抗血小板衍生生长因子受体的核酶对肝星状细胞生物学特性的影响

目的 研究抗血小板衍生生长因子受体β亚单位(PDGFR-β)核酶在肝星状细胞(HSC)内的切割活性及其对HSC生物学特性的影响。 方法 构建抗PDGFR-β核酶的真核表达载体,将其转染入HSC-T6细胞,G418筛选出阳性细胞克隆;分别用northern blot、western blot和免疫细胞化学检测PDGFR-β表达,用MTT法检测细胞增殖,免疫细胞化学检测α-F滑肌肌动蛋白(α-sMA)和Ⅰ、Ⅲ型胶原表达,用流式细胞仪、吖啶噔荧光染色和电镜分析细胞凋亡。 结果 转染核酶的HSC的PDGFR-β在mRNA和蛋白水平的表达量均显著降低,仅为对照组的43％～51％(t≥3.95 7,P<0.05);增殖活性显著低于对照组(t≥3.858,P<0.0 5),且对血小板衍生生长因子(PDGF)促增殖效应的敏感性显著减弱;Ⅰ、Ⅲ型胶原和α-SMA的表达显著减少(t≥6.790,P<0.01);凋亡发生率显著高于对照组(x2≥14.157,P<0.01),电镜下可见典型凋亡细胞。 结论 抗PDGFR-β核酶的真核表达载体可在细胞内稳定表达,能有效切割靶RNA,抑制HSC增殖及胶原合成,并诱导其凋亡。为抗肝纤维化治疗提供了新的靶点和手段。

Effect of ribozyme against platelet-derived growth factor receptor β subunit mRNA on the biological characters of hepatic stellate cells

OBJECTIVE: To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor beta subunit (PDGFR- beta) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs. METHODS: Expression vector of anti-PDGFR- beta ribozyme was constructed and transfected into rat-derived HSC-T6 cells with lipofectin. The positive cell clones were gained by G418 selection. The expression of PDGFR- beta, alpha-smooth muscle actin (alpha-SMA), and type I and type III collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was demonstrated with flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy. RESULTS: The expression of PDGFR- beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSCs only 43% to 51% of that in control cells (t > or = 3.957, P < 0.05), and alpha-SMA expression level, type I and type III collagen synthesis ability were also reduced (t > or = 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t > or = 3.858, P < 0.05), and the proliferation response to PDGF BB was markedly inhibited. However the apoptotic rate was significantly increased in ribozyme-transfected HSCs (chi2 > or = 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy. CONCLUSIONS: The anti-PDGFR- beta ribozyme can be expressed stably in HSCs, cleave the target RNA effectively, inhibit HSCs proliferation and collagen synthesis, and induce HSC apoptosis. The results suggest that inhibiting PDGFR- beta expression in HSCs may be a new therapy for liver fibrosis.