that the secretion of VEGF protein levels and cell proliferation activity were optimal when cultured in a 3% hypoxic environment and pre-activated with the addition of the following concentrations of inflammatory factors: 25ng / ml TNF-a, 10ng / ml IL-1β, 20umol / L LPA, and 20ng / ml HGF.

[0058] Example 2

[0059] This example compares the culture methods of hUCMSCs-P3 and conventional UCMSCs-P3 for high-efficiency ischemic injury repair based on the above Example 1, which specifically includes the following process:

[0060] Preparation of hUCMSCs-P2 cells Referring to Example 1, the hUCMSCs-P2 cells harvested in step 1-⑤ were subcultured and expanded, and the hUCMSCs-P3 cells were subcultured at a ratio of 1:3 according to the cell count, and cultured for 3 days. When the cell fusion rate reached 85%, steps 1-④ were repeated to collect hUCMSCs P3 cells and their cell supernatant.

[0061] The culture methods are compared as follows:

[0062] Experimental group: using a three-gas incubator (providing a hypoxic culture environment, oxygen concentration range: 3%), 30ml / T175cm2 human mesenchymal stem cell serum-free culture medium (Tianjin Haoyang SC2013-GA 450ml, SC2013-GB 50ml fully mixed), inflammatory factor pre-activation composition (25ng / ml TNF-a, 10ng / ml IL-1β, 20umol / L LPA, 20ng / ml HGF), culture period 3 days;

[0063] Control group: CO2 incubator (equipment parameters: 95% air, 5% CO2, oxygen concentration range of about 20%),

[0064] 30ml / T175cm2 human mesenchymal stem cell serum-free culture medium (Tianjin Haoyang SC2013-G-A450ml, SC2013-GB 50ml fully mixed), culture period 3 days.

[0065] 3. Comparison of the effect data of hUCMSCs-P3 and conventional UCMSCs-P3 for high-efficiency ischemic injury repair

[0066] 3.1CCK8 was used to detect the proliferation activity of hUCMSCs cells labeled in the experimental group and the control group.

[0067] (1) Take hUMSs P3 cells from the experimental group and the control group in the logarithmic growth phase and count the cells.

[0068] (2) The cells were seeded into a 96-well plate at a density of 20,000 cells / well and cultured according to the experimental plan of each group.

[0069] (3) After the cells adhered, CCK8 detection was performed after 0, 24, 48, and 72 hours.

[0070] (4) CCK8 detection experimental steps:

[0071] ① At the detection time point, 1 / 10 volume of CCK8 was added to each well of each group of cells and cultured in a 37°C, 5% CO2 incubator for 3 hours;

[0072] ② Select 450nm wavelength, measure the light absorbance of each well on the enzyme-linked immunosorbent assay, record the results, and draw a line graph with time as the horizontal axis and OD value as the vertical axis.

[0073] The test results are shown in Table 1 and Figure 1 shown.

[0074] Table 1 CCK8 method to detect cell OD value

[0075]

[0076] (5) Experimental conclusion: Figure 1 As shown, compared with the control group, the proliferation ability of hUMSCs in the experimental group was upregulated, which was significantly higher than that in the control group.

[0077] 3.2 Nanoflow cytometry and BCA assays were used to detect the exosome particle concentration (particles / mL) and protein content (μg / mL), respectively, and the differences in the ratios of exosome particle number to protein content were compared.

[0078] Exosome particle concentration detection (nanoflow cytometry):

[0079] C.1 Instruments and equipment: C.1.1 Nanoflow cytometer.

[0080] C.2 Reagents and consumables: All reagents used in this method are analytically pure. Unless otherwise specified, the experimental water is first-grade water specified in GB / T6682. C.2.1 Phosphate buffer: pH 7.4. C.2.2 200 nm polystyrene (PS) microspheres, nano-flow cytometry concentration standard (1.99×1010 particles / mL).

