Use of regulatory t cells in the preparation of a medicament for improving survival and quality of survival of subcutaneous fat grafts

Abstract

The application relates to the technical field of subcutaneous fat transplantation, and particularly relates to application of regulatory T cells (Treg) in improving survival rate and survival quality of subcutaneous fat transplantation. The application first finds that the survival rate and survival quality of subcutaneous fat transplantation have a significant relationship with the proportion of regulatory T cells, and the survival rate of subcutaneous fat tissue mixed with regulatory T cells in a specific proportion is obviously improved, so that the application can be used as an innovative fat transplantation technology to improve the existing clinical solution, and has strong clinical transformation value.

Description

Technical Field

[0001] This invention relates to the field of subcutaneous fat transplantation technology, specifically to a novel application of regulatory T cells (Tregs) in subcutaneous fat transplantation. Background Technology

[0002] Subcutaneous fat grafting is a common technique in cosmetic and reconstructive surgery. It involves harvesting fat from areas rich in subcutaneous fat, processing it, and then transplanting (injecting, filling) it into areas such as the face, chest, and soft tissue defects. This improves local tissue volume, resulting in a smoother, fuller, and more aesthetically pleasing appearance. However, subcutaneous fat grafting has a limited survival rate; only about 50-70% of the transplanted fat survives. Furthermore, fibrosis and hematoma organization can occur within the tissue, leading to postoperative nodules, poor sensation, and the need for multiple fat grafts.

[0003] To address the above issues, several technologies can be used to improve the situation, such as the fat centrifugation technique proposed by Coleman and the fat gel technique proposed by Professor Lu Feng in my country. These technologies can significantly improve fat survival rates and are widely used in clinical practice. Existing technologies mainly rely on the mechanical processing of obtained fat cells, filtering / removing impurities such as water and oil from the tissue to improve tissue purity and activity, thereby achieving the goal of increasing the survival rate after fat transplantation.

[0004] However, fat grafting can be considered a form of cell-free transplantation, and the survival of transplanted cells depends on local nutrient supply, local inflammation regulation, and angiogenesis. The tissue surrounding the transplanted fat can provide nutrients to support fat cell survival for a period in the early stages. After this, the transplanted tissue establishes a blood supply with the surrounding tissue through angiogenesis, achieving a stable survival state. In this process, postoperative local inflammation caused by trauma plays a significant double-edged sword. On the one hand, local inflammation can stimulate angiogenesis; on the other hand, excessive inflammation can damage the transplanted tissue, leading to tissue necrosis, fibrosis, and other problems. Therefore, improving the inflammatory state of the transplanted fat tissue, preserving the angiogenesis effect of inflammation while inhibiting its destructive effects, can theoretically significantly improve the survival rate of transplanted fat.

[0005] Chinese patent document CN108721200A discloses a method for preparing and applying an exosome cosmetic preparation derived from human mesenchymal stem cells, and discloses the application of mesenchymal stem cells in assisted fat transplantation.

[0006] However, no new uses for regulatory T cells (Tregs) in the preparation of agents or drugs that promote the survival of subcutaneous transplanted fat have been reported to date. Summary of the Invention

[0007] The purpose of this invention is to provide, based on theoretical analysis and experimental research, a method for mixing a certain proportion of regulatory T cells (Tregs) into subcutaneous adipose tissue to be transplanted, addressing the limitations and deficiencies of existing technologies. This method can reduce tissue fibrosis and hematoma, inhibit the destructive effects of local inflammation without affecting angiogenesis, and significantly improve the survival rate of transplanted fat. Thus, a new technique for subcutaneous fat transplantation is proposed.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] 1) It was found that the survival quality of subcutaneous fat donors from females was significantly better than that from males.

[0010] 2) The survival quality of subcutaneous fat with T cell development defects in the body is significantly reduced, while there is no significant difference between male and female fat.

[0011] 3) The proportion of Tregs in subcutaneous fat derived from females is significantly higher than that from males.

[0012] 4) The survival rate of Treg cells was shown to be related to that of Treg cells after mixing them with subcutaneous adipose tissue with T cell development defects in vivo.

