脂肪间充质干细胞中Noggin下调能够促进GDF5的表达

GDF5 expression increased in adipose-derived stem cells by down-regulating Noggin expression

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 sh RNA),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 sh RNA转染ADSCs;C组为Lv-GDF5转染ADSCs;D组为LvGDF5+Lv-Noggin sh RNA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(collagenⅡ,Col-Ⅱ)m RNA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 m RNA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 m RNA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-Ⅱm RNA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。

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 type Ⅱ （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.