Conditioned medium of TERT-overexpressing brain pericytes and its application in protecting neurons from hypoxia-ischemia

Abstract

The present invention discloses a TERT-overexpressing pericyte conditioned medium and its application in protecting hypoxic-ischemic neurons. The TERT-overexpressing pericyte conditioned medium of the present invention uses a lentivirus containing TERT to transfect second-generation pericytes to construct pericytes overexpressing TERT; the pericytes overexpressing TERT are then added to a serum-free high-glucose DMEM medium for culture, thereby obtaining a TERT-overexpressing pericyte conditioned medium. The present invention proves through experiments that the TERT-overexpressing pericyte conditioned medium increases the secretion of FGF2 that promotes neuronal survival and reduces the secretion of the neuronal toxic molecule APOE4, thereby protecting hypoxic-ischemic neurons, reducing hypoxia-ischemia-induced neuronal apoptosis, and promoting their survival. Therefore, the TERT-overexpressing pericyte conditioned medium can be used to protect hypoxic-ischemic neurons.

Description

Technical Field

[0001] The invention relates to a cell culture fluid and an application thereof, and in particular to a TERT overexpressing brain pericyte culture fluid and an application thereof in protecting hypoxic-ischemic neurons. Background Art

[0002] Neonatal hypoxic–ischemic encephalopathy (HIE) is a neonatal encephalopathy caused by reduced cerebral blood perfusion and / or hypoxemia. The most fundamental measure in current treatment is to maintain the stability of the body's internal environment and the normal functioning of various organs. On this basis, various treatments for reperfusion injury and brain cell metabolism activators are used to gradually restore the metabolism of damaged nerve cells. However, neonatal death or permanent damage to the central nervous system is still common, manifested as mental retardation, epilepsy, cerebral palsy or developmental delay. Studies have shown that the degree of neuronal damage during the development of HIE determines the progression and prognosis of the disease. Exogenous intervention to reduce hypoxic–ischemic neuronal damage and repair neurons is an important measure to alleviate HIE.

[0003] Pericytes are parietal cells embedded in the basement membrane surrounding endothelial cells in brain capillaries and small blood vessels (from precapillary arterioles to postcapillary venules). They can express neurotrophic factors such as nerve growth factor (NGF), basic fibroblast growth factor (FGF2) and brain-derived neurotrophic factor (BDNF), and can also secrete proinflammatory factors such as tumor necrosis factor-α (TNF-α) and neurotoxic molecule apolipoprotein E4 (APOE4), which together construct the neural repair microenvironment after brain injury. Therefore, how to improve the secretion profile of pericytes after brain injury to facilitate neural repair is an important issue that needs to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a telomerase reverse transcriptase overexpressing brain pericyte conditioned culture medium and its application in protecting hypoxic-ischemic neurons.

[0005] The telomerase reverse transcriptase is the catalytic subunit of telomerase, and its English name is telomerase reverse transcriptase. In the description of the specification, telomerase reverse transcriptase is abbreviated as TERT.

[0006] The TERT overexpressing pericyte conditioned medium of the present invention is to transfect the second generation pericytes with a lentivirus containing TERT to construct pericytes overexpressing TERT; and then the pericytes overexpressing TERT are added to a serum-free high-glucose DMEM medium for culture, thereby obtaining a TERT overexpressing pericyte conditioned medium. The amount of serum-free high-glucose DMEM medium added depends on the size of the culture dish.

[0007] The above-mentioned TERT overexpressing brain pericyte conditioned medium, the said TERT overexpressing brain pericyte added to the serum-free high-glucose DMEM medium is cultured in a 37°C constant temperature cell culture incubator for 24h to 48h. The incubator is 5v.% CO 2 、95v.% CO in air 2 Incubator.

[0008] The present invention proves through experiments that TERT overexpression brain pericyte conditioned medium plays a protective role on hypoxic-ischemic neurons by increasing the secretion of FGF2 that promotes neuronal survival and reducing the secretion of neuronal toxic molecule APOE4, thereby reducing hypoxia-ischemia-induced neuronal apoptosis and promoting its survival. Therefore, TERT overexpression brain pericyte conditioned medium can be used to protect hypoxic-ischemic neurons.

[0009] The present invention has the following beneficial results:

[0010] 1. The present invention successfully obtained the conditioned culture medium of TERT-overexpressing brain pericytes.

[0011] 2. The TERT-overexpressing brain pericyte conditioned medium of the present invention provides a new technical solution for the protection of hypoxic-ischemic neurons. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 These are photographs of primary brain microvascular pericytes under an optical microscope; Figure A is a photograph of primary brain microvascular pericytes under a light microscope at 40 times magnification, and Figure B is a photograph of primary brain microvascular pericytes under a light microscope at 100 times magnification.

[0013] Figure 2 These are photos of brain pericytes identified by immunofluorescence staining; among them, Figure A is a photo of brain pericytes positively expressing the relatively specific marker α-SMA, Figure B is a photo of brain pericytes positively expressing the relatively specific marker β-PDGFR, Figure C is a photo of brain pericytes negatively expressing the endothelial cell marker CD31, and Figure D is a photo of brain pericytes negatively expressing the astrocyte GFAP.

[0014] Figure 3 Photographs of immunofluorescence staining to identify neurons in the cerebral cortex.

[0015] Figure 4These are photos of brain pericytes transfected with lentivirus carrying TERT; among them, Figures AB are photos of brain microvascular pericytes 3 days after lentivirus infection and GFP protein label of brain microvascular pericytes infected with lentivirus under an optical microscope; Figure CD is a photo of brain microvascular pericytes infected with lentivirus under an optical microscope when the GFP protein label reaches 90%.

[0016] Figure 5 The figure shows the expression of TERT mRNA in brain pericytes transfected with lentivirus; NC is the negative control group of brain pericytes with lentivirus vector; TERT is the brain pericyte group of rats with TERT overexpression lentivirus.

[0017] Figure 6 The volume ratios of brain pericyte conditioned medium to neuron culture medium were 1:0, 1:1, and 1:2 respectively to intervene in OGD neurons, and CCK8 was used to detect the cell survival rate of neurons.

[0018] Figure 7 OGD neurons were intervened with brain pericyte conditioned medium and neuron culture medium at a volume ratio of 1:0, 1:1, and 1:2, and the expression of neuronal Bax and Bcl-2 was detected by RT-PCR.

[0019] Figure 8 OGD neurons were cultured in brain pericyte conditioned medium, and immunofluorescence staining was used to detect neuronal apoptosis after 24 hours of culture; NC was the negative control group, and TERT was the TERT-overexpressing brain pericyte group; Figure A was the CC3-labeled immunofluorescence image, and Figure B was the statistical graph of neuronal apoptosis stained with immunofluorescence.

[0020] Fig. 9 The OGD neurons were cultured in brain pericyte conditioned medium, and TUNEL staining was used to detect neuronal apoptosis after 24 hours of culture; NC was the negative control group, and TERT was the TERT-overexpressing brain pericyte group; Figure A was the TUNEL staining image, and Figure B was the statistical graph of neuronal apoptosis after TUNEL staining.

[0021] Fig.10 A principal component analysis (PCA) diagram was performed for the expression levels of credible proteins. Each point in the figure represents a repetition in a grouping experiment. The circle is the NC group, the diamond is the TERT group, and the ellipse represents the confidence interval of the sample.

[0022] Fig.11 This is the overall distribution of differentially expressed proteins in the conditioned culture medium of brain pericytes in the negative control NC group and the TERT overexpressing brain pericytes TERT group.

