骨髓间充质干细胞干预异种气管移植免疫排斥反应的实验研究
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摘要
第一部分
异种气管移植动物模型的建立
目的建立一种简单的豚鼠-SD大鼠异种气管移植动物模型,为下一步研究其免疫排斥反应特点及干预措施奠定基础,为解决临床上供体气管来源问题提供新方向。方法采用“加植法”建立了一种新型的短段(5个气管环)异种原位气管移植动物模型,观察术后受体存活时间、移植气管在光镜和电镜下的病理组织学改变,初步分析其免疫排斥反应特点。结果单人操作连续进行20例异种气管移植,手术成功率达100%(20/20),受体手术时间(20.8±1.6)min,无重要手术并发症。受体术后存活最短8.0d,最长12.5d,平均(10.0±1.3)d。移植气管随着时间延长逐渐出现管腔狭窄,黏膜上皮、黏膜下、外膜及软骨逐渐出现不同程度淋巴细胞、嗜酸粒细胞、单核巨噬细胞的浸润以及细胞凋亡现象。
结论以“加植法”建立的短段(5个气管环)异种气管移植动物模型,术后移植段气管的病理变化符合异种气管移植免疫排斥反应的一般特点,是一种简便、经济实用、稳定可靠和易于复制的器官移植研究动物模型。
第二部分SD大鼠骨髓间充质干细胞体外培养、表型鉴定与标记
目的探讨SD大鼠骨髓间充质干细胞(MSCs)的体外分离培养、表型鉴定和标记的方法,为进一步研究MSCs干预异种气管移植的免疫排斥做好准备。方法采用直接贴壁法分离培养MSCs;通过流式细胞仪检测细胞表面标志抗原CD90、CD45的表达率对培养的MSCs进行表型鉴定;DAPI标记第3代MSCs,观察标记效率。结果体外培养的原代MSCs 48h内可见有少量贴壁细胞,7～10d左右达到90%汇合;流式细胞仪检测第3代MSCs表面标记物CD90、CD45的阳性率分别为99.8%、6.8%,提示MSCs表达CD90而不表达CD45;用DAPI进行细胞标记后,荧光显微镜下见几乎所有MSCs均已被标记蓝色荧光,提示DAPI标记法敏感性好,标记效率高。结论贴壁培养法是一种简便易行的培养MSCs方法,操作简单,成功率高,可以作为培养SD大鼠MSCs的常规方法;DAPI标记可作为标记MSCs的一种有效手段。
第三部分骨髓间充质干细胞对异种气管移植免疫排斥反应的影响
目的通过静脉移植的方式将骨髓间充质干细胞(MSCs)输入豚鼠-大鼠异种气管移植后的受体,进一步研究异种气管移植的排斥反应特点及MSCs对其免疫干预作用,为解决临床上气管移植术后抗排斥问题提供新思路。方法实验分成4组:A组(自体移植,n=17);B组(异种移植,n=20);C组(异种移植+CsA,n=25);D组(异种移植+MSCs,n=25)。记录受体存活时间,观察移植气管病理组织学变化及气管通畅度,流式细胞仪检测外周血CD4+、CD8+T淋巴细胞含量的变化,移植气管冷冻标本免疫荧光检查IgG、IgM、C3的沉积,TUNEL法检测移植物细胞凋亡并计算凋亡指数(AI),生化分析仪检测外周血肌酐(CREA)的变化。结果(1)异种移植后各组受体平均存活时间分别为: B组(10.0±1.3)d、C组(17.1±2.7)d、D组(17.6±3.2)d,C、D组生存时间显著长于B组(P<0.01)。(2)各组移植气管病理积分和通畅度的比较:异种移植后的各组与自体移植组相比,病理积分显著增加(P<0.01)而通畅度显著下降(P<0.01),表现均为急性排斥改变;C、D组病理损害比B组轻,说明排斥反应被减轻。(3)外周血CD4+、CD8+T淋巴细胞含量的比较:异种移植后的各组比自体移植组显著增加,随时间持续升高,说明T细胞被激活参与了急性免疫排斥反应;B组增加最明显,C、D组低于B组,说明细胞免疫被抑制。(4)免疫荧光检查结果比较:自体移植组未见IgG、IgM、C3的沉积,异种移植后的各组可见不同程度的IgG、IgM、C3沉积,说明体液免疫和补体系统参与了急性排斥反应;B组IgG、IgM、C3复合物沉积最明显,D组低于B组,说明MSCs对体液免疫和补体系统具有调节作用。(5)移植气管细胞凋亡的比较:自体移植组AI显著低于异种移植后的各组(P<0.01),B组移植物AI最高与C、D组比较差异有显著性(P<0.01)。(6)外周血肌酐(CREA)的比较:C组随时间的延长逐渐增加,其余3组则基本不变。结论细胞免疫、体液免疫均参与了豚鼠-大鼠异种气管移植的急性排斥反应;静脉移植MSCs可明显延长异种气管移植受体的存活时间,增加移植气管通畅度,抑制细胞凋亡,减轻免疫排斥反应;MSCs可能通过对免疫细胞和补体系统的调节等多方面因素干预免疫排斥反应;MSCs抗排斥效力与CsA相当,但没有其增加感染和损害肾功能等副作用。
Development of an animal model for tracheal xenotransplantation Objective To establish a simple guinea pig-to-rat tracheal xenotransplantation animal model, prepare for the advanced research on studying its immunological rejection characteristics and corresponding interventional approaches, and provide new insights into the clinical interventions to increase the tracheal procurement. Methods To establish a new short segment (5 rings) orthotopic tracheal xenotransplantation animal model by“adding-grafting technique”, and observe the survival after operation, and the histopathological changes of the tracheal xenograph using optical microscope and electron microscope. The characteristics of immunological rejection were analyzed.
