第三方骨髓間充質(zhì)干細(xì)胞誘導(dǎo)同種異體移植受體免疫耐受機(jī)制的研究
發(fā)布時(shí)間：2018-07-20 11:05
【摘要】：目的：通過(guò)對(duì)第三方骨髓間充質(zhì)干細(xì)胞(Bone marrow-derived mesenchymal stem cells, BMSCs)注射同種異體皮片移植小鼠受體的研究,驗(yàn)證BMSCs具有延長(zhǎng)受體小鼠皮片存活的作用,并初步研究其MSCs誘導(dǎo)同種異體免疫耐受的作用機(jī)制。 方法：1BMSCs的分離、培養(yǎng)和鑒定：無(wú)菌條件下取SD大鼠股骨,制成骨髓單細(xì)胞懸液。采用全骨髓培養(yǎng)法,接種于含有10%FBS的DMEM的培養(yǎng)瓶中培養(yǎng),間隔3d除去非貼壁細(xì)胞,待細(xì)胞鋪滿至瓶底達(dá)90%時(shí)傳代,取第3代以后的細(xì)胞,調(diào)整濃度為2×106/ml備用。流式細(xì)胞儀鑒定SD大鼠MSCs表面抗原CD29、CD34、CD45和CD90。2動(dòng)物模型的建立：40只雌性C57BL/6小鼠作為供體,麻醉后于其軀干部以鉗夾法制成一約1cm2的圓形皮片；40只雄性BALB/C小鼠作為受體,剪去全層皮膚一塊,形成約1 cm2的圓形創(chuàng)面,建立穩(wěn)定的同種異體皮膚移植模型。3細(xì)胞,藥物輸注與實(shí)驗(yàn)分組：將40只BALB/C小鼠隨機(jī)分為4組：①空白對(duì)照組：只進(jìn)行皮膚移植,未給予其他治療；②環(huán)磷酰胺組(CP組)：大劑量環(huán)磷酰胺(Cyclophosphamide, CP)腹腔注射,200_mg/kg,連用2_d(q.d.)；③單純給予SD-BMSCs移植組(SD-BMSCs組)：移植當(dāng)天自受體小鼠尾靜脈輸注2×106個(gè)SD-BMSCs;④細(xì)胞藥物聯(lián)合運(yùn)用組(CP+ SD-BMSCs組)：大劑量環(huán)磷酰胺組(Cyclophosphamide, CP)腹腔注射,200mg/kg,連用2d(q.d.),并于移植當(dāng)天自受體小鼠尾靜脈輸注2×106個(gè)SD-BMSCs。4移植皮片的大體觀察與HE染色：術(shù)后第7天觀察受體小鼠移植的皮片,并記錄存活時(shí)間。皮片90%結(jié)痂為壞死,若皮片完全變黑、發(fā)硬、脫落視為排斥,進(jìn)行病理分級(jí)；并取每組受體小鼠移植皮片進(jìn)行HE染色觀察。5受體小鼠脾臟調(diào)節(jié)性T細(xì)胞(CD4+、D25+、Foxp3+、Treg細(xì)胞)的檢測(cè)：取移植術(shù)后第7天受體小鼠脾制備單細(xì)胞懸液,用CD4、CD25、Foxp3熒光標(biāo)記抗體檢測(cè)移植后Treg細(xì)胞的含量。6受體小鼠外周血細(xì)胞因子(TGF-p、IL-10、IFN-γ)的檢測(cè)：取移植術(shù)后第7天受體小鼠外周血進(jìn)行TGF-βIL-10、IFN-γ細(xì)胞因子ELISA檢測(cè)。7不同來(lái)源異基因MSCs刺激T淋巴細(xì)胞增殖的情況檢測(cè)：異基因T淋巴細(xì)胞(來(lái)源于C57小鼠或SD大鼠)與經(jīng)60Co照射的不用來(lái)源MSCs共培養(yǎng)后,MTT法測(cè)定異基因T淋巴細(xì)胞增殖的情況。 結(jié)果：1SD-BMSCs的培養(yǎng)觀察與鑒定：全貼壁原代培養(yǎng)的BMSCs_48 h后可見(jiàn)有細(xì)胞貼壁生長(zhǎng),部分形態(tài)變?yōu)樗髽?