摘要
目的 初步探讨CCAAT增强子结合蛋白α(C/EBPα)在肝星状细胞(HSC)激活过程中的作用。 方法 采用免疫细胞化学、western blot及RT-PCR检测原代培养不同时间段HSC内C/EBPα蛋白和mRNA的表达,以及α平滑肌肌动蛋白(α-SMA)、结蛋白、基质金属蛋白酶-2(MMP2)mRNA、Ⅰ型前胶原(α1)mRNA的表达;Lipofect AMINE2000介导的瞬时转染方法将pcDNA3.1(-)-C/EBPα真核表达质粒转染入激活的HSC内,采用免疫细胞化学方法鉴定转染成功及转染后HSC内增殖细胞核抗原(PCNA)的表达;在相差显微镜下观察HSC在原代培养过程中形态的改变。 结果 原代正常HSC中可以检测到C/EBPα mRNA和蛋白的表达,蛋白位于细胞质和细胞核内,但主要位于细胞质内,且除新鲜分离(0d)组以外,随着HSC培养至2、4、7、10d,C/EBPα蛋白和mRNA的表达呈逐步下降的趋势,而α-SMA、MMP2和Ⅰ型前胶原(α1)的表达则逐步增强;转染后24 h,目的基因转染组HSC内C/EBPα的表达明显强于空载体对照组,而PCNA的阳性细胞数较空载体对照组明显减少;转染后36 h,目的基因转染组细胞几乎全部死亡,残存的细胞形态变细,体积缩小,而空载体对照组细胞仍存活。 结论 C/EBPα基因可能参与HSC的激活调控机制,且C/EBPα过表达对HSC的增殖存在抑制作用。
Objective The expression of C/EBP α protein and mRNA during automatically activation process in primary cultures of HSCs were observed in order to explore its possible association with the proliferation and activation of HSCs. Methods Immunocytochemistry, Western blot and RT-PCR were used to evaluated the expression of C/EBP α protein and mRNA; as well as the expression of α-SMA, Desmin, MMP2, type Ⅰ procollagen(α1). The eukaryotic vector harboring the full length cDNA of C/EBP α was transfected into activated HSC, then immunocytochemistry was applied to confirm the transfection and evaluate the effect of transfection on the proliferation of HSC by calculating the PCNA-positive cells. The morphological changes of HSC were observed by use of phase-contrast microscope. Results Constitutive expression of mRNA and protein of C/EBP α were detected in primarily cultured HSCs, and the protein was seen in both nuclei and cytoplasm with the latter being dominant. Their expression levels reached highest at day 2 of the culture, then decreased gradually when continually cultured to the day 4, 7, 10, on the other hand, the expression of α-SMA, MMP2 and Col Ⅰ(α1) increased steadily. Transient transfection was verified by the fact that much more and stronger C/EBP α stain was observed in transfected HSCs than in void-vector transfected cells. In C/EBP α gene transfected HSCs, the number of PCNA-positive cells dramatically decreased compared with the void-vector transfected cells 24 h after transfection. In addition, the C/EBP α gene transfected HSCs died 36h after transfection, a few surviving cells became longer and thinner in morphology, however the void-vector transfected cells almost all remained alive. Conclusions C/EBP α was likely involved in the HSCs activation, and over-expressed C/EBP α by transfection had inhibitory influence on the proliferation of cultured rat HSCs.
出处
《中华肝脏病杂志》
CAS
CSCD
2004年第5期259-262,共4页
Chinese Journal of Hepatology
基金
国家自然科学基金(30170417)