Results 20 consecutive tracheal transplantation procedures had been performed by a single operator. The operation success rate was 100% (20/20). The duration of recipient procedure was (20.8±1.6) minutes. No complication observed. Post-transplantation survival durations ranged from shortest 8.0d to longest 12.0 d, with average (10±1.3) d. With the time increment, the tracheal lumen became narrowing down. Mucosal epithelium, submucosa, adventitia, and cartilage gradually showed various degrees of infiltration of lymphocytes, eosinophils, and macrophages, and cell apoptosis had been observed. Conclusions It is able to establish the short segment (5 tracheal rings) tracheal transplantation animal model by“adding-grafting technique”. The histopathological changes post-transplantation agree with the characteristics of tracheal xenotransplantation immunological rejection. Therefore it is a simple, economic, robust, and repeatable animal model for transplantation research.
PART 2
Culture, Phenotype and Labeling of SD Rat's Mesenchymal Stem Cells in Vitro
Objective To investigate the methods of isolation,culture,phenotype and labeling of mesenchymal stem cells(MSCs) from SD rat in vitro, in order to prepare for the next study on the immunorejection of MSCs on tracheal xenotransplantation intervention. Methods MSCs were isolated and cultivated by adherent methods. The expression of cell surface antigen CD90 and CD45 was analyzed by flow cytometry in order to identify MSCs phenotype. The third generation of MSCs were labeled by DAPI. Results Primary cultured MSCs adhered to plastic surface within 48 hours and reached 90% confluence within 7-10 days. Flow cytometry showed that the positive rates of CD90 and CD45 MSCs at the third generation were 99.8% and 6.8%, suggesting that MSCs expressed CD90 but not CD45. Almost all of the MSCs after labeling by DAPI showed blue fluorescence under fluorescence microscope, suggesting DAPI labeling is sensitive and highly efficient for MSCs. Conclusions Adherent method is simple and easy to isolate and cultivate MSCs and it can become a routine protocol for culturing SD rat's MSCs. DAPI labeling can be used as an efficient method to label MSCs.