[0023] Fig.12It is a volcano plot of differentially expressed proteins in the conditioned medium of brain pericytes of the negative control NC group and the TERT overexpressing brain pericytes TERT group.

[0024] Fig.13 This is a graph showing the survival rate of neuronal cells after using CCK8 to detect different concentrations of recombinant FGF2, APOE4 protein, and FGF2 neutralizing antibodies to intervene in OGD neurons.

[0025] Fig.14 This is a graph showing the concentrations of differentially expressed proteins APP, APOE, csf1, NT-3, HGF, BDNF, and FGF2 in the conditioned medium of brain pericytes of each group detected by ELISA. Among them, DMEM is the concentration of differentially expressed proteins in DMEM medium (the basis for preparing the conditioned medium of brain pericytes); NC is the concentration of differentially expressed proteins in the conditioned medium of brain microvascular pericytes infected with empty lentiviral vectors; TERT is the concentration of differentially expressed proteins in the conditioned medium of brain microvascular pericytes overexpressing TERT.

[0026] Fig.15 This is a diagram of the apoptosis of OGD neurons cultured with recombinant APOE4 protein using immunofluorescence technology; NC is the negative control group OGD neurons cultured in the conditioned medium of pericytes; TERT is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes; TERT+APOE4 is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes with recombinant APOE4 protein added; Figure A is the CC3-labeled immunofluorescence image, and Figure B is the statistical graph of immunofluorescence staining neuronal apoptosis.

[0027] Fig.16 This is a diagram for detecting the apoptosis of OGD neurons cultured with recombinant FGF2 protein using immunofluorescence technology; NC is the negative control group, OGD neurons cultured in the conditioned medium of pericytes; TERT is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes; TERT+FGF2 is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes with recombinant FGF2 protein added; Figure A is the CC3-labeled immunofluorescence image, and Figure B is the statistical graph of immunofluorescence staining neuronal apoptosis.

[0028] Fig.17 This is a diagram of the use of TUNEL staining to detect the apoptosis of OGD neurons cultured with recombinant APOE4 protein; NC is the negative control group, OGD neurons cultured in the conditioned medium of pericytes; TERT is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes; TERT+APOE4 is the OGD neurons cultured in the conditioned medium of TERT-overexpressing pericytes plus recombinant APOE4 protein; Figure A is the TUNEL staining diagram, and Figure B is the statistical diagram of neuronal apoptosis after TUNEL staining.

[0029] Fig.18 The TUNEL staining method was used to detect the apoptosis of OGD neurons cultured with recombinant FGF2 protein; NC was the negative control group cultured with brain pericyte conditioned medium in OGD neurons; TERT was the TERT-overexpressing brain pericyte conditioned medium in OGD neurons; TERT+FGF2 was the TERT-overexpressing brain pericyte conditioned medium added with recombinant FGF2 protein in OGD neurons; Figure A is the TUNEL staining image, and Figure B is the statistical graph of neuronal apoptosis after TUNEL staining. DETAILED DESCRIPTION

[0030] 1. Experimental Materials and Reagents

[0031] 1.1 Experimental animals

[0032] Postnatal day 7 (P7) SPF SD rats were used to extract rat brain pericytes, and postnatal day 1 (P1) SPF SD rats were used to extract rat cortical neurons. All experimental rats were purchased from Chengdu Dashuo Biotechnology Co., Ltd. with a certificate of quality.

[0033] 1.2 Main Reagents

[0034]

[0035]

[0036] 2. Experimental methods

[0037] 2.1 Extraction and identification of rat brain pericytes 2.1.1 Primary culture of rat brain pericytes

[0038] 1) Preparation: One day in advance, sterilize and dry the scissors, tweezers, gun tips, beakers, and filter papers needed for the operation in a high-pressure steam sterilizer.

[0039] 2) Brain extraction: 7-day-old SD rats of either sex were selected and anesthetized by isoflurane. The rats were killed by cervical dislocation and placed in a 75% alcohol (volume fraction) beaker for disinfection for about 3 min. The scalp and skull were cut in layers, and the whole brain was removed. The brain was rolled on sterilized and dried filter paper to remove the pia mater and placed in pre-cooled PBS.

[0040] 3) Separation: Separate the cerebral cortex with microtweezers, remove the vascular membrane, place the obtained cortical tissue in a pre-cooled high-glucose DMEM (5 mL) culture dish, and cut the tissue into 1 mm pieces with ophthalmic scissors. 3 Small pieces, ready to digest.

[0041] 4) Digestion: Add 50 μL 10% collagenase type II (mass fraction) (working concentration 0.1%), 150 μL DNaseI, and 50 ul penicillin / streptomycin double antibody suspension and mix well. Use a pipette to gently blow until there is no large brain tissue. Place in an incubator (37°C 5v% CO 2 ) for 90 min, shaking every 30 min. Add 50 μL 10% collagenase / dispase (mass fraction) (working concentration 0.1%), suspend and mix, and place at 37°C 5% CO 2 Incubator, digest for 60 min, shaking every 30 min.

[0042] 5) Filtration: Mix by gently pipetting and pass through a mesh screen, collect the filtrate and centrifuge (800 g, 5 min, room temperature).

[0043] 6) Purification: Add about twice the volume of 20% BSA (mass fraction) to the precipitate after centrifugation, mix well and centrifuge (1000g, 20min, 4°C), and keep the bottom precipitate. Add DMEM to resuspend the precipitate, use a pipette to gently adhere to the wall and slowly transfer to 33% Percoll density gradient centrifuge (volume fraction), centrifuge again (1000g, 10min, 20°C), and the precipitate after centrifugation is divided into two layers, from bottom to top, respectively, the red blood cell layer and the microvascular fragment enrichment layer (yellow-white).

[0044] 7) Culture: The yellow-white layer of precipitate was aspirated and added to DMEM complete culture medium (containing 10% FBS (volume fraction), 1% double antibody (volume fraction)), pipetted and mixed, inoculated into a culture dish, and cultured in an incubator (37°C 5% CO 2 ).

[0045] 8) Medium replacement: On the second day after primary pericyte extraction, observe the attachment of the extracted brain pericytes under an optical microscope, wash with PBS buffer to remove the pericytes and impurities that are not attached to the wall, and then replace with fresh culture medium. After medium replacement, place in a cell culture incubator (37°C 5% CO 2 ), changing the medium every 3-4 days, and subculturing pericytes after they grew to more than 95% confluence.

[0046] 2.1.2 Passaging of rat brain pericytes

[0047] 1) Digestion: After the brain pericytes grow and become more than 95% confluent, discard the culture medium and rinse twice with PBS buffer, add 1 ml of trypsin, shake the culture dish, observe under a microscope, and wait until the cell processes shrink and gradually become round. After most of the cells are suspended, add 3-6 ml of pericyte complete culture medium and blow to make a single cell suspension, then divide the cells from one culture dish into three culture dishes for passaging.

[0048] 2) Seed plate: First, digest and resuspend the brain pericytes after subculture in the above steps into a single cell suspension, then count the cells, take a sterile EP tube, take 10μl of the cell suspension, add it to the EP tube containing 90ul complete culture medium, mix well, and take 10μl to count under the microscope (the counting base is 1×10^4 cells / ml). After counting, dilute the cell suspension to 6.25×10^4 cells / ml, and inoculate 2ml of the culture system in a 6-well plate for TERT overexpression, and inoculate 0.5ml of the culture system in a 24-well plate containing a cell slide for immunofluorescence identification.

[0049] 2.1.3 Identification of rat brain pericytes by immunofluorescence staining

[0050] 1) Take out the 24-well plate with built-in cell slides from the above seed plate, discard the culture medium, add 1 ml of PBS to each well to rinse the cell slides, rinse for 5 minutes each time, and rinse 3 times.