[0081] C.3 Detection steps: C.3.1 Perform system initialization operations according to the instrument operating procedures, including liquid flow initialization and pipeline bubble exhaustion. C.3.2 Use 200nm polystyrene (PS) microspheres to calibrate the instrument and adjust the instrument to the optimal state. C.3.3 Put the diluted concentration standard on the machine for detection, adjust the detection parameters, and collect data. C.3.4 Use washing liquid and ultrapure water to clean the capillary according to the instrument operating procedures. C.3.5 Use filtered (0.22μm) PBS as the background signal for analysis. Only PBS with a particle count of less than 200 can be used as a blank control for data collection and subsequent sample dilution. C.3.6 Use clean PBS to perform a 10-fold gradient dilution of the exosome sample to be tested, test it on the machine, and collect data under the measurement parameters of the concentration standard. The background baseline of the sample is not significantly higher than that of the blank control. If it is significantly higher, it needs to be diluted, and at the same time, the particle count of the sample can be significantly distinguished from that of the blank control.

[0082] C.4 Result analysis: Use NF Profession software to calculate the particle concentration of the sample to be tested.

[0083] Exosome protein content detection (BCA detection method):

[0084] G.1 Instruments and equipment: G.1.1 High-speed centrifuge, vortex oscillator, metal bath, and microplate reader.

[0085] G.2 Reagents: All reagents used in this method are of analytical grade. Unless otherwise specified, the experimental water is first-grade water specified in GB / T 6682. G.2.1 Phosphate buffer solution: pH 7.4. G.2.2 RIPA tissue / cell lysis buffer, protein quantification kit (BCA method).

[0086] G.3 Experimental steps: G.3.1 RIPA lysis of exosomes. G.3.1.1 Prepare RIPA lysis buffer according to the instructions of RIPA tissue / cell lysis buffer kit. G.3.1.2 Mix the lysis buffer and exosomes in a 1:1 volume ratio and lyse on ice for 20 minutes, vortexing 3 times for 5 minutes each time. G.3.1.3 After lysis, centrifuge the lysate at 13000g in a 4℃ centrifuge for 5 minutes, and take the supernatant as the protein sample. G.3.2 Determine protein concentration by BCA method. G.3.2.1 Prepare BCA working solution and standard protein solution according to the instructions of protein quantification kit. G.3.2.2 Mix 25μL of protein standards of different concentrations and samples to be tested with 200μL of BCA working solution respectively, and react at 60℃ for 30 minutes. G.3.2.3 After the reaction is completed, wait for the reaction tube to cool to room temperature and perform microplate determination using an ELISA reader.

[0087] G.4 Result analysis: Use an enzyme-labeled instrument to measure the OD values ​​of the standard and the sample to be tested at 562 nm, and calculate the protein concentration of the sample to be tested based on the standard curve.

[0088] The test results are as follows Figure 2 The supernatant of P3 UCMSCs cells from the experimental group and the control group were taken, and the concentration of exosome particles (particles / mL) was detected by nanoflow cytometry. The experimental group had 9.69*10 9 , control group 4.87*10 9 ; The protein content (μg / mL) was detected by BCA method. The experimental group was 38.46, and the control group was 23.75. The ratio of exosome particle number to protein amount in the experimental group and the control group was 2.52×10^8 and 2.05×10^8, respectively. Conclusion: The paracrine effect of the experimental group is significantly better than that of the control group.

[0089] 3.3 ELISA method was used to detect the difference in VEGF protein secretion levels between UCMSCs and conventional UCMSCs.

[0090] The cell culture supernatant was collected in a 50ml sterile centrifuge tube. Centrifuge for 20 minutes at 3000 rpm, and the EGF protein level was detected using the (VEGF) ELISA kit. Experimental principle: This kit uses the double antibody sandwich method to determine the level of human vascular endothelial growth factor (VEGF) in the sample. The microwell plate is coated with purified human vascular endothelial growth factor (VEGF) capture antibody to make a solid phase antibody. Human vascular endothelial growth factor (VEGF) is added to the coated microwells in turn, and then combined with the HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme, and converted into the final yellow under the action of acid. The depth of color is positively correlated with the human vascular endothelial growth factor (VEGF) in the sample. The absorbance (OD value) is measured at a wavelength of 450nm using an enzyme reader, and the human vascular endothelial growth factor (VEGF) content in the sample is calculated using the standard curve. The operation steps are as follows:

[0091] 1. Adding standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well.