PART 3 The Effect of Mesenchymal Stem Cells to Tracheal Xenotransplantation Immunological rejection
Objective Through the form of vein graft, injecting the mesenchymal stem cells(MSCs)into the recipient of guinea pig–to-rat tracheal xenotransplantation, further study the rejection reaction characteristics of tracheal xenotransplantation and MSCs’s immune intervention function to it, aim to provide new ideas to solve the clinical problem of anti-rejection after tracheal xenotransplantation. Methods Experimental subjects were divided into 4 groups: A group (Autografting, n=17), B group (Xenotransplantation, n=17), C group(Xenotransplantation+ CsA, n=25), D group (Xenotransplantation+ MSCs, n=25).Recording receptor’s survival time to observe the pathological changes in histology and degree of patency of the tracheal transplant, the expression situation of flow cytometry in peripheral blood CD4+,CD8+T lymphocyte, tracheal transplantation immunofluorescence examination of frozen specimens of IgG, IgM, C3 deposition, TUNEL detection of apoptosis in graft and calculate the apoptotic index (AI), peripheral blood biochemical analyzer creatinine (CREA) changes. Results (1) After xenotransplantation, the average survival time of the recipient in each group were (10.0±1.3) d, (17.1±2.7) d, (17.6±3.2) d in B, C, D group respectively. C , D group’s survival time was significantly longer than B group (P <0.01). (2) Comparison of the pathological integral and degree of patency of the tracheal transplant in different group: compared to autotransplant group, xenotransplant groups showed significant increase in pathological points (P<0.01), and decrease in the degree of tracheal patency(P<0.01), these were due to acute rejection. The histopathological damages in groups C and D were less severe than group B, indicating the reduced immunological rejection. (3) Comparison of peripheral blood CD4 + ,CD8 + T lymphocyte’s expression content: each group significantly increased than the autografting group after xenotransplantation, and kept in sustained increases over time, it illustrated that T cells were activated to involve in acute immunological rejection. B group showed the strongest increase, while C and D groups were less than it, indicating the suppression of T cell immune response.(4)Comparison of the immunofluorescence test results: no IgG, IgM, C3 deposition found in autografting group, different level of IgG, IgM, C3 deposition can be seen in each group after xenotransplantation, it illustrated that humoral immunity and complement system involved in acute rejection reaction; the complex deposition of IgG, IgM, C3 in B group was the most obvious, D group is lower than group B, if revealed that MSCs had a adjustment effcct to the humoral immune system and alexin system. (5)Comparison of the cells apoptosis in tracheal transplantation: AI from autografting group was significantly lower than the each group after xenotransplantation(P<0.01). B group had the lowest AI, and significantly differed from groups C and D (P<0.01). (6)Comparison of the peripheral blood creatinine (CREA): C group showed increase with time, and the remaining 3 groups were basically unchanged. Conclusions Both cellular and humoral immunity involved in the guinea pig-to-rat tracheal xenotransplantion acute rejection. Vein graft MSCs can significantly prolong receptor’s survival time of the tracheal xenotransplantation, increase the patency of tracheal xenotransplantation, inhibit the apoptosis of cells, reduce the immune rejection. MSCs may interfere with immune rejection through the adjustment of immune cells and alexin system etc. factors. MSCs has a same anti-rejection effect with CsA, but it has not the side effects such as increase in infections and damage to renal function.