[0051] 2) Fixation: Add 0.5 ml of 4% paraformaldehyde solution (mass volume fraction) to each well (enough to cover the slide) and fix for 30 minutes at 4°C.

[0052] 3) Discard the paraformaldehyde (collect it into a waste bottle, seal it and discard it), add 1 ml of 1×PBS to each well to rinse the cell slides, once every 5 minutes, for a total of three times.

[0053] 4) Perforation: Aspirate PBS and add 500 μl 0.3% TritonX-100 (volume fraction) to each well at 4°C for 15 min.

[0054] 5) Blocking with fluorescent blocking solution: discard 0.3% Triton (volume fraction), add 500ul fluorescent blocking solution, and incubate in a 4°C refrigerator for 60min.

[0055] 6) Primary antibody incubation: discard the blocking solution, use the blocking solution to prepare the primary antibodies (α-SMA (1:200), β-PDGFR (1:200), CD31 (1:200), GFAP (1:500)) (the volume ratio of the antibody stock solution to the blocking solution is shown in the brackets), add 200 μl of the prepared primary antibody to each well, and incubate at 4°C overnight.

[0056] 7) Discard the primary antibody and add 1×PBS to each well to rinse the slides three times, each time for 5 minutes.

[0057] 8) Secondary antibody incubation: Use blocking solution to prepare the secondary antibody and DAPI of the corresponding species (1:500) (the volume ratio of the antibody stock solution to the blocking solution is shown in the brackets), aspirate PBS, add 50ul of the secondary antibody and DAPI of the corresponding species to each slide, and incubate for 1 hour in the dark.

[0058] 9) Discard the secondary antibody and add 1 ml PBS to each well to rinse the slides for 5 min each time, for a total of three times, keeping away from light.

[0059] 10) Sealing: Add 100ul of sealing agent to the slide, remove the slide and absorb the remaining PBS, place the slide with fixed cells upside down on the slide with sealing agent, press gently to remove residual bubbles, and wait for the sealing solution to dry in a dark place. It can be stored in a 4℃ refrigerator in a dark place.

[0060] 11) Collecting images: After the sealing liquid dries, use a slice scanner to collect images.

[0061] 2.2 Extraction and identification of rat cerebral cortical neurons

[0062] 2.2.1 Primary culture of rat cerebral cortical neurons

[0063] 1) Plate coating: Dilute 100μg / ml poly-lysine with PBS to 10μg / ml. Add the diluted poly-lysine to a 6-well plate at 1ml / well, add the cells to a 24-well plate at 300ul / well poly-lysine, and place it in a cell culture incubator for at least 4 hours. Recover the poly-lysine before use, wash it 3 times with pre-sterilized and cooled cell-grade ultrapure water, open the bottle cap and place it in a biosafety cabinet to dry.

[0064] 2) Preparation: Sterilize and dry the scissors, forceps, gun tips, and beakers required for the operation in a high-pressure steam sterilizer one day in advance. 3) Anesthesia and disinfection: Take 15 1-day-old SD rats in batches and anesthetize them with low temperature, then soak them in a beaker filled with pre-cooled 75% ethanol (volume fraction) for 2 minutes.

[0065] 4) Brain removal: Take out 1 pair of scissors, 1 pair of straight forceps, and 3 curved forceps from the instrument box and set aside. Use the straight forceps to remove the anesthetized and sterilized suckling mouse and place it in a 10 cm dish. After the scissors cut off the head, use 1 curved forceps to fix the head from the orbital plane, and use another curved forceps to separate the skull along the midline of the brain at the foramen magnum. Replace the forceps to remove the intact brain tissue and immediately place it in a 6 cm dish filled with pre-cooled PBS buffer.

[0066] 5) Separation of brain tissue: Take another pair of fine straight forceps and another pair of fine curved forceps, separate the brain tissue into left and right sides along the midline of the brain, remove the olfactory bulb, brain stem, thalamus, etc., retain the cerebral cortex, and immediately place it in a 6 cm dish containing pre-cooled PBS buffer.

[0067] 6) Removing the vascular membrane: Replace one pair of fine straight forceps and one pair of fine curved forceps, carefully remove the vascular membrane, and immediately place the brain tissue into a 6 cm dish containing 4 ml of pre-cooled high-glucose DMEM.

[0068] 7) Digestion: Use ophthalmic scissors to cut the brain tissue in a 6 cm dish soaked in high-glucose DMEM into pieces of approximately 1 mm 3 Add 1 ml of 10 mg / ml papain (working concentration is 2 mg / ml) to the tissue block. Add 50 μl of DNase I. Place in the incubator and digest for 30 minutes, shaking the culture dish every 10 minutes.

[0069] 8) Termination of digestion: Take out the culture dish, add 15 ml of high-glucose DMEM complete culture medium containing 10% FBS (volume fraction) to terminate the digestion, and gently pipette to form a single-cell suspension.

[0070] 9) Filtration: Place a 70 μm filter on a 50 ml centrifuge tube, pipette the single cell suspension onto the filter, collect the filtrate and centrifuge (300 g, 5 min, room temperature).

[0071] 10) Counting: Add 5 ml of Neurobasal culture medium containing 2% B27 (volume fraction), 0.25% glutamine (volume fraction), and 0.5% penicillin / streptomycin (volume fraction) to resuspend the pellet from the previous step. Take a sterile EP tube, pipette 10 μl of the cell suspension, add it to the EP tube containing 90 μl Neurobasal culture medium, mix well, and pipette 10 μl to count under a microscope (the counting base is 1×10^4 cells / ml).

[0072] 11) Inoculation: After counting, dilute the cell suspension to 1.5×10^6 cells / ml with neuronal culture medium, and inoculate 2 ml of the culture system into a 6-well plate for protein or RNA collection, 0.5 ml of the culture system into a 24-well plate containing a cell slide for immunofluorescence identification, and 0.2 ml of the culture system into a 96-well plate for CCK8 detection. 12) Medium change: On the second day of neuronal culture, observe the growth and adhesion of the extracted cells under an optical microscope, and replace with fresh culture medium on the third day. After medium change, place the cells in a cell culture incubator (37°C 5% CO 2 ), and follow-up experiments were performed on the sixth day.

[0073] 2.2.2 Identification of rat cerebral cortical neurons

[0074] The steps are the same as 2.1.3, and the primary antibody is NeuN (1:200) (the volume ratio of the antibody stock solution and the blocking solution is shown in the brackets).

[0075] 2.3 Construction of TERT-overexpressing brain pericytes and determination of transfection efficiency

[0076] 2.3.1 Preparation of TERT overexpressing lentiviral vector and negative control empty vector

[0077] The preparation of TERT overexpressing lentiviral vector and negative control empty vector was carried out by Shanghai Jikai Chemical Technology Co., Ltd. (China). Fluorescent label: gcGFP; Virus titer: negative control (CON335) is 3.0×10^8TU / ml; TERT is 5×10^8TU / ml.

[0078] 2.3.2 Lentiviral transfection of brain pericytes

[0079] 1) Extraction of primary brain pericytes: Same as 2.1.1.

[0080] 2) Digestion: Same as 2.1.2.

[0081] 3) Seed plate: Same as 2.1.2.

[0082] 4) Virus transfection: After the pericytes were plated and adhered to the wall, the corresponding virus volume was added according to the titer of the lentivirus at MOI = 10, and the required virus volume = number of cells × MOI / virus titer (negative control: 3.0 × 10^8TU / ml; TERT: 5 × 10^8TU / ml) and then placed in a constant temperature incubator (37°C 5% CO 2 ) and observe the expression of green fluorescent protein under a fluorescence microscope every 48 hours.

