卵巢颗粒细胞体外培养体系的建立及DEHP、MEHP对其增殖功能的影响

Foundation of primary cultural system of ovarian granulosa cells and effects of di-(2-ethylhexyl) phthalate and mono-(2-ethylhexyl) phthalate on its proliferation in rats

目的：建立卵巢颗粒细胞的原代培养体系，探讨邻苯二甲酸二（2-乙基）己酯（DEHP）及其活性中间产物邻苯二甲酸单（2-乙基）己酯（MEHP）对原代培养的卵巢颗粒细胞增殖作用的影响。方法：选用21～25日龄未成年雌性Wistar大鼠，皮下注射孕马血清（PMSG），48h后断头处死，收集卵巢颗粒细胞，在DMEM—F12培养液中培养。卵巢颗粒细胞原代培养体系建立成功后分别将不同剂量的DEHP（1μM、5μM、25μM、50μM、100μM）和MEHP（1μM、5μM、25μM、50μM、100μM）作用细胞24h及48h，以CCK-8法检测细胞增殖情况。结果：DEHP作用卵巢颗粒细胞24h时，1μM DEHP和100μM DEHP均能明显抑制卵巢颗粒细胞增殖，差异有统计学意义（P〈0．05）。DEHP作用卵巢颗粒细胞48h时，1μM DEHP和100μMDEHP均能明显促进卵巢颗粒细胞增殖，差异有统计学意义（P〈0.05）。而MEHP作用卵巢颗粒细胞24h及48h，MEHP各染毒剂量组均无统计学差异（P〉0．05）。结论：DEHP能促进卵巢颗粒细胞增殖，并且和作用时间长短有关。MEHP在观察剂量下，尚未显示有促进卵巢颗粒细胞增殖的作用。

Objective： To build the primary cultural system of ovarian granulosa cells in rats, and explore the effects of Di- （2- ethylhexyl） phthalate and Mono- （2-ethylhexyl） phthalate on the proliferation of primary culture cells. Methods： 21 -25 days Wister rats were used with subcutaneous injection of PMSG, after 48h ovarian granulosa cells were collected and cultured in the DMEM - F12. After rat granulosa cells were prepared, cells were exposed to different doses of 1μM, 5μM , 25μM, 50μM, 100μM DEHP and 1μM, 5μM , 25μM, 50μM, 100μM MEHP for 24h or 48h respectively, the proliferation rate of cells was analyzed by CCK - 8 assay. Results： After exposed to 1μM and 100μM DEHP for 24h, the proliferation rate of cells decreased significantly （P 〈 0. 05 ）. After exposed to 1μM and 100μM DEHP for 48h, the proliferation rate increased significantly （P 〈 0. 05）. After exposed to MEHP for 24h and 48h, there was no significant change （P 〉 0. 05 ）. Conclusion： DEHP can enhance the proliferation of ovarian granulosa cells, which may be related to the action time. Under the observed doses of MEHP, there was no effectiveness on the proliferation of ovarian granulosa cells.