CTLA4-Ig重组慢病毒引流淋巴结定向表达的研究

发布时间：2018-07-15 19:51

【摘要】：新型免疫抑制药物的研制及发展，为整形外科在异体复合组织移植领域的稳步发展带来了新的机遇。然而，异体复合组织移植（CTA）的发展远远不似内脏器官移植一样迅速，究其根本，是因为异体复合组织移植物组分复杂，并且抗原性不均一。自1998年首次手移植被Dubernard成功实施以来，到目前为止,在临床上已完成超过60例手移植和23例异体颜面移植。第一例异体颜面移植在法国成功施行手术后，曾经发生2次急性排斥反应，第二例患者是在本科室成功实施后，术后也发生了3次急性排斥反应。加大免疫抑制剂用量是使排斥反应得到控制的有效方法，但是往往会出现各种棘手的并发症如急性肾衰、高血压症等。鉴于上述原因，寻找可以使免疫抑制剂用量最小化的方法，已成为CTA移植领域重点研究的项目。免疫应答的一般规律同样适用于异体复合组织移植排斥反应的发生，首先，外来的异体抗原被免疫识别之后激活机体免疫系统，继而产生对移植物的免疫排斥反应，也就是说，阻断免疫应答中的任何一个环节，都可以实现对移植免疫耐受的诱导。细胞毒性T淋巴细胞相关抗原4抗体融合蛋白（CTLA4-Ig）可以阻断CD28-B7途径，能有效诱导免疫耐受的发生。树突状细胞毋庸置疑是最主要的抗原提呈细胞，在其表面可以高水平表达MHC-II类分子以及CD80、CD86等共刺激分子，通过B7分子与CD28的结合，开启第二信号刺激途径，，从而激活初始T细胞。成熟的树突状细胞具有淋巴归巢的特性，因此，CTLA4-Ig重组慢病毒转染的成熟树突状细胞有可能成为细胞回输治疗方案的备选细胞。目的：研究CTLA4-Ig直接淋巴结注射后其表达效率、CTLA4-Ig重组慢病毒转染成熟树突状细胞后相关基因的表达，局部注射转染后的成熟树突状细胞后观察CTLA4-Ig在引流淋巴结内的表达，探索淋巴结直接基因治疗方法的可行性；试图通过局部细胞治疗诱导免疫耐受，降低全身免疫抑制剂的用量，为同种异体复合组织移植探索新途径。方法： 1．大鼠淋巴结内注射CTLA4-Ig重组慢病毒，观察CTLA4-Ig在淋巴结内的表达。 2．贴壁培养法体外诱导成熟树突状细胞，进行形态学鉴定、活性及表型鉴定，体外鉴定其免疫学功能。 3．体外试验研究CTLA4-Ig重组慢病毒转染大鼠成熟树突状细胞，观测CTLA4-Ig的表达时间及表达高峰。 4．体内试验研究CTLA4-Ig重组慢病毒转染的成熟树突状细胞于大鼠引流淋巴结的定向表达。结果： 1．实时荧光定量PCR及冰冻切片示，空白对照及阴性对照组淋巴结内均无CTLA4-Ig的表达。CTLA4-Ig重组慢病毒淋巴结内注射组有明显的CTLA4-Ig表达。 2．光镜见培养的成熟树突状细胞集落分散，悬浮，分布较均匀。扫描电镜见细胞表面大量褶皱与粗细不等的突起。流式细胞术显示成熟树突状细胞表面的DC功能相关抗原MHC II、CD80、CD86均高表达。功能试验检测其分泌IL-12能力较未成熟树突状细胞强，混合淋巴细胞反应显示其有强的刺激T细胞增殖的能力。活细胞率为（80.34±1.25）%。 3．慢病毒转染成熟树突状细胞76h后，于倒置荧光显微镜下可观察到荧光表达。随时间延长，荧光表达增多，细胞形态亦出现老化。转染后第7d细胞明显老化。Q-PCR示CTLA4-Ig重组慢病毒转染成熟树突状细胞第3天，于mRNA水平有CTLA4-Ig的表达，western blotting检测所示结果与Q-PCR一致。 4．距大鼠腋固有淋巴结处2公分皮下缓慢注射所收获的CTLA4-Ig重组慢病毒转染后第3天的DCs，术后第4天取材，Q-PCR显示注射CTLA4-Ig重组慢病毒转染的DCs组于淋巴结内可检测到CTLA4-Ig的表达。结论： 1．CTLA4-Ig重组慢病毒淋巴结内直接注射可行,但范围局限且损伤较大。 2．细胞治疗初步显示可行，其效果需在异体复合组织移植模型中做进一步的验证。
[Abstract]:The development and development of novel immunosuppressive drugs have brought new opportunities for the steady development of orthopedic surgery in the field of allograft transplantation . However , the development of allograft tissue transplantation ( CTA ) is far less than that of internal organ transplantation .

