转化生长因子-β受体抑制剂复合物C促进人胚胎干细胞向视网膜色素上皮细胞定向诱导  

TGF-β receptor inhibitor Compound C promotes the directed differentiation of human embryonic stem cells into retinal pigment epithelial cells

作　　者：杨博宇[1] 杨春波[1] 于敏[2] 李筱荣[1] 

机构地区：[1]天津医科大学眼科医院天津医科大学眼科研究所,300384 [2]天津医科大学公共卫生学院营养与食品卫生学系

出　　处：《中华眼底病杂志》2014年第1期75-79,共5页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金（81273056）;天津市高等学校科技发展基金（20100135、20100103）

摘　　要：目的观察转化生长因子-β（TGF—B）受体抑制剂复合物C对人胚胎干细胞（hESC）向视网膜色素上皮（RPE）细胞定向诱导效率的影响。方法将H1hESC分为对照组和实验组。对照组在细胞过度融合后，以去除碱性成纤维细胞生长因子的血清替代物培养体系诱导RPE细胞定向分化。实验组于诱导分化前6d在培养体系中加入1μmol／l复合物C诱导分化第1、3、5周，采用实时荧光定量逆转录聚合酶链反应（RT—PCR）检测两组人类配对盒基因（PAX6）、小眼球相关转录因子（MITF）、细胞视黄醛结合蛋白（CRAI。BP）、RPE65mRNA的表达。将hESC来源的RPE（hESC—RPE）细胞分离纯化，采用RT—PCR、蛋白免疫印迹法（Westernblot）和细胞免疫荧光法对纯化的hESC-RPE细胞进行鉴定。结果诱导分化第4周，实验组肉眼可见色素团块，对照组无色素团块出现。将实验组的色素细胞分离纯化后，可见100％的细胞表现为多角形色素化。RT—PCR检测结果显示，实验组PAX6mRNA表达在诱导分化第1、3周显著高于对照组，差异有统计学意义（t=28．498、3．141，P〈0．05）；诱导分化第3、5周，实验组MITF（户8．866、10．111）、CRAlBP（t=5．293、5．394）和RPE65（t=9．263、9．504）的表达均明显高于对照组，差异有统计学意义（P〈0．05）。hESC-RPE细胞PAX6、MITF、CRALBP、RPE65mRNA表达均显著高于hESC（t=9．154、17．284、18．204、44．194）和ARPE-19（t=7．605、16．770、18．190、44．190）细胞，差异有统计学意义（P〈0．05）。Westernblot检测结果显示，hESC-RPE细胞高水平表达RPE标志蛋白PAX6、RPE65。荧光显微镜观察发现，hESC—RPE细胞表达RPE细胞特异性标志蛋白PAX6、紧密连接蛋白-1。结论TGF-I？受体抑制剂复合物C能显著提高hESC向RPE定向诱导效率。Objective To observe the effect of TGF-~3 receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells （hESC） into retinal pigment epithelial （RPE） cells. Methods H1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 ~mol/L TGF-i3 receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 （PAX6）, microphthalmia-associated transcription factor （MITF）, cellular retinaldchyde blinding protein （CRALBP）, and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE （hESC-RPE） cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells. Results Pigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group（P^0.05）. Compared with the hESC and ARPEq9 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65（P〈0.05）. High expression level of PAX6 and RPE65 proteins were observed in hESC RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE ceils. Conclusion TGF receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.

关 键 词：TGFβ超家族蛋白质类 拮抗剂和抑制剂 胚胎干细胞 移植 视网膜色素上皮细胞 细 I胞学 

分 类 号：R774.1[医药卫生—眼科]