[0013] Further, the specificity of the Treg effect was verified by mixing non-Treg CD4+ cells with subcutaneous adipose tissue with T cell development defects in vivo.

[0014] Further, the survival rate of Treg cells was measured after mixing them with subcutaneous adipose tissue with T cell development defects in vivo. This verified the role of Treg cells and confirmed whether: 1. a higher Treg content is always better; and 2. whether there is an optimal value for Treg content.

[0015] This invention utilizes animal experiments, employing wild-type Balb / c male and female mice, as well as nude mice with a Balb / c background (lacking T-cell development). Fat transplantation involved mixing subcutaneous fat from different mouse species into a suspension and transplanting it into different mouse species according to the experimental design. Two weeks later, mice were sacrificed to obtain transplanted adipose tissue. Fat survival rate and quality were assessed through wet weighing, histological examination, and analysis. Treg and other cell detection methods included isolating subcutaneous fat from wild-type Balb / c male and female mice, separating it into single cells, and performing staining and flow cytometry analysis. Isolation of Treg and non-Treg CD4+ T cells: A magnetic bead isolation kit was purchased, and mouse spleen single cells were further isolated according to the kit instructions. Detection of inflammatory markers and Treg content in transplanted fat was achieved by obtaining intracellular RNA, transcribing it into cDNA, and performing real-time quantitative PCR (qPCR). Experimental results were analyzed using analysis of variance and t-tests.

[0016] Results: The survival rate of subcutaneous fat from wild-type female Balb / c mice transplanted into female Balb / c mice was better than that from male to male, with significantly reduced intrafacial fibrosis, hematoma, and infiltrating inflammatory cells. The survival rate of subcutaneous fat from wild-type female Balb / c mice transplanted into male Balb / c mice was better than that from male to female, with persistently low levels of intrafacial fibrosis, hematoma, and infiltrating inflammatory cells, indicating a high survival rate of female-derived adipose tissue and low influence from recipient factors. The survival rate of subcutaneous fat from female Balb / c-background nude mice transplanted into female nude mice showed no difference compared to male-to-male nude mice, but intrafacial fibrosis, hematoma, and infiltrating inflammatory cells were significantly worse than those from wild-type mouse-derived subcutaneous fat. Flow cytometry analysis revealed that the total proportion of CD4+ T cells and the content of Tregs in subcutaneous fat derived from wild-type female Balb / c mice were significantly higher than those in males. However, there were no significant sex differences in the proportions of non-Treg CD4+Foxp3-Granzyme B+ effector T cells, total CD8+ T cells, and CD8+Granzyme B+ cytotoxic T cells. After separating Treg cells with 80% purity and mixing them with nude mouse fat, the survival rate, quality, and fibrosis of fat mixed with 500,000 Tregs (CD4+CD25+) were significantly better than those of the 100,000, 200,000, and 1,000,000 groups, and also better than fat mixed with 500,000 non-Treg CD4+ T cells (CD4+CD25-). The analysis indicated that the Treg content in Treg-mixed fat was significantly higher than that of non-Treg CD4+ T cells (CD4+CD25-), and the expression of inflammatory factors was significantly reduced.

[0017] Based on the above technical solution, the first aspect of the present invention provides the use of regulatory T cells (Tregs) in the preparation of reagents or drugs that improve the survival rate and quality of subcutaneous fat transplantation.

[0018] Furthermore, the subcutaneous fat transplantation is an autologous fat transplantation.

[0019] Furthermore, the purity of the regulatory T cells is above 80% (CD4+CD25+ Treg).

[0020] Furthermore, the reagents or drugs used to improve the survival rate and quality of subcutaneous fat grafts are injectables.

[0021] Furthermore, the reagents or drugs used to improve the survival rate and quality of subcutaneous fat grafts include regulatory T cells (Tregs).

[0022] Furthermore, in the aforementioned application, regulatory T cells (Tregs) are mixed in before subcutaneous fat transplantation.

[0023] Furthermore, in the aforementioned application, subcutaneous fat contains regulatory T cells that comprise 1% to 10% of the total number of adipocytes.

