ChREBP基因RNA干扰真核表达载体的构建、筛选及转染
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摘要
目的构建针对大鼠(Rattus Norvegicus )脂肪细胞ChREBP基因的miRNA干扰载体,通过脂质体转染大鼠脂肪细胞,观察不同浓度葡萄糖对转染48小时后的原代培养的脂肪细胞的增殖分化的影响,探索ChREBP在大鼠脂肪细胞生脂调控中的途径。
方法1.根据RNA干扰原理,构建针对大鼠脂肪细胞ChREBP基因的四种质粒pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ;2.将构建的四种质粒和pcDNA6.2-GW/EmGFP-control分别转染大鼠脂肪细胞,用杀稻瘟素筛选得到转染成功的细胞克隆;3、采用荧光显微镜下观察脂肪细胞转染效果,选择较好的构建好的重组质粒;4、采用MTT法检测转染脂肪细胞的增殖情况;5、采用油红O法检测对脂肪细胞的分化的影响。
结果1、成功构建了在大鼠脂肪细胞细胞核中表达pri-miRNA的四种质粒pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ;2、成功转染大鼠脂肪细胞,通过杀稻瘟素筛选稳定的转染细胞克隆。荧光显微镜下简单观察转染效果,观察到pcDNA6.2-GW/EmGFP-ChREBPⅢ转染效果较好,总体转染率都很低;3、不同浓度葡萄糖处理重组质粒pcDNA6.2-GW/EmGFP-ChREBPⅢ转染后48小时后对脂肪细胞增殖分化的影响:20mmol/L葡萄糖浓度处理组对大鼠脂肪细胞分化的影响显著高于0mmol/L、5mmol/L和10mmol/L葡萄糖浓度处理组(p<0.05),对于25mmol/L和15mmol/L没有显著性(P>0.05);在细胞培养的第二天、第四天、第六天和第八天,与培养基中维持细胞生长的葡萄糖浓度组(0mmol/L)相比,10mmol/L、15mmol/L和20mmol/L葡萄糖处理的大鼠脂肪细胞增殖作用显著增强(P<0.05),其中15mmol/L葡萄糖处理的大鼠脂肪细胞增殖作用极显著(p<0.05),而5mmol/L和25mmol/L浓度有增强的趋势,但没有统计学显著性(P>0.05)。
结论结果表明构建的真核表达载体对于大鼠脂肪细胞转染效率不高,其中pcDNA6.2-GW/EmGFP-ChREBPⅢ转染效果较好,不同浓度葡萄糖对转染48小时后的大鼠脂肪细胞增值分化影响与未转染脂肪细胞对其影响相比较变化不大。
Objective: To construct four plasmids which can inhibit expression of ChREBP of rat adipocyte, observe effects of glucose on rat adipocyte multiplication and differentiation by lipofectamine 2000 transfecting and the regulation role of ChREBP on its lipogenesis.
Methods: 1. Four short DNA targetsequence were designed according to gene-silencing technologies principle, chcmically synthesized , and reconstructed to four plasmids: pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte,respectively. 3. Effective transfection recombinant plasmids can be obeseved by fluorescence-microscope. 4. The rate of increase of primary rat adipocyte proliferation were detected by MTT.5. Effects of primary rat adipocyte differentiation by oil red O.
Results: 1. Four plasmids were constracted successfully. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte and was used Blasticidin to gain the cell clones that express stably. Recombinant plasmids pcDNA6.2-GW/EmGFP-ChREBPⅢ’s fluorescent images shows batter than others .Tissue that were not digested were good in transfection unexpected. 3. Effects of glucose on rat preadipocytes differentiation by applications,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h, 20mmol/L glucose(P<0.05) was higher significantly than 0mmol/L、5mmol/L and 10mmol/L glucose, 25mmol/L、15mmol/L was no significant(P>0.05). Effects of glucose on rat proliferation applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h,15mmol/L glucose was more enhancement significantly than 10mmol/L、15mmol/L and 20mmol/L glucose(P<0.05), 5mmol/L、25mmol/L glucose was no significant(P>0.05).
Conclusion: The recombinant plasmids was inefficiency express ChREBP specific RNA,but pcDNA6.2-GW/EmGFP-ChREBPⅢcan more efficiency. Effects of glucose on rattus norvegicus preadipocytes differentiation and proliferation by applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h,has few changes.
