CaMKⅡ对多囊卵巢综合征模型小鼠颗粒细胞中Caspase-3表达的影响

doi:10.13481/j.1671-587X.20230418

摘要/Abstract

摘要：

目的探讨钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）在正常小鼠卵巢和多囊卵巢综合征（PCOS）小鼠卵巢组织中的差异表达，阐明CaMKⅡ对PCOS小鼠卵巢颗粒细胞凋亡的影响。方法用蓖麻油溶解脱氢表雄酮（DHEA）皮下注射诱导PCOS小鼠模型（PCOS组），对照组小鼠注射等体积蓖麻油，采用酶联免疫吸附试验（ELISA）法检测2组小鼠血清中睾酮水平，阴道涂片监测2组小鼠发情周期变化，HE染色观察2组小鼠卵巢组织病理形态表现，免疫荧光染色法检测2组小鼠卵巢组织中CaMKⅡ蛋白定位和荧光强度，Western blotting法检测2组小鼠卵巢组织中CaMKⅡ和含半胱氨酸的天冬氨酸蛋白水解酶3（Caspase-3）蛋白表达水平。采用短发夹RNA（sh-RNA）-CaMKⅡ慢病毒转染人卵巢颗粒细胞（KGN细胞）建立稳转体系，分为空白对照组、sh-CaMKⅡ-1组和sh-CaMKⅡ-2组，Western blotting法检测各组KGN细胞中CaMKⅡ和Caspase-3蛋白表达水平。结果与对照组比较，PCOS组小鼠血清中总睾酮和游离睾酮水平明显升高（P<0.01），发情周期紊乱，停滞于发情中期，卵巢组织中出现颗粒细胞层数较少的空泡状卵泡，表明PCOS小鼠模型建立成功。CaMKⅡ蛋白在小鼠卵巢组织的卵母细胞、颗粒细胞和间质细胞中均有表达；与对照组比较，PCOS组小鼠卵巢组织中CaMKⅡ荧光强度和蛋白表达水平明显降低（P<0.01），Caspase-3蛋白表达水平明显升高（P<0.01）。与空白对照组比较，sh-CaMKⅡ-1和sh-CaMKⅡ-2组KGN细胞中CaMKⅡ蛋白表达水平明显降低（P<0.01），Caspase-3蛋白表达水平明显升高（P<0.01）。结论 PCOS小鼠卵巢组织中CaMKⅡ蛋白表达水平降低诱导Caspase-3蛋白表达水平升高，进而促进颗粒细胞凋亡。

关键词: 钙调蛋白依赖性蛋白激酶Ⅱ, 多囊卵巢综合征, 动物模型, 含半胱氨酸的天冬氨酸蛋白水解酶3

Abstract:

Objective： To discuss the differential expressions of calmodulin- dependent protein kinase Ⅱ （CaMKⅡ） in ovarian tissue of the normal mice and the mice with polycystic ovary syndrome （PCOS），and to clarify the effect of CaMKⅡ on the apoptosis of ovarian granulosa cells of the PCOS mice. Methods The dissolved dehydroepiandrosterone （DHEA） was used to induce the PCOS mouse models by subcutaneous injection， and the mice in control group were injected with the equal volume of castor oil. Enzyme-linked immunosorbent assay（ELISA） method was used to detect the serum testosterone level of mice in two groups； vaginal smear was used to monitor the changes of estrous cycle of the mice in two groups； HE staining was used to observe the pathomorphology of ovarian tissue of the mice in two groups； immunofluorescence staining was used to detect the localization and fluorescence intensity of CaMKⅡ protein in ovarian tissue of the mice in two groups； Western blotting method was used to detect the expression levels of CaMKⅡ and cysteine-containing aspartate protein hydrolase 3 （Caspase-3） proteins in ovarian tissue of the mice in two groups. The short hairpin RNA （sh-RNA）-CaMKⅡ lentivirus was used to transfect the human ovarian granulosa cells （KGN cells） to establish the stable transfection system. The experiment was divided into blank control， sh-CaMKⅡ-1，and sh-CaMKⅡ-2 groups.The expression levels of CaMKⅡ and Caspase-3 proteins in the KGN cells in various groups were detected by Western blotting method. Results Compared with control group， the serum total testosterone and free testosterone levels of the mice in PCOS group were significantly increased （P<0.01）， the estrous cycle was disordered and stagnated in the middle of estrus， and the ovarian tissue showed the vacuolated follicles with fewer layers of granulosa cells， indicating that the PCOS mouse model was successfully established.The CaMKⅡ protein was expressed in the oocytes， granulosa cells， and mesenchymal cells in ovarian tissue； compared with control group， the fluorescence intensity of CaMKⅡ and expression level of protein in ovarian tissue of the mice in PCOS group were significantly decreased （P<0.01）， and the expression level of Caspase-3 protein in ovarian tissue of the mice was significantly increased （P<0.01）.Compared with blank control group， the expression levels of CaMKⅡ protein in the KGN cells in sh-CaMKⅡ-1 and sh-CaMKⅡ-2 groups were significantly decreased （P<0.01）， and the expression levels of Caspase-3 protein were significantly increased （P<0.01）. Conclusion Decreasing the CaMKⅡ protein expression level in ovarian tissue of the PCOS mice induces the increasing of Caspase-3 protein expression level， thereby promoting the apoptosis of granulosa cells.

Key words: Calmodulin-dependent protein kinases Ⅱ, Polycystic ovary syndrome, Animal model, Cysteinyl aspartate specific proteinase-3

中图分类号:

R711.75

谢贤国,马亚博,徐娅秀,张燕,杜长征,徐金瑞,杨易. CaMKⅡ对多囊卵巢综合征模型小鼠颗粒细胞中Caspase-3表达的影响[J]. 吉林大学学报(医学版), 2023, 49(4): 968-974.

Xianguo XIE,Yabo MA,Yaxiu XU,Yan ZHANG,Changzheng DU,Jinrui XU,Yi YANG. Effect of CaMKⅡ on Caspase-3 expression of granulosa cells in mice with polycystic ovary syndrome model[J]. Journal of Jilin University(Medicine Edition), 2023, 49(4): 968-974.

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Group	Total testosterone[ρ_B/(μg·L^－1)]	Free testosterone[ρ_B/(ng·L^－1)]
Control	6.42±1.58	174.62±47.57
PCOS	19.87±3.79^*	2 372.17±172.64^*

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