[0024] Furthermore, in the aforementioned application, subcutaneous fat contains regulatory T cells that comprise 1% to 5% of the total number of adipocytes.

[0025] Further animal experiments revealed that the survival quality was best when the Treg mixture ratio was 5%, the fat survival quality was relatively high when the mixture ratio was 1% and 2%, and the fat survival quality was significantly reduced when the mixture ratio was 10%.

[0026] In one embodiment of the invention, after anesthetizing the donor mouse, the skin was disinfected and kept warm. Inguinal fat was obtained, placed in physiological saline, shredded, and washed three times until the fat became emulsifiable. It was then allowed to stand for 30 minutes. Once the fat and water separated (water at the bottom), the water was drained and set aside. After anesthetizing the recipient mouse, the skin was disinfected and kept warm. 0.1 ml of the processed fat, mixed with or without Treg, was injected subcutaneously into the back of the mouse using a 20G blunt needle. 0.1 ml of adipose tissue contains approximately 10 cells. 7 The Treg content in the subcutaneous fat of normal mice is approximately 10. 5 Animal experiments have shown that when the number of Treg mixtures is 5 × 10⁻⁶, the number of Tregs is 5 × 10⁻⁶. 5 The optimal survival quality is achieved when 5 × 10⁻⁶ adipose tissue is mixed with the adipose tissue. 5 / 10 7 The survival rate of adipose tissue is highest when the percentage is 5%.

[0027] Furthermore, the regulatory T cells reduce fibrosis in subcutaneous transplanted adipose tissue and hematoma in the transplanted adipose tissue, inhibiting the destructive effects of local inflammation without affecting angiogenesis.

[0028] A second aspect of the invention provides the use of regulatory T cells (Tregs) in the preparation of reagents or medicaments for reducing complications of subcutaneous fat grafting surgery.

[0029] In a third aspect, the present invention provides a reagent or drug for improving the survival rate and quality of subcutaneous fat grafts, said reagent or drug comprising regulatory T cells (Tregs) and pharmaceutically acceptable carriers or excipients.

[0030] A fourth aspect of the present invention provides a method for improving the survival rate and quality of subcutaneous fat transplantation by mixing regulatory T cells (Tregs) before subcutaneous fat transplantation.

[0031] A fourth aspect of the present invention provides a method for maintaining normal subcutaneous fat function by mixing regulatory T cells, comprising 1% to 10% of the number of adipocytes, into the subcutaneous fat.

[0032] Furthermore, regulatory T cells, comprising 1% to 5% of the total number of adipocytes, are mixed within the subcutaneous fat.

[0033] The beneficial effects of this invention are as follows:

[0034] 1. This invention is the first to discover that the survival rate and quality of subcutaneous fat transplantation are significantly related to the proportion of Treg cells, which can significantly improve the survival rate and quality of fat.

[0035] 2. This invention is the first to discover that the optimal ratio of Treg cells mixed within subcutaneous fat is not necessarily better the more there are. Combining points 1 and 2, a new technique for Treg-mixed subcutaneous fat transplantation can be proposed, providing new ideas and potential solutions for improving the survival rate and quality of life after fat transplantation and reducing surgical complications.

[0036] 3. This invention is the first to discover that the survival rate and quality of life after subcutaneous fat transplantation are significantly better in females than in males, while the impact on the recipient is lower. This can provide new insights for clinical diagnosis and treatment, namely that male patients require a larger amount of fat per transplantation and a greater number of transplantation sessions compared to female patients. Attached Figure Description

[0037] Figure 1(A) Schematic diagram: Fat was removed from the donor, minced, washed, centrifuged, and collected in a 1ml syringe, then transplanted subcutaneously into the recipient mouse. Samples were collected and analyzed after 2 weeks. (B) Wild-type mouse fat was transplanted into nude mice (same sex, i.e., female to female, male to male). The size of surviving fat in female-to-female transplants was significantly larger than that in male transplants. (C) The wet weight was also significantly greater. (D) H&E histology indicated that female-to-female transplants had better survival quality, while male-to-male transplants showed extensive infiltration of confirmatory cells. (E) Oil Red staining also supported the good results of female-to-female transplants, while the fat droplets in male-to-male transplants were larger and contained many vacuoles. WT: Wild-type; Nude: Nude mouse; **: P<0.01. The large icon bar in the histological images is 500μm, and the small icon bar is 200μm.

