第三方骨髓间充质干细胞诱导同种异体移植受体免疫耐受机制的研究
发布时间：2018-07-20 11:05
【摘要】：目的：通过对第三方骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cells, BMSCs)注射同种异体皮片移植小鼠受体的研究,验证BMSCs具有延长受体小鼠皮片存活的作用,并初步研究其MSCs诱导同种异体免疫耐受的作用机制。 方法：1BMSCs的分离、培养和鉴定：无菌条件下取SD大鼠股骨,制成骨髓单细胞悬液。采用全骨髓培养法,接种于含有10%FBS的DMEM的培养瓶中培养,间隔3d除去非贴壁细胞,待细胞铺满至瓶底达90%时传代,取第3代以后的细胞,调整浓度为2×106/ml备用。流式细胞仪鉴定SD大鼠MSCs表面抗原CD29、CD34、CD45和CD90。2动物模型的建立：40只雌性C57BL/6小鼠作为供体,麻醉后于其躯干部以钳夹法制成一约1cm2的圆形皮片；40只雄性BALB/C小鼠作为受体,剪去全层皮肤一块,形成约1 cm2的圆形创面,建立稳定的同种异体皮肤移植模型。3细胞,药物输注与实验分组：将40只BALB/C小鼠随机分为4组：①空白对照组：只进行皮肤移植,未给予其他治疗；②环磷酰胺组(CP组)：大剂量环磷酰胺(Cyclophosphamide, CP)腹腔注射,200_mg/kg,连用2_d(q.d.)；③单纯给予SD-BMSCs移植组(SD-BMSCs组)：移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs;④细胞药物联合运用组(CP+ SD-BMSCs组)：大剂量环磷酰胺组(Cyclophosphamide, CP)腹腔注射,200mg/kg,连用2d(q.d.),并于移植当天自受体小鼠尾静脉输注2×106个SD-BMSCs。4移植皮片的大体观察与HE染色：术后第7天观察受体小鼠移植的皮片,并记录存活时间。皮片90%结痂为坏死,若皮片完全变黑、发硬、脱落视为排斥,进行病理分级；并取每组受体小鼠移植皮片进行HE染色观察。5受体小鼠脾脏调节性T细胞(CD4+、D25+、Foxp3+、Treg细胞)的检测：取移植术后第7天受体小鼠脾制备单细胞悬液,用CD4、CD25、Foxp3荧光标记抗体检测移植后Treg细胞的含量。6受体小鼠外周血细胞因子(TGF-p、IL-10、IFN-γ)的检测：取移植术后第7天受体小鼠外周血进行TGF-βIL-10、IFN-γ细胞因子ELISA检测。7不同来源异基因MSCs刺激T淋巴细胞增殖的情况检测：异基因T淋巴细胞(来源于C57小鼠或SD大鼠)与经60Co照射的不用来源MSCs共培养后,MTT法测定异基因T淋巴细胞增殖的情况。 结果：1SD-BMSCs的培养观察与鉴定：全贴壁原代培养的BMSCs_48 h后可见有细胞贴壁生长,部分形态变为梭样,之后细胞迅速增长,至7～11d时细胞已增殖至90%融合,呈漩涡状生长。传代细胞24 h贴壁,5～7_d传一代,可稳定传代至少20次；流式细胞仪鉴定经培养的MSCs的结果为：间充质干细胞表面标志CD29、CD44阳性率分别为99.7%和96.7%；造血系干细胞表面标志CD34,CD45阳性率分别为1.6%和1.3%。2移植皮片的存活时间与HE染色结果：CP+ SD-MSCs组皮肤移植物存活时间为(15.7±1.4)d,空白对照组为(6.1±1.1)d,CP组为(12.3±1.5)d,SD-BMSCs组为(12.6±1.8)d,CP+ SD-BMSCs组皮肤移植物存活时间明显比后3组延长(P0.05)；对照组表现为表皮与真皮层完全坏死,细胞数量明显增加,炎症反应明显。药物组和细胞组可见真皮细胞减少,并呈现广泛纤维化。药物和细胞组可见部分表皮缺失,真皮层正常,并可见毛囊结构。3受体小鼠脾脏调节性T细胞(CD4+、CD25+、Foxp3+、Treg细胞)的检测结果：流式细胞仪检测Treg含量SD-BMSCs组和CP+ SD-BMSCs组明显高于空白对照组和CP组(P0.05).4受体小鼠外周血细胞因子(TGF-β、IL-10、IFN-γ)的检测结果ELISA检测受体外周血MSCs组和CP组TGF-β和IL-10明显高于空白对照组,SD-BMSCs和CP组IFN-γ则明显低于空白对照组(P0.05).5不同来源MSCs刺激异基因T淋巴细胞增殖的情况检测：共培养结果显示：来源于C57小鼠和SD大鼠的MSCs可以明显抑制T淋巴细胞的增殖反应(P0.05),而上述两组组间比较差异则无统计学意义(P0.05) 结论：1第三方MSCs能够延长同种异体皮片移植物的存活时间.2第三方MSCs诱导同种异体移植免疫耐受作用可能与诱导受体Treg细胞增殖有关.3第三方MSCs诱导同种异体移植免疫耐受作用可能与促进免疫耐受因子的表达,抑制免疫排斥因子的表达有关.4不同来源的MSCs在抑制异基因T淋巴细胞的作用上没有明显差别
[Abstract]:Objective: To investigate the effect of BMSCs on the survival of Bone marrow-derived mesenchymal stem cells (BMSCs) by injecting allogeneic skin graft to mice, and the mechanism of MSCs induced allogeneic immune tolerance was preliminarily studied.
