Establish The Cultivation Of Ovarian Interstitial Cell In Early Pregnant Rabbit In Vitro

the purposes of the paper are to research the function of the ovarian interstitial cell(OIC) in the ovarian physiology and pathology. We successfully isolate and culture the OIC from early pregnant rabbit by using different dissociating solutions (collagenases and trypsin-EDTA) and methods(dissociation at 37 ℃,20～30 min and at 4 ℃,4～5 h respectly). Growth character of OIC are studied by through primary culture, passage, and identificaton and so on. We discuss the factors of the physiological stage , serum, insulin or ITS and so on that influence the OIC's growth character in vitro. The results as follows:1 When the ovary were digested by the same enzyme at 4℃, we can obtain the cells with high viability, but the amount of the cell is relatively not much and it takes us much time to do so. The OIC dissociated by type Ⅺ collagenase or type Ⅲ collagenase can provide enough dispersed cells for primary culture. But the effect of type Ⅺ collagenase on the cell is apparently better than that of type Ⅲ collagenase and the damage to the OIC of type Ⅺ collagenase is less than that of type Ⅲ collagenase. Twenty-four hours after seeding, the large number of the OIC are adhere to the wall of culture dish. Primary cells can coalesce and form complete monolayer in seven to eight days. While the cells digested by type Ⅲ collagenase can not attactch to the wall in 24 hours.2 The ovary tissue from adult rabbit dissociated twenty minutes by type Ⅺ collagenase can provide the most OICs. The time of digestion can exceeds five to ten minutes. We must grasp the time of digestion , or we can not get enough cells.3 Trypsin-EDTA companying with injector is more appropriate to digestion and passage of the OIC compared with the two kinds collagenases that mentioned above.4 Ham's F-12/DMEM(1:1) supplemented with 10% NBS, 2.5 μg/mL insulin or 1%ITS are the best culture medium compared with other substrate.5 The supplement of NBS, insulin or ITS can evidently augment cells proliferation. Absence of NBS, the OICs which have formed complete monolayer can keep viability for 72 to 96 hours, but its proliferation is ceased. Absence of insulin or ITS, the OICs can not grow, say nothing of forming commplete monolayer.6 In early pregnant stage, the ovary is active in function. The OICs obtained from such ovary are eazier successfully cultured than that of the ovary that is inactive in function. The physiologycal phase of the rabbit effect the culture of the OICs in vitro. 7 The research established rabbit OIC culture system. The results show that using the 0.1% collagenase type Ⅺ and dissociating at 37 ℃ is the best digesting way, that using Ham's F-12/DMEM(1:1) supplemented with 10%NBS, 2.5 μg/mL insulin or 1% ITS is the best culture medium, Which can keep the OICs high viability and provide basal nutrition in vitro.8 We successfully identified the cell by immunocytochemistry. The OIC specificly express both P450 side chain cleavage(P450scc) and 3β-hydroxysteroid dehydrogenase/ Δ5-4-isomerase(3β-HSD). After staining, mitochondria become blue and cytoplasma is red-brown.