Method for prolonging in-vivo survival time of exogenous stem cells and improving biological activity of exogenous stem cells

Abstract

The invention relates to the technical field of cell patches, and discloses a method for prolonging the in-vivo survival time of exogenous stem cells and improving the biological activity of the exogenous stem cells, and the method comprises the following steps: cross-linking a prepared exogenous cell patch in a tannic acid solution before the exogenous cell patch is implanted into a receptor. According to the tannic acid cross-linking method, the prepared cell sheet is soaked in a tannic acid solution for 10-30 S before being implanted into a receptor. In-vitro and in-vivo experimental verification shows that the method provided by the invention can improve the biological activity of the exogenous stem cells on the cell sheet, prolong the in-vivo survival time of the exogenous stem cells, remove reactive oxygen free radicals in an implantation environment of the cell sheet, and provide a better microenvironment for regeneration. The method is easy to operate and good in effect, and a new effective method is provided for improving the repairing effect of the exogenous stem cells in the cell patch.

Description

Technical Field

[0001] The present invention relates to the technical field of cell membrane sheets, and in particular to a method for prolonging the survival time of exogenous stem cells in vivo and enhancing the biological activity thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] In recent years, cell sheet technology based on exogenous stem cells has been widely studied in the field of tissue engineering. In oral restoration, cell sheet technology has been applied to many fields such as periodontal reconstruction and dental pulp regeneration. For example, periodontal ligament stem cells (PDLSCs) have been used for periodontal tissue regeneration and have shown good results. In addition, extracellular matrix derived from mesenchymal stem cells (MSCs) has shown high bone formation differentiation ability and bone defect reconstruction ability in bone regeneration.

[0004] However, studies have shown that after exogenous stem cells are implanted into the recipient, the MHC antigens on the cell surface are recognized by the recipient's immune system, causing a rejection reaction of the immune system, resulting in a low survival rate and short survival time of the implanted exogenous stem cells, and a decrease in regeneration potential. The prior art "Immunomodulatory effect of deferoxamine combined with interferon gamma pretreatment on human dental pulp stem cells" provides a method for enhancing the immunomodulatory effect of human dental pulp stem cells after combined pretreatment with deferoxamine and interferon gamma. After pretreatment, the immunomodulatory effect of human dental pulp stem cells is significantly enhanced, effectively reducing the immune rejection reaction of skin transplantation.

[0005] This comparative document enhances the immunomodulatory activity of human dental pulp stem cells from the source, and enhances the immunomodulatory effect of human dental pulp stem cells and reduces immune rejection after adding appropriate amounts of deferoxamine and interferon gamma to the growth medium of human dental pulp stem cells. Other existing studies have shown that local use of immunosuppressants can also inhibit host immune rejection and effectively prolong the survival time of exogenous stem cells in the body.

[0006] However, local injection of immunosuppressants can only reduce acute rejection reactions, but chronic rejection remains a difficult problem, and chronic rejection is one of the main causes of transplant failure. In addition, immunosuppressive drugs may have adverse effects on the survival, maturation and function of stem cells, and the specific mechanism is not fully understood; and the way of pre-treating cells cannot play an immunomodulatory role in the long term.

[0007] In view of the above problems, it is urgent to develop new methods to enhance the repair effect of exogenous stem cells in cell membranes in vivo. Summary of the invention

[0008] The purpose of the present invention is to provide a method for prolonging the survival time of exogenous stem cells in the body and improving their biological activity, in response to the current urgent need to develop new methods for prolonging the survival time of exogenous stem cells in cell membranes, and to solve the problem of how to load immunomodulators into cell membranes while avoiding the side effects of immunomodulators on the biological activity of stem cells.

[0009] The technical solution of the present invention is as follows:

[0010] On one hand, the present invention provides a method for prolonging the survival time of exogenous stem cells in vivo and enhancing their biological activity, comprising the following steps: cross-linking the prepared exogenous cell membrane in a tannic acid solution before implanting it into a receptor.

[0011] Preferably, the specific method comprises: soaking the prepared cell membrane sheet in a tannic acid solution for 10-30 seconds before implanting it into the receptor.

[0012] Preferably, the concentration of the tannic acid solution is 1%.

