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体外诱导人羊膜上皮细胞分化为角膜上皮样细胞的实验研究

发布时间:2018-03-31 07:01

  本文选题:人羊膜上皮细胞 切入点:自发永生化人角膜上皮细胞 出处:《暨南大学》2013年硕士论文


【摘要】:目的:探索以自发永生化人角膜上皮细胞(S-ihCECs)培养液体外模拟角膜上皮细胞微环境,诱导人羊膜上皮细胞(hAECs)分化为角膜上皮样细胞的可行性。 方法:1、无菌条件下取36~38w人羊膜,以两步酶消化法提取hAECs,倒置相差显微镜观察其生物学特性,流式细胞仪检测其间充质干细胞表面标记物(CD29、CD90、CD73、CD166)、造血干细胞表面标记物(CD34)及HLA-DR的表达水平;复苏培养S-ihCECs,倒置相差显微镜观察其生物学特性,免疫荧光检测其角膜上皮细胞特异性角蛋白CK12、CK3的表达。分别以10ug/ml、20ug/ml、30ug/ml、40ug/ml丝裂霉素处理S-ihCECs2h后常规培养,CCK8于第3、5、7d测OD值,观察各浓度丝裂霉素对S-ihCECs增殖抑制作用的稳定性;以10ug/ml、20ug/ml丝裂霉素处理S-ihCECs后,分别每12h、24h收集细胞培养液用于培养hAECs,,CCK8检测第1、3、5、7、9d的OD值,绘制生长曲线,筛选适宜的细胞培养液条件。2、收集S-ihCECs细胞培养液,制备诱导培养液用以培养hAECs,诱导过程中以倒置相差显微镜及扫描电镜观察其形态变化,qRT-PCR检测第0、4、8、12、16d Oct-4、NANOG、PAX6、CK12等mRNA的表达变化,免疫荧光、westernblot检测CK12、CK3的表达。 结果:1、 hAECs呈三角形或椭圆形,具有很强的立体感,其活性与接种密度高度相关,高密度利于hAECs原代及传代培养,可阳性表达CD29、CD90、CD73、CD166,阴性表达CD34、HLA-DR;S-ihCECs复苏后初呈短梭形,待接近融合后呈多边形,铺路石样外观,生长迅速,可表达角膜上皮细胞特异性标记物CK12、CK3。20ug/ml丝裂霉素对S-ihCECs的增殖抑制作用最显著(P<0.05)且相对稳定持久;以20ug/ml浓度丝裂霉素处理S-ihCECs后每12h收集的细胞培养液对hAECs的促增殖作用最显著(P<0.05),每24h收集的细胞培养液不利于hAECs的长期培养。2、以诱导培养液培养hAECs后,其形态较诱导前更趋于一致,体积变大,核浆比变小,细胞表面微绒毛变短。分化前后qRT-PCR检测Oct-4、NANOG、PAX6均有表达;CK12mRNA的表达水平上调,但随诱导时间的延长,其表达呈递减趋势;免疫荧光及western blot检测CK12、CK3阳性表达,CK12先于CK3表达。 结论:以S-ihCECs培养液制备的诱导培养液可用于模拟角膜上皮细胞微环境,诱导hAECs分化为角膜上皮样细胞,表达角膜上皮细胞特征性角蛋白。
[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.
【学位授予单位】:暨南大学
【学位级别】:硕士
【学位授予年份】:2013
【分类号】:R772.2

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