脂肪间充质干细胞中Noggin下调能够促进GDF5的表达
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南京医科大学科技发展基金(2016NJMUZD069)


GDF5 expression increased in adipose ⁃ derived stem cells by down ⁃ regulating Noggin expression
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    摘要:

    目的:通过抑制细胞中Noggin的表达,观察其对大鼠脂肪间充质干细胞(adipose-derived stem cells,ADSCs)中生长分化因子5(growth differentiation factor 5,GDF-5)表达量的影响,探究两者之间的关系。方法:构建GDF5慢病毒过表达载体(Lv-GDF5)及Noggin干扰载体(Lv-Noggin shRNA),根据目的基因的不同,将实验分成4组,分别使用重组慢病毒转染大鼠ADSCs:A组为Lv-EGFP转染ADSCs(空载体病毒转染组);B组为Lv-Noggin shRNA转染ADSCs;C组为Lv-GDF5转染ADSCs;D组为Lv-GDF5+Lv-Noggin shRNA共转染ADSCs。通过实时定量PCR技术和Western blot技术检测转染后14 d GDF5和Noggin的表达量,进行对比分析。并检测软骨基质二型胶原(collagen Ⅱ,Col-Ⅱ)mRNA的表达,进行组间比较。结果:实时定量PCR结果显示:转染后14 d,A、B、C、D组GDF5 mRNA表达量呈递增趋势,两两之间比较,除B组与C组之间差异无统计学意义外,其余各组间差异均有统计学意义(P < 0.05);各组间Noggin mRNA 表达量比较,A组>C组>B组>D组,两两比较,除A组与C组间差异无统计学意义外,其余各组间差异均有统计学意义(P < 0.05);Col-Ⅱ mRNA表达量呈递增趋势,两两比较,差异均有统计学意义(P < 0.05)。Western blot结果显示:转染后14 d,A、B、C、D组GDF5 蛋白表达量呈递增趋势,两两比较,差异均有统计学意义(P < 0.05);各组间Noggin 蛋白表达量比较,A组>C组>B组>D组,两两比较,A组与C组、C组与B组间差异无统计学意义,其余组间差异均有统计学意义(P < 0.05)。结论:抑制大鼠ADSCs Noggin的表达,GDF5表达量相应增加,两者呈负相关关系。

    Abstract:

    Objective:To explore the influence on expression of growth differentiation factor 5(GDF-5)in adipose-derived stem cells(ADSCs)by inhibiting the expression of Noggin. Methods:The recombinant lentivirus vectors of GDF5 gene(Lv-GDF5)and lentivirus vectors carrying Noggin RNAi sequence(Lv-Noggin shRNA)were constructed. ADSCs were divided into four groups:ADSCs in group A were transfected by empty lentivirus(with enhanced green fluorescent protein,Lv-EGFP);ADSCs in group B were transfected by Lv-Noggin shRNA;ADSCs in group C were transfected by Lv-GDF5;ADSCs in group D were co-transfected by Lv-GDF5 and Lv-Noggin shRNA. Then,the expression of GDF5 and Noggin in ADSCs was determined by real-time PCR and Western blot 14 days after transfection. The capacity of cartilage differentiation was detected through collagen typeⅡ(Col-Ⅱ)by RT-PCR. Finally,the expression quantity was contrastly analyzed. Results:RT-PCR showed that the expression of GDF5 mRNA and protein increased gradually 14 d after transfection from group A to group D. In comparison between each two groups,except the difference between group B and group C was not statistically significant,differences between the other groups were statistically significant(P < 0.05). The expression of Noggin mRNA and protein increased gradually in group D,B,C and A. In comparison between each two groups,except the difference between group A and group C was not statistically significant,the differences between the other groups were statistically significant(P < 0.05). The expression of Col-Ⅱ mRNA increased gradually from group A to group D. In comparison between each two groups,the differences were statistically significant(P < 0.05). The results of Western blot showed that the expression of GDF5 protein increased gradually from group A to group D 14 d after transfection. In comparison between each two groups,the difference was statistically significant(P < 0.05). The expression of Noggin protein decreased gradually in group A,C,B and D. In comparison between each two groups,the differences between group A and group C,as well as group C and group B were not statistically significant,while the differences between the other groups were statistically significant(P < 0.05). Conclusion:The expression of GDF5 in ADSCs increased by inhibiting the expression of Noggin. A negative correlation between GDF5 and Noggin expression quantity was found.

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刘 浩,陆征峰,杨 凯,张圣智,汤峰林,陆 淼,丁 涛.脂肪间充质干细胞中Noggin下调能够促进GDF5的表达[J].南京医科大学学报(自然科学版),2018,(9):1181-1186,1225

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  • 收稿日期:2017-06-11
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  • 在线发布日期: 2018-09-17
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