大鼠卵巢顆粒細(xì)胞的分離方法

大鼠顆粒細(xì)胞的分離方法一：

分離23天的SD大鼠，皮下注射DES（2.5 mg/只），連續(xù)三天。于最后一次注射24 h后，頸椎脫臼處死動(dòng)物。無菌收集兩側(cè)的卵巢，置于盛有PBS的EP管中清洗。將PBS清洗的卵巢放在盛有培養(yǎng)基的平皿中，在體視顯微鏡下，用25號針頭刺破卵泡，釋放出顆粒細(xì)胞。后吸取平皿中的細(xì)胞懸液，鏡下計(jì)數(shù)，接種細(xì)胞。培養(yǎng)基（McCoy's 5A Medium），加谷氨酰氨，用碳酸氫鈉調(diào)節(jié)PH值為7.2。

參考文獻(xiàn)：
1.Down-Regulation of Steroidogenic Response to Gonadotropins in Human and Rat Preovulatory Granulosa Cells Involves Mitogen-Activated Protein Kinase Activation and Modulation of DAX-1 and Steroidogenic Factor-1.J Clin Endocrinol Metab May 2003,88:2288-2299.
2.Activin-A, but not inhibin, regulates 17beta-hydroxysteroid dehydrogenase type 1 activity and expression in cultured rat granulosa cells.J Steroid Biochem Mol Biol, Jul 2000,73(5):203-10.
3.Synergistic effects of activin and FSH on hyperphosphorylation of Rb and G1/S transition in rat primary granulosa cells.Mol Cell Endocrinol.Nov 2003,210(1-2):31-38.
4.Induction of Cyclin D2 in Rat Granulosa Cells Requires FSH-dependent Relief from FOXO1 Repression Coupled with Positive Signals from Smad.J Biol Chem, Mar 2005,280:9135-9148.

大鼠顆粒細(xì)胞的分離方法二：

23天的SD大鼠，皮下注射PMSG10IU 48 h，hCG 10 IU。方法同前。

參考文獻(xiàn)：
1.Proliferation of Rat Granulosa Cells during the Periovulatory Interval Endocrinology, Jan 2005,146:414-422.
2.Transient expression of progesterone receptor messenger RNA in ovarian granulosa cells after the preovulatory luteinizing hormone surge OK Park and KE Mayo Mol.Endocrinol.Jul 1991,5:967.
3.A method for transplantation of luteinizing granulosa cells: evidence for progesterone secretion.Biol Reprod, Dec 1982,27:1261.
4.Estrogen Receptor-? Is Critical to Granulosa Cell Differentiation and the Ovulatory Response to Gonadotropins.Endocrinology, Aug 2005,146:3247-3262.
5.Regulation of Prostaglandin-Endoperoxide Synthase 2 Messenger Ribonucleic Acid Expression in Mouse Granulosa Cells during Ovulation.Endocrinology, Jul 2001,142:3187.

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