α-亚麻酸对水牛卵巢颗粒细胞体外培养的影响

Effects of Alpha-linolenic Acid on Buffalo Ovarian Granulosa Cells Cultured in vitro

【目的】研究α-亚麻酸(ALA)对水牛卵巢颗粒细胞体外培养过程中细胞活力、细胞凋亡、细胞周期相关基因表达及激素分泌功能的影响。【方法】为了筛选体外培养水牛卵巢颗粒细胞ALA最适浓度,在96孔板中加入起始细胞数量为1×104/mL的水牛卵巢颗粒细胞,细胞贴壁12 h后更换含0(对照组)、10、50、100、200μmol/L ALA的培养液,在相同条件下处理24 h,用CCK-8进行细胞活力检测,筛选出最适处理浓度,并用于后续试验。后续试验分为对照组(0μmol/L)和处理组(50μmol/L)两组,用实时荧光定量PCR检测颗粒细胞中B淋巴细胞瘤-2(Bcl-2)、半胱氨酸蛋白酶-3(Caspase-3)、细胞周期蛋白依赖性激酶抑制蛋白1(p21)和增殖细胞核抗原(PCNA)基因的相对表达量,用免疫荧光对颗粒细胞中细胞凋亡相关蛋白Caspase-3和细胞周期相关蛋白p21进行荧光检测和定量分析,用ELISA法检测培养液中水牛颗粒细胞分泌的雌二醇(E 2)和孕酮(P 4)含量。【结果】与对照组相比,水牛卵巢颗粒细胞经ALA处理24 h后,在10~50μmol/L范围内随着ALA浓度升高水牛卵巢颗粒细胞的细胞活力随之升高,处理浓度为50μmol/L时细胞活力相比对照组极显著提高(P<0.01),随着浓度继续增加,水牛卵巢颗粒细胞活力下降,200μmol/L时细胞活力极显著低于对照组(P<0.01),因此后续试验选用ALA的浓度为50μmol/L。与对照组相比,50μmol/L ALA处理后颗粒细胞中抗凋亡基因Bcl-2和增殖细胞核抗原基因PCNA相对表达量均无显著差异(P>0.05),但促凋亡基因Caspase-3和细胞周期基因p 21在mRNA和蛋白水平上的表达量均极显著降低(P<0.01),E 2和P 4水平显著或极显著增高(P<0.05;P<0.01)。【结论】培养液中添加50μmol/L ALA能够提升水牛卵巢颗粒细胞增殖活力、抗凋亡能力及激素分泌能力,本研究为调控水牛卵泡发育提供理论参考。

【Objective】The experiment was to study the effect ofα-linolenic acid(ALA)on cell viability,cell apoptosis,cell cycle-related gene expression and hormone secretion function of buffalo ovarian granulosa cells during in vitro culture.【Method】In order to screen the optimal concentration of ALA for in vitro cultivation of buffalo ovarian granulosa cells,1×104/mL of buffalo ovarian granulosa cells with an initial cell count were added to a 96 well plate.After 12 h of cell adhesion,the cells replaced the culture medium with 0(control group),10,50,100 and 200μmol/L ALA and treated under the same conditions for 24 h,and cell viability was detected using CCK-8 to screen the optimal treatment concentration and use it for subsequent experiments.Subsequent experiments were divided into two groups:The control group(0μmol/L)and the treatment group(50μmol/L).Real-time quantitative PCR was used to detect the relative expression of B-cell lymphoma 2(Bcl-2),cysteinyl aspartate-specific proteinase-3(Caspase-3),cyclin-dependent kinase inhibitor 1(p21),and proliferating cell nuclear antigen(PCNA)genes.Immunofluorescence was used to detect and quantitatively analyze the apoptosis related protein Caspase-3 and cell cycle related protein p21.ELISA was used to detect the levels of estradiol(E 2)and progesterone(P 4)secreted by buffalo granulosa cells in the culture medium.【Result】Compared with the control group,the cell viability of buffalo ovarian granulosa cells increased with the increase of ALA concentration within the range of 10 to 50μmol/L after 24 hours of ALA treatment,the cell viability was extremely significantly higher at a concentration of 50μmol/L compared to the control group(P<0.01).However,as the concentration continued to increase,the activity of buffalo ovarian granulosa cells decreased,and at a concentration of 200μmol/L,the cell viability was extremely significantly lower than that of the control group(P<0.01).Therefore,a concentration of 50μmol/L ALA was selected for subsequent experiments.Compared to the control group,after 50μmol/L ALA treatment,there was no significant difference in the relative expression of anti apoptotic gene Bcl-2 and proliferating cell nuclear antigen gene PCNA(P>0.05),but the expression of pro apoptotic gene Caspase-3 and cell cycle gene p 21 were extremely significantly reduced at both mRNA and protein levels(P<0.01),while E 2 and P 4 levels were significantly or extremely significantly increased(P<0.05 or P<0.01).【Conclusion】Adding 50μmol/L ALA to the culture medium could enhance the proliferation activity,anti apoptotic ability,and hormone secretion ability of buffalo ovarian granulosa cells,and provided a theoretical reference for regulating the development of buffalo follicles.