[0083] 2.3.3 Puromycin selection of brain pericytes stably transfected with TERT

[0084] 1) After lentiviral infection, the growth status of pericytes and the expression of gcGFP were observed under a microscope every day. Under a light microscope, pericytes reached 95% fusion and gcGFP reached 40%. Puromycin was added for stable transfection screening until the green fluorescent protein expression rate reached more than 90%.

[0085] 2) The working concentration of puromycin is 0.5 μg / ml for initial screening and 0.25 μg / ml for continuous screening. During the screening period, the working concentration and screening time can be adjusted according to the cell growth status and the expression of green fluorescent protein.

[0086] 2.3.4 RT-PCR detection of TERT transfection efficiency

[0087] 2.3.4.1 Extraction of total cellular RNA by Trizol method

[0088] 1) The brain pericytes stably transfected with TERT were cultured in a 6-well plate, the supernatant was discarded, and after washing with PBS, 0.4 ml of Trizol was added to each well, and the well bottom was evenly covered. After being placed on ice for 10 minutes, the cells were scraped upside down with a 1 ml gun tip, and the lysis was continued for 5 minutes. The lysis solution was transferred to an EP tube and placed on ice.

[0089] 2) Add 80 μl of chloroform (1 / 5 Trizol volume) to each EP tube, shake for 15 seconds, incubate on ice for at least 10 minutes, and centrifuge at 4°C after liquid stratification, set the speed to 12000 rcf, and centrifuge for 15 minutes.

[0090] 3) Carefully aspirate the supernatant, about 200 μl, add isopropanol equal to the volume of the aspirated supernatant and mix by inversion. Place on ice for 10 min and then centrifuge (4°C, 12000 rcf, 10 min).

[0091] 4) After discarding the supernatant, wash the precipitate with 1 ml of 75% ethanol (prepared with enzyme-free water and 100% ethanol by volume, prepared and used immediately), centrifuge at 4°C, set the speed to 9500 rcf, centrifuge for 10 min, and repeat the washing twice.

[0092] 5) Place the EP tube with the mouth facing downward and use absorbent paper to absorb the excess ethanol. After about 2 minutes, place it on the EP tube rack for about 5-8 minutes. After the ethanol evaporates, add 20 μl of enzyme-free water to dissolve the RNA precipitate, place it on ice, measure and adjust the concentration for reverse transcription operation.

[0093] 2.3.4.2 Reverse transcription reaction

[0094] 1) Remove DNA, reaction conditions: 42°C, 2 min.

[0095]

[0096] 2) Reverse transcription reaction, reaction conditions: ① 50℃, 15min; ② 85℃ 5sec.

[0097]

[0098] After the reaction is completed, the cDNA is diluted to 100 μl with enzyme-free water and PCR is performed (the remaining cDNA can be stored at -40°C for future use).

[0099] 2.3.4.3PCR

[0100] Primer sequences: Primer sequences were synthesized using the NCBI database and PubMed primerblast, and nucleotide sequence synthesis was provided by Qingke Biotechnology (China).

[0101]

[0102] Reaction system:

[0103]

[0104] Reaction procedure:

[0105]

[0106] Calculation: 2- △△CT The relative expression level was determined by T test of two independent samples (△Ct = Ct (target gene) - Ct (GAPDH) 2.4 Collection of brain pericyte conditioned medium

[0107] 1) The primary culture, passaging, plating (6-well plates), lentiviral transfection, and puromycin resistance screening of pericytes are the same as 2.1; 2.2; 2.3.

[0108] 2) After pericytes were stably transfected with TERT, the conditioned medium of brain pericytes was collected: the group infected with empty virus was designated as the NC group, and the group infected with virus carrying TERT plasmid was designated as the TERT group.

[0109] 3) After washing the cells twice with PBS buffer, add an equal amount of serum-free sugar-containing DMEM (5 ml per well) and continue culturing for 24 hours. 4) The serum-free sugar-containing DMEM medium from the NC group and TERT group for 24 hours is the conditioned medium for brain pericytes of the corresponding groups, which are collected in two centrifuge tubes respectively. When culturing neurons, take out the same volume of brain pericyte conditioned medium from the NC group and TERT group from the two centrifuge tubes and add them to the cultured neurons. This operation ensures the consistency of concentration within the group. In repeated experiments, we unified the concentration of each group in different batches after measuring the protein concentration to ensure the repeatability of the experiment.

[0110] 2.5 Effects of brain pericyte conditioned medium on OGD neurons

[0111] 2.5.1 Construction of hypoxic-ischemic neuronal model in vitro

[0112] 1) Place the sugar-free DMEM in a three-gas incubator containing 94v% high-purity nitrogen, 5v% high-purity carbon dioxide and 1v% oxygen for pre-hypoxia for more than 6 hours.

[0113] 2) Primary neurons were observed under an optical microscope. On the sixth day of extraction and culture, the neurons were rinsed twice with PBS buffer.

[0114] 3) Add pre-hypoxic sugar-free DMEM and culture in the above hypoxic incubator for 2 hours to simulate in vitro hypoxia-ischemia to construct an oxygen-glucose deprivation (OGD) model.

[0115] 2.5.2 CCK8 detection of the survival rate of OGD neurons

[0116] 1) Set up groups with a ratio of conditioned medium to neuronal medium of 1:0, 1:1, and 1:2, respectively. Remove the neurons from the 96-well plate that has been subjected to oxygen-glucose deprivation according to the above conditions (Method 2.5.1), add PBS buffer to wash twice, add the above-mentioned culture medium respectively, and culture in an incubator for 24 hours (37°C 5% CO2).

[0117] 2) After culturing the OGD neurons according to step 1), remove the supernatant, add PBS buffer to gently rinse once, add 100 μl of culture medium containing CCK8 to each well (culture medium: CCK8 = 1:9), incubate in a constant temperature incubator for 1.5 h, and measure the absorbance of each group at a wavelength of 450 nm.

[0118] 3) Calculate the cell survival rate = (TERT group-blank) or (NC-blank) × 100%.

[0119] 2.5.3 RT-PCR detection of bax / bcl-2 mRNA in OGD neurons

[0120] 1) Primary neurons were extracted according to method 2.2.1 and inoculated into 6-well plates with 2 ml of culture system for RNA collection.

[0121] 2) Set up groups with a ratio of conditioned medium to neuronal medium of 1:0, 1:1, and 1:2, respectively. Remove the neurons from the 6-well plate that have been subjected to oxygen-glucose deprivation according to the above conditions (Method 2.5.1), wash twice with PBS buffer, add the culture medium in the above ratio, and culture in an incubator for 24 hours (37°C 5% CO2).

[0122] 3) Extract RNA using Trizol method: Same as 2.3.4.1. Reverse transcription: Same as 2.3.4.2.

[0123] 4) PCR: Primer sequence: The primer sequence was synthesized using the NCBI database and PubMed primerblast, and the nucleotide sequence was provided by Qingke Biotechnology (China).

[0124]

[0125] 5) The reaction system and reaction procedure are the same as 2.3.4.3.

[0126] 6) Two independent sample T-tests were used to determine the P value (ΔCt = Ct (target gene) - Ct (β-actin).

[0127] 2.5.4 CC3 staining to detect apoptosis of OGD neurons

[0128] The method was the same as 2.1.3, and the primary antibody was caspase-3 (1:200).

[0129] 2.5.5 TUNEL staining to detect apoptosis of OGD neurons

[0130] 1) The steps for primary culture of neurons are the same as 2.2.1. When wrapping the plate, add crawling sheets for primary culture of neurons.

