端粒酶在大鼠肝卵圆细胞中表达的意义

Expression and its significance of teiomerase in rat hepatic oval cells

目的检测大鼠肝卵圆细胞端粒酶活性，探讨端粒酶表达与卵圆细胞增殖分化的关系。方法采用2-AAF／PH模型诱导大鼠卵圆细胞增殖，改良的胶原酶灌注结合密度梯度离心法分离，用细胞免疫荧光和电镜鉴定卵圆细胞。采用免疫组织化学、RT—PCR、荧光定量PCR等方法检测卵圆细胞端粒酶的表达，组间比较采用t检验分析其意义。结果采用2-AAF／PH模型成功诱导大鼠卵圆细胞增殖。分离的卵圆细胞核大，卵圆形，胞质少，呈铺路石样生长。电镜发现细胞核质比大，胞质内细胞器少且发育不成熟。细胞免疫荧光结果表达OV-6、AFP、CK-19、albumin、c-kit。端粒酶逆转录酶（TERT）表达在门静脉周围增殖的卵圆细胞核内，随着卵圆细胞逐渐向肝细胞方向分化，TERT阳性细胞数逐渐减少。大鼠正常肝组织TERT mRNA表达水平最高，为LE-6卵圆细胞的2．27倍；分离的卵圆细胞TERT mRNA表达水平为LE-6卵圆细胞的1．26倍；采用2μg和4μg细胞提取物分析细胞的端粒酶活性，随着LE-6卵圆细胞传代次数的增加，由24代传至40代，端粒酶活性由△A=1．05、1．15降低到△A=0．25、0．45（t=17．74，12．38，P〈0．05）。结论卵圆细胞具有端粒酶活性，端粒酶可能是卵圆细胞维持其增殖能力和多分化潜能的一个必要条件。

Objective To detect the telomerase activity in rat hepatic oval cells, and to explore the relationship between telomerase expression and the proliferation and differentiation of oval cells. Methods The 2-acetamidofluorene/partial hepatectomy （2-AAF/PH） rat model was used to induce the proliferation of oval ceils. Oval cells were isolated by modified collagenase perfusion and gradient centrifugation. Electron microscope examination and immunofluorescence were adopted to identify oval cells. Immunohistochemistry, RT-PCR and fluorescence quantitative PCR were used to detect the expression of telomerase in oval ceils. All the data collected were analyzed by t test. Results The proliferation of oval cells was successfully induced by 2-AAF/PH rat model. Freshly isolated oval cells showed a large and ovoid nuclei, a small proportion of cytomplasm and a cobblestone appearance. When viewed by electron microscopy, there were few and immature organelles, and the nucleus/ cytoplasm ratio was high. Immunofluorescence staining showed that oval ceils expressed OV-6, alpha-fetoprotein, cytokeratin-19, albumin and c-kit. Telomerase reverse transcriptase （TERT） was located in the nuclei of oval cells which were around the portal areas. As oval cells differentiated into small hepatocytes, the number of TERT- positive cells decreased significantly. The expression level of TERT mRNA in normal rat liver tissue was 2.27 times higher than that in LE-6 oval cells; the expression level of TERT mRNA in the isolated oval cells was 1.26 times higher than that in LE-6 oval cells. The telomerase activity decreased gradually （from △A = 1.05, 1.15 to △A = 0.25, 0.45） as the increase of oval cells passage （from passage 24 to passage 40） （ t = 17.74, 12.38, P 〈 0.05 ）. Conclusions Oval cells have telomerase activity. Telomerase may be indispensable for maintaining the proliferative and multi-directional differentiation abilities of oval cells.