[0013] Another aspect of the present invention provides a method for reducing immune rejection of exogenous stem cell membrane transplantation, comprising the following steps: cross-linking the prepared exogenous cell membrane in a tannic acid solution before implanting it into a receptor.

[0014] Preferably, the specific method comprises: soaking the prepared cell membrane sheet in a tannic acid solution for 10-30 seconds before implanting it into the receptor.

[0015] Preferably, the concentration of the tannic acid solution is 1%.

[0016] Another aspect of the present invention provides a method for preparing a cell membrane sheet with a long survival time, comprising the following steps:

[0017] Step (1): preparing a basic cell membrane sheet;

[0018] Step (2): Soak the basic cell membrane sheet in a tannic acid solution for 10-30 seconds to obtain the cell membrane sheet with a long survival time of the present application.

[0019] Preferably, the concentration of the tannic acid solution in step (2) is 1%.

[0020] According to a preferred embodiment, the step (1) specifically comprises: placing 5×10 5 The cells were cultured at a density of 10 cells / mL for 2 weeks to form a basic cell membrane sheet.

[0021] According to a preferred embodiment, the culture medium of the cell membrane sheet is an alpha culture medium comprising 10% fetal bovine serum, 1% penicillin / streptomycin and 50 μg / ml ascorbic acid.

[0022] According to a preferred embodiment, the cross-linking time between tannic acid and the cell membrane sheet is before the cell membrane sheet is implanted into the desired receptor.

[0023] Preferably, in step (1), the cells used in the cell membrane are allogeneic mesenchymal stem cells, such as human dental follicle stem cells, periodontal ligament stem cells, SD rat dental follicle stem cells, etc.

[0024] Another aspect of the present invention provides a cell membrane sheet with a long survival time prepared by the method for preparing a cell membrane sheet with a long survival time as described above.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. A method for prolonging the survival time of exogenous stem cells in vivo and enhancing their biological activity. The present invention ingeniously utilizes the following functions of polyphenols-tannic acid from natural plants to successfully solve the above technical problems: (1) the anti-inflammatory function of tannic acid. Tannic acid can utilize its abundant phenolic hydroxyl groups to remove reactive oxygen free radicals, thereby inhibiting inflammatory reactions; (2) Tannic acid and collagen are rapidly cross-linked through hydrogen bonds. The extracellular matrix in the cell membrane is rich in collagen. Before the cell membrane is implanted in the body, a simple cross-linking / immersion in a tannic acid solution for 20-30 seconds can enrich a large number of tannic acid molecules on the surface of the membrane. After implantation in the body, the tannic acid molecules can be slowly released for 3-7 days, effectively inhibiting the inflammatory response in the early stage of cell membrane implantation: inhibiting the infiltration of immune cells around the membrane and inhibiting the secretion of pro-inflammatory cytokines by immune cells, thereby avoiding these factors from causing damage to the stem cells in the membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of cell proliferation after cross-linking of cell membrane sheets treated in different groups in Example 1 and cultured in vitro for 3, 7 and 14 days;

[0028] Figure 2 It is a data graph of the free radical scavenging ability of cell membranes treated by different groups in Example 1;

[0029] Figure 3 This is a live and dead cell staining image of cells extracted from cell membranes treated in different groups in the rat subcutaneous implantation experiment in Example 2;

[0030] Figure 4 The percentage of apoptosis observed by TUNEL and DAPI staining in Example 2;

[0031] Figure 5 This is a data graph of the cell activity of cell membranes treated in different groups in the rat subcutaneous implantation experiment in Example 2;

[0032] Figure 6The results of Western blot analysis in Example 2 are as follows;

[0033] Figure 7 The results of bioluminescence imaging at 1, 3, 7, 10 and 14 days after subcutaneous implantation in Example 2;

[0034] Figure 8 The semi-quantitative results of bioluminescence fluorescence at 1, 3, 7, 10 and 14 days after subcutaneous implantation in Example 2;

[0035] Fig. 9 The immunofluorescence image in Example 2 shows the pluripotency and cytoskeleton morphology of exogenous stem cells;

[0036] Fig.10 The data diagram of the results of detecting the expression of aging genes and Western blot analysis after subcutaneous implantation of cell membranes treated in different groups in Example 2;

[0037] Fig.11 The immunohistochemical staining in Example 3 shows the expression of markers related to immune regulatory factors;

