RNA干扰沉默CTGF表达对人成骨样MG63细胞Ⅰ型胶原、ALP mRNA表达的影响

Inhibition of connective tissue growth factor expression decreases the expression of type 1 collagen and alkaline phosphatase mRNA in human osteoblastic MG 63 cells

目的本研究将利用RNA干扰技术,阻断CTGF在人成骨样MG63细胞中的表达,观察CTGF降表达后对人成骨样MG63细胞Ⅰ型胶原、碱性磷酸酶 mRNA表达的影响。方法针对人CTGF mRNA440、875、910位点设计、合成3对21核苷酸siRNA(siRNA1、siRNA2、siRNA3);在阳离子脂质体介导下转染人成骨样MG63细胞,以空白及非特异性siRNA作为对照,转染48h后收集细胞。采用Northern杂交研究CTGF mRNA表达水平的改变,半定量RT-PCR观察Ⅰ型胶原、ALP mRNA表达的改变,Western blotting观察CTGF蛋白表达的变化。MTT法测定RNA干扰后人成骨样MG63细胞活力的改变。结果空白对照组相比,转染siRNA1、siRNA3的MG63细胞CTGF mRNA和蛋白表达明显下调,转染siRNA2及非特异性siRNA的MG63细胞CTGF的表达无明显变化。转染siRNA1、siRNA3的MG63细胞Ⅰ型胶原、ALP mRNA表达明显下调,细胞活力明显降低。结论针对人CTGF mRNA设计、合成的siRNA可有效抑制人成骨样MG63细胞CTGF的转录和表达;CTGF表达下调可抑制MG63细胞表型标志物Ⅰ型胶原、ALP mRNA的表达,降低MG63细胞活力,这说明CTGF可能在维持骨代谢平衡中具有重要的作用。

Objective To investigate the inhibition effect of small interfering RNA（siRNA） on the expression of connective tissue growth factor（ CTGF）, and to observe the effects of down-regulation of CTGF on the expression of type 1 collagen and alkaline phosphatase mRNA in human osteoblastic MG 63 cells. Methods Three pairs of 21- nueleotide CTGF siRNAs directed to human CTGF mRNA 440,875and 910 targets were differently transfected into human osteoblastic MG 63 cells with DMRIE-C reagent package. Untreated and transfected scramble siRNA served as the blank control and nonspecific siRNA control respectively. Total RNA and protein of the cells after 48h incubation were extracted. The expression of CTGF in mRNA and protein level were assessed by northern blot and western blot, and the expression of type 1 collagen and alkaline phosphatase mRNA in human osteoblastic MG 63 cells were evaluated by semiquantitive RT-PCR, cell viability was tested by MTT assay. Results Compared with blank control group, the expression of CTGF mRNA and protein were markedly down-regulated by siRNA directed to 440 and 910 target of human CTGF mRNA, siRNA directed to 875 target and scramble siRNAs show no effect on the expression of CTGF. The expression of type 1 collagen and alkaline phosphatase mRNA was reduced in human osteoblastie MG 63 cells transfected with siRNAs directed to 440 and 910 targets, and cell viability significantly decreased in human osteoblastic MG 63 cells transfected with siRNAs directed to 440 and 910 targets. Conclusions CTGF siRNA can effectively reduced the expression of CTGF mRNA and protein. Inhibition of CTGF expression decreases the expression of type 1 collagen and alkaline phosphatase mRNA as well as cell viability in human osteoblastic MG 63 cells.