分离、培养仔猪关节软骨细胞,从中提取总RNA,用RT-PCR法扩增出TGF-β的cDNA,与pM D 18-T载体相连,转化JM 109大肠杆菌,提取质粒,鉴定所扩增片断为目的片断后,培养仔猪关节软骨细胞,并在培养液中添加不同水平的铜,分别在0、12、24、48 h...分离、培养仔猪关节软骨细胞,从中提取总RNA,用RT-PCR法扩增出TGF-β的cDNA,与pM D 18-T载体相连,转化JM 109大肠杆菌,提取质粒,鉴定所扩增片断为目的片断后,培养仔猪关节软骨细胞,并在培养液中添加不同水平的铜,分别在0、12、24、48 h培养结束时,收集细胞,提取细胞总RNA。采用RT-PCR法扩增出TGF-β的cDNA,以-βactin为内参,用凝胶分析系统对扩增出的cDNA进行扫描分析,以TGF-β的cDNA的光密度值与-βactin的cD-NA的光密度值之比作为TGF-βmRNA基因表达的相对水平。结果表明,细胞培养液中添加不同水平的铜能促进软骨细胞中TGF-βmRNA基因表达,其中以31.2μm o l/L效果最佳。展开更多
目的:观察高糖高脂刺激下人肾小球系膜细胞增殖分化及FN、TGF-β mRNA和蛋白活性表达变化,探究益气解毒活络中药有效成分防治糖尿病肾病的分子机制,讨论正交配伍中最优药物组合。方法:体外培养人肾小球系膜细胞株,传代3~5代后接种于96...目的:观察高糖高脂刺激下人肾小球系膜细胞增殖分化及FN、TGF-β mRNA和蛋白活性表达变化,探究益气解毒活络中药有效成分防治糖尿病肾病的分子机制,讨论正交配伍中最优药物组合。方法:体外培养人肾小球系膜细胞株,传代3~5代后接种于96孔培养板,采用4因素3水平正交设计方案分为正常对照组,高糖高脂模型组、中药1、2、3、4、5、6、7、8、9组,以相应药物对HMCs干预24、48、72h。MTT法检测24、48、72 h各时间点HMCs增殖分化情况;以qRT-PCR法检测48 h FN、TGF-βmRNA表达;以ELISA法检测48 h FN、TGF-β蛋白活性表达。结果与结论:(1)高糖高脂环境刺激作用下HMCs及胞外基质均能够显著增殖,且在48h达到顶峰;(2)益气解毒活络中药有效成分防治DN作用机制与通过下调高糖高脂作用下HMCs TGF-β、FN mRNA及蛋白活性表达,进而抑制高糖高脂刺激HMCs增殖相关;(3)益气解毒活络中药有效成分最佳配伍为黄芪甲苷中剂量、盐酸小檗碱低剂量、水蛭素低剂量、木犀草苷低剂量。展开更多
This study was conducted to examine the activity of TGF α and EGF R mRNA using the RT PCR technique in the early embryonic development of the mouse. Expression of TGF α mRNA was initially observed in the 8 cell stag...This study was conducted to examine the activity of TGF α and EGF R mRNA using the RT PCR technique in the early embryonic development of the mouse. Expression of TGF α mRNA was initially observed in the 8 cell stage embryo of the mouse. The level of expression gradually increased in the morula and blastula. Expression of EGF R mRNA was initially observed in the 4 cell stage embryo of the mouse and 8 cell stage embryo;morula and blastula all expressed EGF R mRNA and the levels of expression had not changed. These results confirm that epidermal growth factors play a key role in early embryonic development of the mouse, especially TGF α which can stimulate and regulate embryonic development via the autocrine system.展开更多
文摘分离、培养仔猪关节软骨细胞,从中提取总RNA,用RT-PCR法扩增出TGF-β的cDNA,与pM D 18-T载体相连,转化JM 109大肠杆菌,提取质粒,鉴定所扩增片断为目的片断后,培养仔猪关节软骨细胞,并在培养液中添加不同水平的铜,分别在0、12、24、48 h培养结束时,收集细胞,提取细胞总RNA。采用RT-PCR法扩增出TGF-β的cDNA,以-βactin为内参,用凝胶分析系统对扩增出的cDNA进行扫描分析,以TGF-β的cDNA的光密度值与-βactin的cD-NA的光密度值之比作为TGF-βmRNA基因表达的相对水平。结果表明,细胞培养液中添加不同水平的铜能促进软骨细胞中TGF-βmRNA基因表达,其中以31.2μm o l/L效果最佳。
文摘目的:观察高糖高脂刺激下人肾小球系膜细胞增殖分化及FN、TGF-β mRNA和蛋白活性表达变化,探究益气解毒活络中药有效成分防治糖尿病肾病的分子机制,讨论正交配伍中最优药物组合。方法:体外培养人肾小球系膜细胞株,传代3~5代后接种于96孔培养板,采用4因素3水平正交设计方案分为正常对照组,高糖高脂模型组、中药1、2、3、4、5、6、7、8、9组,以相应药物对HMCs干预24、48、72h。MTT法检测24、48、72 h各时间点HMCs增殖分化情况;以qRT-PCR法检测48 h FN、TGF-βmRNA表达;以ELISA法检测48 h FN、TGF-β蛋白活性表达。结果与结论:(1)高糖高脂环境刺激作用下HMCs及胞外基质均能够显著增殖,且在48h达到顶峰;(2)益气解毒活络中药有效成分防治DN作用机制与通过下调高糖高脂作用下HMCs TGF-β、FN mRNA及蛋白活性表达,进而抑制高糖高脂刺激HMCs增殖相关;(3)益气解毒活络中药有效成分最佳配伍为黄芪甲苷中剂量、盐酸小檗碱低剂量、水蛭素低剂量、木犀草苷低剂量。
文摘This study was conducted to examine the activity of TGF α and EGF R mRNA using the RT PCR technique in the early embryonic development of the mouse. Expression of TGF α mRNA was initially observed in the 8 cell stage embryo of the mouse. The level of expression gradually increased in the morula and blastula. Expression of EGF R mRNA was initially observed in the 4 cell stage embryo of the mouse and 8 cell stage embryo;morula and blastula all expressed EGF R mRNA and the levels of expression had not changed. These results confirm that epidermal growth factors play a key role in early embryonic development of the mouse, especially TGF α which can stimulate and regulate embryonic development via the autocrine system.