[0038] Fig.12 The semi-quantitative data diagram of immunohistochemical staining showing immunomodulatory factor-related markers in Example 3;

[0039] Fig.13 H&E staining images of cell membrane sheets on day 3 and day 7 in Example 3;

[0040] Fig.14 Statistical analysis of the thickness of the fiber capsule around the cell membrane sheet on the 7th day in Example 3;

[0041] Fig.15 Schematic diagram of the distribution of T lymphocytes (CD3) and B lymphocytes (CD20) in the tissue surrounding the transplant on day 7 in Example 3;

[0042] Fig.16 The distribution data of T lymphocytes (CD3) and B lymphocytes (CD20) in the tissues surrounding the transplant on the 7th day in Example 3;

[0043] Fig.17 The figure is a schematic diagram showing the expression of inflammation-related markers TNF-α, IL-1β and IFN-γ on the 3rd and 7th days by immunohistochemical staining in Example 3;

[0044] Fig.18 The data graph of the expression of inflammation-related markers TNF-α, IL-1β and IFN-γ on the 3rd and 7th days shown by immunohistochemical staining in Example 3;

[0045] Fig.19The expression of three pro-inflammatory factors and three anti-inflammatory factors in different groups was analyzed by ELISA in Example 3;

[0046] Fig. 20 The micro-CT imaging of the periodontal ligament after 4 weeks of cell sheet treatment in Example 4, and the statistical analysis of the new bone volume in the samples;

[0047] Fig.21 H&E staining of the periodontal defect section in Example 4. (PDL: periodontal ligament, NB: new bone, Defect: periodontal defect);

[0048] Fig. 22 IF staining of osteogenic markers COL1, OCN, PERIOSTIN and CEMP1 in Example 4;

[0049] Fig.23 It is the quantitative analysis based on micro-CT scanning in Example 4, including BV / TV, Tb.N, Tb.Th, Conn.D and Tb.Sp in periodontal defects receiving three types of hydrogels 28 days after surgery (n=6, data are expressed as mean ± standard deviation);

[0050] Fig.24 This is a semi-quantitative analysis of the osteogenic markers COL1, OCN, PERIOSTIN and CEMP1 in Example 4. DETAILED DESCRIPTION

[0051] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the purport of the present invention.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0053] 1. Materials

[0054] Tannic acid (TA) and 2,7-dichlorofluorescein diacetate (DCFHDA) were purchased from Sigma-Aldrich (USA). Fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA), 2,2-diphenyl-1-pyridinium hydrazine (DPPH), 2,2'-naphthalene-3-ethylbenzothiazole-6-sulfonate (ABTS), and 2-phenyl 4,4,5,5-tetramethylimidazolinyloxy-1-oxyl-3-oxide (PTIO) were all from Aladdin Chemical Co., Ltd., China. Calcein AM, propidium iodide (PI), CCK-8 kit, enzyme-linked immunosorbent assay (ELISA) kit, and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) reagent were from Elabscience (China). All reagents used in cell experiments were purchased from Gibco (USA). Antibodies used for immunostaining and western blotting were purchased from Abcam and Invitrogen, USA. – GFP-LUC encoding lentivirus and puromycin were from Shanghai Hanheng Company, China. All chemicals were used according to their commercial sources.

[0055] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0056] Example 1 Preparation of TA-treated stem cell membrane

[0057] 1. In vitro culture experiment: SD rat dental follicle stem cells were cultured in 6-well plates at 5×10 5 The cells were cultured at a density of 10 cells / ml for 14 days to form dental follicle stem cell sheets. The culture medium was α medium containing 10% fetal bovine serum, 1% penicillin / streptomycin and 50 μg / ml ascorbic acid.

[0058] The formed dental follicle stem cell membranes were cross-linked in tannic acid solution for 10S, 20S and 30S respectively before implantation into animals. According to the different cross-linking times, they were divided into TA-0 group (cell membranes not cross-linked with TA), TA-10 group, TA-20 group and TA-30 group, and each group corresponded to the duration of TA cross-linking.

[0059] Specifically, dental follicle stem cell sheets were cross-linked in 1% sterile tannic acid solution for different times and gently rinsed with sterile PBS to remove excess tannic acid (TA). Cell sheets of TA-0, TA-10, TA-20, and TA-30 groups were cultured in vitro for 3, 7, and 14 days, and the number of cells was detected. Figure 1 As shown, the cells in each experimental group continued to proliferate, but the proliferation rate decreased slightly with the extension of cross-linking time.

