吲哚胺2,3-双加氧酶基因防治卵巢移植排斥反应的实验研究

发布时间：2018-07-10 10:54

本文选题：吲哚胺2 + 3-双加氧酶　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:通过动物实验,用IDO酶的基因来防治同种异基因卵巢移植所致的排斥反应,恢复移植卵巢的生殖和内分泌功能。方法:1.选取9-11周龄的Lewis雌性大鼠进行自体移植(A组);同种异体移植选取9-11周龄的Lewis雌性大鼠作为供体,9-11周龄的SD雌性大鼠作为受体,随机分为4组:B组(空白对照组)、C组(环孢素A组)、D组(含IDO基因的重组慢病毒组)、E组(空慢病毒组),每组20只,按组序给予不同处理,观察术后各组大鼠的存活情况。2.比较各组大鼠移植卵巢的成功率。3.术后第5天开始,每日行阴道涂片检查后HE染色,根据涂片中无核角化细胞的比例来判断各组大鼠是否出现动情周期及出现动情周期的时间,动情周期恢复的大鼠与同系雄性大鼠同笼,观察受孕情况。4.术后40天、70天取血检测FSH、E2的水平。5.术后40天随机处死5只受鼠进行组织学检查,剩余受鼠术后70天处死,取卵巢组织进行组织学病理检查;将D组的新鲜卵巢组织快速冰冻切片,荧光显微镜下观察病毒的感染情况。6.各组采用免疫组化方法检测IDO的表达情况。结果:1.术后各组受鼠的存活率分别为 A 组 95%(19/20)、B 组 55%(11/20)、C 组 80%(16/20)、D 组 95%(19/20)、E 组 60%(12/20),D 组的存活率比 B 组、E 组高(p0.05),与A组比较无明显差异(p0.05)。2.各组大鼠卵巢移植成功率分别为A组94.74%(18/19)、B组27.27%(3/11)、C组37.5%(6/16)、D 组73.68%(14/19)、E组25.00%(3/12),D组存活率比B组、C组、E组高(p0.05),与A组比较无明显差异(p0.05)。3.D组出现动情周期时间早于B组、C组、E组(p0.05),与A组比较无明显差异(p0.05),术后A组、D组分别有1只大鼠自然受孕并分娩,而其他3组均未受孕。4.术后40天,各组分别取血检测FSH、E2水平,D组的FSH水平明显低于B组、C组、E组(p0.05),D组的E2水平明显高于B组、C组、E组(p0.05),但与A组比较无明显差异(p0.05);术后70天,D组的FSH及E2水平与其他4组比较无显著性差异。5.术后组织病理切片结果显示:B组、E组卵巢组织间见少量卵泡及黄体,C组卵泡及黄体数量较B组、E组稍多,A组、D组见中等量发育卵泡及黄体;D组行新鲜组织快速冰冻切片,荧光显微镜下观察可见大量的绿色荧光。6.各组免疫组化结果显示:正常卵巢组织中IDO基本不表达,IDO在移植卵巢中表达的位置主要在黄体及卵泡细胞浆中,D组中IDO表达最强,均高于其余4组。结论:1.重组慢病毒携带的IDO基因能成功地转染大鼠移植的卵巢,并在一段时间内,能在移植卵巢中稳定表达。2.初步证实了 IDO的表达可能会提高同种异体卵巢移植的成功率,同时恢复移植卵巢的生殖和内分泌功能。
[Abstract]:Aim: to prevent allogeneic ovarian allograft rejection and restore reproductive and endocrine function of allogenic ovary by using IDO enzyme gene in animal experiments. Method 1: 1. Lewis female rats aged 9-11 weeks were selected for autologous transplantation (group A) and Lewis female rats aged 9-11 weeks were used as recipients. They were randomly divided into 4 groups: group B (blank control group), group C (cyclosporine A group) and group D (recombinant lentivirus containing IDO gene) group E (empty lentivirus group), 20 rats in each group were given different treatments according to group order, the survival of rats in each group was observed. The success rate of ovarian transplantation in each group was compared. 3. 3. From the 5th day after operation, he staining was performed daily after vaginal smear examination. According to the proportion of nucleated keratinocytes in the smear, the estrous cycle and the time of estrous cycle were determined in each group. The rats recovered from estrous cycle were caged with the male rats of the same line, and the conception was observed. 4. 4. Blood samples were taken from 40 days to 70 days after operation to detect the level of E2. On the 40th day after operation, 5 recipients were randomly killed for histological examination, and the remaining recipients were killed 70 days after operation, and the ovarian tissues were taken for histopathological examination. Fluorescence microscope to observe the infection of the virus. 6. Immunohistochemical method was used to detect the expression of IDO in each group. The result is 1: 1. The survival rate of group A was 95% (19 / 20) and that of group B was 55% (11 / 20). The survival rate of group D was 95% (19 / 20). The survival rate of group D was 60% (12 / 20). There was no significant difference between group A and group A (p0.05). The successful rate of ovarian transplantation was 94.74% (18 / 19) in group A and 27.27% (3 / 11) in group B respectively. The survival rate of group D was 73.68% (14 / 19). The survival rate of group D was 25.00% (3 / 12). The survival rate of group D was higher than that of group B (p0.05). There was no significant difference between group A and group A (p0.05). 3.The time of estrous cycle in group D was earlier than that in group B (p0.05). There was no significant difference between the two groups (p0.05). The other three groups were not pregnant. 4. Forty days after surgery, The level of FSH in group D was significantly lower than that in group B (p0.05) and the level of E2 in group D was significantly higher than that in group B (p0.05), but there was no significant difference between group A and group A (p0.05), and the levels of FSH and E2 in group D were significantly higher than those in group A (p0.05) 70 days after operation, the levels of FSH and E2 in group D were significantly lower than those in group B (p0.05). There was no significant difference in his four groups. The results of histopathological sections showed that the number of follicles and corpus luteum in group C was a little more than that in group B, group A and group D were slightly more than that in group B and group D, and rapid frozen sections of fresh tissue were observed in group E and group D, respectively. A large amount of green fluorescence can be observed under fluorescence microscope. The results of immunohistochemistry showed that the expression of IDO in normal ovarian tissues was mainly in luteal body and follicular cytoplasm, and the expression of IDO in group D was higher than that in other 4 groups. Conclusion 1. The IDO gene carried by the recombinant lentivirus could be successfully transfected into the transplanted ovary of rats and expressed stably in the transplanted ovary for a period of time. It was preliminarily confirmed that the expression of IDO might increase the success rate of ovarian allotransplantation and restore the reproductive and endocrine functions of the transplanted ovary.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R713.6

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