之后細(xì)胞迅速增長(zhǎng),至7～11d時(shí)細(xì)胞已增殖至90%融合,呈漩渦狀生長(zhǎng)。傳代細(xì)胞24 h貼壁,5～7_d傳一代,可穩(wěn)定傳代至少20次；流式細(xì)胞儀鑒定經(jīng)培養(yǎng)的MSCs的結(jié)果為：間充質(zhì)干細(xì)胞表面標(biāo)志CD29、CD44陽(yáng)性率分別為99.7%和96.7%；造血系干細(xì)胞表面標(biāo)志CD34,CD45陽(yáng)性率分別為1.6%和1.3%。2移植皮片的存活時(shí)間與HE染色結(jié)果：CP+ SD-MSCs組皮膚移植物存活時(shí)間為(15.7±1.4)d,空白對(duì)照組為(6.1±1.1)d,CP組為(12.3±1.5)d,SD-BMSCs組為(12.6±1.8)d,CP+ SD-BMSCs組皮膚移植物存活時(shí)間明顯比后3組延長(zhǎng)(P0.05)；對(duì)照組表現(xiàn)為表皮與真皮層完全壞死,細(xì)胞數(shù)量明顯增加,炎癥反應(yīng)明顯。藥物組和細(xì)胞組可見(jiàn)真皮細(xì)胞減少,并呈現(xiàn)廣泛纖維化。藥物和細(xì)胞組可見(jiàn)部分表皮缺失,真皮層正常,并可見(jiàn)毛囊結(jié)構(gòu)。3受體小鼠脾臟調(diào)節(jié)性T細(xì)胞(CD4+、CD25+、Foxp3+、Treg細(xì)胞)的檢測(cè)結(jié)果：流式細(xì)胞儀檢測(cè)Treg含量SD-BMSCs組和CP+ SD-BMSCs組明顯高于空白對(duì)照組和CP組(P0.05).4受體小鼠外周血細(xì)胞因子(TGF-β、IL-10、IFN-γ)的檢測(cè)結(jié)果ELISA檢測(cè)受體外周血MSCs組和CP組TGF-β和IL-10明顯高于空白對(duì)照組,SD-BMSCs和CP組IFN-γ則明顯低于空白對(duì)照組(P0.05).5不同來(lái)源MSCs刺激異基因T淋巴細(xì)胞增殖的情況檢測(cè)：共培養(yǎng)結(jié)果顯示：來(lái)源于C57小鼠和SD大鼠的MSCs可以明顯抑制T淋巴細(xì)胞的增殖反應(yīng)(P0.05),而上述兩組組間比較差異則無(wú)統(tǒng)計(jì)學(xué)意義(P0.05) 結(jié)論：1第三方MSCs能夠延長(zhǎng)同種異體皮片移植物的存活時(shí)間.2第三方MSCs誘導(dǎo)同種異體移植免疫耐受作用可能與誘導(dǎo)受體Treg細(xì)胞增殖有關(guān).3第三方MSCs誘導(dǎo)同種異體移植免疫耐受作用可能與促進(jìn)免疫耐受因子的表達(dá),抑制免疫排斥因子的表達(dá)有關(guān).4不同來(lái)源的MSCs在抑制異基因T淋巴細(xì)胞的作用上沒(méi)有明顯差別
[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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
本文編號(hào)：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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李富榮;鄧春艷;王新根;齊暉;任莉莉;周漢新;;骨髓間充質(zhì)干細(xì)胞聯(lián)合胰島移植對(duì)受體鼠樹(shù)突狀細(xì)胞成熟和功能的影響[J];細(xì)胞與分子免疫學(xué)雜志;2010年07期
，本文編號(hào)：2133320
本文鏈接：http://sikaile.net/xiyixuelunwen/2133320.html
最近更新
教材專著