[0060] 2. The DPPH method was used to evaluate the ability of cross-linked tannic acid (TA) to scavenge ROS in cell membranes.

[0061] The cell membranes of the TA-0, TA-10, TA-20 and TA-30 groups were immersed in 1 ml of 0.4 mM DPPH ethanol solution and stored in a dark place. The absorbance was measured at 517 nm using a spectrophotometer. The calculation formula for DPPH free radical scavenging ability is:

[0062] DPPH scavenging ability (%) = (Ac-As) / Ac×100%,

[0063] Where Ac is the absorbance of the control (DPPH· solution only), and As is the absorbance of the DPPH· solution and TA sample. This method is also applicable to other free radicals (ABTS·, PTIO·).

[0064] The measurement results are as follows Figure 2 shown.

[0065] The ability of cell membranes to scavenge hydrogen peroxide after cross-linking tannic acid (TA) was evaluated using a fluorescent hydrogen peroxide assay kit. Then, the samples were washed with PBS and the medium was replaced with serum-free DCFH-DA at 37°C for 30 minutes. Intracellular ROS levels were assessed using a confocal laser scanning microscope (CLSM, Olympus FV1000, Japan). The relative mean fluorescence intensity (MFI) was quantified by measuring the fluorescence intensity within a single cell area using ImageJ software.

[0066] The calculation results are as follows Figure 2 shown. Figure 2 It can be seen that the cell membranes of each treatment group in this embodiment can scavenge the reactive oxygen free radicals in the environment where the cell membrane is implanted, thereby regulating the local immune microenvironment. The scavenging ability increases with the increase of cross-linking time, especially the TA-20 group and the TA-30 group. There is no significant difference between TA-20 and TA-30, which indicates that the surface-enriched TA molecules have reached the saturation threshold.

[0067] Example 2 Cell activity test of TA-treated stem cell membrane

[0068] 1. Expression of stem cell marker CD90, pluripotency genes (Sox-2, Nanog) and senescence marker (P16) in cell membrane culture in vitro

[0069] Total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit (Vazyme) and then reverse transcribed into cDNA using the HiScriptll 1st Strand cDNA Synthesis Kit (Vazyme, China). Real-time qPCR was performed on the QuantStudio6FlexSystem (Applied Biosystems, USA) using SYBRGreenPCRmix (Bio-Rad, USA) to detect pluripotency genes (Sox-2, Nanog) and aging markers (P16). Relative gene expression was calculated by the 2-ΔΔCT method and normalized to GAPDH as a reference gene.

[0070] To evaluate the protective effect of the TA shell on stem cells on the cell membrane sheet, we incubated the re-extracted stem cells in culture medium for 48 hours. After incubation, the cells were fixed with 4% glutaraldehyde for 10 minutes, permeabilized with 0.1% Tween X-100 in PBS for 10 minutes, and then washed with PBS. Blocked with 1% bovine serum albumin, the cells were incubated with primary antibodies (CD90, Abcam) at 4°C overnight. After this, secondary antibodies were applied, as well as phalloidin and DAPI staining solutions. The cells were observed under a confocal fluorescence microscope. The results are shown in Fig. 9 Western blotting was performed using specific primary antibodies: anti-β-actin (1:5000), anti-Nanog (1:5000) and anti-p16 (1:5000).

[0071] The experimental results are as follows Fig.10 As shown in Figure 3, the expression of the stem cell marker CD90 was significantly increased in the TA shell group compared with the TA-0 group. In addition, in the TA shell group, the expression of genes associated with stem cell properties, including Sox-2 and Nanog, was upregulated, while the expression of the aging-related gene P16 was downregulated. These findings were further confirmed by Western blot analysis, which showed that the expression of Nanog was increased in the TA shell group, while the level of P16 protein was decreased.

[0072] 2. Detection of stem cell viability after cell membrane implantation for different days

[0073] We used a rat subcutaneous implantation model. Cell membranes in the TA-0, TA-10, TA-20, and TA-30 groups were implanted subcutaneously in rats, and samples were taken on the third day. Live / dead staining was used to evaluate the viability of stem cells in the cell membranes. Six images were randomly taken in each group, and the images were imported into ImageJ software for cell counting. The live / dead staining results showed that the proportion of surviving exogenous stem cells in the TA shell group was significantly higher than that in the TA-0 group.

