论文部分内容阅读
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。
OBJECTIVE: To observe the effect of EGF and bFGF combined with the induction of nestin, NF M and telomerase reverse transcriptase (ERK) on the differentiation of human umbilical cord blood mesenchymal stem cells (MSCs) hTERT) mRNA changes. Methods: Umbilical cord blood of healthy spontaneous childbirth and mononuclear cells (MNCs) were separated by density gradient centrifugation. The cells were washed twice with PBS and then resuspended in DMEM / F12 containing 20% fetal bovine serum. (Cell density 1 × 106ml-1), a group of cytokines EGF and bFGF, the final concentration of 10μg / L, the other without cytokines, the remaining cells were frozen with liquid nitrogen. Incubate all the liquid for 24 hours to remove the non-adherent cells, after every 3 days a full amount of fluid exchange, under inverted microscope to observe the cell morphology. Adherent cells and cryopreserved cells were collected on day 1, day 4, day 7, day 14 respectively. The expression of nestin, NF M and hTERT mRNA was detected by RT-PCR. Results: The untreated cord blood MNCs (containing MSCs) nestin, NF M, hTERT mRNA were positive. After cultured, the expression of hTERT mRNA in nestin decreased and could not be detected by the 7th day. The expression of NF MmRNA increased with the prolongation of culture time. Compared with the control group, the expression of NF MmRNA in cytokines group was higher, and the decline of the expression of nestin and hTERTmRNA was delayed. Conclusion: EGF and bFGF can down-regulate the expression of hTERTmRNA in the process of UC-MSCs differentiation into neural cells.