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

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

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。

Objectives To study the cleavage activity of hammerhead ribozyme targeting at platelet-derived growth factor receptor β subunit (PDGFR- β) mRNA in hepatic stellate cells (HSCs) and its effect on the biological characters of HSCs. Methods Expression vector of anti-PDGFR- p 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-β , α -smooth muscle actin (α -SMA), and type I and type in collagen was detected by means of northern blot, Western blot and immunocytochemical staining respectively. The cell proliferation was determined with MTT colori-metric assay. The cell apoptosis was demonstrated with flow cytometry. acridine orange fluorescence vital staining and transmission electron microscopy. Results The expression of PDGFR- BBBBB at mRNA and protein level was markedly reduced in ribozyme-transfected HSCs only 43%-51% of that in control cells (t ≥ 3.957, P < 0.05), and α-SMA expression level, type Ⅰ and type Ⅱ collagen synthesis ability were also reduced (t ≥ 6.790, P < 0.01). The proliferation of ribozyme-transfected HSCs was significantly decreased (t ≥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 ( x2 ≥ 14.157, P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy. Conclusions The anti-PDGFR- β 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- β expression in HSCs may be a new therapy for liver fibrosis.