黄芪诱导人脐带MSCs向神经样细胞分化过程中NSE、nestin和NF-H的表达变化
发布时间：2018-08-21 07:29
【摘要】：中枢神经系统疾病一直以来都是临床治疗上难点。随着现代生物技术的发展,利用干细胞移植技术来治疗神经系统疾病成为可能的治疗策略。人脐带Wharton's jelly源性间充质干细胞(Wharton's jelly mesenchymal stem cells, WJ-MSCs)是来源于分娩废弃脐带的一种成体干细胞,具有自我更新和多向分化潜能,且有独特的免疫调节特性。在体内、外适当的诱导条件下,WJ-MSCs具有分化为神经样细胞的潜能。在诱导体系中,中国传统中药的应用备受瞩目。黄芪是一味扶正固本、补中益气之要药,在传统中药配方中被广泛使用。已有的研究表明,黄芪对神经组织具有一定的保护作用,且可诱导WJ-MSCs分化为神经样细胞。本课题组已建立培养WJ-MSCs的技术平台,观察基本的生物学特性,用免疫组化方法检测到nestin和NF在黄芪诱导WJ-MSCs向神经样细胞分化的过程中有表达,但其在mRNA水平的表达情况尚不清楚。目的：探讨WJ-MSCs在黄芪诱导下分化为神经样细胞的潜能以及钙离子阻断剂的干预作用,观察黄芪对WJ-MSCs表达NSE、nestin和NF-H的影响。方法：本研究采用组织块贴壁培养法获取WJ-MSCs,用胰酶消化传代来纯化细胞,取P3-P5代生长状态良好的细胞进行诱导分化实验；用RT-PCR检测黄芪在不同浓度(5ug/ml、50ug/ml、500ug/ml、5000ug/ml、25000ug/ml)和不同时间(3h、6h、24h、48h和72h)下,诱导NSE、nestin和NF-H基因的表达变化,并观察钙离子通道阻断剂LY294002和氯化锰(MnCl2)对其表达的影响。另外,用蛋白质免疫印迹(Western blot)检测黄芪诱导WJ-MSCs分化过程中NSE蛋白表达的时效和量效关系。本研究对阐明黄芪的药效以及WJ-MSCs的分化潜能具有重要意义,为建立WJ-MSCs分化为神经样细胞的诱导体系提供重要信息,以期为深入研究黄芪的诱导机制奠定理论基础。结果：用组织块贴壁培养法获取的WJ-MSCs为长梭形或成纤维细胞样细胞,密集排列呈漩涡状或束状交织,细胞增殖能力强,折光性好。RT-PCR检测结果提示,NSE、nestin和NF-H基因在对照组和黄芪诱导组中均有表达。NSE基因在黄芪诱导WJ-MSCs分化48h和72h组中表达增加,有量效关系,与对照组比较,差异具有显著性(P0.05)。NF-H基因在5ug/ml、50ug/ml和25000ug/ml黄芪诱导WJ-MSCs分化48h组中表达增加,与对照组比较,差异具有显著性(P0.05)。在72h组中,NF-H基因在5ug/ml、50ug/ml黄芪诱导WJ-MSCs分化为神经样细胞的过程中表达增加,与对照组比较,差异具有显著性(P0.05)。Nestin基因在5000ug/ml、25000ug/ml黄芪诱导WJ-MSCs分化24h组中表达增加,与对照组比较,差异具有显著性(P0.05)。给钙离子通道阻断剂(LY294002和Mncl2), nestin和NF-H基因分别在6h组、24h组中表达减弱,与50ug/ml黄芪诱导组相比,差异有显著性(P0.05)。但对NSE基因的表达无明显影响。用Western blot检测到NSE蛋白在不同浓度黄芪(12.5ug/ml-62.5ug/ml)诱导WJ-MSCs分化24h,50ug/ml为最佳浓度。另外,在不同时间点(6h、12h、24h、36h和48h),NSE蛋白在50 ug/ml黄芪诱导WJ-MSCs 6h的表达量高。结论：1、NSE、nestin和NF-H基因在黄芪诱导WJ-MSCs前后均有表达。2、黄芪诱导WJ-MSCs分化过程中NSE、nestin和NF-H基因及NSE蛋白表达上调,LY294002和MnCl2下调nestin和NF-H基因表达。提示黄芪具有诱导WJ-MSCs分化为神经样细胞的潜能,且与钙离子通路有关系。
[Abstract]:With the development of modern biotechnology, stem cell transplantation has become a possible treatment strategy for nervous system diseases. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from human umbilical cord are derived from childbirth waste. Adult stem cells abandoning umbilical cord have the potential of self-renewal and multi-differentiation, and have unique immunoregulatory properties. WJ-MSCs have the potential to differentiate into nerve-like cells in vivo and in vitro under appropriate induction conditions. In the induction system, the application of traditional Chinese medicine has attracted much attention. Astragalus membranaceus is widely used in traditional Chinese medicine formulas.Previous studies have shown that Astragalus membranaceus has a protective effect on nerve tissue and can induce WJ-MSCs to differentiate into neuron-like cells.Our team has established a technical platform for culturing WJ-MSCs to observe the basic biological characteristics and detected nestin and NF in Astragalus membranaceus-induced WJ-MSCs by immunohistochemistry. Objective: To investigate the potential of WJ-MSCs to differentiate into neuron-like cells induced by Astragalus membranaceus and the effect of calcium blockers on the expression of NSE, nestin and NF-H in WJ-MSCs. WJ-MSCs were obtained by adherent culture and purified by trypsin digestion and passage, and the cells of P3-P5 generation with good growth status were induced to differentiate. The expression of NSE, nestin and NF-H genes were detected by RT-PCR at different concentrations (5ug/ml, 50ug/ml, 500ug/ml, 5000ug/ml, 25000 ug/ml) and at different times (3h, 6h, 