Methods: isolation, culture and identification of 1BMSCs: taking SD rat femur under aseptic condition to make single cell suspension of bone marrow, using full bone marrow culture, inoculated in culture bottle containing DMEM containing 10%FBS, removing non adherent cells at intervals of 3D and spreading to the bottom of the bottle at the bottom of the bottle at 90%, and taking the cells after third generations to adjust the concentration to 2 * 106/ml Reserve. Flow cytometry identification of SD rat MSCs surface antigen CD29, CD34, CD45 and CD90.2 animal models: 40 female C57BL/6 mice as donors, after anesthesia, the body of the body with a clamp method into a round piece of circular skin 1cm2; 40 male BALB/C mice as a receptor, cut off the whole layer of skin, forming a round wound of about 1 cm2. Establish a stable allograft skin transplantation model.3 cell, drug infusion and experimental grouping: 40 BALB/C mice were randomly divided into 4 groups: (1) blank control group: only skin transplantation, no other treatment; (group CP): high dose of cyclophosphamide (Cyclophosphamide, CP) intraperitoneal injection, 200_mg/kg, 2_d (q.d.). SD-BMSCs transplantation group (group SD-BMSCs): 2 x 106 SD-BMSCs from recipient mice tail vein on the day of transplantation; (CP+ SD-BMSCs group): intraperitoneal injection of large dose of cyclophosphamide group (Cyclophosphamide, CP), 200mg/kg, and 2D (q.d.), and 2 x 106 infusion from the recipient mouse tail vein on the day of transplantation. The gross observation and HE staining of the SD-BMSCs.4 skin graft: seventh days after the operation, the skin graft of the recipient mice was observed and the survival time was recorded. The 90% crusts of skin slices were necrotic. If the skin slices were completely black, hard and exfoliated as rejection, the pathological grading was carried out, and the splenic regulation of.5 receptor mice was observed by HE staining in each group of receptor mice. Detection of sexual T cells (CD4+, D25+, Foxp3+, Treg cells): seventh days after transplantation, a single cell suspension was prepared in the spleen of the recipient mice, and CD4, CD25, and Foxp3 fluorescent labeling antibodies were used to detect the content of the peripheral blood cytokines in the.6 receptor mice (TGF-p, IL-10, and gamma) after the transplantation. The peripheral blood of the recipient mice was taken seventh days after transplantation. Beta IL-10 and IFN- gamma cytokine ELISA were used to detect the proliferation of T lymphocytes stimulated by different sources of MSCs from.7. The proliferation of allogeneic T lymphocytes (from C57 mice or SD rats) was co cultured with 60Co irradiated non source MSCs, and the proliferation of allogeneic lymphocytes was measured by MTT method.
