山羊FST慢病毒载体构建及其对卵巢卵泡颗粒细胞增殖的影响

Construction of Goat FST Lentivirus Vectors and Its Effect on the Proliferation of Ovarian Follicular Granulosa Cells

研究卵泡抑素(FST)对山羊卵巢卵泡颗粒细胞增殖的影响。采集山羊卵巢组织、分离培养原代卵巢卵泡颗粒细胞,RT-PCR克隆山羊FST,设计shRNA片段,构建慢病毒过表达载体和干扰载体,感染原代卵巢卵泡颗粒细胞,qPCR技术检测过表达与干扰的效果, CCK-8检测细胞的增殖效果。结果显示:分离培养的山羊卵巢卵泡颗粒细胞经促卵泡素受体(FSHR)免疫荧光鉴定,阳性率为95%;慢病毒感染颗粒细胞时荧光蛋白的表达占比80%;qPCR验证FST过表达和干扰慢病毒载体的效果均显著(P<0.05);过表达FST显著(P<0.05)抑制卵巢卵泡颗粒细胞的增殖,干扰FST显著(P<0.05)促进卵巢卵泡颗粒细胞增殖。笔者分离出山羊卵巢卵泡颗粒细胞,成功构建FST过表达和干扰两种慢病毒载体,感染结果表明FST能够抑制山羊卵巢卵泡颗粒细胞的增殖,为进一步研究FST在哺乳动物卵泡发育调控中的作用及其机制提供了基础资料。

This experiment was conducted to study the effect of follistatin(FST) on the proliferation of goat ovarian follicular granulosa cells. Primary ovarian follicular granulosa cells were isolated and cultured from goat ovarian tissue. The goat FST was cloned by RT-PCR, shRNA fragment was designed, and lentiviral overexpression vector and interference vector were constructed. The effect of overexpression and interference were detected by qPCR technique, and the proliferation effect of the cells was detected by CCK-8 assay. Goat ovarian follicular granulosa cells were identified by immunofluorescence of follicle stimulating hormone receptor(FSHR), and the positive rate was 95%. After the lentivirus infected granulosa cells, the expression of fluorescent protein accounted for 80%. The effect of overexpression of FST and interference with lentiviral vector were both significant(P<0.05) by qPCR. Overexpression of FST significantly(P<0.05) inhibited the proliferation of granulosa cells, and interfered with FST significantly(P<0.05) promoted granulosa cell proliferation. The ovarian follicular granulosa cells were isolated;the FST overexpression and interference vectors were successfully constructed. The infection results indicated that FST could inhibit the proliferation of goat ovarian follicular granulosa cells. It provides basic data for further study of the role of FST in the regulation of mammalian follicular development and its mechanism.