24h, 48h and 72h). The effects of calcium channel blocker LY294002 and manganese chloride (MnCl2) on the expression of NSE protein were observed. In addition, the expression of NSE protein in WJ-MSCs induced by Astragalus membranaceus was detected by Western blot. This study is of great significance to clarify the pharmacodynamics of Astragalus membranaceus and the differentiation potential of WJ-MSCs. Results: WJ-MSCs obtained by tissue-block adherent culture method were spindle-shaped or fibroblast-like cells, which were densely arranged in a vortex or bundle-like interweave, with strong cell proliferation and good refraction. The results showed that NSE, nestin and NF-H genes were expressed in both the control group and the astragalus-induced group. The expression of NSE gene was increased in the astragalus-induced WJ-MSCs differentiation 48 h and 72 h groups, with a dose-effect relationship. There was a significant difference between the two groups (P 0.05). The expression of NF-H gene was increased in the 5 ug/ml, 50 ug/ml and 25 000 ug/ml astragalus-induced WJ-MSCs differentiation 48 h group. In 72 h group, the expression of NF-H gene increased during the differentiation of WJ-MSCs into neuron-like cells induced by 5 ug/ml and 50 ug/ml Astragalus membranaceus, and there was a significant difference between the two groups (P 0.05). The expression of Nestin gene increased in 5 000 ug/ml and 25 000 ug/ml Astragalus membranaceus-induced WJ-MSCs differentiation 24 h group, compared with the control group. Compared with the 50 ug/ml Astragalus induction group, the expression of NSE gene was significantly lower in the calcium channel blockers (LY294002 and Mncl2), nestin and NF-H genes in the 6h and 24h groups, respectively, but not in the 50ug/ml Astragalus induction group (P 0.05). The expression of NSE protein in different concentrations of Astragalus membranaceus was detected by Western blot. In addition, at different time points (6h, 12h, 24h, 36h and 48h), the expression of NSE protein in WJ-MSCs induced by 50 ug/ml was high. Conclusion: 1, NSE, nestin and NF-H genes were expressed before and after induction of WJ-MSCs by Astragalus membranaceus. LY294002 and MnCl2 down-regulated nestin and NF-H gene expression due to the up-regulation of NSE protein expression, suggesting that Astragalus membranaceus has the potential to induce WJ-MSCs to differentiate into neuron-like cells and is related to calcium ion pathway.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R741
本文编号：2194996
[Abstract]:With the development of modern biotechnology, stem cell transplantation has become a possible treatment strategy for nervous system diseases. Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) from human umbilical cord are derived from childbirth waste. Adult stem cells abandoning umbilical cord have the potential of self-renewal and multi-differentiation, and have unique immunoregulatory properties. WJ-MSCs have the potential to differentiate into nerve-like cells in vivo and in vitro under appropriate induction conditions. In the induction system, the application of traditional Chinese medicine has attracted much attention. Astragalus membranaceus is widely used in traditional Chinese medicine formulas.Previous studies have shown that Astragalus membranaceus has a protective effect on nerve tissue and can induce WJ-MSCs to