[0092] 2. Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μl of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μl of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0093] 3. Add enzyme: Add 100 μl of enzyme-labeled reagent to each well, except for the blank well.

[0094] 4. Incubation: Seal the plate with a sealing film and incubate at 37°C for 60 minutes.

[0095] 5. Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0096] 6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0097] 7. Color development: First add 50μl of color developer A to each well, then add 50μl of color developer B, gently shake and mix, and color development at 37℃ in the dark for 15 minutes.

[0098] 8. Termination: Add 50 μl of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0099] 9. Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0100] 10. Calculation: Draw a standard curve on the coordinate paper with the concentration of the standard as the horizontal axis and the OD value as the vertical axis. Find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply it by the dilution factor; or use the concentration of the standard and the OD value to calculate the linear regression equation of the standard curve, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0101] The supernatant of P3 UCMSCs cells in the experimental group and the control group were taken, and the ELISA method was used to detect the secretion of VEGF protein level. The results were 483.76±92.34pg / ml in the experimental group and 321.46±67.51pg / mL in the control group. Experimental conclusion: The secretion of VEGF protein level of hUMSCs in the experimental group was significantly better than that in the control group.

[0102] In summary, the embodiment of the present invention provides an efficient hUCMSCs for ischemic injury repair and its preparation method and application. The present invention, by subculture and amplification culture of the P2 generation hUCMSCs cells under the specific conditions of an oxygen concentration volume fraction range of 1%-10% and the addition of an inflammatory factor pre-activation composition, enables UCMSCs to adapt to the pathological microenvironment of hypoxia in the body during the in vitro induction culture stage, induces UCMSCs to remember the damaged microenvironment, protects UCMSCs against adverse microenvironments, and maintains gene stability. Maintaining the undifferentiated state of UCMSCs promotes the proliferation and survival of UCMSCs, and at the same time promotes the enhancement of its paracrine function, such as the increase in the secretion of angiogenic factors VEGF, HGF and basic fibroblast growth factor bFGF, EGF, etc., so that it exhibits better characteristics and has a better repair effect of lower limb ischemic injury, solves the problems of low content of paracrine angiogenic and inflammatory inhibitory cytokines in existing hUCMSCs, poor ability to adapt to the microenvironment in the body, and makes up for the deficiencies of the prior art.

[0103] Various embodiments of the present invention may be presented in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, the range description should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integer) within the indicated range.

[0104] The foregoing is merely a specific embodiment of the present invention, which enables those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing hUCMSCs with high efficiency in repairing ischemic damage, characterized in that: The preparation method comprises the following steps: Obtain P2 generation hUCMSCs cells; Under the condition of an oxygen concentration volume fraction range of 1%-10% and the addition of an inflammatory factor pre-activation composition, the P2 generation hUCMSCs cells are subcultured and expanded to obtain the hUCMSCs with high efficiency in repairing ischemic damage; Wherein, the inflammatory factor pre-activation composition comprises the following components: 10-50ng / ml TNF-a, 10-50ng / ml IL-1β, 0-20umol / L LPA, and 10-100ng / ml HGF.

2. The method for preparing hUCMSCs with high efficiency in repairing ischemic damage according to claim 1, characterized in that: The steps of obtaining P2 hUCMSCs cells include: isolating from umbilical cord tissue and obtaining P2 hUCMSCs cells by adherent culture method.