[0074] Subsequently, the cell membrane was lysed using a membrane lysis buffer (according to the manufacturer's instructions), and the stem cells were extracted and counted, and then re-cultured. The growth and proliferation of the re-extracted stem cells were observed under an optical microscope on the 3rd and 5th days of re-culture. Cell migration was assessed using Transwelll. After 24 hours of serum starvation, the cell suspension (100 μL; 1×10 6 mL -1 ) and incubated at 37°C for 12 hours. The cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 1% crystal violet. Finally, the proliferation capacity of the re-extracted stem cells was evaluated by the CCK-8 method, and the optical density (OD) value at 450nm was measured using a spectrophotometer from Thermo Fisher Scientific, USA. The results are shown in Figure 3 and Figure 5 As shown in (A)-(C), on day 3, the number of reattached exogenous stem cells in the TA shell group was significantly higher than that in the TA-0 group. In addition, the cell migration and proliferation abilities in the TA shell group were enhanced compared with those in the TA-0 group.

[0075] Taken together, these results indicate that the TA shell effectively maintains the biological activity of exogenous stem cells by reducing cell death, inhibiting apoptosis, and maintaining cell migration and proliferation abilities.

[0076] Samples were collected on days 3 and 7 after subcutaneous implantation and stained for exogenous stem cells using a one-step TUNEL in situ apoptosis detection kit according to the manufacturer's instructions. All samples were examined under a laser scanning confocal microscope. Figure 4 and Figure 5 As shown in (D), the percentage of apoptotic cells was lower in the TA shell group.

[0077] Western blotting was used to detect the expression of apoptosis-related proteins. Cell lysate proteins were extracted with RIPA Buffer, separated by 10% SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and probed with specific primary antibodies: anti-β-actin (1:5000) and anti-Bax (1:5000) overnight at 4°C. After washing three times, the membrane was incubated with HRP-conjugated secondary antibody. Protein bands were visualized using electrochemiluminescence (ECL) Western Blotting substrate reagent, and images were captured using a chemiluminescence detector. Band intensity was quantified using ImageJ software, and the normalized value was β-ACTIN. The results are shown in Figure 2. Figure 5 (F) and Figure 6 shown.

[0078] To track cells in vivo, bioluminescence imaging was used to assess the retention of exogenous stem cell implants. Stem cells were labeled with lentivirus encoding GFP, and the signal changes on the cell sheet were monitored using an in vivo imaging system (IVIS). Bioluminescence imaging was performed on days 1, 3, 7, 10, and 14 after subcutaneous implantation, and semi-quantitative statistical analysis of fluorescence intensity was performed. Figure 7 and Figure 8 As shown, the bioluminescent signal of the TA-0 group disappeared within 5 days, while in the TA-20 group, the signal lasted for more than 14 days. This result indicates that cross-linking TA significantly improves the survival rate of exogenous stem cells and prolongs the survival time of transplanted exogenous stem cells.

[0079] The treatment of the cell membrane by each group of the present application can protect the activity state, proliferation and migration ability of the implanted stem cells by inhibiting the local immune response, while protecting their stemness, prolonging their survival time and improving their immune regulation ability.

[0080] Example 3 TA-treated stem cell membrane regulates the immune microenvironment

[0081] 1. Rat subcutaneous implantation model to detect the effects of different groups of cell membranes on the infiltration of inflammatory factors in vivo

[0082] To evaluate the in vivo immunoprotective effect of tannic acid (TA) on cell membranes, we used a rat subcutaneous implantation model. Two samples were implanted in each rat; one sample was collected on day 3 and the other on day 7 (n = 4). The samples were fixed in 4% paraformaldehyde overnight, then dehydrated in a series of ethanol solutions and embedded in paraffin. Paraffin sections were prepared and immunohistochemical (IHC) staining was performed to detect the infiltration of inflammatory factors, including TGF-β (1:200 dilution, Abcam, UK) and IL-10 (1:100 dilution, Abcam, UK). PBS was used as a negative control instead of the primary antibody.

