【作者】 刘佳；

【导师】 沈群；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2007， 硕士

【摘要】 慢性髓细胞白血病（chronic myelogenous leukemia，CML）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的bcr／abl融合基因，转录翻译生成分子量为210kDa的融合蛋白p210^bcr／abl。该蛋白具有很强的酪氨酸激酶（tvrosine kinase，TK）活性，是CML形成的分子基础。而bcl-2基因、p53基因、死亡受体及其配体Fas／FasL都是与细胞凋亡密切相关的重要信号分子。血复康是江苏省中医院血液科1989年根据中医解毒活血法独创的复方制剂，临床用于治疗CML已逾十年，疗效确切。课题组既往的研究表明：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，通过部分抑制bcr／abl融合基因及P210蛋白的表达，降低蛋白酪氨酸激酶（protein tyrosine kinase，PTK）的活性，诱导细胞凋亡而发挥疗效。本研究试图从mRNA和蛋白水平进一步分别探究血复康及各组分青黛、葛根、莪术提取物对K562细胞增殖、凋亡的影响及可能的分子机制。实验目的：1．研究不同浓度青黛、葛根、莪术提取物对K562细胞增殖、凋亡的影响；P210^bcr／abl以及细胞凋亡相关信号分子bcl-2、P53、Fas／FasL表达的变化，分别探讨血复康各组分诱导K562细胞凋亡可能的分子机制。2．在课题组既往研究取得良好结果的基础上，进一步探讨5mg／ml血复康处理K562细胞后不同时段对细胞增殖、凋亡及细胞凋亡相关基因表达水平的影响。实验方法：以K562细胞株为研究对象，观察①不同浓度青黛、葛根、莪术提取物（0mg／ml、2.5mg／ml、5mg／ml、10 mg／ml和20mg／ml）处理24小时后②5mg／ml血复康处理0、24、48、72、96小时后光镜下的细胞形态学变化；MTT法检测药物处理对K562细胞的生长抑制作用；Hoechst 33258染色检测细胞凋亡；FITC-AnnexinV／PI双染法检测细胞凋亡率；半定量逆转录聚合酶链反应（semi-quantitative reversetranscription p01ymerase chain reaction，SQ-RT-PCR）检测药物处理后K562细胞bcr／abl融合基因、bcl-2基因的变化；Western blot检测P210、bcl-2、p53、Fas／FasL蛋白的变化。实验结果：1．细胞形态学改变：①青黛、葛根、莪术三药均有抑制K562细胞增殖的作用，且呈浓度依赖性改变，表现为细胞渐现大小不均，形态不规则，胞膜折光性下降，细胞破碎、失去原有结构；②血复康呈时间依赖性抑制K562细胞增殖。2．Hoechst染色结果：①低浓度（2.5 mg／ml）青黛、葛根、莪术处理K562细胞24小时后，便可检测到明显的细胞凋亡，以20mg／ml处理组更为明显；②5mg／ml血复康处理K562细胞后，细胞增殖受抑及细胞凋亡呈时间依赖性增加。3．流式细胞仪检测细胞凋亡率：①不同浓度青黛、葛根、莪术处理K562细胞24小时后，细胞凋亡率与浓度呈正相关；②5mg／ml血复康处理K562细胞0、24、48、72、96小时后，细胞凋亡率渐次增加。4．半定量RT-PcR和Western blot结果：①青黛、葛根处理组bcr／abl融合基因和P210蛋白的表达呈浓度依赖性下降，而莪术处理组则无明显变化；各处理组bcl-2在mRNA和蛋白水平的表达均无统计学意义的变化；P53蛋白在青黛、葛根处理组的表达呈浓度依赖性上升，而莪术处理组则无相应变化；Fas、FasL蛋白在莪术处理组的表达呈浓度依赖性上调，青黛、葛根处理组则无明显变化。②血复康处理组bcr／abl融合基因和P210蛋白呈时间依赖性下调；bcl-2基因的表达无变化；P53蛋白和Fas／FasL蛋白呈时间依赖性上升趋势。结论：1．血复康及组分青黛、葛根、莪术均能抑制K562细胞增殖并诱导细胞凋亡，但作用的分子机制不尽相同。2．血复康及组分青黛、葛根部分抑制K562细胞bcr／abl融合基因及P210蛋白的表达，降低酪氨酸激酶活性，抑制K562细胞增殖，诱导细胞凋亡；部分上调K562细胞P53蛋白的表达。3．血复康及组分莪术上调K562细胞Fas／FasL蛋白的表达，通过激活死亡受体及其配体，诱导细胞凋亡。4．各处理组抗凋亡基因bcl-2的表达均无明显变化。更多还原