3. The method for preparing hUCMSCs with high efficiency in repairing ischemic damage according to claim 2, characterized in that: The steps of obtaining P2 hUCMSCs cells include the following process: Step 1: Cut and clean the gelatin and place it in a culture dish for later use; Step 2: Inoculate the treated tissue blocks in a culture bottle with a spacing of 1-2 cm between tissue blocks, and incubate at 37°C and 5.0% CO2; Step 3: Add human mesenchymal stem cell serum-free culture medium, inoculate labeled hUCMSCs P0, observe cell growth regularly and change the medium; Step 4: hUCMSCs P0 cells are grown to a confluence of 80%-90%, and the P0 cells are digested and collected. The tissue blocks are removed by centrifugation to obtain P0 umbilical cord mesenchymal stem cells hUCMSCs P0; Step 5: Inoculate hUCMSCs P1 at 1*10^6-2*10^6 / T75cm2 flask, culture to 80%-90% confluence and then collect again, subculture and collect cells to obtain P2 generation hUCMSCs cells.

4. The method for preparing hUCMSCs with high efficiency in repairing ischemic damage according to claim 3, characterized in that: The step 4 includes the following process: culturing for 12-15 days, the hUCMSCs P0 cells grow to a confluence of 80%-90%, discarding the supernatant culture medium, adding 3-5 ml / bottle of physiological saline to wash 1-2 times, using 0.25% trypsin / EDTA digestion solution, 0.5-1 ml / bottle to digest the crawling cells and tissue blocks, and using 1-3 ml / bottle of serum-free trypsin stop solution to stop digestion; using 50 ml to collect tissue blocks and cell suspension, centrifuging at 1000-2000 rpm for 5 minutes, discarding the supernatant, adding an appropriate amount of physiological saline to mix, filtering with a 70-100 μm filter to remove tissue blocks, and obtaining P0 generation umbilical cord mesenchymal stem cells hUCMSCs P0.

5. The method for preparing hUCMSCs with high efficiency in repairing ischemic damage according to claim 4, characterized in that: The step 5 includes the following process: inoculating hUCMSCs P1 at 1*10^6-2*10^6 / T75cm2 bottle, adding 10-20ml human mesenchymal stem cell serum-free culture medium to each bottle, placing it in a 37°C 5.0% CO2 incubator and culturing it every 2-4 days until the cell fusion rate reaches 80%-90%, repeating step 4 to collect hUCMSCs P1 generation cells, and passing hUCMSCs P2 by cell counting at 1:

3. When the cell fusion rate reaches 80%-90%, repeating step 4 to collect hUCMSCs P2 generation cells.

6. The method for preparing hUCMSCs with high efficiency in repairing ischemic damage according to claim 5, characterized in that: Under the condition of oxygen concentration volume fraction range of 1%-10% and adding inflammatory factor pre-activation composition, the P2 generation hUCMSCs cells are subcultured and expanded to obtain the hUCMSCs with high efficiency in ischemic injury repair, which comprises the following process: hUCMSCs-P2 generation cells were subcultured and expanded using a three-gas incubator with an oxygen volume fraction concentration range of 1%-10%, 20-30ml / T175cm2 human mesenchymal stem cell serum-free culture medium, an inflammatory factor pre-activation composition, and hUCMSCs-P3 were subcultured at a ratio of 1:3 according to cell count. The cells were cultured for 2-4 days. When the cell fusion rate reached 80%-90%, step 4 was repeated to collect hUCMSCs P3 generation cells and their cell supernatant.

7. A highly efficient hUCMSCs for repairing ischemic damage, characterized in that: The hUCMSCs with high efficiency in repairing ischemic damage are prepared by the preparation method described in any one of claims 1 to 6.

8. The hUCMSCs for highly efficient ischemic damage repair according to claim 7, characterized in that: The VEGF protein secretion level of the hUCMSCs with high efficiency in repairing ischemic damage was 483.76±92.34 pg / ml.

9. Use of the hUCMSCs with high efficiency in repairing ischemic damage according to any one of claims 1 to 8 in the preparation of a drug for treating limb artery ischemic diseases.

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