本文選題：脂肪干細(xì)胞　切入點(diǎn)：微囊泡　出處：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:探討脂肪干細(xì)胞(adipose-derived stem cells,ADSCs)和脂肪干細(xì)胞來源的微囊泡(microvesicles,MVs)在體外環(huán)境中對老年人成纖維細(xì)胞的抗衰老作用。分析比較MVs與ADSCs對老年人成纖維細(xì)胞抗衰老能力的差異,以及單一供體來源的MVs與多個(gè)供體來源的混合MVs對成纖維細(xì)胞抗衰老能力的差異。由此為MVs應(yīng)用于臨床抗衰老提供一定的實(shí)驗(yàn)基礎(chǔ)和理論依據(jù)。方法:1、從3名20~30歲青年人的脂肪抽吸手術(shù)中獲取脂肪組織,使用酶消化法分離、培養(yǎng)出細(xì)胞。在顯微鏡下觀察該細(xì)胞的形態(tài),檢測細(xì)胞表面標(biāo)記物CD34、CD44、CD45、CD73、CD90、CD105,并進(jìn)行成脂、成骨分化誘導(dǎo),以鑒定該細(xì)胞是否為ADSCs。2、采用低溫超高速離心法從ADSCs培養(yǎng)上清中分離提取MVs,通過透射電鏡觀察、鑒定MVs,儲存?zhèn)溆谩?、從3名60~80歲老年人手術(shù)中切取的健康皮膚組織,分離培養(yǎng)成纖維細(xì)胞,光鏡下觀察細(xì)胞形態(tài)。4、將ADSCs和MVs分別與成纖維細(xì)胞通過Transwell小室建立間接共培養(yǎng):(1)實(shí)驗(yàn)組Ⅰ:單一供體來源的MVs;(2)實(shí)驗(yàn)組Ⅱ:多個(gè)供體來源的混合MVs;(3)陽性對照組:ADSCs;(4)空白對照組:空白培養(yǎng)基。5、共培養(yǎng)3天后在顯微鏡下觀察各組成纖維細(xì)胞共培養(yǎng)后形態(tài)的變化,并進(jìn)行以下實(shí)驗(yàn):A.使用CCK-8法檢測共培養(yǎng)后各組成纖維細(xì)胞的活性情況;B.進(jìn)行β-半乳糖苷酶(SA-β-gal)染色,檢測成纖維細(xì)胞的衰老情況;C.分析測定各組成纖維細(xì)胞內(nèi)活性氧(ROS)的含量;D.利用ELISA法測定共培養(yǎng)后成纖維細(xì)胞上清液中Ⅰ型前膠原蛋白含量;E.采用Western Blot檢測各組成纖維細(xì)胞衰老相關(guān)蛋白p16INK4a、p21Cip1以及p53的表達(dá)水平。結(jié)果:1、從脂肪組織中提取的細(xì)胞呈長梭形,細(xì)胞表面標(biāo)記物CD44、CD73、CD90和CD105表達(dá)陽性,CD34、CD45表達(dá)陰性。在誘導(dǎo)下細(xì)胞可成功向成脂和成骨方向分化。2、與ADSCs或MVs共培養(yǎng)后,部分老年人成纖維細(xì)胞逐漸由不規(guī)則的扁平星狀、條索狀轉(zhuǎn)變?yōu)榍逦?guī)則的長梭形。3、CCK-8以及ELISA檢測的實(shí)驗(yàn)結(jié)果提示:MVs與ADSCs與空白對照相比均可增強(qiáng)老年人細(xì)胞增殖以及合成I型前膠原蛋白的活性,但是ELISA檢測結(jié)果提示ADSCs提高成纖維細(xì)胞蛋白合成的能力優(yōu)于MVs。單一供體來源的MVs與多個(gè)供體來源的混合MVs增強(qiáng)成纖維細(xì)胞增殖的能力沒有顯著差別。4、SA-β-Gal染色計(jì)數(shù)、ROS測定和Western Blot檢測衰老相關(guān)蛋白p16INK4a、p21Cip1、p53的結(jié)果顯示:與空白對照相比,MVs和ADSCs都具有降低細(xì)胞老化相關(guān)生物指標(biāo)的作用,但是ADSCs降低細(xì)胞衰老相關(guān)指標(biāo)的能力優(yōu)于MVs。單一供體來源的MVs與多個(gè)供體來源的混合MVs降低細(xì)胞老化指標(biāo)的能力沒有明顯區(qū)別。結(jié)論:1、MVs與ADSCs對成纖維細(xì)胞均有一定的抗衰老作用,此外ADSCs的抗衰老作用強(qiáng)于MVs。2、單一供體來源的MVs與多個(gè)供體來源的混合MVs對成纖維細(xì)胞抗衰老的效果無明顯差異。
[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，

本文編號：1683793