| 摘要: |
| 【目的】探讨沉默信息调节因子1(Silent information regulator1，SIRT1)对牛卵泡颗粒细胞增殖及细胞周期的影响。【方法】选取牛卵巢上直径2～6 mm的卵泡，用注射器抽取卵泡中的颗粒细胞，进行体外培养。根据牛SIRT1基因的核苷酸序列，构建了4条干扰载体（shRNA-SIRT1-1597、shRNA-SIRT1-1471、shRNA-SIRT1-1186、shRNA-SIRT1-1306）及1条阴性对照载体（shRNA-NC），利用脂质体将质粒转入牛卵泡颗粒细胞，通过实时荧光定量PCR法筛选出干扰效果最佳的干扰载体用于后续试验。以转染shRNA-SIRT1-1471的颗粒细胞为干扰组，转染sh-RNA-NC的颗粒细胞为阴性对照组，不做处理的颗粒细胞为空白对照组，采用CCK-8法检测转染后24，36，48和60 h各组颗粒细胞增殖情况，用流式细胞术检测转染后24，36和48 h各组颗粒细胞的细胞周期变化情况。【结果】转染后48 h shRNA-SIRT1-1471干扰的颗粒细胞SIRT1表达量显著低于其他处理组（P<0.05），故选用shRNA-SIRT1-1471作为最佳干扰载体。转染后24，36，48和60 h，干扰组颗粒细胞增殖效果显著高于空白对照组和阴性对照组（P<0.05）；转染后24~48 h干扰组与空白对照组和阴性对照组相比G1期细胞比例显著降低，S期细胞比例显著升高，从而使更多细胞进入M期进行正常的有丝分裂，促进细胞增殖。【结论】SIRT1能抑制牛卵泡颗粒细胞的增殖，并阻滞细胞周期的进程。 |
| 关键词: SIRT1基因 牛卵泡颗粒细胞 细胞增殖 细胞周期 |
| DOI： |
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| 基金项目:国家高技术研究发展计划(“863”计划)项目(2013AA102505) |
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| Effects of SIRT1 gene on proliferation and cell cycle of bovine granulosa cells |
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CHEN Yang,SHAN Xuesong,ZHAO Zhihui,et al
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| Abstract: |
| 【Objective】The effects of SIRT1 (silent information regulator1，SIRT1) gene on the proliferation and cell cycle of granulosa cells were discussed.【Method】The 2-6 mm ovarian follicles were selected and the granular cells were extracted by syringe and culturedin vitro.Four interference vectors (sh-RNA-SIRT1-1597,shRNA-SIRT1-1471,shRNA-SIRT1-1186,and shRNA-SIRT1-1306) and one negative control vector (shRNA-NC) were constructed according to the nucleotide sequence of the SIRT1 gene of cattle.The plasmids were transformed into bovine follicular granulosa cells by liposomes,and the interference vector with the best interference effect was screened by real time fluorescence quantitative PCR for subsequent experiments.The granulosa cells transfected with shRNA-SIRT1-1471 were interference group,the transfected shRNA NC granulosa cells were control group,and the untreated granulosa cells were blank group.Proliferation of CCK-8 was used to detect the proliferation of granulosa cells 24,36,48 and 60 h after transfection.The changes of cell cycle of granulosa cells in each group were detected by flow cytometry 24,36 and 48 h after transfection.【Result】SIRT1 expression in granulosa cells 48 h after sh-RNA-SIRT1-1471 transfection was significantly lower than that in other treatment groups.Thus,shRNA-SIRT1-1471 was the best interference vector.The proliferation of granulosa cells in the interference group 24,36,48 and 60 h after shRNA-SIRT1-1471 transfection was significantly better than that in the blank control group and the negative control group (P<0.05).Compared with the blank group and the control group,the proportion of G1 phase cells was significantly decreased and the proportion of S phase cells was increased.Therefore,more cells could enter M phase for normal mitosis and promote cell proliferation.【Conclusion】SIRT1 can inhibit proliferation of bovine granulosa cells and block cell cycle progression. |
| Key words: SIRT1 gene bovine granulosa cells cell proliferation cell cycle |
