本文選題：吲哚胺2 + 3-雙加氧酶　； 參考：《貴州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過動(dòng)物實(shí)驗(yàn),用IDO酶的基因來防治同種異基因卵巢移植所致的排斥反應(yīng),恢復(fù)移植卵巢的生殖和內(nèi)分泌功能。方法:1.選取9-11周齡的Lewis雌性大鼠進(jìn)行自體移植(A組);同種異體移植選取9-11周齡的Lewis雌性大鼠作為供體,9-11周齡的SD雌性大鼠作為受體,隨機(jī)分為4組:B組(空白對照組)、C組(環(huán)孢素A組)、D組(含IDO基因的重組慢病毒組)、E組(空慢病毒組),每組20只,按組序給予不同處理,觀察術(shù)后各組大鼠的存活情況。2.比較各組大鼠移植卵巢的成功率。3.術(shù)后第5天開始,每日行陰道涂片檢查后HE染色,根據(jù)涂片中無核角化細(xì)胞的比例來判斷各組大鼠是否出現(xiàn)動(dòng)情周期及出現(xiàn)動(dòng)情周期的時(shí)間,動(dòng)情周期恢復(fù)的大鼠與同系雄性大鼠同籠,觀察受孕情況。4.術(shù)后40天、70天取血檢測FSH、E2的水平。5.術(shù)后40天隨機(jī)處死5只受鼠進(jìn)行組織學(xué)檢查,剩余受鼠術(shù)后70天處死,取卵巢組織進(jìn)行組織學(xué)病理檢查;將D組的新鮮卵巢組織快速冰凍切片,熒光顯微鏡下觀察病毒的感染情況。6.各組采用免疫組化方法檢測IDO的表達(dá)情況。結(jié)果:1.術(shù)后各組受鼠的存活率分別為 A 組 95%(19/20)、B 組 55%(11/20)、C 組 80%(16/20)、D 組 95%(19/20)、E 組 60%(12/20),D 組的存活率比 B 組、E 組高(p0.05),與A組比較無明顯差異(p0.05)。2.各組大鼠卵巢移植成功率分別為A組94.74%(18/19)、B組27.27%(3/11)、C組37.5%(6/16)、D 組73.68%(14/19)、E組25.00%(3/12),D組存活率比B組、C組、E組高(p0.05),與A組比較無明顯差異(p0.05)。3.D組出現(xiàn)動(dòng)情周期時(shí)間早于B組、C組、E組(p0.05),與A組比較無明顯差異(p0.05),術(shù)后A組、D組分別有1只大鼠自然受孕并分娩,而其他3組均未受孕。4.術(shù)后40天,各組分別取血檢測FSH、E2水平,D組的FSH水平明顯低于B組、C組、E組(p0.05),D組的E2水平明顯高于B組、C組、E組(p0.05),但與A組比較無明顯差異(p0.05);術(shù)后70天,D組的FSH及E2水平與其他4組比較無顯著性差異。5.術(shù)后組織病理切片結(jié)果顯示:B組、E組卵巢組織間見少量卵泡及黃體,C組卵泡及黃體數(shù)量較B組、E組稍多,A組、D組見中等量發(fā)育卵泡及黃體;D組行新鮮組織快速冰凍切片,熒光顯微鏡下觀察可見大量的綠色熒光。6.各組免疫組化結(jié)果顯示:正常卵巢組織中IDO基本不表達(dá),IDO在移植卵巢中表達(dá)的位置主要在黃體及卵泡細(xì)胞漿中,D組中IDO表達(dá)最強(qiáng),均高于其余4組。結(jié)論:1.重組慢病毒攜帶的IDO基因能成功地轉(zhuǎn)染大鼠移植的卵巢,并在一段時(shí)間內(nèi),能在移植卵巢中穩(wěn)定表達(dá)。2.初步證實(shí)了 IDO的表達(dá)可能會(huì)提高同種異體卵巢移植的成功率,同時(shí)恢復(fù)移植卵巢的生殖和內(nèi)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.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R713.6

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