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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。
Objective To study the cleavage activity of PDGFR-β ribozyme in hepatic stellate cells (HSC) and its effect on the biological characteristics of HSC. Methods The eukaryotic expression vector of PDGFR-β ribozyme was constructed and transfected into HSC-T6 cells. The positive clones were screened by G418. The expression of PDGFR-β was detected by northern blot, western blot and immunocytochemistry respectively. Α-sMA and collagen type Ⅰ, Ⅲ were detected by immunocytochemical staining. Apoptosis was analyzed by flow cytometry, acridine staining and electron microscopy. Results The expression of PDGFR-β at the mRNA and protein levels of HSC transfected with ribozyme was significantly lower than that of the control group (43% -51%, t ≥ 3.95 7, P <0.05) (T≥3.858, P <0.0 5), and the sensitivity to PDGF-induced proliferation was significantly weakened. The expressions of typeⅠ, type Ⅲ collagen and α-SMA were significantly decreased (t≥6.790, P < 0.01). The incidence of apoptosis was significantly higher than that of the control group (x2≥14.157, P <0.01). Typical apoptotic cells were observed under electron microscope. Conclusion The eukaryotic expression vector of anti-PDGFR-β ribozyme can be stably expressed in the cells, effectively cutting target RNA, inhibiting HSC proliferation and collagen synthesis, and inducing apoptosis. It provides a new target and method for anti-liver fibrosis treatment.