[0131] 2) Add 50ul 0.2% Triton X-100 (volume fraction) to each slide to break the membrane, incubate at 4°C for 15min, and then wash with PBS for 5min×3 times.

[0132] 3) After absorbing all the PBS, add 50 μl of TUNEL reaction solution to each slide, incubate at 37°C for 1 hour, add 2×SSC to terminate the reaction, and wash with PBS for 5 minutes × 3 times.

[0133] 4) Aspirate all PBS and carefully add 50ul DAPI staining solution to each slide and incubate at room temperature in the dark for 5 minutes.

[0134] 5) Sealing: Wash the slides with PBS 3 times, 5 minutes each time, take out the slides, wipe off the excess water gently, and seal the slides with anti-fluorescence quenching sealing agent. After the sealing agent is dry, shake the slides slightly dry and seal the slides with anti-fluorescence quenching sealing agent. Observe the results using a 3D slice scanner under a laser confocal microscope.

[0135] 2.6 Proteomic detection of brain pericyte conditioned medium and screening and verification of differentially expressed proteins

[0136] 2.6.1 Proteomic detection of brain pericyte conditioned medium Proteomic detection was performed by LuMing Biomedical Company, using tandem mass tags (TMT) technology to perform qualitative and quantitative analysis of proteins in conditioned medium. The proteins that have been qualitatively and quantitatively analyzed were searched in the database. After quality assessment and pretreatment, expression level analysis and functional analysis were performed respectively, and the data were organized into raw data tables. 2.6.1.1 Protein extraction of brain pericyte conditioned medium

[0137] 1) Redissolve the sample in a water bath at room temperature.

[0138] 2) Transfer 10 mL each time to a 3KD ultrafiltration centrifuge tube.

[0139] 3) Centrifuge (4°C, 6400 g, 40 min), collect the retentate, and repeat the previous step until all the sample is used up.

[0140] 4) Transfer out the retentate and rinse the ultrafiltration tube three times with 200 μL of chromatographic water, and mix it with the retentate after rinsing.

[0141] 5) Centrifuge the mixture (4°C, 12000 g, 15 min) and take the supernatant for later use.

[0142] 6) Lyophilize the supernatant overnight.

[0143] 7) Dissolve in SDS lysis buffer at room temperature for 3 h.

[0144] 8) The solution was centrifuged (room temperature, 12000 g, 10 min), the supernatant was retained, and the centrifugation was repeated, and the supernatant was retained again to completely remove insoluble impurities.

[0145] 9) The protein concentration of the supernatant was measured and stored in aliquots (-80°C).

[0146] 2.6.1.2 BCA protein concentration determination

[0147] 1) Prepare the colorimetric solution according to the BCA kit instructions, with the ratio of buffer A:Buffer B = 50:1 (v / v).

[0148] 2) Take out part of the protein solution and dilute it with ddH2O.

[0149] 3) Take a 96-well plate and add the following volumes of pre-prepared standard protein solution to each well: 0, 1, 2, 4, 8, 12, 16, 20 μL, and add ddH2O to make up the volume to 20 μL.

[0150] 4) Add 2 μL of the protein to be tested to each well, and add ddH2O to make up the volume to 20 μL. Set up three replicate wells for each sample.

[0151] 5) Add 200 μL of color development solution to each of the standard protein well and the test protein well, and react at 37°C for 30 min.

[0152] 6) Adjust the wavelength of the microplate reader to 562 nm and measure the absorbance value.

[0153] 7) Plot the absorbance value as the horizontal axis and the known concentration of the standard as the vertical axis to obtain the formula related to concentration and absorbance. Substitute the absorbance value of the protein to be tested into it and calculate the protein concentration value.

[0154] 2.6.1.3 SDS-polyacrylamide gel electrophoresis

[0155] 1) 10 μg of protein was taken from each group of samples and electrophoresed using 12% SDS-PAGE.

[0156] 2) The gel was stained with Coomassie Brilliant Blue using the eStain LG protein stainer.

[0157] 3) The stained gel was imaged using a digital gel image analysis system.

[0158] 2.6.1.4 Trypsin digestion

[0159] 1) Take 50 μg of protein from each sample group and dilute the sample with lysis buffer to adjust to the same concentration and volume.

[0160] 2) Add DTT to a final concentration of 5 mM, mix well and incubate (55° C., 30 min).

[0161] 3) After cooling, add iodoacetamide to adjust the final concentration to 10 mM, mix thoroughly and incubate (room temperature, 15 min, avoid light).

[0162] 4) Add 6 volumes of acetone (place at -20°C for more than four hours to precipitate the protein).

[0163] 5) Centrifuge (4°C, 8000 g, 10 min), collect the precipitate and leave it vacant for several minutes to evaporate the acetone.

[0164] 6) Add 100 μL TEAB (200 mM) to dissolve the precipitate, then add trypsin again to digest overnight at 37°C (the amount of trypsin used is 1 mg / ml trypsin-TPCK, which is 1 / 50 of the sample mass. The enzymatically hydrolyzed sample is freeze-dried and stored in a -80°C refrigerator).

[0165] 2.6.1.5 Peptide labeling

[0166] 1) Add 50 μL of 100 mM TEAB buffer to the above enzymatically hydrolyzed freeze-dried sample, mix thoroughly, and perform labeling reaction (performed in a 1.5 mL Eppendorf tube).

[0167] 2) Take out the TMTpro reagent, equilibrate to room temperature, add 20 μL of anhydrous acetonitrile, vortex for 5 minutes and then centrifuge.

[0168] 3) Add 10 μL of TMTpro reagent to the sample, mix thoroughly, place at room temperature for 60 min, add 5 μL of 5% hydroxylamine to terminate the reaction, and freeze-dry after 15 min (store in a -80°C refrigerator).

[0169] 2.6.1.6 Reversed-phase chromatography separation

[0170] Liquid chromatography: Agilent 1100HPLC.

[0171] Chromatographic column: Agilent Zorbax Extend–C18 narrow bore column, 2.1×150 mm, 5 μm.

[0172] Detection wavelength: UV 210nm and 280nm.

[0173] Mobile phase A: ACN-H2O (2:98, v / v), pH adjusted to 10 with aqueous ammonia.

[0174] Mobile phase B: ACN-H2O (90:10, v / v), pH adjusted to 10 with aqueous ammonia.

[0175] Flow rate: 300 μL / min.

[0176] Gradient elution conditions: 0-8 min, 98% A; 8-8.01 min, 98%-95% A; 8.01-48 min, 95%-75% A; 48-60 min, 75-60% A; 60-60.01 min, 60-10% A; 60.01-70 min, 10% A; 70-70.01 min, 10-98% A; 70.01-75 min, 98% A.

[0177] Collect samples from 8 to 60 minutes, collect the eluent into centrifuge tubes numbered 1 to 15 every one minute, and then collect samples repeatedly in the order of 1 to 15. After collecting the samples, freeze-dry them in a vacuum, store them in a frozen state, and perform mass spectrometry in the next step.

[0178] 2.6.1.7 Chromatographic and mass spectrometry conditions

[0179] <Chromatographic conditions>

[0180] The sample was loaded onto a pre-column Acclaim PepMap100, 100 μm×2 cm (RP-C18, Thermo Fisher) at a flow rate of 300 nL / min and then separated on an analytical column Acclaim PepMap RSLC, 75 μm×15 cm (RP-C18, Thermo Fisher).

[0181] Mobile phase A: H2O-FA (99.9:0.1, v / v).