Results: 1SD-BMSCs culture observation and identification: after the full adherent primary culture of BMSCs_48 h, there was cell wall growth, some form became shuttle, and then the cells grew rapidly, and the cells proliferated to 90% fusion and whirlpool like 7 to 11d. The passages of the cells were 24 h to the wall and 5 to 7_d passed the first generation, which could stabilize the passage for at least 20 times; flow finely The results of the cytosmeter identification were that the surface markers of mesenchymal stem cells were CD29, CD44 positive rates were 99.7% and 96.7%, the hematopoietic stem cell surface markers were CD34, CD45 positive rates were 1.6% and 1.3%.2 graft survival time and HE staining results: CP+ SD-MSCs group skin skin graft survival time was (15.7 + 1.4) D, blank pair The group was (6.1 + 1.1) d, group CP was (12.3 + 1.5) d, SD-BMSCs group was (12.6 + 1.8) d, and the survival time of skin graft in CP+ SD-BMSCs group was significantly longer than that in the latter 3 groups (P0.05). The control group showed that the epidermis and dermis were completely necrotic, the number of cells increased obviously, and the inflammatory reaction was obvious. Fibrosis. Drug and cell groups showed partial epidermis deletion, dermis normal, and detection results of spleen regulatory T cells (CD4+, CD25+, Foxp3+, Treg cells) of the hair follicle structure.3 receptor mice: the flow cytometry was significantly higher in the Treg content SD-BMSCs and CP+ SD-BMSCs groups than in the blank control group and CP group (P0.05) recipient mice peripheral blood The detection results of cytokine (TGF- beta, IL-10, IFN- gamma) were significantly higher than that of the blank control group, TGF- beta and IL-10 in the MSCs group and the CP group were significantly higher than that of the blank control group. The SD-BMSCs and CP group IFN- gamma was significantly lower than that of the blank control group (P0.05). MSCs in rats and SD rats could significantly inhibit the proliferation reaction of T lymphocytes (P0.05), but the difference between the two groups was not statistically significant (P0.05).
Conclusion: 1 third party MSCs can prolong the survival time of allograft graft,.2 third party MSCs induced allograft immune tolerance may be related to the proliferation of.3 third party induced by.3 third party, which may promote the expression of immune tolerance factor and inhibit the immune rejection. There was no significant difference in the expression of MSCs between.4 and T from different sources.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
本文编号：2133320
[Abstract]:Objective: To investigate the effect of BMSCs on the survival of Bone marrow-derived mesenchymal stem cells (BMSCs) by injecting allogeneic skin graft to mice, and the mechanism of MSCs induced allogeneic immune tolerance was preliminarily studied.
Methods: isolation, culture and identification of 1BMSCs: taking SD rat femur under aseptic condition to make single cell suspension of bone marrow, using full bone marrow culture, inoculated in culture bottle containing DMEM containing 10%FBS, removing non adherent cells at intervals of 3D and spreading to the bottom of the bottle at the bottom of the bottle at 90%, and taking the cells after third generations to adjust the concentration to 2 * 106/ml Reserve. Flow cytometry identification of SD rat MSCs surface antigen CD29, CD34, CD45 and CD90.2 animal models: 40 female C57BL/6 mice as donors, after anesthesia, the body of the body with a clamp method into a round piece of circular skin 1cm2; 40 male BALB/C mice as a receptor, cut off the whole layer of skin, forming a round wound of about 1 cm2. Establish a stable allograft skin transplantation model.3 cell, drug infusion and experimental grouping: 40 BALB/C mice were randomly divided into 4 groups: (1) blank control group: only skin transplantation, no other treatment; (group CP): high dose of cyclophosphamide (Cyclophosphamide, CP) intraperitoneal injection, 200_mg/kg, 2_d (q.d.). SD-BMSCs transplantation group (group SD-BMSCs): 2 x 106 SD-BMSCs from recipient mice tail vein on the day of transplantation; (CP+ SD-BMSCs group): intraperitoneal injection of large dose of