人AAT在胰岛β细胞移植中对自身免疫与排斥反应的作用

发布时间：2018-05-11 09:56

本文选题：α1-抗胰蛋白酶 + β细胞移植　；参考：《暨南大学》2013年硕士论文

【摘要】：目的：利用人AAT（α1-抗胰蛋白酶）基因导入胰岛β细胞构建NIT-hAA系，将NIT-hAA移植到7周龄的雌性NOD小鼠左肾包膜下，利用环磷酰胺促进糖尿病发病，观察hAAT是否有效阻止胰岛自身免疫破坏和对移植β细胞免疫保护的作用。方法：已成功构建NIT-hAAT细胞系，Western Blotting技术鉴定hAAT在NIT－1细胞能稳定表达。将NIT-hAAT细胞和NIT－1细胞分别移植到7周龄未发病的雌性NOD小鼠左肾包膜下，腹腔注射环磷酰胺促进糖尿病发病。首先，进行血糖水平动态监测、葡萄糖耐量实验、统计糖尿病发病时间和发病率，评估NIT-hAAT移植能否有效阻止NOD小鼠糖尿病发病；其次，在不同时间点对NOD鼠胰腺、移植部位进行病理观察，并用TUNEL检测凋亡胰岛细胞，观察NIT-hAAT移植对β细胞免疫保护的作用；最后，采用ELISA动态检测小鼠血清hAAT浓度、小鼠血清Th1细胞因子（TNF-α、IFN-γ）与Th2细胞因子（IL-4、IL-10）水平，流式细胞术分析小鼠脾淋巴细胞Treg/Th17水平，分析hAAT对移植的β细胞免疫保护的作用机制。结果：在NIT-hAAT移植后21天内，小鼠体内hAAT水平维持在164.33-340.33μg/ml之间，hAAT可推迟NOD小鼠糖尿病发病时间和降低发病率。病理观察发现hAAT明显减轻自身胰岛细胞炎症浸润，降低胰岛炎症评级，减少胰岛细胞凋亡；同时，，明显减少移植部位β细胞炎症浸润。在免疫保护机制方面，hAAT通过增加Treg细胞数量和减少Th17细胞数量；上调Th2细胞因子和下调Th1细胞因子，减轻NOD小鼠体内炎症反应。hAAT可抑制自身免疫对自体胰岛免疫破坏和移植β细胞免疫排斥，具有双重免疫保护作用。但遗憾的是，NIT-hAAT细胞分泌hAAT的量在移植14天后开始下降，49天时已无法检测。移植后期，自体胰岛与移植β细胞被免疫破坏，细胞凋亡数量增加，移植部位炎性细胞浸润逐渐增加，同时，Treg细胞数量与Th2细胞因子水平逐渐下降，Th17细胞数量与Th1细胞因子水平逐渐上升，在移植后49d后自体胰岛细胞和移植β细胞被免疫完全破坏，血糖水平和糖尿病发病率近似NIT-1组与糖尿病水平。结论：NIT-hAAT移植后可表达hAAT，在短期内明显改善NOD小鼠体内炎症反应，抑制自身免疫对自体胰岛细胞和移植β细胞的破坏，发挥对自身胰岛和移植细胞的双重免疫保护作用。但在移植后期由于hAAT表达量不足，无法诱导形成长期免疫耐受，不能完全阻止自身免疫对胰岛细胞破坏和机体对移植β细胞免疫排斥双重作用。
[Abstract]:Objective: to construct a NIT-hAA cell line by introducing human AAT- 伪 1-antitrypsin gene into islet 尾 cells and transplant NIT-hAA into the left renal capsule of 7-week-old female NOD mice. Cyclophosphamide (cyclophosphamide) was used to promote the pathogenesis of diabetes mellitus. To observe whether hAAT can effectively prevent islet autoimmune damage and protect 尾-cells from transplantation. Methods: NIT-hAAT cell line was successfully constructed to identify the stable expression of hAAT in NIT-1 cells by Western Blotting. NIT-hAAT cells and NIT-1 cells were transplanted into the left renal capsule of 7 week-old female NOD mice, and cyclophosphamide was injected intraperitoneally to promote the onset of diabetes. First, blood glucose levels were dynamically monitored, glucose tolerance test was performed to calculate the onset time and incidence of diabetes, and to evaluate whether NIT-hAAT transplantation could effectively prevent diabetes in NOD mice. Secondly, pancreas of NOD mice was treated at different time points. The transplantation site was observed pathologically, the apoptotic islet cells were detected by TUNEL, and the protective effect of NIT-hAAT transplantation on 尾 -cell immunity was observed. Finally, the serum hAAT concentration of mice was dynamically detected by ELISA. The levels of Th1 cytokine TNF- 伪 (IFN- 纬) and Th2 cytokine (IL-4 / IL-10) in serum of mice were determined by flow cytometry, and the level of Treg/Th17 in spleen lymphocytes of mice was analyzed by flow cytometry, and the mechanism of hAAT on the immunological protection of transplanted 尾 cells was analyzed. Results: within 21 days after NIT-hAAT transplantation, maintaining the level of hAAT between 164.33-340.33 渭 g/ml in mice could delay the onset of diabetes and reduce the incidence of diabetes in NOD mice. Pathological observation showed that hAAT could obviously reduce the inflammatory infiltration of islet cells, reduce the inflammatory grade of islet cells and decrease the apoptosis of islet cells, at the same time, the inflammatory infiltration of 尾 cells in transplantation site was significantly reduced. In immune protection mechanism, hAAT increased the number of Treg cells and reduced the number of Th17 cells, upregulated Th2 cytokines and down-regulated Th1 cytokines. Attenuating inflammation in NOD mice, hAAT can inhibit autoimmune damage to autoimmune islets and immune rejection of transplanted 尾 cells, which has double immune protective effect. Unfortunately, the amount of hAAT secreted by NIT-hAAT cells began to decrease 14 days after transplantation and could not be detected at 49 days after transplantation. In the late stage of transplantation, autogenous islets and transplanted 尾 cells were destroyed by immunity, the number of apoptotic cells increased, and the infiltration of inflammatory cells gradually increased. At the same time, the number of Treg cells and the level of Th2 cytokines decreased gradually. The number of Th17 cells and the level of Th1 cytokines increased gradually, and the autogenous islet cells and transplanted 尾 cells were completely destroyed after 49 days of transplantation. The level of blood sugar and the incidence of diabetes were similar to those of NIT-1 group and diabetes mellitus group. ConclusionWhen NIT-hAAT can express hAAT. in a short period of time, it can obviously improve the inflammatory reaction in NOD mice, inhibit the destruction of autologous islet cells and transplanted 尾 cells by autoimmunity, and play a dual protective effect on autoimmune islets and transplanted cells. However, at the late stage of transplantation, due to the insufficient expression of hAAT, it was unable to induce long-term immune tolerance and could not completely prevent the destruction of islet cells by autoimmunity and the immune rejection of transplanted 尾 cells.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392.11

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