[0038] Figure 2 (A) When wild-type mouse fat was transplanted into nude mice (different sexes, i.e., female to male, male to female), the surviving fat from female mice was significantly larger than that from male mice. (B) The wet weight was also significantly greater. (C) H&E histology indicated that the female-to-male transplant had better survival quality, while a large number of verification cells were observed to infiltrate the tissue from male-to-female transplants. WT: wild-type; Nude: nude mouse; *: P<0.05. The large icon bar in the histological images is 500 μm, and the small icon bar is 200 μm.

[0039] Figure 3 (A) When fat was transplanted from nude mice to nude mice (same sex, i.e., female to female, male to male), there were no significant differences in the size of the surviving fat, (B) wet weight, and (C) H&E histology. Histology showed extensive infiltration of inflammatory cells. (D) When fat was transplanted from nude mice to nude mice (different sexes, i.e., female to male, male to female), there were no significant differences in the size of the surviving fat, (E) wet weight, and (F) H&E histology. Histology showed extensive infiltration of inflammatory cells. Nude: nude mouse; ns: no significant difference. The large icon bar in the histological images is 500 μm, and the small icon bar is 200 μm.

[0040] Figure 4(A) Subcutaneous fat was obtained from wild-type female and male mice, and the proportions of CD4+ and CD8+ T cells in lymphocytes were detected. (B) The proportion of CD4+ T cells was higher in females than in males, while (C) the proportion of CD8+ T cells showed no significant difference. Based on the gates in Figure (D), namely Foxp3+ Tregs and Foxp3- CD4+ effector T cells (Teff), analysis (E) showed that the proportion of Tregs in all lymphocytes and CD4+ subsets in female fat was significantly higher than that in males (F&G). However, there was no significant difference in Granzyme B expression in Teff cells (H) (I). (J) Further analysis of Granzyme B expression in gated CD8+ T cells indicated no significant difference between males and females (K). WT: Wild-type; ♀: Female; ♂: Male.

[0041] Figure 5 (A) Schematic diagram: Flow cytometry sorting of CD4+CD25+Treg and CD4+CD25-Teff cells using Miltenyi magnetic beads revealed that Treg purity reached 80%, and Teff purity was close to 100%. Treg or Teff cells were mixed with subcutaneous fat from nude mice, transplanted, and analyzed. (B) Mixing 100,000, 200,000, 500,000, and 1,000,000 Treg cells with adipocytes, followed by transplantation and tissue sampling, revealed that the 500,000 Treg group showed the best fat survival size, fat treatment, and (C) wet weight. Comparing the 500,000 Treg-mixed fat and the 500,000 Teff-mixed fat groups, (D) the Treg group showed significantly better fat survival wet weight and (E) tissue quality than the Teff group, while a large number of inflammatory cells were observed to infiltrate the Teff-mixed fat. When qPCR was used to detect surviving fat, it was found that the gene expression of (F) Foxp3, a Treg-specific transcription factor, was significantly higher in the Treg-mixed fat group than in the Teff group; while the expression of the inflammatory factor TNFα was significantly lower in the Treg group. *: P < 0.05; **: P < 0.01. The large icon scale in histological images is 500 μm, and the small icon scale is 200 μm. Detailed Implementation

[0042] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0043] Example 1

[0044] I. Materials and Methods

[0045] 1. Obtaining mouse fat grafts

[0046] BALB / c mice weighing 18-22g and nude mice weighing 15-20g used in this experiment were obtained from the Ninth People's Hospital affiliated with Shanghai Jiao Tong University School of Medicine. Adipose tissue was obtained from the bilateral inguinal region. After washing (using phosphate-buffered saline, PBS, HyClone, Logan, UT, USA and 1% penicillin / streptomycin, P / S, Gibco, Grand Island, NY, USA), excess liquid was absorbed with gauze. The adipose tissue was then minced, centrifuged, and stored on ice for later use.

