Chen Yue-xiang, Lu Cui-hua, Xie Wei-fen, Zhang Xing-rong, Zhang Zhong-bing, Wei Li-xin, Jin You-xin, Guo Ya-jun
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Chin Med J (Engl). 2005 Jun 20;118(12):982-8.
Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC.
Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy.
The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy.
The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.
肝星状细胞（HSC）的激活和增殖在肝纤维化的发生和发展过程中起关键作用。对HSC最具活性的生长因子是血小板衍生生长因子受体（PDGF），而PDGF受体β亚基（PDGFR-β）是PDGF的主要信号转导途径，在活化的HSC中过度表达。本研究探讨了靶向PDGFR-β mRNA的锤头状核酶对HSC的切割活性及其对HSC生物学特性的影响。
构建抗PDGFR-β核酶表达载体，并用脂质体转染大鼠活化的HSC。通过G418筛选获得阳性细胞克隆。分别采用Northern印迹、Western印迹和免疫细胞化学染色检测PDGFR-β、α-平滑肌肌动蛋白以及I型和III型胶原的表达。用MTT比色法测定细胞增殖。采用流式细胞术、吖啶橙荧光活体染色和透射电子显微镜分析细胞凋亡。
核酶转染的HSC中，PDGFR-β在mRNA和蛋白水平的表达均显著降低，降低幅度为49% - 57%（P < 0.05 - 0.01）。核酶转染的HSC的增殖及α-平滑肌肌动蛋白表达显著降低（P < 0.05 - 0.01），I型和III型胶原合成也减少（P < 0.01）。此外，核酶转染的HSC对PDGF BB的增殖反应受到显著抑制。另外，核酶转染的HSC中凋亡细胞显著增加（P < 0.01），透射电子显微镜下可发现典型的凋亡细胞。
抗PDGFR-β核酶能有效切割靶RNA并显著抑制其表达，在受体水平阻断PDGF的信号转导，抑制HSC增殖和胶原合成，并诱导HSC凋亡。这些结果表明，抑制HSC的PDGFR-β表达可能是肝纤维化治疗的新靶点，核酶可能是抑制PDGFR-β表达的有效工具。
