联合共培养系统诱导骨髓间充质干细胞分化为视网膜色素上皮样细胞
연합공배양계통유도골수간충질간세포분화위시망막색소상피양세포
Differentiation of bone marrow mesenchymal stem cells into retinal pigment epithelial-like cells by a systematic induction in vitro
  • 万方数据
    中华眼视光学与视觉科学杂志 중화안시광학여시각과학잡지
    CHINESE JOURNAL OF OPTOMETRY OPHTHALMOLOGY AND VISUAL SCIENCE
    2011年 2期 88-93 ,共6页

    郑学栋%徐国兴%侯泽江%陈金国

    정학동%서국흥%후택강%진금국

    间质干细胞,骨髓%色素上皮,眼%细胞分化%细胞培养技术 間質榦細胞,骨髓%色素上皮,眼%細胞分化%細胞培養技術
    간질간세포,골수%색소상피,안%세포분화%세포배양기술
    Mesenchymal stem cells,bone marrow%Pigment epithelium of eye%Cell differentiation%Cell cultivation techniques
    目的 探讨添加脑源性神经生长因子(BDNF)、表皮生长因子(EGF)、碱性成纤维细胞生长因子(bFGF)的培养基联合人类视网膜色素上皮细胞(HRPECs)共培养对骨髓间充质干细胞(BMSCs)定向诱导分化的影响.方法 实验研究.实验分三组:HRPECs+BDNF、EGF、bFGF+BMSCs共培养组、BDNF、EGF、bFGF+BMSCs共培养组和对照组(单独BMSCs).第一组取第3代HRPECs接种在双层培养板的上层,将第3代人BMSCs接种于下层培养板中,在双层六孔板的每孔中加入混合有20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF及10%胎牛血清(FBS)的DMEM-LG培养液(需做免疫细胞化学染色应同时在下层放入18 mm×18 mm盖玻片进行细胞爬片).第二组取第3代人BMSCs接种在六孔培养板中,每孔中加入含20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF及10%FBS的DMEM-LG培养液.第三组将第3代人BMSCs接种于六孔培养板中,加入10%FBS的DMEM-LG培养液.在倒置相差显微镜下观察细胞形态学变化.2周后停止培养,采用免疫细胞化学染色法和RT-PCR检测角蛋白18、RPE65蛋白存诱导细胞中的表达.数据采用Holm-Sidak法进行分析.结果 诱导2周后,第一组BMSCs细胞呈圆形、类圆形、不规则形、短棒状外观,细胞内有色素颗粒形成,其他两组没有类似改变.三组间免疫细胞化学染色法检测RPE65蛋白、角蛋白18,光密度值结果显示第一组和第二组间、第一组和第三组间差异有统计学意义(RPE65:t=37.416、36.236,P＜0.05;角蛋白18:t=38.611、37.532,P＜0.05).而第二组和第三组间差异没有统计学意义(RPE65:t=1.180,P＞0.05;角蛋白18:t=1.079,P＞0.05).RT-PCR检测相对mRNA表达量,结果显示第一组和第二组间、第一组和第三组间差异有统计学意义(RPE65/β-actin:t=176.110、174.820,P＜0.05;角蛋白18/β-actin:t=243.230、241.560,P＜0.05).而第二组和第三组间差异无统计学意义(RPE65/β-actin:t=1.283.P＞0.05;角蛋白18/β-actin:t=1.670,P＞0.05).结论 利用BDNF、EGF、bFGF联合HRPECs共培养可以使BMSCs分化为视网膜色素上皮样细胞.
    목적 탐토첨가뇌원성신경생장인자(BDNF)、표피생장인자(EGF)、감성성섬유세포생장인자(bFGF)적배양기연합인류시망막색소상피세포(HRPECs)공배양대골수간충질간세포(BMSCs)정향유도분화적영향.방법 실험연구.실험분삼조:HRPECs+BDNF、EGF、bFGF+BMSCs공배양조、BDNF、EGF、bFGF+BMSCs공배양조화대조조(단독BMSCs).제일조취제3대HRPECs접충재쌍층배양판적상층,장제3대인BMSCs접충우하층배양판중,재쌍층륙공판적매공중가입혼합유20 ng/mlbFGF、20 ng/ml EGF、20 ng/ml BDNF급10%태우혈청(FBS)적DMEM-LG배양액(수주면역세포화학염색응동시재하층방입18 mm×18 mm개파편진행세포파편).제이조취제3대인BMSCs접충재륙공배양판중,매공중가입함20 ng/ml bFGF、20 ng/ml EGF、20 ng/ml BDNF급10%FBS적DMEM-LG배양액.제삼조장제3대인BMSCs접충우륙공배양판중,가입10%FBS적DMEM-LG배양액.재도치상차현미경하관찰세포형태학변화.2주후정지배양,채용면역세포화학염색법화RT-PCR검측각단백18、RPE65단백존유도세포중적표체.수거채용Holm-Sidak법진행분석.결과 유도2주후,제일조BMSCs세포정원형、류원형、불규칙형、단봉상외관,세포내유색소과립형성,기타량조몰유유사개변.삼조간면역세포화학염색법검측RPE65단백、각단백18,광밀도치결과현시제일조화제이조간、제일조화제삼조간차이유통계학의의(RPE65:t=37.416、36.236,P＜0.05;각단백18:t=38.611、37.532,P＜0.05).이제이조화제삼조간차이몰유통계학의의(RPE65:t=1.180,P＞0.05;각단백18:t=1.079,P＞0.05).RT-PCR검측상대mRNA표체량,결과현시제일조화제이조간、제일조화제삼조간차이유통계학의의(RPE65/β-actin:t=176.110、174.820,P＜0.05;각단백18/β-actin:t=243.230、241.560,P＜0.05).이제이조화제삼조간차이무통계학의의(RPE65/β-actin:t=1.283.P＞0.05;각단백18/β-actin:t=1.670,P＞0.05).결론 이용BDNF、EGF、bFGF연합HRPECs공배양가이사BMSCs분화위시망막색소상피양세포.