[0182] Mobile phase B: ACN-H2O-FA (80:19.9:0.1, v / v / v).

[0183] Gradient elution conditions: 0-63 min, 5-45% B; 63-65 min, 45-90% B; 65-75 min, 90% B.

[0184] <Mass spectrometry conditions>

[0185] The primary MS mass resolution was set to 60000, the automatic gain control value was set to 3e6, and the maximum injection time was 50ms; the mass spectrometer scan was set to a full scan of the charge-to-mass ratio m / z range of 350-1500, and the 20 highest peaks were scanned by MS / MS; all MS / MS spectra were acquired using high-energy collision fragmentation in data-dependent positive ion mode, with a collision energy of 32; the MS / MS resolution was set to 45000, the automatic gain control was set to 2e5, and the maximum ion injection time was 80ms; the dynamic exclusion time was set to 30s.

[0186] 2.6.1.8 Database Search

[0187] The data were analyzed using Proteome Discover 2.4 (Thermofisher). The protein database used was uniprot-proteome_UP000002494-20200817.fasta. Trypsin was used as a digestion enzyme, and the number of missed cuts was set to 2; dynamic modifications were oxidation (M), acetyl (N-terminus), and fixed modifications were TMT (N-terminus, K); carbamidomethyl (C); and the tolerances for MS1 and MS2 were set to 10 ppm and 0.02 Da, respectively.

[0188] 2.6.2 Screening and verification of differentially expressed proteins

[0189] 1) After LuMing Biomedical Company conducted proteomics testing, it searched the database for qualitative and quantitative data, and after quality assessment and preprocessing, performed expression level analysis and functional analysis respectively. Functional annotation analysis of the identified proteins was performed using multiple common databases. GO (Gene Ontology) analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis of the screened differential proteins were performed. In the GO enrichment, protein functions were found to include descriptions of aging, neurons, synapses, dendrites, axons, cell growth, death, and nervous system development. Conditions were set for the original results to screen proteins with a fold change greater than 1.2 times and a p-value < 0.05, and molecules that may be involved in the regulation of neuronal activity were locked in through bioinformatics analysis and literature retrieval.

[0190] 3) In the KEGG pathway analysis, proteins that are in the same pathway and meet the above conditions are searched to obtain the final differential protein screening results. Amyloid precursor protein (APP), apolipoprotein E (APOE), and colony stimulating factor 1 (csf1) are enriched in the Alzheimer's disease pathway, and their expression levels are further detected using the corresponding ELISA kits.

[0191] 4) Through previous literature [7,8] Pericytes can produce NT-3, HGF, BDNF, FGF2 and other proteins through paracrine secretion. Among them, FGF2 was upregulated in the TERT group compared with the NC group in this proteomics detection, and its expression was verified using an ELISA kit. 2.6.3 ELISA method was used to detect the concentrations of APP, APOE, csf1, NT-3, HGF, BDNF, and FGF2 in the conditioned medium of each group

[0192] 1) Take out the kit in advance and equilibrate it to room temperature. Melt the conditioned medium of each group on ice and mix it by vortexing. Prepare the washing solution, standard working solution with different concentration gradients, biotinylated antibody working solution, and enzyme conjugate working solution according to the instructions.

[0193] 2) Set up standard wells, blank wells, and sample wells on the ELISA plate. Add 100μl of standard working solution with different concentration gradients to each standard well, add 100ul of standard diluent to each blank well, and add 100μl of NC group and TERT group conditioned culture solution to each sample well (set up 4 replicates each). Cover the ELISA plate with a film and incubate at 37℃ for 90min. (The sample addition time is less than 10min). 3) Shake out the liquid in the well and pat it dry on clean paper, add 100μl of biotinylated antibody working solution to each well, cover with a film and incubate at 37℃ for 1h.

[0194] 4) Shake out all the liquid in the wells and pat dry on clean paper. Add 350 μl of washing solution to each well, soak for 1 min, shake out all the liquid in the wells, pat dry, and repeat washing 3 times.

[0195] 5) Add 100 μl of enzyme conjugate working solution to each well, cover the membrane, and incubate at 37°C for 30 min.

[0196] 6) Shake off all liquid in the wells and pat dry on clean paper, wash the plate 5 times, using the same method as step 4.

[0197] 7) Add 100 μl of substrate solution to each well, cover the membrane, and incubate at 37°C in the dark for 15 min.

[0198] 8) Add 100 μl of stop solution to each well to terminate the reaction (in the same order as adding substrate solution).

[0199] 9) Set the wavelength of the microplate reader to 450 nm and measure the optical density (OD value) of each well.

[0200] 10) The average OD value of the standard or sample duplicate wells minus the OD value of the blank wells = the calibration value. With the OD value as the horizontal axis and the concentration as the vertical axis, the corresponding function was established using Excel software to calculate the concentrations of APP, APOE, and csf1 in the conditioned culture medium of each group.

[0201] 2.7 APOE4 and FGF2 recombinant proteins and FGF2 neutralizing antibodies intervene in OGD neurons

[0202] 2.7.1CCK8 detection of OGD neuron survival rate

[0203] 1) Five groups were set up: conditioned medium of brain pericytes in the NC group, conditioned medium of brain pericytes in the TERT group, and conditioned medium of brain pericytes in the TERT group + 1 / 10 / 20 (μg / ml) APOE4; five groups were set up: conditioned medium of brain pericytes in the NC group, conditioned medium of brain pericytes in the TERT group, and conditioned medium of brain pericytes in the TERT group + 10 / 100 / 1000 (ng / ml) FGF2; conditioned medium of brain pericytes in the NC group, conditioned medium of brain pericytes in the TERT group, and conditioned medium of brain pericytes in the TERT group There were five groups in total, including brain pericyte conditioned medium + 50 / 100 / 200 (ng / ml) FGF2 neutralizing antibody in the ERT group. Neurons in the 96-well plate that had been subjected to oxygen-glucose deprivation according to the above conditions (Method 2.5.1) were removed and washed twice with PBS buffer. 100 μl of the above-mentioned conditioned medium and recombinant protein were added to each well, and the plates were cultured in a 37°C 5v% CO2 incubator. The APOE4 group was cultured for 72 h, and the FGF2 group and the FGF2 neutralizing antibody group were cultured for 24 h.

[0204] 2) After culturing the OGD neurons according to step 1), remove the supernatant, gently rinse once with PBS buffer, and then add 100 μl of pre-prepared culture medium containing CCK8 to each well (corresponding ratio of culture medium:CCK8=1:9).

[0205] 3) Incubate in a constant temperature incubator for 1.5 h and measure the optical density of each group at a wavelength of 450 nm.

[0206] 4) Calculate cell survival rate = (TERT group - blank) or (NC - blank) × 100%. (This part of the experiment selected the appropriate experimental concentration, and the subsequent experiments used 20 μg / ml APOE4, 1000 ng / ml FGF2, and 50 ng / ml FGF2 neutralizing antibody for verification).

[0207] 2.7.2 CC3 staining to detect apoptosis of OGD neurons

[0208] The method is the same as 2.5.3.

[0209] 2.7.3 TUNEL staining to detect apoptosis of OGD neurons

[0210] The method is the same as 2.5.4.