differentiate into neuron-like cells.Our team has established a technical platform for culturing WJ-MSCs to observe the basic biological characteristics and detected nestin and NF in Astragalus membranaceus-induced WJ-MSCs by immunohistochemistry. Objective: To investigate the potential of WJ-MSCs to differentiate into neuron-like cells induced by Astragalus membranaceus and the effect of calcium blockers on the expression of NSE, nestin and NF-H in WJ-MSCs. WJ-MSCs were obtained by adherent culture and purified by trypsin digestion and passage, and the cells of P3-P5 generation with good growth status were induced to differentiate. The expression of NSE, nestin and NF-H genes were detected by RT-PCR at different concentrations (5ug/ml, 50ug/ml, 500ug/ml, 5000ug/ml, 25000 ug/ml) and at different times (3h, 6h, 24h, 48h and 72h). The effects of calcium channel blocker LY294002 and manganese chloride (MnCl2) on the expression of NSE protein were observed. In addition, the expression of NSE protein in WJ-MSCs induced by Astragalus membranaceus was detected by Western blot. This study is of great significance to clarify the pharmacodynamics of Astragalus membranaceus and the differentiation potential of WJ-MSCs. Results: WJ-MSCs obtained by tissue-block adherent culture method were spindle-shaped or fibroblast-like cells, which were densely arranged in a vortex or bundle-like interweave, with strong cell proliferation and good refraction. The results showed that NSE, nestin and NF-H genes were expressed in both the control group and the astragalus-induced group. The expression of NSE gene was increased in the astragalus-induced WJ-MSCs differentiation 48 h and 72 h groups, with a dose-effect relationship. There was a significant difference between the two groups (P 0.05). The expression of NF-H gene was increased in the 5 ug/ml, 50 ug/ml and 25 000 ug/ml astragalus-induced WJ-MSCs differentiation 48 h group. In 72 h group, the expression of NF-H gene increased during the differentiation of WJ-MSCs into neuron-like cells induced by 5 ug/ml and 50 ug/ml Astragalus membranaceus, and there was a significant difference between the two groups (P 0.05). The expression of Nestin gene increased in 5 000 ug/ml and 25 000 ug/ml Astragalus membranaceus-induced WJ-MSCs differentiation 24 h group, compared with the control group. Compared with the 50 ug/ml Astragalus induction group, the expression of NSE gene was significantly lower in the calcium channel blockers (LY294002 and Mncl2), nestin and NF-H genes in the 6h and 24h groups, respectively, but not in the 50ug/ml Astragalus induction group (P 0.05). The expression of NSE protein in different concentrations of Astragalus membranaceus was detected by Western blot. In addition, at different time points (6h, 12h, 24h, 36h and 48h), the expression of NSE protein in WJ-MSCs induced by 50 ug/ml was high. Conclusion: 1, NSE, nestin and NF-H genes were expressed before and after induction of WJ-MSCs by Astragalus membranaceus. LY294002 and MnCl2 down-regulated nestin and NF-H gene expression due to the up-regulation of NSE protein expression, suggesting that Astragalus membranaceus has the potential to induce WJ-MSCs to differentiate into neuron-like cells and is related to calcium ion pathway.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R741
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