F蛋白诱导原位肝移植大鼠免疫耐受的实验研究

发布时间：2018-04-16 02:16

本文选题：原位肝移植 + F蛋白　；参考：《天津医科大学》2012年硕士论文

【摘要】：目的：寻找肝脏F蛋白诱导免疫耐受的新靶标,探讨肝移植免疫耐受的可能机制,为人类肝移植特异性免疫耐受的成功诱导提供实验依据和理论支持。方法：留取空白对照组(未经原位肝移植的Wistar大鼠,立即处死,n=6)、免疫排斥组(移植术后7d,Wistar→SD,n=6)和免疫耐受组(术前1周胸腺内注射供体源性F蛋白0.4mg,移植后100d,Wistar→SD,n=6)受体大鼠的肝脏组织,Trizol法提取肝组织总RNA,逆转录PCR合成cDNA,采用荧光定量PCR技术检测移植肝脏内FLJ11565、MGC45806、DKFZp434K1210、F蛋白基因等免疫耐受相关基因的表达水平；采用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法检测移植大鼠肝组织浸润淋巴细胞的凋亡情况；利用氨基酸分析技术和液质联用技术分析肝脏匀浆液的氨基酸、中小分子代谢物等代谢组学的变化。结果：1、排斥组的免疫耐受相关基因表达水平较空白组低,耐受组的目的基因表达水平远高于排斥组,且较空白组有所升高(P0.05)。2、空白组、排斥组和耐受组浸润淋巴细胞凋亡指数分别为(8.83±0.43)%、(11.32±1.29)%和(19.00±1.96)%,任意两组的凋亡指数均有统计学差异(P0.05)。3、与排斥组相比,耐受组的牛磺酸、谷氨酸、缬氨酸、甲硫氨酸、赖氨酸和色氨酸含量明显降低(P0.05),甘氨酸含量明显升高(P0.05)；移植肝脏代谢轮廓分析筛选出有意义的特征离子47个,其中排斥组和耐受组均升高的离子5个,耐受组较正常组和排斥组升高的离子2个,降低的离子2个。经HMDB数据库搜索和结构推导初步鉴定出不同代谢物18种,包括D-塔格糖、尿刊酸、本胆烷醇酮等。结论：FLJ11565、MGC45806、DKFZp434K1210和F蛋白基因在调节特异性免疫耐受的过程中可能起着重要作用；移植物中浸润淋巴细胞的细胞凋亡分析有助于直观判断移植物处于排斥或耐受状态,移植物中浸润淋巴细胞的凋亡有助于诱导移植物免疫耐受；谷氨酰胺、甘氨酸和甲硫氨酸等免疫营养氨基酸在肝脏移植免疫反应中起重要的调节作用,各种免疫营养氨基酸的综合作用对于诱导原位肝移植术后大鼠免疫耐受非常重要,D-塔格糖、尿刊酸和本胆烷醇酮等代谢物可能影响免疫耐受的诱导。
[Abstract]:Objective: to search for a new target of liver F protein to induce immune tolerance, and to explore the possible mechanism of liver transplantation immune tolerance, and to provide experimental basis and theoretical support for successful induction of human liver transplantation specific immune tolerance.Methods: blank control group (Wistar rats without orthotopic liver transplantation) was used.Immediately put to death the liver tissue of the recipient rats, the immunological rejection group (7 days after transplantation) and the immune tolerance group (intrathymic injection of donor-derived F protein 0.4 mg, 100 d after transplantation). The liver tissue of the recipient rats was extracted by Trizol method. Reverse transcriptional PCR was obtained from the liver tissue.The expression of protein genes related to immune tolerance, such as the protein gene DKFZp434K1210F, was detected by fluorescence quantitative PCR technique in the transplanted liver of FLJ11565MGC45806 and DKFZp434K1210F.Deoxyribonucleotide terminal transferase mediated Nick end labeling method was used to detect apoptosis of infiltrating lymphocytes in liver tissue of transplanted rats, and amino acids in liver homogenate were analyzed by amino acid analysis and liquid-mass spectrometry.Changes in metabolites such as metabolites of small and medium molecules.Results the expression level of immune tolerance related genes in the rejection group was lower than that in the blank group, the expression level of the target gene in the tolerance group was much higher than that in the rejection group, and it was higher than that in the blank group.The apoptotic index of infiltrating lymphocytes in the rejection group and the tolerance group were 11.32 卤1.29% and 19.00 卤1.96%, respectively. The apoptotic index of any two groups was significantly different (P0.05 路3). Compared with the rejection group, taurine, glutamate, valine, methionine, taurine, glutamate, valine, methionine in the tolerance group were higher than those in the control group.The contents of lysine and tryptophan decreased significantly (P 0.05), the content of glycine increased significantly (P 0.05), and the metabolic profile analysis of transplanted liver screened out 47 significant characteristic ions, among which 5 were increased in both rejection group and tolerance group.Compared with the normal group and the rejection group, the tolerance group had 2 elevated ions and 2 decreased ions.By HMDB database search and structure derivation, 18 different metabolites were identified, including D- tag sugar, uranic acid, procholanolone and so on.Conclusion the MGC45806 DKFZp434K1210 and F protein genes may play an important role in the regulation of specific immune tolerance, and the analysis of apoptosis of infiltrating lymphocytes in grafts may be helpful in judging the rejection or tolerance of the grafts.Apoptosis of infiltrating lymphocytes in grafts contributes to the induction of graft immune tolerance. Immune nutritious amino acids such as glutamine, glycine and methionine play an important role in the immune response to liver transplantation.The comprehensive effects of various immuno-nutritive amino acids on the induction of immune tolerance after orthotopic liver transplantation in rats are very important. The metabolites such as D-tagose, urachinic acid and native cholanolone may affect the induction of immune tolerance.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.4

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