青海牦牛卵巢颗粒细胞体外培养及kisspeptin-10对其孕酮分泌的影响和机制研究  

In vitro culture of ovarian granulosa cells from Qinghai yak and effects of kisspepin-10 on progesterone secretion and related mechanism in the cells

作　　者：张琳钦 张君[2] 徐尚荣[2] 彭巍[2] 黄荣[2] 舒适 关凯文 赵明旺 ZHANG Linqin;ZHANG Jun;XU Shangrong;PENG Wei;HUANG Rong;SHU Shi;GUAN Kaiwen;ZHANG Mingwang(College of Agriculture and Animal Husbandry,Qinghai University,Xining 810016,China;College of Animal Husbandry and Veterinary Science,Qinghai University,Xining 810016,China)

机构地区：[1]青海大学农牧学院,青海西宁810016 [2]青海大学畜牧兽医科学院,青海西宁810016

出　　处：《畜牧与兽医》2020年第3期17-22,共6页Animal Husbandry & Veterinary Medicine

基　　金：青藏高原牦牛高效安全养殖技术应用示范(2018YFD0502304);青海省高原牦牛研究开发中心能力建设(2017-GX-G06-1)。

摘　　要：体外培养牦牛卵巢颗粒细胞,并研究Kisspeptin-10对其孕酮分泌的作用和可能机制。于屠宰场选取适宜卵巢后在4h内运回实验室,抽吸法提取细胞,并通过卵泡刺激素受体(FSHR)抗体验证培养的颗粒细胞纯度,HE染色观察细胞形态,CCK测细胞生长曲线;不同浓度的Kisspeptin-10、Verapamil单独或共同处理细胞24 h后,分别收集细胞和上清,通过流式细胞仪检测细胞内Ca2+浓度,ELLSA测上清内孕酮含量。结果显示,FSHR阳性率>97%,验证了细胞为卵巢颗粒细胞;使用含10%胎牛血清和1%双抗的DMEM/F12培养液培养,可获得生长良好的牦牛卵巢颗粒细胞。颗粒细胞呈多角形或梭形,并伸出伪足,呈放射状生长。培养24 h后,颗粒细胞进入对数生长期,于72 h后进入平台期;Kisspeptin-10处理可显著增加颗粒细胞的活力,100 nmol·L^-1时可显著促进孕酮的分泌;钙离子阻断剂Verapamil会降低孕酮的分泌,于20、50 nmol·L^-1时,达显著降低水平(P<0.05);100 nmol·L^-1 Kisspeptin-10与不同浓度的Verapamil共同处理时,孕酮含量有所提升,但不能逆转其对孕酮分泌的抑制作用(P>0.05);流式细胞仪检测胞内Ca2+浓度,其结果与检测的颗粒细胞孕酮分泌水平相一致。综上,Kisspeptin-10能促进体外培养的牦牛卵巢颗粒细胞孕酮分泌,其机制可能与胞内Ca2+浓度相关。Ovarian granulosa cells from Qinghai yaks were cultured in vitro,and the effect of Kisspeptin-10 on progesterone secretion and possible mechanism in the cells were studied.Appropriate ovaries were obtained in the slaughterhouse,and were transported to the laboratory within 4 hours.Cells were extracted by suction.The purity of the cultured granulosa cells was verified by the FSHR antibody.Cell morphology was observed by HE staining,and the cell growth curve was measured by CCK.The cells were treated with Kisspeptin-10 and Verapamil at different concentrations individually or jointly for 24 hours,and then the cells and supernatants were collected respectively.The intracellular Ca^2+concentration was detected by flow cytometry,and the progesterone content in the supernatant was measured by ELLSA.The results showed that the positive rate of FSHR was>97%,which proved that the cells were ovarian granulosa cells.Using DMEM/F12 culture medium containing 10%fetal bovine serum and 1%double antibody,the ovarian granulosa cells with good growth could be obtained.The granulosa cells were polygonal or spindle-shaped and produced pseudopodia growing radially.The granulosa cells entered the logarithmic phase after 24 hours of culture and the plateau phase after 72 hours of culture.Kisspeptin-10 treatment significantly increased the activity of the cells,and 100 nmol·L^-1 significantly promoted the secretion of progesterone in them.Verapamil,a calcium ion blocker,reduced the secretion of progesterone,reaching a significant reduction level at 20 and 50 nmol·L^-1(P<0.05).When 100 nmol·L^-1 Kisspeptin-10 was treated with Verapamil at different concentrations,the progesterone content increased,but its inhibitory effect on the progesterone secretion could not be reversed(P>0.05).Flow cytometry was used to detect intracellular Ca^2+concentration,and the results were consistent with the progesterone secretion level in the supernatant medium.In conclusion,Kisspeptin-10 promoted progesterone secretion of ovarian granulosa cells from ya

关 键 词：青海牦牛 颗粒细胞 Kisspeptin-10 孕酮 钙离子 

分 类 号：S823.85[农业科学—畜牧学]