异种气管移植动物模型的建立
目的建立一种简单的豚鼠-SD大鼠异种气管移植动物模型,为下一步研究其免疫排斥反应特点及干预措施奠定基础,为解决临床上供体气管来源问题提供新方向。方法采用“加植法”建立了一种新型的短段(5个气管环)异种原位气管移植动物模型,观察术后受体存活时间、移植气管在光镜和电镜下的病理组织学改变,初步分析其免疫排斥反应特点。结果单人操作连续进行20例异种气管移植,手术成功率达100%(20/20),受体手术时间(20.8±1.6)min,无重要手术并发症。受体术后存活最短8.0d,最长12.5d,平均(10.0±1.3)d。移植气管随着时间延长逐渐出现管腔狭窄,黏膜上皮、黏膜下、外膜及软骨逐渐出现不同程度淋巴细胞、嗜酸粒细胞、单核巨噬细胞的浸润以及细胞凋亡现象。
结论以“加植法”建立的短段(5个气管环)异种气管移植动物模型,术后移植段气管的病理变化符合异种气管移植免疫排斥反应的一般特点,是一种简便、经济实用、稳定可靠和易于复制的器官移植研究动物模型。
第二部分SD大鼠骨髓间充质干细胞体外培养、表型鉴定与标记
目的探讨SD大鼠骨髓间充质干细胞(MSCs)的体外分离培养、表型鉴定和标记的方法,为进一步研究MSCs干预异种气管移植的免疫排斥做好准备。方法采用直接贴壁法分离培养MSCs;通过流式细胞仪检测细胞表面标志抗原CD90、CD45的表达率对培养的MSCs进行表型鉴定;DAPI标记第3代MSCs,观察标记效率。结果体外培养的原代MSCs 48h内可见有少量贴壁细胞,7～10d左右达到90%汇合;流式细胞仪检测第3代MSCs表面标记物CD90、CD45的阳性率分别为99.8%、6.8%,提示MSCs表达CD90而不表达CD45;用DAPI进行细胞标记后,荧光显微镜下见几乎所有MSCs均已被标记蓝色荧光,提示DAPI标记法敏感性好,标记效率高。结论贴壁培养法是一种简便易行的培养MSCs方法,操作简单,成功率高,可以作为培养SD大鼠MSCs的常规方法;DAPI标记可作为标记MSCs的一种有效手段。
第三部分骨髓间充质干细胞对异种气管移植免疫排斥反应的影响
目的通过静脉移植的方式将骨髓间充质干细胞(MSCs)输入豚鼠-大鼠异种气管移植后的受体,进一步研究异种气管移植的排斥反应特点及MSCs对其免疫干预作用,为解决临床上气管移植术后抗排斥问题提供新思路。方法实验分成4组:A组(自体移植,n=17);B组(异种移植,n=20);C组(异种移植+CsA,n=25);D组(异种移植+MSCs,n=25)。记录受体存活时间,观察移植气管病理组织学变化及气管通畅度,流式细胞仪检测外周血CD4+、CD8+T淋巴细胞含量的变化,移植气管冷冻标本免疫荧光检查IgG、IgM、C3的沉积,TUNEL法检测移植物细胞凋亡并计算凋亡指数(AI),生化分析仪检测外周血肌酐(CREA)的变化。结果(1)异种移植后各组受体平均存活时间分别为: B组(10.0±1.3)d、C组(17.1±2.7)d、D组(17.6±3.2)d,C、D组生存时间显著长于B组(P<0.01)。(2)各组移植气管病理积分和通畅度的比较:异种移植后的各组与自体移植组相比,病理积分显著增加(P<0.01)而通畅度显著下降(P<0.01),表现均为急性排斥改变;C、D组病理损害比B组轻,说明排斥反应被减轻。(3)外周血CD4+、CD8+T淋巴细胞含量的比较:异种移植后的各组比自体移植组显著增加,随时间持续升高,说明T细胞被激活参与了急性免疫排斥反应;B组增加最明显,C、D组低于B组,说明细胞免疫被抑制。(4)免疫荧光检查结果比较:自体移植组未见IgG、IgM、C3的沉积,异种移植后的各组可见不同程度的IgG、IgM、C3沉积,说明体液免疫和补体系统参与了急性排斥反应;B组IgG、IgM、C3复合物沉积最明显,D组低于B组,说明MSCs对体液免疫和补体系统具有调节作用。(5)移植气管细胞凋亡的比较:自体移植组AI显著低于异种移植后的各组(P<0.01),B组移植物AI最高与C、D组比较差异有显著性(P<0.01)。(6)外周血肌酐(CREA)的比较:C组随时间的延长逐渐增加,其余3组则基本不变。结论细胞免疫、体液免疫均参与了豚鼠-大鼠异种气管移植的急性排斥反应;静脉移植MSCs可明显延长异种气管移植受体的存活时间,增加移植气管通畅度,抑制细胞凋亡,减轻免疫排斥反应;MSCs可能通过对免疫细胞和补体系统的调节等多方面因素干预免疫排斥反应;MSCs抗排斥效力与CsA相当,但没有其增加感染和损害肾功能等副作用。
Development of an animal model for tracheal xenotransplantation Objective To establish a simple guinea pig-to-rat tracheal xenotransplantation animal model, prepare for the advanced research on studying its immunological rejection characteristics and corresponding interventional approaches, and provide new insights into the clinical interventions to increase the tracheal procurement. Methods To establish a new short segment (5 rings) orthotopic tracheal xenotransplantation animal model by“adding-grafting technique”, and observe the survival after operation, and the histopathological changes of the tracheal xenograph using optical microscope and electron microscope. The characteristics of immunological rejection were analyzed.
Results 20 consecutive tracheal transplantation procedures had been performed by a single operator. The operation success rate was 100% (20/20). The duration of recipient procedure was (20.8±1.6) minutes. No complication observed. Post-transplantation survival durations ranged from shortest 8.0d to longest 12.0 d, with average (10±1.3) d. With the time increment, the tracheal lumen became narrowing down. Mucosal epithelium, submucosa, adventitia, and cartilage gradually showed various degrees of infiltration of lymphocytes, eosinophils, and macrophages, and cell apoptosis had been observed. Conclusions It is able to establish the short segment (5 tracheal rings) tracheal transplantation animal model by“adding-grafting technique”. The histopathological changes post-transplantation agree with the characteristics of tracheal xenotransplantation immunological rejection. Therefore it is a simple, economic, robust, and repeatable animal model for transplantation research.