[0211] 3. Experimental results

[0212] 3.1 Cultivation and identification of brain microvascular pericytes

[0213] 3.1.1 Observation of primary brain microvascular pericytes under optical microscope

[0214] After two digestions with different digestive enzymes and one density gradient centrifugation, viable brain microvascular pericytes can be isolated. After 24 hours, pericytes can be seen adhering to the wall. After 72 hours, the cell body can be seen to be enlarged and have irregular multi-sided protrusions. The initial number of cells is different, and the 97% fusion state can be reached in different time periods (7-14 days), with more protrusions, larger cell bodies, and branched ( Figure 1 AB). 3.1.2 Identification of brain pericytes by immunofluorescence

[0215] In order to identify the extracted primary cells and their purity, immunofluorescence technology was used to detect the molecular markers α-SMA and β-PDGFR that were specifically expressed. The results showed that the positive staining of α-SMA and β-PDGFR and DAPI co-staining reached more than 95%, and both were expressed in the cytoplasm ( Figure 2 AB), while the endothelial cell marker CD31 ( Figure 2 C) astrocyte GFAP ( Figure 2 D) Negative staining. The results showed that the purity of the brain microvascular pericytes cultured after two different digestion enzyme digestions, one density gradient centrifugation, and DMEM+10% FBS+penicillin / streptomycin double antibody culture was high.

[0216] 3.2 Culture and identification of rat cerebral cortical neurons

[0217] The cerebral cortical neurons were cultured in Neurobasal medium containing 2% B27, 0.25% glutamine, and 0.5% penicillin / streptomycin double antibodies using papain digestion. The cells were initially round, and after 24 hours of culture, the neurons were star-shaped with fewer neurites. During the culture process, the neurites gradually became longer. After 5 days of culture, the neurons were identified by immunofluorescence, and the co-staining of NeuN and DAPI reached 95% (see Figure 3). The results showed that the neurons digested with papain and cultured in Neurobasal medium had high purity. 3.3 Transfection of pericytes with lentivirus carrying TERT and determination of transfection efficiency

[0218] 3.3.1 Transfection of pericytes with lentivirus carrying TERT

[0219] Pericytes that reached 97% fusion were digested with trypsin and counted. After being inoculated in a six-well plate and attached to the wall, pericytes were infected with lentiviral empty vector negative control and lentivirus carrying TERT, respectively. Puromycin resistance was used to screen cells stably transfected with TERT (initial screening concentration 0.5μg / ml, continuous screening concentration 0.25μg / ml). The expression of the green fluorescent protein (GFP) tag carried by the GV492 vector was observed using an inverted fluorescence microscope. The results showed that 3 days after lentiviral infection, pericytes reached 95% fusion under a light microscope, and cells expressing green fluorescent protein accounted for about 40% ( Figure 4 AB). After adding puromycin for stable transfection screening, the expression of green fluorescent protein can reach more than 90% ( Figure 4 CD).

[0220] 3.3.2 TERT expression

[0221] The lentiviral empty vector negative control and the lentivirus carrying TERT were used to infect brain microvascular pericytes, respectively. Puromycin resistance was used to screen cells stably transfected with TERT (initial screening concentration 0.5 μg / ml, continuous screening concentration 0.25 μg / ml). RT-PCR was used to detect the expression of TERT mRNA. The results showed that compared with the NC group, the expression of TERT mRNA in brain microvascular pericytes in the TERT group was upregulated by 4.8 times (P<0.001). Figure 5 .

[0222] 3.4 Effects of TERT-overexpressing pericyte conditioned medium on oxygen-glucose deprived neurons

[0223] 3.4.1CCK8 detection of neuronal survival rate

[0224] The ratio of brain pericyte conditioned medium to neuron culture medium was 1:0, 1:1, and 1:2, respectively. The neurons that had completed oxygen-glucose deprivation in 96-well plates were cultured for 24 hours, and the survival rate of neurons was detected using CCK8. Compared with the NC group, when the ratio of brain pericyte conditioned medium to neuron culture medium was 1:0 and 1:1, the survival rate of neurons in the TERT group was higher, and P<0.05, which was statistically significant ( Figure 6). The results showed that when the ratio of brain pericyte conditioned medium to neuron culture medium was 1:0 and 1:1, the survival rate of TERT neurons was significantly increased.

[0225] 3.4.2 PCR detection of apoptosis indicators of oxygen-glucose deprived neurons cultured in conditioned medium with different ratios of brain pericytes

[0226] The ratio of brain pericyte conditioned medium to neuron culture medium was 1:0, 1:1, and 1:2, respectively. The neurons that had completed oxygen-glucose deprivation in 6-well plates were cultured for 24 hours. The expression of Bax and Bcl-2 in neurons was detected by RT-PCR, and the ratio of Bax to Bcl-2 was calculated, recorded as Bax / Bcl-2. Compared with the NC group, when the ratio of brain pericyte conditioned medium to neuron culture medium was 1:0, the Bax / Bcl-2 of neurons in the TERT group was significantly reduced, and P<0.001, which was statistically significant. Figure 7 The results showed that when the ratio of brain pericyte conditioned medium to neuron culture medium was 1:0, TERT neuronal apoptosis was significantly reduced. 3.4.3 CC3 staining to detect apoptosis of OGD neurons cultured with brain pericyte conditioned medium

[0227] After CCK8 and RT-PCR detection, the ratio of brain pericyte conditioned medium to neuron culture medium was 1:0, that is, complete conditioned medium was used to culture OGD neurons. Immunofluorescence staining was used to detect neuronal apoptosis after 24 hours of culture. The results are as follows Figure 8 As shown: Compared with the NC group, the proportion of apoptotic cells in neurons in the TERT group was reduced, P < 0.001, and the difference was statistically significant.

[0228] 3.4.4 TUNEL staining to detect apoptosis of OGD neurons cultured in brain pericyte conditioned medium

[0229] TUNEL staining was used to detect the apoptosis of OGD neurons cultured in brain pericyte conditioned medium for 24 hours. Fig. 9 As shown: Compared with the NC group, the proportion of apoptotic cells in neurons in the TERT group was reduced, P < 0.001, and the difference was statistically significant.

[0230] 3.5 Proteomic detection of brain pericyte conditioned medium and screening of differentially expressed proteins

[0231] Brain microvascular pericytes were transfected with empty lentiviral vectors and lentivirus carrying TERT to overexpress TERT. After stable transfection of TERT, the cells were cultured in serum-free DMEM for 24 hours, and the conditioned medium of brain pericytes, namely the NC and TERT groups, was collected. Shanghai Luming Biotechnology Co., Ltd. undertook proteomic detection of conditioned medium, and functional analysis was performed on the differentially expressed proteins in the conditioned medium of brain microvascular pericytes in the NC and TERT groups. Proteins related to neuronal protection or death were screened out based on protein expression difference fold, differential protein GO functional enrichment analysis, differential protein KEGG pathway analysis, and literature review.

[0232] 3.5.1 Analysis of authentic proteins in brain pericyte conditioned medium

[0233] The conditioned medium of brain pericytes in the NC group and TERT group was labeled with TMT (Tandem Mass Tags) for qualitative and quantitative analysis, and the expression of credible proteins was analyzed by principal component analysis (PCA) to show the relationship between samples from different dimensions. Fig.10 As shown in the figure, each point represents a repetition in a grouping experiment, the circle is the NC group, the diamond is the TERT group, and the ellipse represents the confidence interval of the sample. The position of the coordinate point on the score map is proportional to the difference between the samples. The results show that all samples are within the confidence interval, and the positions of the two groups are far apart, and the difference between the samples is significant, indicating that the secretory function of brain microvascular pericytes changes greatly after overexpressing TERT.

[0234] 3.5.2 Analysis of differential protein expression in brain pericyte conditioned medium

[0235] Based on the reliable proteins, two standards were selected to calculate the differences between samples. Foldchange represents the fold change of the expression level of a protein between groups; the P value calculated by the t-test represents the significance of the change between protein groups. The difference screening conditions are: Foldchange = 1.0 times and P < 0.05. The results are as follows Fig.11 As shown, compared with the NC group, a total of 551 proteins were upregulated and 761 proteins were downregulated in the TERT group.