【Abstract】 Chronic myeloid leukemia (CML) is a hematopoietic disorder characterized by themalignant expansion of bone marrow stem cells. Its cytogenetic hallmark is a reciprocalt(9;22)(q34;q11) chromosomal translocation that creates a derivative 9_q~+ and a small 22_q~-,known as the Philadelphia (Ph) chromosome. The latter harbors the bcr-abl fusion geneencoding a chimeric bcr-abl protein with a deregulated tyrosine kinase activity, theexpression of which has been shown to be necessary and sufficient for the transformedphenotype of CML cells. Otherwise, such as bcl-2 gene family, wt-p53 gene and Fas/FasLare also important genes to influence apoptosis.Xue Fu Kang is a new Chinese medicine, which is composed of Qingdai, Gegen andEzhu. It has been used to treat CML for more than a decade in Jiang Su province hospital ofTCM. It is effective and safety, and also well-tolerated by most patients. Previous study ofour group showed that Xue Fu Kang partially inhibited proliferation as well as inducedapoptosis of K562 cells. Expression of bcr/abl and P210 protein were down-regulated in adose dependent manner. This paper would further investigate molecular mechanisms ofsingle composition of Xue Fu Kang respectively.Objective: To study the effects of single composition of Xue Fu Kang respectively onproliferation and apoptosis of K562 cells, as well as on expression of bcr/abl and bcl-2 gene,the level of P210, bcl-2, wt-p53 and Fas/FasL protein.Methods: K562 cells were handled with different concentrations of Qingdai, Gegen,Ezhu(0 mg/ml、2.5 mg/ml、5 mg/ml、7.5 mg/ml、10 mg/ml、20 mg/ml)and were harvestedin 24 hours. In addition, they were also treated with different time(0、24、48、72、96 h)of 5 mg/ml Xue Fu Kang in K562 cells. The morphological changes were observed throughmicroscope; the changes of cells proliferation was examined by MTT; apoptotic cells weredyed by Hoechest 33258; apoptosis rate was determined with FITC-annexin V/PI doublestaining by flow cytometry; the changes of transcript and protein level of bcr/abl, bcl-2,wt-p53, Fas/FasL were determined by semi-quantitative RT-PCR and Western blot analysisrespectively.Results: 1. Morphological changes showed that with increase of Qingdai, Gegen, Ezhuconcentration, we observed inhibited proliferation and apoptosis characteristics in K562cells. Hoechest exhibited typical morphological changes of apoptotic cells underfluorescent microscope. 2. After treatment with different concentrations Qingdai andGegen, the expression of both bcr/abl mRNA and P210 proteins were decreased in aconcentration-dependent manner in K562 cells, and in a time-dependent decrease with XueFu Kang alone. 3. Both of the transcript and protein level of bcl-2 had no change indifferent treated groups. 4. The wt-p53 protein was up-regulated in Qingdai and Gegengroups, while Fas/FasL in Ezhu group in a concentration-dependent manner.Conclusion: Xue Fu Kang and the single compositions partially inhibitedproliferation and induced apoptosis of K562 cells. Qingdai, Gegen and Ezhu inducedapoptosis in a concentration-dependent manner. The mechanisms of Qingdai and Gegenmight be involved in down-regulation of bcr/abl fusion gene expression and P210 proteinlevel, as well as up-regulation of wt-p53 protein level, while Ezhu up-regulation ofFas/FasL expression. In summary, it suggested that Xue Fu Kang and the singlecompositions induce K562 apoptosis by different signaling pathways.更多还原