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目的 :检测重组人表皮生长因子 (EGF)和碱性成纤维生长因子 (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端粒酶活性的变化 ,以探讨人脐血MSCs的增殖性和安全性 ,为其临床应用提供实验和理论依据。方法 :采集健康自然分娩产妇志愿捐献的脐血 ,密度梯度离心分离单个核细胞 ,接种于含体积分数为2 0 %胎牛血清的DMEM/F12中 ,对照组不加细胞因子 ,实验组加入EGF和bFGF ,终浓度均为 10 μg/L ,剩余细胞用液氮冻存。培养 2 4h后倾去全部液体以去除未贴壁细胞 ,以后每 3d全量换液 1次。分别收集培养 1d、4d、7d、10d、14d、2 1d的贴壁细胞和冻存细胞 ,将细胞密度调整一致 ,采用反转录 -多聚酶链反应 (RT -PCR)方法检测脐血MSCs培养前后各时间段的巢蛋白 (nestin)和神经丝亚单位M(NF -M)mRNA表达 ;采用端粒重复序列扩增 -酶联免疫吸附实验 (TRAP -ELISA)法检测人脐血MSCs向神经细胞分化过程中的端粒酶活性变化。结果 :RT -PCR检测发现 ,未培养的脐血细胞nestin、NF -MmRNA均呈阳性表达 ;培养后NF -MmRNA的表达随培养时间延长而增强(P <0 .0 1) ;实验组与对照组相比 ,细胞因子能明显促进NF -MmRNA的表达 (P <0 .0 1)。而nestinmRNA的表达随培养时间延长而下降 (P <0 .0 1) ,至第 7d已不能检出 ;T
OBJECTIVE: To detect the changes of telomerase activity during the differentiation of human umbilical cord blood mesenchymal stem cells (MSCs) into neural cells by the combination of recombinant human epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) Proliferation and safety of blood MSCs provide the experimental and theoretical basis for its clinical application. Methods: Umbilical cord blood collected from healthy spontaneous childbirth mothers and mononuclear cells were separated by density gradient centrifugation and inoculated into DMEM / F12 medium containing 20% fetal bovine serum. The control group received no cytokines. The experimental group was given EGF And bFGF, the final concentration was 10 μg / L, the remaining cells were stored with liquid nitrogen. Twenty-four hours after incubation, pour all the liquid to remove non-adherent cells, after a full volume change every 3d. Adherent cells and cryopreserved cells were collected and cultured for 1d, 4d, 7d, 10d, 14d and 21d respectively, and the cell density was adjusted to be consistent. The levels of MSCs in cord blood were detected by reverse transcription-polymerase chain reaction The expressions of nestin and NF-M mRNA in each time period were detected by RT-PCR. The expression of nestin and NF-M mRNA in human umbilical cord blood was detected by TRAP-ELISA. Changes in telomerase activity during differentiation. Results: The expression of nestin and NF-MmRNA in umbilical cord blood cells were detected by RT-PCR. The expression of NF-M mRNA in cultured umbilical cord blood cells was increased with the prolongation of culture time (P <0.01) In contrast, cytokines significantly enhanced NF-M mRNA expression (P <0.01). However, the expression of nestin mRNA decreased with the prolongation of culture time (P <0.01), and could not be detected by the 7th day. T