Smad3促进大鼠卵巢颗粒细胞自噬

申春艳 董静霞 徐健

doi:10.3969/j.issn.0529-1356.2014.01.021

解剖学报 ›› 2014, Vol. 45 ›› Issue (1) : 109-113. DOI: 10.3969/j.issn.0529-1356.2014.01.021

组织学胚胎学发育生物学

Smad3促进大鼠卵巢颗粒细胞自噬

申春艳董静霞徐健*

作者信息 +

Smad3 promotes the autophagy of granulosa cells in rats

SHEN Chun-yan DONG Jing-xia XU Jian*

Author information +

文章历史 +

摘要

目的探讨Smad3基因对大鼠卵巢颗粒细胞自噬的影响。方法 21d SD雌性大鼠腹腔注射孕马血清20 IU/只，48h后收集卵巢颗粒细胞进行原代培养。培养细胞分为3组：空白对照组：培养液中不加任何处理因素；敲低实验组：培养液中加入Smad3基因特异性的SiRNA及转染试剂RNAiMAX；过表达实验组：培养液中加入Smad3真核表达质粒及转染试剂Lipo 2000。免疫细胞化学方法鉴定培养细胞纯度；Western blotting方法检测Smad3蛋白表达变化，检测转染效率。Smad3基因敲低及过表达后，Western blotting方法检测自噬体膜形成标志蛋白LC3B及自噬调控相关蛋白Bcl-2的蛋白表达变化。结果 Smad3敲低时，LC3B蛋白Ⅱ型与Ⅰ型的比值(LC3BⅡ/Ⅰ)变化不明显，Bcl-2蛋白表达明显降低；Smad3过表达时，LC3BⅡ/Ⅰ明显增加，Bcl-2蛋白表达明显增加。结论 Smad3基因特异的SiRNA及真核表达质粒可以有效地转入大鼠卵巢颗粒细胞中，Smad3基因促进大鼠卵巢颗粒细胞自噬，其机制可能与Bcl-2蛋白相关。

Abstract

Objective To study the effect of Smad3 on the autophagy of the ovarian granulosa cells (GCs) in the rat. Methods Ovarian granulosa cells were cultured and treated in the following conditions: control group in which GCs were cultured without any treatment; knockdown group in which GCs were transfected by specific Smad3 siRNA with Lipo RNAiMAX; overexpression group in which GCs were transfected by p3×FLAG-CMV-Smad3 plasmid with Lipo 2000. Immunocytochemistry was used to identify cell purity. To determine the efficiency of transfection, the expression of Smad3 protein was evaluated by Western blotting; Western blotting analysis was also used to detect the expression of autophagy associated gene 8(LC3), B cell lymphoma2(Bcl-2) proteins of ovarian GCs. Results Western blotting results showed that: in knockdown group, the ratio of LC3B II/I had no significant difference and the expression intensity of Bcl-2 protein was significantly decreased, compared to the control group. In overexpression group, the ratio of LC3BII/I was significantly increased and the expression intensity of Bcl-2 protein was significantly increased, compared to the control group. Conclusion Both Smad 3 SiRNA and P3×FLA-CMV-Smad 3 can be effectively transfected to GCs. The mechanism, by which Smad 3 promotes the autophagy of GCs, may be related to the expression of Bcl-2 protein.

导出引用

申春艳董静霞徐健. Smad3促进大鼠卵巢颗粒细胞自噬[J]. 解剖学报. 2014, 45(1): 109-113 https://doi.org/10.3969/j.issn.0529-1356.2014.01.021

SHEN Chun-yan DONG Jing-xia XU Jian*. Smad3 promotes the autophagy of granulosa cells in rats[J]. Acta Anatomica Sinica. 2014, 45(1): 109-113 https://doi.org/10.3969/j.issn.0529-1356.2014.01.021

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