The general rule of immune response is also applicable to the occurrence of allograft rejection reaction , firstly , the foreign alloantigen is immune - recognized to activate the immune system of the organism , then the immune rejection reaction to the graft is generated , that is , the induction of transplantation immune tolerance can be realized by blocking any one of the immune responses .

The cytotoxic T - lymphocyte - associated antigen - 4 antibody fusion protein can block the CD28 - B7 pathway . It can effectively induce immune tolerance . Dendritic cells can express MHC - II molecules at a high level , and then activate the second signal stimulation pathway through the combination of B7 molecules and CD28 .

Purpose :

The expression efficiency and the expression of the related genes after the transfection of IL - 4 - Ig into mature dendritic cells were investigated . The expression of 4 - Ig in draining lymph nodes was observed after local injection of mature dendritic cells transfected into mature dendritic cells , and the feasibility of direct gene therapy for lymph nodes was explored .
Attempts to induce immune tolerance by local cell therapy , decrease the dosage of systemic immune inhibitor , and explore new ways for the transplantation of allogeneic composite tissue .

Method :

1 . In the lymph node of rats , the recombinant lentivirus was injected into the lymph node , and the expression of 4 - Ig in lymph node was observed .

2 . Induction of mature dendritic cells in vitro by adherent culture method , morphological identification , activity and phenotype identification , and in vitro identification of its immunological function .

3 . In vitro experiments were carried out to investigate the expression time and peak expression of 4 - Ig in mature dendritic cells transfected with the recombinant lentivirus .

4 . In vivo experiments were carried out to study the directional expression of the mature dendritic cells transfected with the recombinant lentivirus in rat draining lymph nodes .

Results :

1 . Real - time fluorescence quantitative PCR and frozen section showed that there were no IL - 4 - Ig expression in lymph nodes of blank control and negative control group .

2 . Light microscopy showed that the cultured mature dendritic cells were dispersed , suspended and distributed uniformly . The scanning electron microscope showed that there was a large number of wrinkles on the surface of mature dendritic cells . The expression of MHC II , CD80 and CD80 on the surface of mature dendritic cells was detected by flow cytometry . The function test showed that the IL - 12 ability of mature dendritic cells was higher than that of immature dendritic cells . The mixed lymphocyte reaction showed that it had strong ability to stimulate T cell proliferation .

3 . The fluorescent expression was observed under the inverted fluorescence microscope after the slow virus transfection of mature dendritic cells for 76h . The fluorescence expression increased with time , and the cell morphology was also aging . After transfection , the cells were aged significantly . Q - PCR showed that the recombinant lentivirus was transfected into mature dendritic cells on the third day , and the mRNA level was expressed by 4 - Ig . The results indicated by western blotting were consistent with Q - PCR .

4 . DCs were injected subcutaneously at 2 cm from the axillary lymph node of the rat , and the DCs were harvested at 3 days post - transfection . On the 4th day after operation , the DCs transfected with the recombinant lentivirus were detected by Q - PCR , and the expression of IL - 4 - Ig was detected in the lymph nodes .

Conclusion :

1 . Direct injection is feasible in the lymph node of the recombinant Lentivirus 4 - Ig , but the range is limited and the damage is large .

2 . The initial display of cell therapy is feasible , and its effect is to be further verified in the allograft model .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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