血复康对K562细胞bcr/abl及JWA基因表达的研究

【作者】 戴海萍；

【导师】 沈群；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2005， 硕士

【摘要】 慢性粒细胞白血病(chronic myelogenous leukemia／chronic granulocytic leukemia；CML／CGL)简称慢粒，是一种获得性多能造血干细胞的克隆增殖性疾病，Ph染色体t(9；22)(q34；q11)为其特征性遗传学标志。这一特异的染色体异常产生bcr／abl融合基因，转录翻译生成分子量为210kDa(P210)的bcr／abl融合蛋白。该蛋白具有很强的酪氨酸激酶(tyrosine kinase，TK)活性，是CML形成的分子基础。 血复康是江苏省中医院血液科于1989年根据中医解毒活血法独创的复方制剂，主要由青黛、莪术、葛根等中药组方。临床用于治疗CML已逾十年，疗效确切，取得了良好的社会效益和经济效益。课题组既往细胞水平的研究已经发现：血复康能抑制K562细胞(人慢性粒细胞白血病红白血病变细胞株，表达b3a2融合基因)增殖及诱导细胞凋亡。本研究试图从mRNA水平进一步研究血复康治疗CML确切的分子机制，为该药的进一步推广使用提供科学的理论依据。 实验目的：研究不同浓度血复康对K562细胞株增殖、凋亡及bcr／abl融合基因、JWA基因表达水平的影响，为临床制订治疗方案提供理论依据。 实验方法：以K562细胞株为研究对象，观察不同浓度血复康(0mg／ml、2.5mg／ml、5mg／ml、7.5mg／ml、10mg／ml、20mg／ml)作用24小时后光镜下的细胞形态学变化；采用半定量反转录聚合酶链式反应(semi-quantitative reverse transcription polymerase chain reaction，SQ-RT-PCR)技术检测不同浓度血复康作用24小时后K562细胞bcr／abl融合基因及JWA基因在mRNA水平表达的变化。 实验结果：1．细胞形态学改变：随血复康作用浓度的升高，K562细胞呈现浓度依赖性改变，即随药物浓度的增加，细胞增殖抑制及凋亡更趋明显。血复康5mg／ml时，便可在低倍镜下观察到细胞增殖逐渐受抑制，以20mg／ml组最明显；油镜观察显示，经2.5mg／ml作用后，就发现细胞出现大小不均，形态不规则，细胞呈现典型的凋亡形态学变化：出现诸如核固缩、破碎，核染色质变粗等。2．半定量RT-PCR结果：各浓度组bcr／abl融合基因及JWA基因均有不更多还原

【Abstract】 Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by the malignant expansion of bone marrow stem cells. Its cytogenetic hallmark is a reciprocal t(9;22)(q34;q11) chromosomal translocation that creates a derivative 9q~+ and a small 22q~-, known as the Philadelphia (Ph) chromosome~1. The latter harbors the bcr-abl fusion gene encoding a chimeric bcr-abl protein with a deregulated tyrosine kinase activity, the expression of which has been shown to be necessary and sufficient for the transformed phenotype of CML cells.Xue Fu Kang is a new Chinese medicine, which has been used to treat CML for more than a decade in Jiang Su province hospital of TCM. It is effective and safety, and also well-tolerated by most patients. Previous study of our group showed that on a cellular basis, Xue Fu Kang inhibited cell proliferation and induced apoptosis. This paper would further investigate molecular mechanisms of this medicine.Objective: To study the effects of Xue Fu Kang on proliferation and apoptosis of K562 cells, as well as on expression of bcr/abl and JWA mRNA.Methods: K562 cells were handled with different concentrations ofXue Fu Kang(0mg/ml、 2.5mg/ml、 5mg/ml、 7.5mg/ml、 10mg/ml、 20mg/ml) and were harvested in 24 hours. Then morphological changes更多还原