骨髓间充质干细胞条件培养基对人自发永生化Müller细胞系增生、黏附和分化的促进作用

Promoting effect of conditioned medium of human bone mesenchymal stem cells on proliferation,adhesion and neuronal differentiation of immortalized human Müller cell line

目的探讨骨髓间充质干细胞(BMSCs)来源的条件培养基对人永生化Müller细胞系MIO-M1细胞增生、黏附和分化的作用。方法BMSCs传至第3代进行成骨、成软骨及成脂诱导培养基诱导分化,并分别使用茜素红、阿利辛蓝及油红O染色进行分化鉴定;采用流式细胞仪检测细胞中间充质干细胞标志物CD73、CD90和CD105以及造血干细胞标志物CD34、CD45和人类白细胞DR抗原(HLA-DR)表达。采用免疫荧光染色法检测MIO-M1细胞中Müller细胞标志物SOX9、谷氨酰胺合成酶(GS)、vimentin和胞内视黄醛结合蛋白(CRALBP),视网膜干细胞标志物SOX2、nestin和CHX10,以及细胞增生标志物细胞周期蛋白D3(CCND3)的表达。将MIO-M1细胞分为标准培养基组、293T条件培养基组和BMSC条件培养基组,分别在标准培养基、293T培养上清液和BMSC培养上清液中培养。定量分析各组细胞面积、圆度、伸长系数、周长等形态参数;采用流式细胞仪检测细胞周期,采用成球实验和5-乙炔基-2'脱氧尿嘧啶核苷(EdU)染色法检测细胞增生情况。采用酶联免疫吸附测定(ELISA)法检测各组培养上清液中血管细胞黏附分子1(VCAM-1)的表达;采用荧光定量PCR法检测各组细胞中VCAM-1 mRNA表达。采用免疫荧光染色法和荧光定量PCR法分别检测各组细胞诱导分化培养后视网膜神经元标志物蛋白激酶C(PKCα)、Rhodopsin、微管相关蛋白2(MAP2)和β-微管蛋白(Tuj1)的表达变化。结果培养的BMSCs高表达CD73、CD90和CD105,低表达CD34、CD45和HLA-DR,并可成功诱导分化为成骨细胞、软骨细胞和脂肪细胞。MIO-M1细胞表达SOX9、GS、vimentin、CRALBP、SOX2、CHX10、nestin和CCND3。与标准培养基和293T条件培养基组比较,BMSC条件培养基组的MIO-M1细胞形态发生改变,形成细长的纺锤形或多极形态,且细胞面积减少,伸长系数增加,圆度降低,差异均有统计学意义(F=6.973、12.370、6.311,均P<0.01);与标准培养基和293T条件培养基组比较,不同时间点BMSC条件培养基组MIO-M1细胞形成的神经球面积明显增大,差异均有统计学意义(F_(组别)=134.300,P=<0.001;F_(时间)=82.910,P<0.001);与标准培养基和293T条件培养基组比较,BMSC条件培养基组MIO-M1细胞的EdU阳性率和细胞增生指数均显著提高,差异均有统计学意义(F=6.973、74.110,均P<0.05);与标准培养基和293T条件培养基组比较,BMSC条件培养基组的细胞上清液中VCAM-1蛋白质量浓度和细胞中的VCAM-1 mRNA相对表达量均显著升高,差异均有统计学意义(F=13.720、7.896,均P<0.05);MIO-M1细胞在分化条件下,BMSC条件培养基组的MIO-M1细胞在mRNA水平的PKCα、Rhodopsin、Tuj1和MAP2相对表达量较标准培养基组和293T条件培养基组均明显升高,差异均有统计学意义(F=14.490、5.424、14.330、7.405,均P<0.05)。结论BMSC条件培养基可以改变Müller细胞的形态,并促进其增生、黏附和向视网膜神经元的分化。

Objective To explore the effects of conditioned medium of human bone marrow mesenchymal stem cells(BMSCs)on the proliferation,adhesion and differentiation of immortalized human Müller cell line(MIO-M1).Methods The differentiation was induced in the third-passage BMSCs with osteogenic,chondrogenic and adipogenic medium and identified by alizarin red,alcian blue and oil red O staining,respectively.The expression levels of mesenchymal stem cell markers CD73,CD90 and CD105 and hematopoietic cell markers CD34,CD45 and human leukocyte antigen-DR(HLA-DR)were assayed by flow cytometry.The expressions levels of Müller cell markers SOX9,glutamine synthetase(GS),vimentin and cellular retinaldehyde-binding protein(CRALBP),retinal stem cell markers SOX2,nestin and CHX10,and cell proliferation marker cyclin D3(CCND3)in MIO-M1 cells were detected by immunofluorescence staining.The MIO-M1 cells were divided into standard medium group,293T conditioned medium group,and BMSC conditioned medium group and were incubated in the medium according to grouping.The cellular area,circularity,elongation factor and perimeter were analyzed quantitatively.The cell cycle was detected by flow cytometry,and the cell proliferation was determined by neurospora experiment and 5-ethynyl-2'-deoxyuridine(EdU)staining.The expression of vascular cell adhesion molecule 1(VCAM-1)at protein and mRNA levels in the culture supernatant was detected by enzyme linked immunosorbent assay(ELISA)and quantitative real-time PCR(qRT-PCR),respectively.The expression of retinal neuron markers protein kinase C(PKCα),Rhodopsin,microtubule-associated protein 2(MAP2)andβ-tubulin(Tuj1)was detected by immunofluorescence staining and qRT-PCR.Results CD73,CD90,CD105 showed an enhanced expression,and CD34,CD45 and HLA-DR showed weakened expression in the BMSCs.The BMSCs differentiated into osteoblasts,chondrocytes and adipocytes.Expression of SOX9,GS,vimentin and CRALBP,SOX2,CHX10,nestin and CCND3 was found in the MIO-M1 cells.Compared with standard medium group and 293T conditioned medium group,MIO-M1 cells cultured in BMSC conditioned medium group changed into an elongated spindle-shaped or multipolar morphology with reduced cell area,increased elongation index and decreased circularity,showing statistically significant differences among them(F=6.973,12.370,6.311;all at P<0.01).There were increased neurospheres formed by MIO-M1 cells in BMSC conditioned medium group compared with standard medium group and 293T conditioned medium group at different time points(F_(group)=134.300,P<0.001;F_(time)=82.910,P<0.001).Compared with the standard medium group and 293T conditioned medium group,the EdU-positive rate and proliferation index of MIO-M1 cells in BMSC conditioned medium group were significantly increased,with statistically significant differences(F=6.973,74.110;all at P<0.05);the VCAM-1 protein expression in cell supernatant and the relative expression level of VCAM-1 mRNA in BMSC conditioed medium group were significantly increased(F=13.720,7.896;all at P<0.05);the mRNA expression levels of PKCα,Rhodopsin,Tuj1 and MAP2 were higher in MIO-M1 cells of BMSC conditioned medium group under the condition of differentiation(F=14.490,5.424,14.330,7.405;all at P<0.05).Conclusions BMSCs conditioned medium can change the morphology of MIO-M1 cells and promote their proliferation,adhesion and differentiation into retinal neurons.