本文選題：Crb3 + 睪丸間質(zhì)細胞��； 參考：《南昌大學》2014年碩士論文

【摘要】：目的：檢測TM3(小鼠睪丸間質(zhì)細胞)中是否有Crb3基因的表達，如有表達，則通過轉(zhuǎn)染，使其Crb3表達上調(diào)，檢測基因表達調(diào)控后睪酮分泌功能的變化。探索Crb3對睪丸間質(zhì)細胞睪酮分泌功能影響的相關(guān)性。為臨床中睪酮缺乏綜合征的治療提供向?qū)А?方法： 1、通過培養(yǎng)傳代細胞，從中提取RNA。將RNA反轉(zhuǎn)錄成C-DNA，利用普通PCR檢測Crb3在睪丸間質(zhì)細胞中的基因表達。 2、利用Western-Blot檢測Crb3在睪丸間質(zhì)細胞中蛋白翻譯表達情況，證明Crb3在TM3中翻譯蛋白。 3、轉(zhuǎn)染細胞，使Crb3在TM3細胞中過表達，設(shè)置轉(zhuǎn)染組、空轉(zhuǎn)組、非轉(zhuǎn)染組，利用熒光定量PCR檢測Crb3基因轉(zhuǎn)錄表達改變。 4、同時以化學發(fā)光法檢測轉(zhuǎn)染組、空轉(zhuǎn)組、非轉(zhuǎn)染組培養(yǎng)基中睪酮的值。 5、統(tǒng)計學分析：利用spss19軟件系統(tǒng)建立數(shù)據(jù)庫和進行統(tǒng)計學分析，多樣板均數(shù)間比較采用方差分析，P0.05認為差異用統(tǒng)計學意義。 結(jié)果： 1、睪丸間質(zhì)細胞中Crb3的DNA的表達及其意義普通PCR結(jié)果顯示：Crb3在睪丸間質(zhì)細胞中呈現(xiàn)出低表達。 2、睪丸間質(zhì)細胞中Crb3的蛋白質(zhì)的翻譯及其臨床意義 Western-blot結(jié)果顯示：Crb3在睪丸間質(zhì)細胞中，Crb3蛋白質(zhì)翻譯亦呈現(xiàn)出低表達，，正好符合Crb3DNA的表達情況。 3、轉(zhuǎn)染實驗結(jié)果顯示：Crb3過表達轉(zhuǎn)染組與空轉(zhuǎn)組及正常未轉(zhuǎn)染組相比，轉(zhuǎn)染組CT小于其他兩組，可見DNA表達明顯高于空轉(zhuǎn)組與未轉(zhuǎn)染組，且有統(tǒng)計學意義（P0.05） 4、轉(zhuǎn)染實驗結(jié)果顯示：Crb3過表達轉(zhuǎn)染組與空轉(zhuǎn)組及正常未轉(zhuǎn)染組相比，轉(zhuǎn)染組培養(yǎng)基中睪酮含量低于空轉(zhuǎn)組與未轉(zhuǎn)染組，且有統(tǒng)計學意義（P0.05） 結(jié)論：Crb3蛋白基因在睪丸間質(zhì)細胞中的過表達將導致睪丸間質(zhì)細胞分泌睪酮功能下降。
[Abstract]:Aim: to detect the expression of Crb3 gene in mouse testicular stromal cells (TM3) and, if so, to up-regulate the expression of Crb3 by transfection, and to detect the changes of testosterone secretion after the regulation of gene expression. To explore the correlation between Crb3 and testosterone secretion in stromal cells of testis. To provide guidance for the treatment of testosterone deficiency syndrome. Methods: 1. RNA was extracted from the cultured cells. RNA was reverse transcribed into C-DNA, and the expression of Crb3 gene in Leydig cells of testis was detected by ordinary PCR. 2. Western-Blot was used to detect the protein translation and expression of Crb3 in Leydig cells of testis, which proved that Crb3 translated protein in TM3. (3) Crb3 was overexpressed in TM3 cells after transfection. The transcriptional changes of Crb3 gene were detected by fluorescence quantitative PCR in transfection group, empty group and non-transfection group. 4. At the same time, the testosterone in the culture medium of transfection group, empty group and non-transfection group was detected by chemiluminescence method. 5, statistical analysis: using spss19 software system to establish database and statistical analysis, the comparison of multi-sample mean using ANOVA (P0.05) that the difference is statistically significant. Results: 1. The expression of Crb3 DNA in Leydig cells of testis and its significance. The results of ordinary PCR showed that the expression of Crb3 was low in Leydig cells of testis. Translation of Crb3 protein in Leydig cells of Testis and its Clinical significance Western-blot results showed that the translation of Crb3 protein in Leydig cells of testis also showed a low expression, which coincided with the expression of Crb3DNA. 3. The results of transfection experiment showed that the DNA expression in the transfected group was lower than that in the other two groups, and the expression of DNA in the transfected group was significantly higher than that in the empty group and the untransfected group, and there was statistical significance (P0.05). 4. The results of transfection experiment showed that the testosterone content in the culture medium of the transfected group was lower than that of the empty group and the untransfected group, and had statistical significance (P0.05). Conclusion the overexpression of the 1: Crb3 protein gene in the Leydig cells of the testis may lead to the decrease of testosterone secretion in the stromal cells of the testis.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R698

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