cyclophosphamide group (Cyclophosphamide, CP), 200mg/kg, and 2D (q.d.), and 2 x 106 infusion from the recipient mouse tail vein on the day of transplantation. The gross observation and HE staining of the SD-BMSCs.4 skin graft: seventh days after the operation, the skin graft of the recipient mice was observed and the survival time was recorded. The 90% crusts of skin slices were necrotic. If the skin slices were completely black, hard and exfoliated as rejection, the pathological grading was carried out, and the splenic regulation of.5 receptor mice was observed by HE staining in each group of receptor mice. Detection of sexual T cells (CD4+, D25+, Foxp3+, Treg cells): seventh days after transplantation, a single cell suspension was prepared in the spleen of the recipient mice, and CD4, CD25, and Foxp3 fluorescent labeling antibodies were used to detect the content of the peripheral blood cytokines in the.6 receptor mice (TGF-p, IL-10, and gamma) after the transplantation. The peripheral blood of the recipient mice was taken seventh days after transplantation. Beta IL-10 and IFN- gamma cytokine ELISA were used to detect the proliferation of T lymphocytes stimulated by different sources of MSCs from.7. The proliferation of allogeneic T lymphocytes (from C57 mice or SD rats) was co cultured with 60Co irradiated non source MSCs, and the proliferation of allogeneic lymphocytes was measured by MTT method.
Results: 1SD-BMSCs culture observation and identification: after the full adherent primary culture of BMSCs_48 h, there was cell wall growth, some form became shuttle, and then the cells grew rapidly, and the cells proliferated to 90% fusion and whirlpool like 7 to 11d. The passages of the cells were 24 h to the wall and 5 to 7_d passed the first generation, which could stabilize the passage for at least 20 times; flow finely The results of the cytosmeter identification were that the surface markers of mesenchymal stem cells were CD29, CD44 positive rates were 99.7% and 96.7%, the hematopoietic stem cell surface markers were CD34, CD45 positive rates were 1.6% and 1.3%.2 graft survival time and HE staining results: CP+ SD-MSCs group skin skin graft survival time was (15.7 + 1.4) D, blank pair The group was (6.1 + 1.1) d, group CP was (12.3 + 1.5) d, SD-BMSCs group was (12.6 + 1.8) d, and the survival time of skin graft in CP+ SD-BMSCs group was significantly longer than that in the latter 3 groups (P0.05). The control group showed that the epidermis and dermis were completely necrotic, the number of cells increased obviously, and the inflammatory reaction was obvious. Fibrosis. Drug and cell groups showed partial epidermis deletion, dermis normal, and detection results of spleen regulatory T cells (CD4+, CD25+, Foxp3+, Treg cells) of the hair follicle structure.3 receptor mice: the flow cytometry was significantly higher in the Treg content SD-BMSCs and CP+ SD-BMSCs groups than in the blank control group and CP group (P0.05) recipient mice peripheral blood The detection results of cytokine (TGF- beta, IL-10, IFN- gamma) were significantly higher than that of the blank control group, TGF- beta and IL-10 in the MSCs group and the CP group were significantly higher than that of the blank control group. The SD-BMSCs and CP group IFN- gamma was significantly lower than that of the blank control group (P0.05). MSCs in rats and SD rats could significantly inhibit the proliferation reaction of T lymphocytes (P0.05), but the difference between the two groups was not statistically significant (P0.05).
Conclusion: 1 third party MSCs can prolong the survival time of allograft graft,.2 third party MSCs induced allograft immune tolerance may be related to the proliferation of.3 third party induced by.3 third party, which may promote the expression of immune tolerance factor and inhibit the immune rejection. There was no significant difference in the expression of MSCs between.4 and T from different sources.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【参考文献】
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1 李富荣;邓春艳;王新根;齐晖;任莉莉;周汉新;;骨髓间充质干细胞联合胰岛移植对受体鼠树突状细胞成熟和功能的影响[J];细胞与分子免疫学杂志;2010年07期
，本文编号：2133320
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