GnIH过表达载体构建及其对小鼠睾丸间质细胞的作用研究

本试验通过构建GnIH过表达载体，探究其对小鼠睾丸间质细胞（TM3细胞系）的凋亡效应及睾酮合成的调节作用。从6~8周雄性小鼠睾丸组织中提取RNA，反转录获得cDNA样本，利用PCR扩增并纯化GnIH基因，应用T4连接酶将其连入PLVX-IRES-ZsGreen1载体，进一步转化到感受态菌中，通过PCR、双酶切及测序鉴定后提取质粒。随后将构建好的重组质粒转染TM3细胞系72 h，分为空质粒组和GnIH过表达组，通过显微镜观察细胞荧光、实时荧光定量PCR（qRT-PCR）法、ELISA法和流式细胞术检测GnIH表达水平、细胞凋亡率及相关凋亡基因表达变化、睾酮分泌水平和睾酮合成酶基因表达水平。结果显示，GnIH扩增片段序列与参考序列一致，将GnIH过表达质粒转染至TM3细胞系72 h后，可见高强度绿色荧光，过表达组中GnIH基因水平极显著升高（P<0.01），表明GnIH过表达载体构建成功且在TM3细胞系中高表达。转染72 h后，与空质粒组相比，过表达组中细胞凋亡率极显著升高（P<0.01），凋亡基因P53和Bax/Bcl-2表达量比值也极显著升高（P<0.01）。与空质粒组相比，过表达组中睾酮浓度极显著降低（P<0.01）。与此同时，睾酮合成相关酶基因P450 scc mRNA表达量极显著降低（P<0.01），StAR、3β-HSD和P450c17 mRNA表达量显著降低（P<0.05），17β-HSD mRNA表达量在两组间无显著性差异（P>0.05）。综上所述，在体外培养的TM3细胞中过表达GnIH能诱导TM3细胞凋亡、抑制睾酮分泌且降低睾酮合成相关酶的基因表达水平，推断GnIH在小鼠睾丸间质细胞生长和睾酮合成过程中发挥负调控作用。本研究可为揭示GnIH在雄性哺乳动物生殖调控过程中的作用提供科学理论依据。

Construction of GnIH Overexpression Vector and Its Effect on Mouse Leydig Cells

In this present study, in order to investigate the effect of GnIH on the apoptosis and testosterone synthesis of mouse Leydig cells, its overexpression vector was constructed. RNA was extracted from the testis tissues of male mice at 6-8 weeks, reverse transcribed into cDNA. The GnIH gene was amplified and purified by PCR, and connected to the PLVX-IRES-ZsGreen1 vector through T4 ligase. Then, the recombinant plasmid was transformed into the competent cells, and extracted the plasmid after identified by PCR, double enzyme digestion and sequencing. Subsequently, the plasmid was transfected into the TM3 cell line for 72 h. The experiment was divided into two groups:the empty plasmid group and the GnIH overexpression group. Cell fluorescence was observed under a microscope. The mRNA level of GnIH, apoptosis gene and testosterone synthase gene in the TM3 cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), apoptotic rate and testosterone secretion level were detected by flow cytometry and ELISA, respectively. The results showed that the GnIH amplified sequence was consistent with the reference sequence. After transfecting the GnIH overexpression plasmid into the TM3 cell line for 72 h, high-intensity green fluorescence was observed, and the expression levels of GnIH was significantly increased in the overexpression group (P<0.01). These results indicated that GnIH overexpression vector was successfully constructed and highly expressed in the TM3 cell line. After 72 h of transfection, compared with the empty plasmid group, the apoptosis rate in the overexpression group was significantly increased (P<0.01), and the expression ratio of apoptosis genes P53 and Bax/Bcl-2 was also significantly increased (P<0.01). Compared with the empty plasmid group, the concentration of testosterone was extremely significantly cut down in the overexpression group (P<0.01). Meanwhile, the expression of the testosterone synthesis-related enzyme genes, P450 scc mRNA expression was extremely significantly decreased (P<0.01); and the expressions of the StAR, 3β-HSD and P450c17 mRNA were obviously reduced (P<0.05). There was no significant difference of 17β-HSD mRNA expression between the two groups (P>0.05). In summary, overexpression of GnIH in TM3 cells could induce apoptosis of TM3 cells, inhibit testosterone secretion, and decrease the gene expression levels of testosterone synthesis-related enzymes. We can infer that GnIH plays a negative regulatory role in the process of testosterone synthesis in mice. This study can provide a scientific basis for revealing the role of GnIH in the reproductive regulation of male mammals.