PDGF-B反义寡脱氧核苷酸对大鼠肝星状细胞增殖及Ⅰ型胶原合成的影响

Effects of proliferation of rat hepatic stellate cells and collagen-Ⅰ production by antisense oligodeoxynucleotides against platelet-derived growth factor B-chain

目的:探讨PDGF-B反义寡脱氧核苷酸(ASODN)对大鼠肝星状细胞(HSCs)增殖及Ⅰ型胶原合成的影响。方法:合成了特异性的PDGF-B硫代磷酸ASODN及其对照错义寡脱氧核苷酸(MSODN),并将其加入至培养的HSCs。采用MTT法检测PDGF-B ASODN对HSCs的增殖抑制作用,RT-PCR检测PDGF-B和collagen-ⅠmRNA的表达,流式细胞仪与ELISA法分别检测PDGF-B的表达和Ⅰ型胶原的合成。结果:PDGF-BASODN在终浓度为10μmol/L,作用HSCs-T6细胞48 h时,能明显地抑制其增殖,降低PDGF-B和collagen-ⅠmRNA的表达;FCM与ELISA分析表明,HSCs表达PDGF-B和合成Ⅰ型胶原都明显低于对照组,而对照组寡脱氧核苷酸在相同浓度下未见抑制效应。结论:PDGF-B ASODN可以抑制HSCs的增殖、Ⅰ型胶原的合成、内源性PDGF-B的表达,可能成为一种有用的抗肝纤维化基因治疗的药物。

AIM： To investigate the effect of antisense oligodeoxynucleotides against platelet- derived growth factor B - chain （ PDGF - B） in the proliferation of cultured rat hepatic stellate cells and collagen - Ⅰ production. METHODS： The phosphorothioate antisense oligodeoxynucleotides and missense oligodeoxynucleotides were synthesized. The rat hepatic stellate cells （HSCs） were treated with these oligodeoxynucleotides at different concentrations, respectively. The inhibition of the proliferating of cultured rat hepatic stellate cells was assayed by MTr method. The expressions of PDGF - B and collagen - Ⅰ mRNA were detected by means of reverse transcription - polymerase chain reaction （ RT - PCR）. The expression of endogenous PDGF- B was determined by flow cytometry （FCM）. Collagen- Ⅰ in the supematant was determined by ELISA. RESULTS ： The results showed that the ASODN against the PDGF - B at a final concentration of 10 μmol/L inhibited the HSCs proliferation, the expressions of PDGF - B and collagen - Ⅰ mRNA. FCM and ELISA demonstrated that the expression of endogenous PDGF - B and the production of collagen - Ⅰ in HSCs were significantly lower than those in controls, whereas the controls （ missense oligodeoxynucleotides） had no effect at the same concentration. CONCLUSION：PDGF- B ASODN inhibits the proliferation of cultured rat hepatic stellate cells, the endogenous expression of PDGF - B and the production of collagen. PDGF - B ASON might be useful for gene therapy in liver fibrosis.