[0083] Cell membranes were collected for histological analysis and immunohistochemical staining for TGF-β and IL-10 was performed to evaluate the immunomodulatory capacity of DFSCs. Fig.11 and Fig.12 shown.

[0084] like Fig.13 As shown, there were a large number of inflammatory cells around the TA-0 group, while only a small number of inflammatory cells were found around the TA armored group. On day 7, the TA-0 group showed a thicker fibrous capsule with extensive inflammatory cell infiltration, indicating the presence of ongoing chronic inflammation. In contrast, the TA-20 group showed a significant decrease in inflammatory cell infiltration and a thinner fibrous capsule ( Fig.14). Immunofluorescence staining for T lymphocytes (CD3) and B lymphocytes (CD20) further demonstrated that the cell membrane protected by the TA shell layer was more effective in inhibiting lymphocyte infiltration ( Fig.15 and Fig.16 Immunohistochemical (IHC) staining on days 3 and 7 showed that the cell membrane sheets protected by the TA shell were more effective in inhibiting the production of proinflammatory cytokines such as TNF-α, IL-1β, and IFN-γ ( Fig.17 and Fig.18 ). On day 3, the TA-10 group showed the highest expression levels of TGF-β and IL-10, while there were no significant differences between the TA-0, TA-20, and TA-30 groups. In both the TA-20 and TA-30 groups, excessive suppression of inflammation by sufficiently enriched TA molecules may have resulted in too low concentrations of proinflammatory cytokines to effectively stimulate the anti-inflammatory function of exogenous stem cells. In contrast, the TA-10 group showed the best level of inflammation, promoting the best anti-inflammatory response. On day 7, the TA-20 group showed the highest expression of TGF-β and IL-10, reflecting the sustained effect of TA.

[0085] 2. Effects of different groups of cell membranes on the expression levels of anti-inflammatory and pro-inflammatory factors detected by air pouch model

[0086] To create the air pouch model, the fur was shaved from the left and right lower backs of the rats, and air was injected subcutaneously at these sites. 3 ml of air was injected on the first day, and 2 ml of air was injected on the second and third days. On the seventh day, the injection sites were checked for obvious signs of inflammation, including redness, swelling, pus, nodules, and exudation, confirming that the air pouch was successfully established. Subsequently, cell membrane sheets of different groups were implanted, and the eye bag fluid was collected on the third day for analysis using an ELISA kit. This analysis focused on detecting three anti-inflammatory factors (TGF-β, IL-10, IL-4) and three proinflammatory factors (IL-1β, TNF-α, IFN-γ). The results are shown in Fig.19 As shown. Compared with the TA-0 group, the expression levels of pro-inflammatory cytokines in the TA shell group were significantly downregulated, while the expression levels of anti-inflammatory cytokines were upregulated. These results together indicate that the TA shell group significantly enhanced the immunomodulatory function of ESCs, thereby promoting their regenerative potential by creating a favorable anti-inflammatory microenvironment.

[0087] In summary, the cell membrane prepared in the present application can further regulate the immune microenvironment around the implant, reduce the infiltration of inflammatory cells, reduce the secretion of pro-inflammatory factors, increase the level of anti-inflammatory factors, and provide a better microenvironment for regeneration.

[0088] Example 4 The therapeutic effect of TA-treated stem cell membrane on periodontal multiple tissue defects.

[0089] 1. Evaluation of new bone formation by implanting different groups of cell membranes into the rat periodontal tissue defect model

[0090] Eighteen 8-week-old Sprague-Dawley rats, weighing 200-220 g each, were randomly divided into blank, TA-0, and TA-20 groups, with 6 rats in each group. After anesthesia and disinfection, the buccal alveolar bone, periodontal ligament, and root surface of the mandibular first molar were removed, and a periodontal defect of 3 mm in length × 2 mm in width × 1 mm in depth was formed with a circular thorn. After the periodontal defect was established, the cell membrane sheets of different groups were cross-linked and implanted into the defect site according to the method described above. After 4 weeks, the rats were killed by overdose of anesthesia, and the bone specimens were taken and fixed with 4% paraformaldehyde for further analysis.