[0047] 2. In vivo experiment A (animal surgery)

[0048] In this experiment, the animals were randomly divided into 8 groups (n=6): Group A, female BALB / c transplanted into female nude mice; Group B, male BALB / c transplanted into male nude mice; Group C, female BALB / c transplanted into male nude mice; Group D, male BALB / c transplanted into female nude mice; Group E, female nude mice transplanted into male nude mice; Group F, female nude mice transplanted into male nude mice; and Group G, male nude mice transplanted into female nude mice.

[0049] Preoperatively, mice were anesthetized with isoflurane inhalation (oxygen flow rate of 400 ml / min), and the skin was cleaned. Approximately 0.1 mL of fat (mixed or unmixed with Treg or Teff) was injected subcutaneously into the back of each mouse, one on each side, and the wet weight was recorded. Two weeks later, the mice were sacrificed and the transplanted fat was harvested. Gross photographs of the grafts were taken, and the wet weight was measured. Half of the tissue was stored at -80°C, and the other half was internally fixed in 4% paraformaldehyde. A small fraction of the sample (approximately 20 mg) was also aliquoted and stored at -80°C for real-time quantitative PCR (qRT-PCR) analysis (details see 7).

[0050] 3. Isolate and purify Tregs

[0051] We used an IL-2 complex (IL-2C) to expand Treg cell numbers. Specifically, we mixed 2 μg of recombinant mouse IL-2 (Biolegend) and 10 μg of IL-2 monoclonal antibody (mAb, JES6-1, BioXcell), followed by incubation at 37°C for 30 minutes. BALB / c mice were injected with L-2C daily for 3 consecutive days. After a 2-day interval, mouse spleens were harvested and single cells were isolated, resuspended in PBS + 1% fetal bovine serum FBS (ScienCell Research Laboratories, San Diego, CA, USA). After erythrocyte lysis, the single cells were placed statically in FBS. Cells were then isolated using Miltenyi Biotec's CD4+CD25+ Treg cell isolation kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions. The purity was then verified using flow cytometry, ensuring a purity of >80% for CD4+CD25+Treg cells and nearly 100% for CD4+CD25- effector T cells.

[0052] 4. Single-cell isolation and flow cytometry analysis of adipose tissue

[0053] Subcutaneous inguinal fat was directly obtained from wild-type Balb / c mice, isolated into single cells, and then stained and analyzed by flow cytometry. Antibodies used were anti-CD4, -CD8α (BD PharMingen, San Diego, CA) and anti-foxp3, Granzyme B, Foxp3 (eBioscience, San Diego, CA). All assays were performed according to the product instructions and instrument operating guidelines. Flow cytometry results were analyzed using FlowJo 8 software (Tree-Star).

[0054] 5. In vivo experiment B (Treg mixed fat transplantation)

[0055] In this experiment, animals were randomly divided into 5 groups (n=6): Group A, 100,000 Tregs mixed with male nude mouse fat transplanted into male nude mice; Group B, 200,000 Tregs mixed with male nude mouse fat transplanted into male nude mice; Group C, 500,000 Tregs mixed with male nude mouse fat transplanted into male nude mice; Group D, 1,000,000 Tregs mixed with male nude mouse fat transplanted into male nude mice; Group E, 500,000 Teffs mixed with male nude mouse fat transplanted into male nude mice. The fat transplantation surgery and harvesting methods were the same as in section 2.

[0056] 6. Histological analysis

[0057] After paraformaldehyde-fixed tissues were embedded in paraffin and cut into 5 μm sections, they were stained with H&E and Masson staining, and the size of fat droplets, hematoma, microdispersion, and inflammatory cell infiltration were observed under a microscope. Evaluation was performed by two researchers.

[0058] In Oil Red staining, fixed tissue is directly frozen into 5μm sections, then stained with 60% Oil Red dye in the dark for 10 minutes. The sections are then washed with pure water and 60% isopropanol, then immersed in hematoxylin for 3 minutes before mounting and observation.

