脂肪干细胞微囊泡抗皮肤成纤维细胞衰老作用的研究

发布时间：2018-03-30 01:36

  本文选题：脂肪干细胞　切入点：微囊泡　出处：《江苏大学》2017年硕士论文

【摘要】：目的:探讨脂肪干细胞(adipose-derived stem cells,ADSCs)和脂肪干细胞来源的微囊泡(microvesicles,MVs)在体外环境中对老年人成纤维细胞的抗衰老作用。分析比较MVs与ADSCs对老年人成纤维细胞抗衰老能力的差异,以及单一供体来源的MVs与多个供体来源的混合MVs对成纤维细胞抗衰老能力的差异。由此为MVs应用于临床抗衰老提供一定的实验基础和理论依据。方法:1、从3名20~30岁青年人的脂肪抽吸手术中获取脂肪组织,使用酶消化法分离、培养出细胞。在显微镜下观察该细胞的形态,检测细胞表面标记物CD34、CD44、CD45、CD73、CD90、CD105,并进行成脂、成骨分化诱导,以鉴定该细胞是否为ADSCs。2、采用低温超高速离心法从ADSCs培养上清中分离提取MVs,通过透射电镜观察、鉴定MVs,储存备用。3、从3名60~80岁老年人手术中切取的健康皮肤组织,分离培养成纤维细胞,光镜下观察细胞形态。4、将ADSCs和MVs分别与成纤维细胞通过Transwell小室建立间接共培养:(1)实验组Ⅰ:单一供体来源的MVs;(2)实验组Ⅱ:多个供体来源的混合MVs;(3)阳性对照组:ADSCs;(4)空白对照组:空白培养基。5、共培养3天后在显微镜下观察各组成纤维细胞共培养后形态的变化,并进行以下实验:A.使用CCK-8法检测共培养后各组成纤维细胞的活性情况;B.进行β-半乳糖苷酶(SA-β-gal)染色,检测成纤维细胞的衰老情况;C.分析测定各组成纤维细胞内活性氧(ROS)的含量;D.利用ELISA法测定共培养后成纤维细胞上清液中Ⅰ型前胶原蛋白含量;E.采用Western Blot检测各组成纤维细胞衰老相关蛋白p16INK4a、p21Cip1以及p53的表达水平。结果:1、从脂肪组织中提取的细胞呈长梭形,细胞表面标记物CD44、CD73、CD90和CD105表达阳性,CD34、CD45表达阴性。在诱导下细胞可成功向成脂和成骨方向分化。2、与ADSCs或MVs共培养后,部分老年人成纤维细胞逐渐由不规则的扁平星状、条索状转变为清晰规则的长梭形。3、CCK-8以及ELISA检测的实验结果提示:MVs与ADSCs与空白对照相比均可增强老年人细胞增殖以及合成I型前胶原蛋白的活性,但是ELISA检测结果提示ADSCs提高成纤维细胞蛋白合成的能力优于MVs。单一供体来源的MVs与多个供体来源的混合MVs增强成纤维细胞增殖的能力没有显著差别。4、SA-β-Gal染色计数、ROS测定和Western Blot检测衰老相关蛋白p16INK4a、p21Cip1、p53的结果显示:与空白对照相比,MVs和ADSCs都具有降低细胞老化相关生物指标的作用,但是ADSCs降低细胞衰老相关指标的能力优于MVs。单一供体来源的MVs与多个供体来源的混合MVs降低细胞老化指标的能力没有明显区别。结论:1、MVs与ADSCs对成纤维细胞均有一定的抗衰老作用,此外ADSCs的抗衰老作用强于MVs。2、单一供体来源的MVs与多个供体来源的混合MVs对成纤维细胞抗衰老的效果无明显差异。
[Abstract]:Objective: to investigate the anti-aging effects of adipose-derived stem cells (ADSCs) and microvesiclesserine (MVss) on elderly fibroblasts in vitro, and to compare the anti-aging ability of MVs and ADSCs on senile fibroblasts. And the difference of anti-aging ability between MVs from single donor and mixed MVs from multiple donor sources. This provides a certain experimental and theoretical basis for the application of MVs in clinical anti-aging. Adipose tissue was obtained from fat aspiration surgery in young people aged 18 years. The cells were isolated and cultured by enzyme digestion method. The morphology of the cells was observed under microscope, and the surface marker CD34, CD4, CD45, CD73, CD90, CD90, CD105 were detected, and the cells were induced by lipogenesis, osteogenesis, differentiation, osteogenesis, osteogenesis, osteogenesis, osteogenesis, osteogenesis, osteogenesis, osteogenesis, osteogenesis, osteogenesis, and osteogenesis. To determine whether the cell was ADSCs.2MVswere isolated and extracted from the supernatant of ADSCs culture by ultracentrifugation at low temperature and high speed. MVswere identified by transmission electron microscope (TEM), and stored in reserve. 3 healthy skin tissues were removed from 3 patients aged 60 to 80 years old. The fibroblasts were isolated and cultured. Observe the morphology of cells under light microscope. ADSCs and MVs were cocultured indirectly with fibroblasts through Transwell chamber. Experiment group 鈪，

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