目的:探索骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)定向分化成光感受器样细胞所需的微环境。方法:采用淋巴细胞分离液梯度离心法分离纯化人BMSCs,培养第3代的细胞以碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)及脑源性神经营养因子(BDNF)3种因子首先进行向神经前体细胞诱导分化,应用免疫细胞化学检测巢蛋白及微管相关蛋白-2,当巢蛋白阳性表达率达到最高时更换诱导因子,加入色素上皮衍生因子(PEDF)及牛磺酸(taurine)继续诱导2~3wk,用免疫细胞化学及RT-PCR法检测视紫红质的表达情况。结果:诱导第3d开始能检测到巢蛋白表达,第12d阳性表达率达到最高,达(90.9±2.6)%。微管相关蛋白-2在诱导后第6d检测到阳性表达。部分细胞形态呈神经元细胞样。第12d诱导因子更换为PEDF及taurine继续诱导2~3wk,检测到有视紫红质表达,第2wk阳性率为(20.7±3.8)%,第3wk阳性率为(89.8±3.7)%。结论:采用分阶段诱导法,应用因子bFGF,EGF,BDNF及PEDF,taurine能在体外诱导BMSCs表达光感受器细胞标志物视紫红质。

AIM:To explore the microenvironment in which human bone marrow mesenchymal stem cells(BMSCs)differentiated into photoreceptor cells under nutrilits induction medium in vitroMETHODS:High-purity BMSCs could be obtained using lymphocyte separation medium by density gradient centrifugation.In the first stage the third passage cells were induced to neural precursor cells with medium including FGF,EGF and BDNF.The expression of the Nestin and MAP-2 were detected by immunocytochemistry.PEDF and taurine instead of FGF,EGF and BDNF in medium cultured the induced BMSCs furthermore 2 or 3 weeks when the positive expression rate of Nestin reached the highest level.Rhodopsin was researched during this second stage by immunocytochemistry and RT-PCR.RESULTS:Nestin could be detected from the third day after inducing differentiation,and reached its peak on the 12th day(90.9±2.6)%.MAP-2 also could be detected since the sixth day after inducing.Rhodopsin was detected during the farther inducing,and the positive rate was(20.7±3.8)% and(89.8±3.7)% in the second and third week respectively.CONCLUSION:BMSCs can differentiate into photoreceptor cells which express Rhodopsin under the condition by stages induction with FGF,EGF,BDNF,PEDF and taurine.

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中国国家自然科学基金重点资助项目(No.60827002);中国福建省重点科学研究基金资助项目（No.2008Y0040）