RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响

doi:10.16431/j.cnki.1671-7236.2019.03.019

摘要/Abstract

摘要：

本研究旨在探讨敲低MTPN基因对体外培养水牛颗粒细胞增殖、凋亡及雌激素、孕酮分泌的影响。应用RNAi技术敲低颗粒细胞中MTPN基因的表达水平，通过实时荧光定量PCR方法检测体外培养水牛颗粒细胞中MTPN基因及增殖和周期相关基因的表达情况，CCK-8法检测细胞增殖，借助流式细胞仪检测细胞周期的分布，采用ELISA试剂盒检测细胞培养液中雌激素与孕酮含量。结果显示，经siRNA（si-MTPN）转染颗粒细胞后，MTPN基因相对表达量下降60%（P<0.01）；细胞增殖受到显著抑制（P<0.05），G1期细胞数量下降，S期细胞数量上升，G2期细胞数量极显著上升（P<0.01），细胞被阻滞在G2期；增殖与周期相关基因Cyclin D2、Cytochrom C表达量显著上升（P<0.05），Caspase9、Fas基因表达量极显著上升（P<0.01）；ELISA检测雌激素和孕酮分泌水平均显著下降（P<0.05）。综上表明，敲低MTPN基因能通过调控相关基因的表达抑制体外培养水牛颗粒细胞的增殖及雌激素、孕酮的分泌水平，为阐明MTPN基因参与家畜卵泡发生的分子机制提供参考。

关键词: 水牛; MTPN基因; 颗粒细胞; 增殖; 凋亡; 雌激素; 孕酮

Abstract:

This study was aimed to investigate the effect of MTPN gene knockdown on proliferation and apoptosis of buffalo granulosa cells in vitro and estrogen and progesterone secretion.The expression of MTPN gene in granulosa cells was knocked down by RNAi technology.The expression of MTPN gene and proliferation and cell cycle-related genes were detected by Real-time PCR,the cell proliferation and cell cycle distribution were detected by CCK-8 assay and flow cytometry,respectively.The estrogen and progesterone levels in the cell culture medium were determined by ELISA kit.The results showed that the relative expression of MTPN gene was decreased by 60% after siRNA (si-MTPN) transfection (P<0.01).The cell proliferation was significantly inhibited (P<0.05),the number of cells in G1 phase decreased,and the number of cells in S phase increased,the number of cells in G2 phase extremely significantly increased (P<0.01),and the cells were blocked in G2 phase.The expressions of Cyclin D2 and Cytochrom C genes were significantly up regulated (P<0.05),and the expressions of Caspase9 and Fas genes were extremely significantly up regulated (P<0.01).ELISA results showed that the levels of estrogen and progesterone secretion were significantly decreased (P<0.05).In conclusion,knockdown of MTPN gene could inhibit the proliferation of buffalo granulosa cells in vitro and the secretion of estrogen and progesterone by regulating the expression of related genes,which laid a reference for elucidating the molecular mechanism of MTPN gene involved in follicular development of livestock.

Key words: buffalo; MTPN gene; granulosa cells; proliferation; apoptosis; estrogn; progesterone

中图分类号:

S814.8

陈超, 李俊, 华丽萍, 安志高, 胡祥维, 刘深贺, 杨利国. RNA干扰水牛MTPN基因对颗粒细胞增殖、凋亡及激素分泌的影响[J]. 《中国畜牧兽医》, 2019, 46(3): 800-807.

CHEN Chao, LI Jun, HUA Liping, AN Zhigao, HU Xiangwei, LIU Shenhe, YANG Liguo. Effect of RNA Interference Buffalo MTPN Gene on Proliferation, Apoptosis of Granulosa cells and Hormone Secretion[J]. , 2019, 46(3): 800-807.

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