[0091] New bone formation was analyzed using micro-computed tomography (micro-CT, kyscan1172, Bruker-microct, Belgium). The newly formed bone area in the micro-CT images was evaluated using the CT Analyser program (SkyScan company). CATn software was used for quantitative analysis of bone volume / tissue volume (BV / TV), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Fixed specimens were decalcified, dehydrated in an ascending ethanol gradient, embedded in paraffin, and sectioned with a microtome (SP1600, Leica, Germany). Continuous 5 μm thick horizontal sections of the defect area were stained with H&E and Masson's trichrome staining (Beyotime, China) to evaluate new bone formation. The results are shown in Figure 2. Figure 20-23 As shown. Quantitative evaluation from micro-CT images showed that the TA-20 group displayed significantly increased bone formation around the tooth root. Compared with the blank group and the TA-0 group, the TA-20 group had significantly increased trabecular number (Tb.N) and trabecular thickness (Tb.Th), and significantly reduced trabecular separation (Tb.Sp). In addition, the TA-20 group had the highest bone volume / tissue volume (BV / TV) and trabecular connection density (Conn.D). Together, these results emphasize the superior regenerative potential of the TA-20 group.

[0092] 2. Immunofluorescence detection of antibodies

[0093] For immunofluorescence (IF), the following antibodies were used: OCN (1:300 dilution, Zenbio, China), OPG (1:200 dilution, Abcam, UK), COL-1 (1:400 dilution, Abcam, UK) and periostin (1:300 dilution, Santa Cruz, USA).

[0094] The results are as follows Fig.24As shown, the density of osteocalcin (OCN)-positive cells was higher in the TA-20 and TA-0 groups compared with the control group. The expression of osteoprotegerin (OPG) was significantly upregulated in the TA-20 group compared with the TA-0 and control groups, indicating enhanced bone formation potential and sustained periodontal tissue formation over time. Immunofluorescence staining for PDL tissue-specific markers COL-1 and periapical osteoprotegerin showed strong positive reactions around the experimental and natural tooth roots, confirming the presence of PDL fibers in the newly formed soft tissue. Taken together, these results clearly demonstrate the superior regenerative capacity of the TA-20 group, which can be attributed to the protective effect of TA in protecting cell survival.

[0095] In summary, this example further verified the survival time and regenerative potential of stem cells through the SD rat periodontal defect animal model.

[0096] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A method for prolonging the survival time of exogenous stem cells in vivo and enhancing their biological activity, characterized in that: The method comprises the following steps: cross-linking the prepared exogenous cell membrane sheet in a tannic acid solution before implanting it into a receptor.

2. A method for prolonging the survival time of exogenous stem cells in vivo and enhancing their biological activity according to claim 1, characterized in that: The specific method includes: soaking the prepared cell membrane sheet in a tannic acid solution for 10-30 seconds before implanting it into the receptor.

3. A method for prolonging the survival time of exogenous stem cells in vivo and enhancing their biological activity according to claim 1, characterized in that: The concentration of the tannic acid solution is 0.5-1.5%.

4. A method for preparing a cell membrane sheet with a long survival time, characterized in that: The steps include: Step (1): preparing a basic cell membrane sheet; Step (2): Soak the basic cell membrane sheet in a tannic acid solution for 10-30 seconds to obtain the cell membrane sheet with a long survival time of the present application.

5. The method for preparing a cell membrane sheet with a long survival time according to claim 4, characterized in that: The concentration of the tannic acid solution is 0.5-1.5%.

6. The method for preparing a cell membrane sheet with a long survival time according to claim 4, characterized in that: The step (1) specifically comprises: in a 6-well plate, 5×10 5 The cells were cultured at a density of 10 cells / mL for 2 weeks to form a basic cell membrane sheet.

7. The method for preparing a cell membrane sheet with a long survival time according to claim 6, characterized in that: The culture medium of the cell membrane sheet is an alpha culture medium including 10% fetal bovine serum, 1% penicillin / streptomycin and 50 μg / ml ascorbic acid.

8. The method for preparing a cell membrane sheet with a long survival time according to claim 7, characterized in that: The cross-linking time between tannic acid and the cell membrane is immediately before the cell membrane is implanted into the receptor.

9. The method for preparing a cell membrane sheet with a long survival time according to claim 7, characterized in that: The cells used in the cell membrane in step (1) are allogeneic mesenchymal stem cells.

10. A cell membrane sheet with a long survival time prepared by the method for preparing a cell membrane sheet with a long survival time as claimed in any one of claims 4 to 9.

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