方法1.根据RNA干扰原理,构建针对大鼠脂肪细胞ChREBP基因的四种质粒pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ;2.将构建的四种质粒和pcDNA6.2-GW/EmGFP-control分别转染大鼠脂肪细胞,用杀稻瘟素筛选得到转染成功的细胞克隆;3、采用荧光显微镜下观察脂肪细胞转染效果,选择较好的构建好的重组质粒;4、采用MTT法检测转染脂肪细胞的增殖情况;5、采用油红O法检测对脂肪细胞的分化的影响。
结果1、成功构建了在大鼠脂肪细胞细胞核中表达pri-miRNA的四种质粒pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ;2、成功转染大鼠脂肪细胞,通过杀稻瘟素筛选稳定的转染细胞克隆。荧光显微镜下简单观察转染效果,观察到pcDNA6.2-GW/EmGFP-ChREBPⅢ转染效果较好,总体转染率都很低;3、不同浓度葡萄糖处理重组质粒pcDNA6.2-GW/EmGFP-ChREBPⅢ转染后48小时后对脂肪细胞增殖分化的影响:20mmol/L葡萄糖浓度处理组对大鼠脂肪细胞分化的影响显著高于0mmol/L、5mmol/L和10mmol/L葡萄糖浓度处理组(p<0.05),对于25mmol/L和15mmol/L没有显著性(P>0.05);在细胞培养的第二天、第四天、第六天和第八天,与培养基中维持细胞生长的葡萄糖浓度组(0mmol/L)相比,10mmol/L、15mmol/L和20mmol/L葡萄糖处理的大鼠脂肪细胞增殖作用显著增强(P<0.05),其中15mmol/L葡萄糖处理的大鼠脂肪细胞增殖作用极显著(p<0.05),而5mmol/L和25mmol/L浓度有增强的趋势,但没有统计学显著性(P>0.05)。
结论结果表明构建的真核表达载体对于大鼠脂肪细胞转染效率不高,其中pcDNA6.2-GW/EmGFP-ChREBPⅢ转染效果较好,不同浓度葡萄糖对转染48小时后的大鼠脂肪细胞增值分化影响与未转染脂肪细胞对其影响相比较变化不大。
Objective: To construct four plasmids which can inhibit expression of ChREBP of rat adipocyte, observe effects of glucose on rat adipocyte multiplication and differentiation by lipofectamine 2000 transfecting and the regulation role of ChREBP on its lipogenesis.
Methods: 1. Four short DNA targetsequence were designed according to gene-silencing technologies principle, chcmically synthesized , and reconstructed to four plasmids: pcDNA6.2-GW/EmGFP-ChREBPⅠ、pcDNA6.2-GW/EmGFP-ChREBPⅡ、pcDNA6.2-GW/EmGFP-ChREBPⅢ、pcDNA6.2-GW/EmGFP-ChREBPⅣ. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte,respectively. 3. Effective transfection recombinant plasmids can be obeseved by fluorescence-microscope. 4. The rate of increase of primary rat adipocyte proliferation were detected by MTT.5. Effects of primary rat adipocyte differentiation by oil red O.
Results: 1. Four plasmids were constracted successfully. 2. Four recombinant plasmids and control plasmid were transfected to primary rat adipocyte and was used Blasticidin to gain the cell clones that express stably. Recombinant plasmids pcDNA6.2-GW/EmGFP-ChREBPⅢ’s fluorescent images shows batter than others .Tissue that were not digested were good in transfection unexpected. 3. Effects of glucose on rat preadipocytes differentiation by applications,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h, 20mmol/L glucose(P<0.05) was higher significantly than 0mmol/L、5mmol/L and 10mmol/L glucose, 25mmol/L、15mmol/L was no significant(P>0.05). Effects of glucose on rat proliferation applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h,15mmol/L glucose was more enhancement significantly than 10mmol/L、15mmol/L and 20mmol/L glucose(P<0.05), 5mmol/L、25mmol/L glucose was no significant(P>0.05).
Conclusion: The recombinant plasmids was inefficiency express ChREBP specific RNA,but pcDNA6.2-GW/EmGFP-ChREBPⅢcan more efficiency. Effects of glucose on rattus norvegicus preadipocytes differentiation and proliferation by applications RNAi,that were transfected by pcDNA6.2-GW/EmGFP-ChREBPⅢ48 h,has few changes.
引文
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