细胞因子诱导人脐血间充质干细胞分化过程中细胞巢蛋白、神经丝亚单位和端粒酶逆转录酶mRNA的表达

Expression of nestin,NF-M, hTERT mRNA during differentiation of mesenchymal stem cells derived from human umbilical cord blood in vitro

目的:观察细胞因子EGF和bFGF联合诱导人脐血间充质干细胞(MSCs)向神经细胞分化过程中细胞 巢蛋白(nestin)、神经丝亚单位M(NF M)和端粒酶逆转录酶(hTERT)mRNA的变化。方法:采集健康自然分娩产 妇脐血,密度梯度离心分离单个核细胞(MNCs),PBS洗涤2次后重悬于含体积分数20%胎牛血清的DMEM/F12 中,接种于T 75培养瓶内(细胞密度为1×106ml-1),一组加入细胞因子EGF和bFGF,终浓度各为10μg/L,另一 组不加细胞因子,剩余细胞用液氮冻存。培养24h倾去全部液体以除去未贴壁细胞,以后每3d全量换液1次,倒 置显微镜下观察细胞形态。分别收集培养1d、4d、7d、14d贴壁细胞和冻存细胞,采用RT -PCR方法检测nestin、 NF M、hTERTmRNA的表达。结果:未培养的脐血MNCs(内含MSCs)nestin、NF M、hTERTmRNA均表达阳性。培 养后nestin、hTERTmRNA表达下降,至第7d已不能检出;而NF MmRNA的表达随培养时间延长而增强。与对照 组相比,细胞因子组NF MmRNA表达较高,nestin、hTERTmRNA表达的下降趋势延迟。结论:细胞因子EGF和 bFGF在联合诱导脐血MSCs分化为神经细胞的过程中,能下调hTERTmRNA的表达。

Aim: To evaluate the expression of nestin,NF-M, hTERT mRNA during differentiation of mesenchymal stem cells (MSCs) derived from human umbilical cord blood in vitro. Method:Mononuclear cells (MNCs) were prepared from heparinized human umbilical cord blood (50~100 ml) by density grade centrifugation and were divided into three parts. One part was cryopreserved in liquid nitrogen, the other two parts were resuspended in DMEM/F12 media containing 20% fetal bovine serum and allowed to adhere to T-75 tissue culture flasks in humidified atmosphere with 5% CO_2 at 37 ℃. After 24 hours, the non-adherent cells were removed, and adherent cells were subsequently cultured for 1, 4, 7 and 14 days in the media with or without recombinant human EGF and bFGF. Phenotypic changes were monitored by light microscopy. The cryopreserved MNCs and harvested cells on day 1, 4, 7 and 14 respectively were subjected to detect nestin, neurofilament subunit M (NF-M) and hTERT mRNA using RT-PCR. Results:Compared to the cryopreserved MNCs, higher expression of NF-M mRNA was found in the cultured cells along with culture time, especially in the presence of EGF and bFGF; but lower levels of nestin and hTERT mRNA were detected and downregulated in the course of culture, especially in the cultured cells without EGF and bFGF. On day 7, no detectable nestin and hTERT mRNA expression were found. Conclusion:The combination of human recombinant EGF and bFGF could promote the differentiation of MSCs derived from human umbilical cord blood into neuron-like cells and downregulate the expression of hTERT mRNA.