大鼠视网膜干细胞与神经干细胞体外增殖、分化特点比较

目的 通过对大鼠视网膜干细胞和神经干细胞体外增殖、分化特点进行比较,进一步明确视网膜于细胞自我更新及向神经细胞方向分化的能力.方法 实验研究.分离出生10 d大鼠睫状体区组织及新生大鼠脑组织,分别应用酶消化法将两种组织制成单细胞悬液,放入含有20 μg/L碱性成纤维细胞生长因子、20μg/L表皮生长因子及1×B27添加剂的无血清DMEM/F12培养液中培养,观察并比较视网膜干细胞及神经干细胞体外增殖的特点.分别取第2代细胞,应用免疫细胞化学染色法检测干细胞特异性抗原神经巢蛋白（nestin）及细胞分裂增殖标志物5-溴脱氧尿核苷（BrdU）的表达.同时,应用含50 ml/L的胎牛血清及1×N2添加剂的培养基促进其分化,观察两种细胞向神经细胞方向分化的特点：分别应用免疫细胞化学染色法对分化后的细胞nestin、神经无标志物神经元特异性烯醇酶（NSE）、神经胶质细胞标志物神经胶质酸性蛋白（GFAP）进行检测,并应用卡方检验对两种干细胞分化后的NSE及GFAP阳性细胞数比例进行比较.结果 两种细胞原代培养48 h后均可见小的球状细胞团悬浮生长,折光性强.原代培养约7 d后传代,传代后细胞能重新形成球状细胞团,应用免疫细胞化学染色方法均可检测到细胞内nestin及BrdU的阳性表达,视网膜干细胞体外增殖的能力较神经干细胞弱.两种细胞均可在血清诱导条件下转变为贴壁生长,并分化出具有神经细胞形态的细胞.诱导第7天进行免疫细胞化学染色,检测出两种干细胞表达nestin阳性细胞数比例分别为（9.5±3.5）%及（9.1±0.7）%.视网膜干细胞分化后NSE及GFAP的阳性细胞数比例分别为（11.2±2.8）%及（18.9＋2.1）%,低于神经干细胞分化后两种标志物阳性细胞数比例（34.1±63）%及（41.9±3.3）%],NSE、GFAP在两组表达的差异均有统计学意义（x2=103.23,P＜0.05;x2=74.36,P＜0.05）.结论 来源于睫状体区的大鼠视网膜干细胞形态、体外增殖及可分化特性与神经干细胞相似,但其增殖及分化为神经细胞的能力较神经干细胞弱.

Comparison of the characteristics of cell proliferation and differentiation between rat retinal stem cells and neural stem cells

Objective To study the abilities of cell renewal and neuronal differentiation of retinal stem cells (RSCs) by comparing the characteristics with neural stem cells (NSCs) in aspects of cell proliferation and differentiation. Methods Experimental study. Cells of the ciliary body zone of postnatal 10 d rat and the brain of neonate rat were isolated respectively by enzymatic digestion, then were cultivated with serum-free DMEM/FI2 medium, 20 μg/L basic fibrulast growth factor (bFGF), 20 μg/L epithelial growth factor (EGF) and 1×B27 Supplement, the characteristics of proliferation of two kinds of cells were observed and compared. These two kinds of cells were identified by detection of nestin and 5-bromodeoxyuridine (BrdU) by immunocytochemistry staining, differentiation of RSCs and NSCs was induced by DMEM/F12 medium with 50 ml/L fetal bovine serum and 1 ×N2Supplement. The differentiated characteristics of two kinds of cells were observed, the marker nestin was detected after cell differentiation, the neuron marker neurone specific enolase (NSE) and glial marker glial fibrillary acidic protein (GFAP) of two kinds of cells were also detected by immunocytochemistry staining. A x2 test was used to test data. Results In the primary cultivation of both RSCs and NSCs, cell spheres with high refraction were observed 48 hour after isolation and cultivation. The passage of the cells was about in the 7th day of cultivation, and the cells of the next passage could also form the spherical shape. The expression of marker nestin and BrdU could be detected both in RSCs and NSCs by immunocytochemistry staining. The proliferation ability of RSCs was lower than NSCs. Both RSCs and NSCs became adherent to the bottom of the culture dish in the induced condition with serum, and some of the cells differentiated into cells with neuronal cell shapes. In the 7th day after differentiation, the ratio of nestin-positive cells was 9.5%±3.5% in RSCs,and which was 9.1%±0.7% in NSCs. The ratio of NSE-positive cells (11.2%+2.8%), and the ratio of GFAP-positive cells (18.9%+2.1%) in the differentiated RSCs were significantly lower than those in the differentiated NSCs (34.1%±6.3% and 41.9%±3.3%) respectively, the differences were significant (x2=103.23, 74.36, P＜0.05 for both). Conclusion The characteristics of the shape, proliferation and differentiation of RSCs from ciliary body zone are similar comparing with NSCs, but the abilities of proliferation and neuronal differentiation of RSCs are lower than NSCs.