本文選題：人羊膜上皮細胞　切入點：自發(fā)永生化人角膜上皮細胞　出處：《暨南大學》2013年碩士論文

【摘要】：目的：探索以自發(fā)永生化人角膜上皮細胞（S-ihCECs）培養(yǎng)液體外模擬角膜上皮細胞微環(huán)境，誘導人羊膜上皮細胞（hAECs）分化為角膜上皮樣細胞的可行性。 方法：1、無菌條件下取36~38w人羊膜，以兩步酶消化法提取hAECs，倒置相差顯微鏡觀察其生物學特性，流式細胞儀檢測其間充質(zhì)干細胞表面標記物（CD29、CD90、CD73、CD166）、造血干細胞表面標記物（CD34）及HLA-DR的表達水平；復蘇培養(yǎng)S-ihCECs，倒置相差顯微鏡觀察其生物學特性，免疫熒光檢測其角膜上皮細胞特異性角蛋白CK12、CK3的表達。分別以10ug/ml、20ug/ml、30ug/ml、40ug/ml絲裂霉素處理S-ihCECs2h后常規(guī)培養(yǎng)，CCK8于第3、5、7d測OD值，觀察各濃度絲裂霉素對S-ihCECs增殖抑制作用的穩(wěn)定性；以10ug/ml、20ug/ml絲裂霉素處理S-ihCECs后，分別每12h、24h收集細胞培養(yǎng)液用于培養(yǎng)hAECs，，CCK8檢測第1、3、5、7、9d的OD值，繪制生長曲線，篩選適宜的細胞培養(yǎng)液條件。2、收集S-ihCECs細胞培養(yǎng)液，制備誘導培養(yǎng)液用以培養(yǎng)hAECs，誘導過程中以倒置相差顯微鏡及掃描電鏡觀察其形態(tài)變化，qRT-PCR檢測第0、4、8、12、16d Oct-4、NANOG、PAX6、CK12等mRNA的表達變化，免疫熒光、westernblot檢測CK12、CK3的表達。 結(jié)果：1、 hAECs呈三角形或橢圓形，具有很強的立體感，其活性與接種密度高度相關(guān)，高密度利于hAECs原代及傳代培養(yǎng)，可陽性表達CD29、CD90、CD73、CD166，陰性表達CD34、HLA-DR；S-ihCECs復蘇后初呈短梭形，待接近融合后呈多邊形，鋪路石樣外觀，生長迅速，可表達角膜上皮細胞特異性標記物CK12、CK3。20ug/ml絲裂霉素對S-ihCECs的增殖抑制作用最顯著（P＜0.05）且相對穩(wěn)定持久；以20ug/ml濃度絲裂霉素處理S-ihCECs后每12h收集的細胞培養(yǎng)液對hAECs的促增殖作用最顯著（P＜0.05），每24h收集的細胞培養(yǎng)液不利于hAECs的長期培養(yǎng)。2、以誘導培養(yǎng)液培養(yǎng)hAECs后，其形態(tài)較誘導前更趨于一致，體積變大，核漿比變小，細胞表面微絨毛變短。分化前后qRT-PCR檢測Oct-4、NANOG、PAX6均有表達；CK12mRNA的表達水平上調(diào)，但隨誘導時間的延長，其表達呈遞減趨勢；免疫熒光及western blot檢測CK12、CK3陽性表達，CK12先于CK3表達。 結(jié)論：以S-ihCECs培養(yǎng)液制備的誘導培養(yǎng)液可用于模擬角膜上皮細胞微環(huán)境，誘導hAECs分化為角膜上皮樣細胞，表達角膜上皮細胞特征性角蛋白。
[Abstract]:Aim: to explore the feasibility of inducing human amniotic epithelial cells (hAECs) to differentiate into corneal epithelioid cells by imitating the microenvironment of corneal epithelial cells in vitro using spontaneous immortalized human corneal epithelial cells (S-ihCECs). Methods Human amniotic membrane was extracted by two-step enzyme digestion under aseptic condition. The biological characteristics of hAECs were observed by inverted phase-contrast microscope. Flow cytometry was used to detect the expression of CD29, CD90, CD73, CD34, and HLA-DR, and S-ihCECswere resuscitated to observe their biological characteristics. The expression of specific keratin CK12 CK3 in corneal epithelial cells was detected by immunofluorescence. The OD value of CCK8 was measured on the 3rd day after treated with 10ug- / ml 20ug- / ml 30ugrml mitomycin, respectively. The stability of mitomycin concentration on S-ihCECs proliferation inhibition was observed. After S-ihCECs was treated with 10ugml / ml mitomycin, the cell culture medium was collected every 12 h and CCK8 was used to detect the OD value of the first cell culture medium. The growth curve was drawn, and the suitable condition of cell culture medium was screened. The S-ihCECs cell culture medium was collected. The morphological changes of hAECs were observed by inverted phase contrast microscope and scanning electron microscope during the induction. The expression of mRNA, such as Oct-4, NANOGN, PAX6, CK12, and CK12CK3 were detected by reverse phase contrast microscope and scanning electron microscope. The expression of CK12CK3 was detected by Western blot. Results the hAECs was triangular or elliptical and had a strong stereosensitivity. The activity of hAECs was highly correlated with the inoculation density. The high density was beneficial to the primary and passage culture of hAECs. The positive expression of CD29, CD90, CD73, CD166, negative expression of CD34, HLA-DRS-ihCECs, was short spindle after resuscitation, and the negative expression of CD34-DRS-ihCECs was short fusiform at the beginning after the resuscitation of HLA-DRS-ihCECs. The keratoepithelial cell specific marker CK12 CK3.20ugr / ml showed the most significant inhibitory effect on S-ihCECs proliferation (P < 0.05) and was relatively stable and lasting. The cell culture medium collected every 12 hours after treatment with 20ug/ml mitomycin had the most significant effect on the proliferation of hAECs (P < 0.05). The cell culture medium collected every 24 hours was not conducive to the long-term culture of hAECs. The expression of CK12 mRNA was up-regulated by qRT-PCR before and after differentiation, but the expression of CK12 mRNA decreased with the prolongation of induction time. The positive expression of CK12 and CK3 was detected by immunofluorescence and western blot. The expression of CK12 was prior to that of CK3. Conclusion: the induced medium prepared by S-ihCECs can be used to mimic the microenvironment of corneal epithelial cells and induce hAECs to differentiate into corneal epithelioid cells and express keratin characteristic of corneal epithelial cells.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R772.2

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