[0236] Such as volcano map ( Fig.12 ), the horizontal axis is log2 (FC), the farther the value is from 0, the greater the difference, the right side is up-regulated, and the left side is down-regulated. The vertical axis is -log10 (P-value), the farther the vertical axis value is from 0, the greater the difference. The results show that overexpression of TERT has a significant effect on the secretory function of brain microvascular pericytes.

[0237] 3.5.3 Screening and verification of differentially expressed proteins

[0238] In the GO enrichment raw data, according to the protein function description, it includes protein functions related to aging, neurons, synapses, dendrites, axons, cell growth, death, and nervous system development. Proteins that meet the difference Foldchange greater than 1.2 times and p-value < 0.05 were analyzed in detail. Combined with bioinformatics analysis and literature review, 12 proteins related to neuronal protection or death were screened, of which 11 proteins were downregulated and 1 protein was upregulated (Table 1). Further KEGG pathway analysis showed that the differential proteins APP, APOE, and csf1 were simultaneously classified into the pathway related to the neurodegenerative disease Alzheimer's disease, and APP, APOE, and csf1 were found to exist at the same time. In addition, previous literature studies have found that pericytes can secrete proteins such as NT-3, HGF, BDNF, and FGF2 to produce neuroprotective effects, and proteomics results show that FGF2 also has differential expression.

[0239] Table 1. Screened differentially expressed proteins

[0240]

[0241]

[0242]

[0243] 3.5.4 ELISA method to detect the concentration of key differential proteins in the conditioned medium of brain pericytes in each group

[0244] The concentrations of APP, APOE, csf1, NT-3, HGF, BDNF, and FGF2 in the conditioned medium of brain pericytes in each group were detected by ELISA. The protein content in the conditioned medium of pericytes in the NC group and the TERT group was detected by ELISA kits of the corresponding proteins. Fig.14As shown: Among the three proteins with protective significance after downregulation, the contents of APP and APOE in the conditioned medium of brain pericytes in the TERT group were significantly reduced compared with those in the NC group, and P < 0.001, while csf1 did not change significantly. Among the four proteins with protective effects after upregulation in previous literature (Gaceb, A., et al., The pericyte secretome: Potential impact on regeneration. Biochimie, 2018.155: p.16-25.), FGF2 in the conditioned medium of brain pericytes in the NC group increased compared with the DMEM group, and P < 0.001; compared with the NC group, the content of FGF2 in the conditioned medium of brain pericytes in the TERT group was significantly increased, and P < 0.001, both of which were statistically significant. However, NT-3, HGF, and BDNF did not change significantly. The results suggest that brain pericytes may secrete FGF2 to protect OGD neurons, and after overexpression of TERT, brain pericytes may play a neuroprotective role by increasing FGF2, reducing APOE secretion, and downregulating APP expression.

[0245] 3.6 APOE4 and FGF2 recombinant proteins and FGF2 neutralizing antibodies intervene in OGD neurons

[0246] 3.6.1CCK8 detection of the survival rate of OGD neurons under different intervention conditions

[0247] CCK8 was used to detect the survival rate of neurons after different concentrations of recombinant FGF2, APOE4 protein, and FGF2 neutralizing antibody intervention in OGD neurons. The results are as follows Fig.13As shown: Compared with the NC group and the DMEM group, the neuronal survival rate in the TERT group was higher, P < 0.05, and the difference was statistically significant. Compared with the TERT group, the neuronal survival rate was higher after intervention with 10ng / ml, 100ng / ml, and 1000ng / ml FGF2, P < 0.05, and the difference was statistically significant, and with the increase of concentration, the neuronal survival rate increased. After intervention with 50ng / ml, 100ng / ml, and 200ng / ml FGF2 neutralizing antibodies, the neuronal survival rate decreased, P < 0.05, and the difference was statistically significant, but with the increase of concentration, the neuronal survival rate did not change significantly. After intervention with 1μg / ml, 10μg / ml, and 20μg / ml recombinant protein APOE4, the neuronal survival rate decreased, P < 0.05, and the difference was statistically significant, but with the increase of concentration, the neuronal survival rate did not change significantly. The results showed that recombinant FGF2 protein can increase the survival rate of OGD neurons, and as the concentration increases, the protective effect on OGD neurons is more obvious. After intervention with FGF2 neutralizing antibodies and recombinant protein APOE4, the protective effect of brain pericyte conditioned medium in the TERT group on neurons decreased, but as the concentration increases, the reduction effect does not increase significantly. Therefore, we selected 1μg / ml recombinant APOE4 protein, 1000ng / ml recombinant FGF2 protein and 50ng / ml FGF2 neutralizing antibody as the late intervention concentration.

[0248] 3.6.2 CC3 staining to detect recombinant APOE4 protein and recombinant FGF2 to intervene in the apoptosis of OGD neurons

[0249] After exploring the concentration of CCK8 recombinant protein, we selected 1 μg / ml recombinant APOE4 protein and 1000 ng / ml recombinant FGF2 protein to culture OGD neurons. Immunofluorescence technology was used to detect the apoptosis of cultured OGD neurons. The results are as follows Fig.15 As shown in the figure: compared with the NC group, the proportion of apoptotic cells in the TERT group neurons decreased, P < 0.05, the difference was statistically significant, and 1 μg / ml recombinant APOE4 protein and 1000 ng / ml recombinant FGF2 were added to the conditioned medium of brain pericytes in the TERT group, respectively. Compared with the TERT group, the apoptotic cells in the intervention group with 1 μg / ml recombinant APOE4 protein increased (as shown in Figure 2A). Fig.15 ), adding 1000ng / ml recombinant FGF2 intervention group neuronal apoptosis decreased (such as Fig.16 ), P<0.05, the differences were statistically significant.

[0250] 3.6.3 TUNEL staining to detect the apoptosis of OGD neurons by recombinant APOE4 protein and recombinant FGF2

[0251] The TUNEL kit was used to detect the apoptosis of OGD neurons cultured with 1 μg / ml recombinant APOE4 protein and 1000 ng / ml recombinant FGF2 protein. The results of TUNEL staining showed that compared with the NC group, the proportion of apoptotic cells in the neurons of the TERT group was reduced, P < 0.05, and the difference was statistically significant. In addition, after 1 μg / ml recombinant APOE4 protein and 1000 ng / ml recombinant FGF2 were added to the conditioned medium of brain pericytes in the TERT group to intervene in OGD neurons, compared with the TERT group, the apoptotic cells in the intervention group with 1 μg / ml recombinant APOE protein increased (e.g. Fig.17 ), adding 1000ng / ml recombinant FGF2 intervention group neuronal apoptosis decreased (such as Fig.18 ), P<0.05, the differences were statistically significant.

Claims

1. Use of TERT-overexpressing pericyte conditioned medium and recombinant protein FGF2 in the preparation of a drug for protecting hypoxic-ischemic neurons, wherein the TERT-overexpressing pericyte conditioned medium is used to transfect the second-generation pericytes cultured in vitro with a lentivirus containing TERT to construct pericytes overexpressing TERT, and then the pericytes overexpressing TERT are added to serum-free high-glucose DMEM medium for culture, and the TERT-overexpressing pericytes conditioned medium is collected to obtain.

2. The use according to claim 1, characterized in that The brain pericytes overexpressing TERT were added to serum-free high-glucose DMEM medium and cultured in a 37°C constant temperature cell culture incubator for 24h~48h.

Images