本文關(guān)鍵詞：人脂肪干細(xì)胞誘導(dǎo)分化為角膜內(nèi)皮樣細(xì)胞的研究　出處：《大連醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人脂肪間充質(zhì)干細(xì)胞 角膜內(nèi)皮細(xì)胞 共培養(yǎng) 誘導(dǎo)分化

【摘要】：目的：體外培養(yǎng)人脂肪間充質(zhì)干細(xì)胞和兔角膜內(nèi)皮細(xì)胞，通過將培養(yǎng)的人脂肪間充質(zhì)干細(xì)胞和兔角膜內(nèi)皮細(xì)胞共培養(yǎng)，探討人脂肪間充質(zhì)干細(xì)胞體外誘導(dǎo)分化為角膜內(nèi)皮樣細(xì)胞的可行性，為組織工程角膜內(nèi)皮細(xì)胞重建尋求一種新的種子細(xì)胞來源，為組織工程角膜內(nèi)皮細(xì)胞重建奠定基礎(chǔ)。 方法：（1）分別采用酶消化法和組織塊法提取人脂肪間充質(zhì)干細(xì)胞和兔角膜內(nèi)皮細(xì)胞，用倒置顯微鏡、流式細(xì)胞儀、免疫組化法分別觀察鑒定兩種細(xì)胞。（2）Transwell共培養(yǎng)，取原代兔角膜內(nèi)皮細(xì)胞細(xì)胞懸浮液（接種密度1×10~5/ml）（0.4μm孔徑）接種于Transwell小室中，將第3代人脂肪干細(xì)胞（接種密度為1×10~5/ml）接種于Transwell小室下培養(yǎng)板中，放入37℃、飽和度5%的CO2的孵育箱中培養(yǎng),隔日換液，共培養(yǎng)10天。用免疫組化染色法鑒定被誘導(dǎo)人脂肪間充質(zhì)干細(xì)胞的水通道蛋白的表達(dá)。 結(jié)果：（1）體外培養(yǎng)的人脂肪干細(xì)胞大部分細(xì)胞伸展為梭形，，部分區(qū)域呈集落樣生長(zhǎng)，呈典型的成纖維細(xì)胞樣，局部排列具有一定的方向性，第3代人脂肪間充質(zhì)干細(xì)胞表面抗原測(cè)定結(jié)果顯示人脂肪間充質(zhì)干細(xì)胞表面抗原對(duì)CD44陽性率93.7%，CD34陽性率1.6%。（2）體外培養(yǎng)的兔角膜內(nèi)皮細(xì)胞形態(tài)多為六邊形或多邊形，片狀生長(zhǎng)。免疫組化染色顯示神經(jīng)特異烯醇化酶表達(dá)陽性。（3）經(jīng)體外transwell共培養(yǎng)誘導(dǎo)后的人脂肪間充質(zhì)干細(xì)胞形態(tài)變化不明顯，可見部分細(xì)胞體積變大，折光性增強(qiáng)，部分hADSCs皺縮凋亡，生長(zhǎng)速度減慢。免疫組化染色顯示水通道蛋白表達(dá)陽性。 結(jié)論：（1）人脂肪間充質(zhì)干細(xì)胞和兔角膜內(nèi)皮細(xì)胞能夠在體外成功培養(yǎng)并傳代。（2）體外培養(yǎng)的人脂肪干細(xì)胞在適宜的條件下具有向角膜內(nèi)皮細(xì)胞分化的潛能。
[Abstract]:Objective: to culture human adipose mesenchymal stem cells and rabbit corneal endothelial cells in vitro and co-culture human adipose mesenchymal stem cells and rabbit corneal endothelial cells. To explore the feasibility of inducing human adipose mesenchymal stem cells to differentiate into corneal endothelioid cells in vitro, and to seek a new seed cell source for tissue engineered corneal endothelial cell reconstruction. To lay the foundation for tissue engineering corneal endothelial cell reconstruction. Methods Human adipose mesenchymal stem cells and rabbit corneal endothelial cells were extracted by enzyme digestion method and tissue mass method, respectively. Flow cytometry and inverted microscope were used to extract human adipose mesenchymal stem cells and rabbit corneal endothelial cells. Immunohistochemical method was used to observe and identify the co-culture of two kinds of cells. The primary rabbit corneal endothelial cell suspension (seeding density 1 脳 10 ~ (5) / ml ~ (4) 渭 m) was inoculated into the Transwell chamber. The third generation of human adipose stem cells (inoculation density 1 脳 10 ~ (5) / ml) was cultured in the incubator of CO2 (37 鈩

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