卵泡基膜中IV型胶原、层粘连蛋白来源的初步探讨

Major source of type IV collagen and laminin in ovarian basement membrane

通过离体培养猪、大鼠卵泡颗粒细胞、内泡膜细胞，应用免疫组化技术，观察细胞外基质与卵泡细胞结构和功能的关系，初步探讨卵泡基膜中ＩＶ型胶原、层粘连蛋白（ＬＮ）的主要来源及其生物学意义。结果表明：内泡膜细胞合成、分泌大量的ＩＶ型胶原，颗粒细胞不合成ＩＶ型胶原；颗粒细胞和泡膜细胞合成、分泌大量ＬＮ。由于ＩＶ型胶原属于不溶性蛋白，ＬＮ属于微溶性蛋白，因此卵泡基膜中的ＬＮ主要来源于基膜两侧的颗粒细胞、内泡膜细胞，ＩＶ型胶原主要来源于内泡膜细胞。体外无血清培养大鼠颗粒细胞，单独铺有细胞外基质的主要成分纤粘连蛋白（ＦＮ）、ＬＮ、ＩＶ型胶原时，明显促进颗粒细胞的增殖、分化，孕酮分泌增加；在同时铺有这三个细胞外基质成分时，上述作用最强，颗粒细胞分泌ｃＡＭＰ明显增加，说明ＦＮ、ＬＮ、ＩＶ型胶原具有协同作用，同时说明它们促进颗粒细胞分泌孕酮作用，是通过刺激ｃＡＭＰ水平升高实现的。

Objectives:To investigate the major source of type IV collagen and laminin(LN) in ovarian basement membrane and their effect on steroidogenesis. Design: In vitro experimental and immunohistochemical studies. Settings: Endocrinology laboratory in National Research Institute for Family Planning. Methods:The theca cells and granulosa cells isolated from porcine oraries;and granulosa cells also isolated from 25 day old Sprague Dawley(SD) rats were cultured under a serum free McCoy's 5a medium for 3 days. The distribution of type IV collagen,LN and fibronectin(FN) in the cultured cells was studied by immunohistochemistry. The granulosa cells isolated from SD rats were cultured on glass or plastic coverslips for 48 hours. The coverslips were separately coated with FN, LN, type IV collagen or with combined FN, LN and type IV collagen. Samples of incubation medium were collected and stored at -20℃ for measurements of cAMP and progesterone. Results: A large amount of type IV collagen was produced only in theca cells;granulose cells did not synthesize type IV collagen. Theca cells and granulosa cells generated both LN and FN. The cultured cells on glass coverslips coated with FN, LN,type IV collagen or combined FN, LN and type IV collagen secreted more progesterone than in the control group. The progesterone level in the medium of control group was 113±1.2 fmol/10 5 cells,those in the experimental groups were 146± 1.3 fmol/10 5 cells, 144±1.2 fmol/10 5 cells, 161±1.3 fmol/10 5 cells, 176±1.1 fmol/10 5 cells respectively The cells cultured on plastic coverslips coated with LN or combined FN, LN and type IV collagen secreted more cAMP than in the control group ( P <0.05 or P <0.01). Conclusion: Type IV collagen secreted from theca cells is a major source of type IV collagen in ovarian basement membrane. LN secreted from both theca cells and granulosa cells participate in the basement membrane formation. They increased cAMP levels in promoting progesterone secretion. Type IV collagen, LN and FN stimulated proliferation and differentiation of granulosa cells through their synergistic action.