PART 2
Culture, Phenotype and Labeling of SD Rat's Mesenchymal Stem Cells in Vitro
Objective To investigate the methods of isolation,culture,phenotype and labeling of mesenchymal stem cells(MSCs) from SD rat in vitro, in order to prepare for the next study on the immunorejection of MSCs on tracheal xenotransplantation intervention. Methods MSCs were isolated and cultivated by adherent methods. The expression of cell surface antigen CD90 and CD45 was analyzed by flow cytometry in order to identify MSCs phenotype. The third generation of MSCs were labeled by DAPI. Results Primary cultured MSCs adhered to plastic surface within 48 hours and reached 90% confluence within 7-10 days. Flow cytometry showed that the positive rates of CD90 and CD45 MSCs at the third generation were 99.8% and 6.8%, suggesting that MSCs expressed CD90 but not CD45. Almost all of the MSCs after labeling by DAPI showed blue fluorescence under fluorescence microscope, suggesting DAPI labeling is sensitive and highly efficient for MSCs. Conclusions Adherent method is simple and easy to isolate and cultivate MSCs and it can become a routine protocol for culturing SD rat's MSCs. DAPI labeling can be used as an efficient method to label MSCs.
PART 3 The Effect of Mesenchymal Stem Cells to Tracheal Xenotransplantation Immunological rejection
Objective Through the form of vein graft, injecting the mesenchymal stem cells(MSCs)into the recipient of guinea pig–to-rat tracheal xenotransplantation, further study the rejection reaction characteristics of tracheal xenotransplantation and MSCs’s immune intervention function to it, aim to provide new ideas to solve the clinical problem of anti-rejection after tracheal xenotransplantation. Methods Experimental subjects were divided into 4 groups: A group (Autografting, n=17), B group (Xenotransplantation, n=17), C group(Xenotransplantation+ CsA, n=25), D group (Xenotransplantation+ MSCs, n=25).Recording receptor’s survival time to observe the pathological changes in histology and degree of patency of the tracheal transplant, the expression situation of flow cytometry in peripheral blood CD4+,CD8+T lymphocyte, tracheal transplantation immunofluorescence examination of frozen specimens of IgG, IgM, C3 deposition, TUNEL detection of apoptosis in graft and calculate the apoptotic index (AI), peripheral blood biochemical analyzer creatinine (CREA) changes. Results (1) After xenotransplantation, the average survival time of the recipient in each group were (10.0±1.3) d, (17.1±2.7) d, (17.6±3.2) d in B, C, D group respectively. C , D group’s survival time was significantly longer than B group (P <0.01). (2) Comparison of the pathological integral and degree of patency of the tracheal transplant in different group: compared to autotransplant group, xenotransplant groups showed significant increase in pathological points (P<0.01), and decrease in the degree of tracheal patency(P<0.01), these were due to acute rejection. The histopathological damages in groups C and D were less severe than group B, indicating the reduced immunological rejection. (3) Comparison of peripheral blood CD4 + ,CD8 + T lymphocyte’s expression content: each group significantly increased than the autografting group after xenotransplantation, and kept in sustained increases over time, it illustrated that T cells were activated to involve in acute immunological rejection. B group showed the strongest increase, while C and D groups were less than it, indicating the suppression of T cell immune response.(4)Comparison of the immunofluorescence test results: no IgG, IgM, C3 deposition found in autografting group, different level of IgG, IgM, C3 deposition can be seen in each group after xenotransplantation, it illustrated that humoral immunity and complement system involved in acute rejection reaction; the complex deposition of IgG, IgM, C3 in B group was the most obvious, D group is lower than group B, if revealed that MSCs had a adjustment effcct to the humoral immune system and alexin system. (5)Comparison of the cells apoptosis in tracheal transplantation: AI from autografting group was significantly lower than the each group after xenotransplantation(P<0.01). B group had the lowest AI, and significantly differed from groups C and D (P<0.01). (6)Comparison of the peripheral blood creatinine (CREA): C group showed increase with time, and the remaining 3 groups were basically unchanged. Conclusions Both cellular and humoral immunity involved in the guinea pig-to-rat tracheal xenotransplantion acute rejection. Vein graft MSCs can significantly prolong receptor’s survival time of the tracheal xenotransplantation, increase the patency of tracheal xenotransplantation, inhibit the apoptosis of cells, reduce the immune rejection. MSCs may interfere with immune rejection through the adjustment of immune cells and alexin system etc. factors. MSCs has a same anti-rejection effect with CsA, but it has not the side effects such as increase in infections and damage to renal function.
引文
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