    Objective To investigate the differentiation features of bone marrow mesenchymal stem cells (BMSCs) cultivated with human retinal pigment epithelial cells (HRPECs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) in vitro. Methods Experimental study. Three experimental groups were established based on the following criteria: HRPECs+EGF, BFGF, BDNF+BMSCs group (group Ⅰ), EGF, BFGF, BDNF+BMSCs group (group Ⅱ) and human BMSCs only (group Ⅲ). For group Ⅰ, HRPECs at passage 3were inoculated at the upper layer of a Transwell 6-well double layer culture plate, and human BMSCs at passage 3 were seeded at the lower layer of the culture plate; a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% fetal bovin serum (FBS) was added to each well (for immunocytochemical analysis, a 18 mm×18 mm cover slip was placed under the lower layer to allow cells to grow on it). For group Ⅱ, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 20 ng/ml bFGF, 20 ng/ml EGF, 20 ng/ml BDNF and 10% FBS was added to each well. For group III, human BMSCs at passage 3 were seeded at the 6-well culture plate, and a DMEM-LG medium containing 10% FBS was added to each well. Cell morphology was monitored under an inverted microscope. After 2 weeks, cells were collected for immunocytochemistry and RT-PCR analysis. A Holm-Sidak test was used to analyze data. Results After induction, the cells in group Ⅰ presented a similar appearance to retinal pigment epithelial cells and intracellular pigment particles were visible. However, similar changes did not occur in group Ⅱ and group Ⅲ. Immunocytochemical analysis of RPE65 and keratin-18 showed the optical density value differences between group Ⅰ and group Ⅱ, and between group Ⅰ and group Ⅲwere statistically significant (RPE65: t=37.416, 36.236, P＜0.05; keratin-18: t=38.611,37.532, P＜0.05),while the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65: t=1.180,P＞0.05; keratin-18: t=1.079, P＞0.05). The expression of RPE65 and keratin-18 was examined by RT-PCR using cell extracted from the 6-well plate, and mRNA expression was calculated after a computer software analysis and a comparison to the internal control β-actin. The inter-group difference showed that the differences between group Ⅰ and group Ⅱ, and between group Ⅰand group Ⅲ were statistically significant (RPE65/β-actin: t=176.110, 174.820, P＜0.05; keratin-18/β-actin:t=243.230, 241.560, P＜0.05), but the difference between group Ⅱ and group Ⅲ was not statistically significant (RPE65/β-actin: t=1.283, P＞0.05; keratin-18/β-actin: t=1.670, P＞0.05). Conclusion BMSCs can be induced into retinal pigment epithelium-like cells when cocultured with HRPECs and BDNF, EGF, bFGF.
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