[0059] 7. Real-time quantitative PCR

[0060] Treg-related gene expression was detected using quantitative real-time PCR (qRT-PCR). All RNA was extracted using the Tissue RNA Purification Kit (EZB-RN001A) (EZBioscience, Roseville, MN, USA) according to the manufacturer's instructions. RNA purity was assessed using the A260 / A280 ratio (between 1.8 and 2.1). RNA was then transcribed into cDNA using a 4× Reverse Transcription Kit (A0010) (EZBioscience). qRT-PCR was performed using cDNA as a template and a SYBR kit (EZBioscience) under the following conditions: denaturation (5 min, 95°C), and 40 cycles of amplification (10 s at 95°C and 30 s at 60°C) designed using a StrataGene Mx3000p (Agilent Technologies, Inc., Santa Clara, CA). Primer synthesis was performed by Sangon Biotech Co. (Shanghai, China), and target gene expression was normalized and quantified using Ct calculations. The primers used are shown in Table 1 below.

[0061] Table 1

[0062]

[0063] 8. Statistical Analysis

[0064] Statistical analysis was performed using SPSS software (version 16.0, SPSS Inc., Chicago, IL). Differences between variables were analyzed using two-tailed t-tests based on the concentration values ​​obtained in step 4. One-way ANOVA (using the F-distribution) was used for comparisons of three or more samples. When the data did not follow a Gaussian distribution, the Wilcoxon paired-signed-rank test and the Mann-Whitney test were used as two nonparametric tests. All measurement data are expressed as mean ± SD. P < 0.05 was considered statistically significant.

[0065] II. Results

[0066] 1. The quality of subcutaneous fat transplanted from wild-type females into same-sex nude mice was superior to that transplanted from wild-type males into male nude mice.

[0067] according to Figure 1 Schematic diagram A: Fat was harvested from the donor, minced, washed, centrifuged, and collected in a 1ml syringe, then transplanted subcutaneously into the recipient mouse. Samples were collected and analyzed two weeks later. Figure 1 B shows that when wild-type mouse fat is transplanted into nude mice (same sex, i.e., female to female, male to male), the surviving fat grafts from female to female are significantly larger than those from males. Furthermore ( Figure 1 C) The wet weight was also significantly higher (P<0.01), Figure 1 D) H&E histology suggests that female-to-female survival quality is good, while male-to-male tissue shows abundant infiltration of confirmatory cells. (And...) Figure 1 E) Oil red staining also supports the conclusion that female-to-female transplantation has a better effect, while male-to-male tissues have larger fat droplets and more vacuoles.

[0068] 2. The quality of subcutaneous fat transplanted from wild-type females to male nude mice was superior to that transplanted from wild-type males to female nude mice.

[0069] Figure 2 A suggests that when fat from wild-type mice was transplanted into nude mice (different sexes, i.e., female to male, male to female), the surviving fat from the female mice was significantly larger than that from the male mice. (And...) Figure 2 B) The wet weight is also significantly greater. Figure 2 C) H&E histology suggests that the female-to-male survival quality is good, and a large number of verification cells can be seen infiltrating the male-to-female tissue.

[0070] 3. Fat survival in nude mice was not related to the sex of the donor and recipient.

[0071] Figure 3 A suggests that fat grafts were transplanted from one nude mouse to another (of the same sex, i.e., female to female, male to male), and the size of the surviving fat grafts was [not specified]. Figure 3 B) Wet weight and ( Figure 3 C) H&E histological examination showed no significant differences, but extensive inflammatory cell infiltration was observed. Figure 3 Fat grafts from type D nude mice were transplanted into nude mice (different sexes, i.e., female to male, male to female). The size of the surviving fat grafts was... Figure 3 E) wet weight and ( Figure 3 F)H&E histology showed no significant differences, but histology revealed extensive inflammatory cell infiltration.

[0072] 4. In wild-type mice, the Treg content in the subcutaneous fat of females was significantly higher than that of males.

