雌激素对大鼠脂肪干细胞诱导成骨成脂分化影响的研究

发布时间：2018-05-30 18:32

本文选题：雌激素 + 增龄　；参考：《第四军医大学》2010年硕士论文

【摘要】： 雌激素参与骨调节代谢及脂肪组织的分布,分解及合成、沉积。中老年女性增龄性变化通常伴随表现有骨质疏松以及雌激素水平的下降,随之机体脂肪组织增加造成中心性肥胖。对于妇女的机体变化既要考虑性腺功能衰退,也要考虑年龄因素造成的身体机能的下降。骨脂代谢中的成骨细胞和脂肪细胞可能是由同一前体细胞即干细胞分化而来,二者的分化平衡可能受相同因素影响。脂肪干细胞(adipose-derived stem cells,ADSCs)由于其自身具备的诸多优点成为了近年干细胞研究的热点。目前,雌激素和增龄性改变对大鼠ADSCs诱导分化中的作用尚不清楚。本研究以3月和18月龄雌性SD大鼠来源的ADSCs为对象,拟观察雌激素与增龄性因素对ADSCs体外增殖及诱导成骨,成脂肪分化能力的影响,探讨雌激素与增龄改变在ADSCs成骨成脂分化调控平衡中可能的作用。研究内容包括: 1.脂肪干细胞的分离,纯化培养鉴定: 采用密度梯度离心和自然贴壁法分别从3月和18月龄雌性SD大鼠腹股沟脂肪处分离纯化培养ADSCs,倒置显微镜观察细胞形态,流式细胞仪对其表面标志物进行检测,具有基质干细胞表面抗原CD29和CD105阳性,CD34阴性而排除其为造血干细胞可能。同时经由成骨,成脂的诱导阳性结果也逆向证实培养分离的细胞为ADSCs。 2.雌激素对脂肪干细胞生长增殖的影响: 通过MTT法检测不同月龄ADSCs生长增殖以及雌激素对其影响。结果发现,3月龄较18月龄ADSCs生长增殖能力强,雌激素处理后能促进细胞的生长增殖。 3.雌激素对于不同月龄大鼠ADSCs体外诱导成骨分化能力的影响: 分别取3月龄和18月龄第三代ADSCs预设为(1)单纯成骨诱导组,(2)成骨诱导雌激素处理组,含雌激素终浓度为10-7M(3)普通单纯培养非诱导组。通过茜素红染色及OCN检测不同月龄和雌激素处理下ADSCs体外成骨分化的能力。结果显示:成骨诱导条件下第21天同月龄雌激素处理组茜素红染色阳性面积大于单纯诱导组,相同诱导条件下茜素红染色阳性面积3月龄组大于18月龄组; RT-PCR半定量检测显示成骨诱导下同月龄雌激素组较单纯诱导组OCN表达强烈,相同成骨诱导条件下3月龄组较18月龄组OCN表达强烈;非诱导组染色及OCN检测阴性。结论:雌激素能增强大鼠ADSCs体外诱导成骨分化能力,其诱导成骨分化能力随增龄性变化下降。 4.雌激素对于不同月龄ADSCs体外诱导成脂分化能力的影响: 方法:分别取3月龄和18月龄第三代ADSCs预设为(1)单纯成脂诱导组,(2)雌激素处理成脂诱导组,含雌激素终浓度为10-7M(3)单纯培养非诱导组。油红O染色及LPL检测雌激素处理和不同月龄的ADSCs体外成脂分化能力的区别。结果显示:成脂诱导第5天同月龄雌激素处理组油红O染色阳性面积大于单纯诱导组,RT-PCR检测雌激素处理组较单纯诱导组LPL表达强烈;相同诱导条件下3月龄组油红O染色阳性面积大于18月龄组,RT-PCR检测3月龄组LPL表达较18月龄组强烈;而单纯培养非诱导组油红染色及LPL检测均为阴性。结论:雌激素能增强大鼠ADSCs体外诱导成脂分化能力,其体外诱导成脂分化能力随着增龄性变化下降。
[Abstract]:The effects of estrogen and aging on the proliferation of ADSCs in vitro and in the induction of osteogenic and adipogenic differentiation of ADSCs can be considered . The effects of estrogen and aging factors on the proliferation of ADSCs in vitro and the ability to induce bone formation and adipogenesis are also investigated .

The research content includes :

1 . Isolation , Purification and Identification of Adipose Stem Cells :

ADSCs were isolated and purified from the inguinal fat of female SD rats in March and 18 months by density gradient centrifugation and natural adherent method respectively . The morphology of the cells was observed by an inverted microscope . The flow cytometry was used to detect the surface markers .

2 . Effect of estrogen on growth and proliferation of fat stem cells :

The effects of estrogen on growth and proliferation of ADSCs were detected by MTT assay . The results showed that the proliferation of ADSCs was stronger than that in 18 months of age , and the growth and proliferation of ADSCs could be promoted after estrogen treatment .

3 . Effects of estrogen on the osteodifferentiation ability of ADSCs in vitro in rats with different ages :

The results showed that the positive area of alizarin red staining was higher than that in the simple induction group . The results showed that the positive area of alizarin red stain in the same induction group was higher than that in the simple induction group . The results showed that the positive area of alizarin red stain in the same induction group was higher than that of the 18 month old group .

4 . Effects of estrogen on the ability of ADSCs to induce adipogenesis in vitro in vitro :

Methods : The third generation ADSCs of 3 - month - old and 18 - month - old were pre - set to be ( 1 ) pure fat - induced group , ( 2 ) estrogen treated as fat - induced group . The positive area of oil - red O - stained positive was higher than that in simple induction group .
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【参考文献】