PRP基因在睾丸中的表达及其生物学功能初步研究

发布时间：2018-11-11 13:20

【摘要】：目的: Proliferin related protein(PRP)最初被认为是小鼠胎盘血管生成抑制因子,据文献报道PRP仅表达于雌性胎盘组织。因此,尽管此基因发现至今已有20多年,但一直被排除在雄性之外,直接的实验研究仅见于几篇文献报道。我们在前期实验中偶然发现PRP表达于睾丸,因此本研究拟探索PRP在睾丸中的表达定位, PRP是否在雄性动物表达,及其在雄性生殖功能中的作用,PRP对人类肿瘤细胞生长的影响。方法: 采用原位杂交和免疫组化方法对PRP进行组织学定位, Real-Time RT-PCR方法研究PRP在大鼠睾丸表达水平与年龄的关系以及人参皂苷对PRP表达的影响。利用BLOCK-iTTM HiPerform TM Lentiviral Pol II miR RNAi Expression System with EmGFP试剂盒制备和包装PRP-RNAi慢病毒,以miRNA方式干扰PRP表达。用Real-Time RT-PCR方法检测PRP沉默后对睾酮分泌相关基因的影响,及对HCG诱导睾酮分泌是否有影响,ELISA方法检测细胞睾酮产生情况。构建PRP基因荧光表达载体,重组质粒转染TM3细胞,流式细胞术分析TM3细胞周期分布。为了研究PRP对肿瘤细胞生长和增殖的影响,转染SGC-7901肿瘤细胞,分析肿瘤细胞增殖改变和周期分布。结果: 1免疫组化和原位杂交结果显示PRP阳性免疫染色位于睾丸间质细胞。免疫荧光染色显示PRP在TM3细胞胞浆中表达。 2 PRP在大鼠睾丸中的表达模式为随年龄增加而增加。有趣的是,人参皂苷可以显著下调老年大鼠睾丸PRP表达水平。HCG通过PKA途径显著上调TM3细胞中PRP表达水平。 3慢病毒介导PRP沉默减弱HCG刺激睾酮产生的作用。另外,PRP沉默后抑制HCG刺激PRLR mRNA增加的作用,且PRL促进HCG诱导睾酮产生的作用也因为PRP沉默而减弱,表明了PRP可能通过影响PRLR表达而参与到睾酮合成。 4对细胞生长和细胞周期的研究表明PRP可能对细胞增殖有抑制作用。结论: 1我们首次发现PRP在睾丸间质细胞表达。 2人参皂甙可以显著下调老年大鼠睾丸PRP表达水平。 3 PRP基因沉默后减弱了HCG刺激睾酮产生的作用。 4 PRP对肿瘤细胞增殖有抑制效应。
[Abstract]:Objective: Proliferin related protein (PRP) was initially thought to be a mouse placental angiogenesis inhibitor. It was reported that PRP was expressed only in female placenta. Therefore, although the gene has been discovered for more than 20 years, it has been excluded from males. We found that PRP was expressed in testis by chance in previous experiments. Therefore, we intend to explore the expression of PRP in testis, whether PRP is expressed in male animals and its role in male reproductive function, and the effect of PRP on the growth of human tumor cells. Methods: the histological localization of PRP was performed by in situ hybridization and immunohistochemistry. The relationship between the expression of PRP in rat testis and age and the effect of ginsenoside on the expression of PRP were studied by Real-Time RT-PCR method. PRP-RNAi lentivirus was prepared and packaged by BLOCK-iTTM HiPerform TM Lentiviral Pol II miR RNAi Expression System with EmGFP kit and interfered with PRP expression by miRNA. Real-Time RT-PCR was used to detect the effect of PRP silencing on testosterone secretion related genes and whether HCG induced testosterone secretion. ELISA method was used to detect testosterone production in cells. The fluorescent expression vector of PRP gene was constructed, and the recombinant plasmid was transfected into TM3 cells. The cell cycle distribution of TM3 cells was analyzed by flow cytometry. In order to study the effect of PRP on the growth and proliferation of tumor cells, SGC-7901 tumor cells were transfected and the cell proliferation and cell cycle distribution were analyzed. Results: 1Immunohistochemistry and in situ hybridization showed that PRP positive immunostaining was located in interstitial cells of testis. Immunofluorescence staining showed that PRP was expressed in the cytoplasm of TM3 cells. 2 the expression pattern of PRP in testis of rats increased with age. Interestingly, ginsenoside could significantly down-regulate the expression of PRP in testis of aged rats. HCG significantly up-regulated the expression of PRP in TM3 cells through PKA pathway. Lentivirus mediated PRP silencing attenuates the effect of HCG stimulation on testosterone production. In addition, PRP silencing inhibits the increase of PRLR mRNA stimulated by HCG, and the effect of PRL on HCG induced testosterone production is weakened by PRP silencing, which suggests that PRP may participate in testosterone synthesis by affecting PRLR expression. 4 the study of cell growth and cell cycle showed that PRP may inhibit cell proliferation. Conclusion: 1 We found that PRP was expressed in Leydig cells of testis for the first time. 2 ginsenosides can significantly down-regulate the expression of PRP in the testis of aged rats. 3 PRP gene silencing attenuates the effect of HCG on testosterone production. 4 PRP could inhibit the proliferation of tumor cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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