[0073] like Figure 4 As shown in Figure A, we first obtained subcutaneous fat from wild-type female and male mice and detected the ratio of CD4+ and CD8+ T cells in the lymphocytes, among which ( Figure 4 B) CD4+ T cells are more abundant in females than in males, while ( Figure 4 C) The proportion of CD8+ T cells did not differ significantly. Figure 4 The gate of D is the Foxp3+ Treg and the Foxp3- CD4+ effector T cell (Teff), which are analyzed ( Figure 4 E) The proportion of Treg cells in all lymphocytes and CD4+ subsets of cells in female adipose tissue was significantly higher than that in males. Figure 4 F&G). And in Teff cells ( Figure 4 There was no significant difference in the expression of H) Granzyme B. Figure 4 I). Figure 4 Further analysis of Granzyme B expression in J-ring CD8+ T cells showed no significant difference between males and females. Figure 4 K).

[0074] 5. Verification showed that mixing Tregs into fat cells significantly improved fat survival rate.

[0075] like Figure 5 As shown in Schematic A, flow cytometry sorting of CD4+CD25+Treg and CD4+CD25-Teff cells using Miltenyi magnetic beads revealed that Treg purity reached 80% and Teff purity was close to 100%. Treg or Teff cells were mixed with subcutaneous fat from nude mice, transplanted, and then analyzed. Figure 5 In B, we mixed 100,000, 200,000, 500,000, and 1,000,000 Treg cells with adipocytes, transplanted them, and harvested samples. We found that the 500,000 Treg group had better fat survival size, better fat treatment outcomes, and better fat grafting results. Figure 5 C) The optimal wet weight was achieved. However, comparing the 500,000 Treg mixed fat group and the 500,000 Teff mixed fat group revealed ( Figure 5 D) Treg group fat survival wet weight and ( Figure 5 E) Tissue quality was significantly better than Teff, with abundant inflammatory cell infiltration observed in Teff mixed adipose tissue. When we used qPCR to detect surviving adipose tissue, we found that the Treg mixed adipose tissue group had significantly better tissue quality than the Teff group. Figure 5 F) The gene expression of Foxp3, a Treg-specific transcription factor, was significantly higher in the F group than in the Teff group; while the expression of the inflammatory factor TNFα was significantly lower in the F group than in the Teff group.

[0076] III. Conclusion

[0077] This invention found that subcutaneous fat transplanted from wild-type female mice had a higher survival rate than that from male mice, and this difference was not significantly different from the recipient's sex. When we replaced the donor mice with nude mice, we found that both the fat survival rate and the quality of survival decreased, and there was no difference between sexes. This indicates that T cells in fat play a role in fat survival. Therefore, further analysis revealed that the proportion of Tregs in the subcutaneous fat of female mice was significantly higher than that of males. In further validation experiments, mixing Tregs with nude mouse fat showed that mixing in a certain proportion (not the higher the better) of Tregs significantly improved the fat survival rate. This effect was caused by the anti-inflammatory effect resulting from the higher Treg retention rate. The above results indicate that subcutaneous fat tissue with a specific proportion of Tregs has a significantly improved survival rate and can serve as an innovative fat transplantation technique to improve existing clinical solutions, possessing strong clinical translational value.

[0078] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application. sequence list <110> Shanghai Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine <120> Application of regulatory T cells in the preparation of reagents or drugs to improve the survival rate and quality of life of subcutaneous fat grafts <130> / <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> Artificial sequence <400> 1 gaccctcaca ctcagatcat cttct 25 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <400> 2 cctccacttg gtggtttgct 20 <210> 3 <211> 19 <212> DNA <213> Artificial sequence <400> 3 ggcccttctc caggacaga 19 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 gctgatcatg gctgggttgt 20 <210> 5 <211> 20 <212> DNA <213> Artificial sequence <400> 5 cccgtagaca aaatggtgaa 20 <210> 6 <211> 20 <212> DNA <213> Artificial sequence <400> 6 tgccgtgagt ggagtcatac 20

Claims

1. The application of regulatory T cells in the preparation of drugs to improve the survival rate and quality of subcutaneous fat transplantation; wherein the subcutaneous fat transplantation is autologous fat transplantation; wherein the purity of the regulatory T cells is above 80%; wherein the drug to improve the survival rate and quality of subcutaneous fat transplantation includes regulatory T cells accounting for 5% of the number of adipocytes.

Images