雌性大鼠骨髓干细胞离体从头合成雌二醇的研究

发布时间：2018-01-05 21:03

本文关键词：雌性大鼠骨髓干细胞离体从头合成雌二醇的研究　出处：《南昌大学》2010年硕士论文　论文类型：学位论文

【摘要】： 目的: 研究雌性大鼠骨髓干细胞(female bone marrow stem cells, FBMSCs)在含10%胎牛血清的高糖DMEM离体培养条件下细胞形态的变化,检测能否分化为产生雌二醇(estradiol,E2)及表达芳香化酶(aromatase, P450arom)的性激素生成细胞,为卵巢早衰、老年人骨质疏松症等疾病的治疗提供新的可能途径。方法: 全骨髓贴壁法分离成年雌性SD大鼠的骨髓干细胞,用含10%胎牛血清的高糖DMEM培养增殖,传代至第3代,加或不加全反式视黄酸(all-trans retinoic acid, ATRA)诱导分化,分为对照组(不加ATRA)和ATRA组(加ATRA 10-5 mol/L),观察两组细胞的形态学变化;放射免疫法(radioimmunoassay, RIA)/酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)检测两组细胞培养液中E2、睾酮(testosterone, T)/雄烯二酮(androstenedione,ASD)的浓度;RT-PCR检测两组细胞中芳香化酶基因CYP19 mRNA的表达和FSHR mRNA的表达;免疫细胞化学检测两组细胞中芳香化酶蛋白的表达情况。结果: 1.离体培养的第3代雌性大鼠骨髓干细胞,主要表现为大小均一、梭形的贴壁细胞。 2. DMEM高糖培养基加胎牛血清培养第3代雌性大鼠骨髓干细胞,RT-PCR示对照组和ATRA组从培养第1天开始均表达合成雌二醇的关键酶芳香化酶CYP19 mRNA和颗粒细胞标志基因FSHR mRNA,免疫细胞化学示两组细胞从培养第2天到第4天都有芳香化酶蛋白的表达,阳性细胞呈簇状分布,形态呈多角形或梭形,细胞核呈卵圆形,染深蓝色。从培养1天开始在两组细胞培养液中都检测到较高水平的E2。 3.对照组和ATRA组都能产生T和ASD,提示雌性大鼠骨髓干细胞在离体培养条件下能从头合成E2。结论: 雌性大鼠骨髓干细胞在离体培养条件下能从头合成E2及表达芳香化酶,分化为性激素生成细胞。
[Abstract]:Objective: To study female bone marrow stem cells of female rat bone marrow stem cells. The morphological changes of high glucose DMEM containing 10% fetal bovine serum were observed in vitro to determine whether the cells could differentiate into estradiol producing estradiol. E2) and the expression of aromatase, The sex hormone producing cells of P450 aromb provide a new way for the treatment of premature ovarian failure and osteoporosis in the elderly. Methods: Bone marrow stem cells from adult female SD rats were isolated by whole bone marrow adherent method. The stem cells were cultured and proliferated with high glucose DMEM containing 10% fetal bovine serum. Differentiation was induced by adding or without all-trans retinoic acid (ATRA). The cells were divided into two groups: the control group (without ATRAA) and the ATRA group (with ATRA 10-5 mol / L). The morphological changes of the two groups were observed. Radioimmunoassay. Enzyme linked immunosorbent assay (Elisa) was used to detect E _ 2 in the culture medium of the two groups. Testosterone testosterone, TX / androstenedione ASD; The expression of aromatase gene CYP19 mRNA and FSHR mRNA were detected by RT-PCR. The expression of aromatase protein was detected by immunocytochemistry. Results: 1. The third generation female rat bone marrow stem cells cultured in vitro mainly showed uniform size and fusiform adherent cells. 2. The third generation female rat bone marrow stem cells were cultured in DMEM high sugar medium and fetal bovine serum. RT-PCR showed that both the control group and the ATRA group expressed CYP19 mRNA, the key enzyme of estradiol synthesis, and the granulosa cell marker gene FSHR mRNA from the first day of culture. Immunocytochemistry showed that aromatase protein was expressed in the two groups from the second day to the fourth day of culture. The positive cells were distributed in clusters with the shape of polygonal or fusiform and the nucleus was oval. High levels of E2 were detected in both groups of cell cultures from the first day of culture. 3. Both control group and ATRA group can produce T and ASD, suggesting that female rat bone marrow stem cells can synthesize E2 from scratch in vitro. Conclusion: Female rat bone marrow stem cells can synthesize E2 and express aromatase from scratch in vitro and differentiate into sex hormone producing cells.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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