【作者】 何於娟；

【导师】 涂植光； 蒋纪恺；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2002， 博士

【摘要】 K562细胞是分化极差的白血病细胞，具有多向分化的潜能。苦参碱作为常用中药苦参中的主要活性成分之一，具有抗肿瘤的药理作用。受国家自然科学基金资助的苦参碱对白血病K562细胞逆转机制的前期研究结果表明，一定浓度的苦参碱能致K562细胞增殖受抑并向成熟方向分化。为进一步研究其诱导分化的方向和分子机制，本文从观察不同浓度的苦参碱作用K562细胞不同时间后的细胞形态学、细胞表面分化抗原表型、原癌基因表达变化到探寻作用早期的基因表达改变等方面进行了进一步研究，其中重点探寻与增殖和分化相关的早期差异表达基因，再对未知差异表达基因的开放阅读编码框ORF进行原核和真核表达研究，以期了解其生物学功能，确定其是否为新基因。本实验研究的第一部分，通过苦参碱作用K562细胞的浓度-时间曲线与乳酸脱氢酶活性测定，发现0.2mg/ml的苦参碱作用K562细胞三天后可抑制细胞增殖。以此增殖抑制有效浓度的苦参碱作用K562细胞三天后，光学显微镜观察细胞形态发现细胞向成熟方向分化，并且九天后有凋亡发生。结果提示，0.2mg/ml的苦参碱可以抑制K562细胞的增殖，并诱导该细胞向成熟方向分化。<WP=7>为明确苦参碱对K562细胞的诱导分化方向，在本实验研究的第二部分，采用流式细胞技术检测0.2mg/ml的苦参碱作用K562细胞六天后细胞表面分化抗原CD34、CD33、CD14、CD15、CD20、CD27、CD41、HIR2表型的改变，结果表明：0.2mg/ml的苦参碱作用K562细胞六天后，可诱导红细胞系、粒细胞系表面标志物表达升高，支持形态学的实验结果。在证实一定浓度的苦参碱可诱导K562细胞多向分化的基础上，我们运用分子生物学技术在基因水平上对其分子机制作了进一步研究。细胞的增殖分化受多因素调控，其中癌基因是一类主要的增殖分化调控因子。因此，本文第三部分，运用RT-PCR技术半定量检测了0.2mg/ml的苦参碱作用K562细胞三小时后，原癌基因c-myc、c-jun、H-ras、P21和HNF-1α表达的改变。结果发现，苦参碱作用K562细胞三小时后c-myc、c-jun、HNF-1α表达降低，H-ras、P21表达增高，这些与细胞增殖分化相关的原癌基因在苦参碱诱导作用早期即发生明显变化。由此，我们推测苦参碱对K562细胞的诱导分化作用是受基因水平的多因素、多途径、多方面的网络体系调控，可能涉及到DNA复制、转录、细胞信号传导等多方面。正是由于细胞增殖分化机制的复杂性，使肿瘤发生与分化的研究非常困难。为进一步研究苦参碱诱导K562细胞分化的分子机制，试图在上述研究基础上，从进一步扩大筛选参与K562细胞增殖分化的相关基因，以及寻找未知新基因的目的出发，本文第四部分，运用了能检出低丰度表达基因的差异显示逆转录PCR技术(DDRT-PCR)，进行苦参碱作用K562细胞后的早期mRNA的差异显示；差示cDNA片段的克隆与测序；测序结果与网上生<WP=8>物资源的同源性比较以及结构与功能的预测。通过以上实验研究，可望发现一些与K562细胞增殖分化相关的基因，研究其功能，并期盼有新基因的参与和发现。结果表明在苦参碱作用K562细胞后，有一些蛋白或蛋白酶的基因在RNA水平有表达量的改变，这种改变可能与细胞的增殖与分化有关。同时发现并克隆了一个未知差示cDNA片段，GenBank的登录编号为BM077298、dbEST ID 10245338（网址为http://www.ncbi.nlm.nih.gov/ Genbank/index.html，我们暂命名为KH基因）。由于条件限制，我们仅对KH基因进行了进一步的研究，并对未知差示cDNA片段（KH基因）进行了11种不同来源组织、细胞的RT-PCR与12种正常人体组织的Northern blot分析，以了解该差示片段的组织、细胞分布情况。RT-PCR与Northern blot的分析结果表明，该片段主要分布于白血病细胞及正常人的脑组织。进一步用cDNA片段的快速末端扩增法（RACE）对cDNA片段进行了全长克隆，但由于目前全长克隆方法尚不成熟，我们选用了多种方法和国外公司的试剂盒，均未获得KH基因的全长片段，估计该基因表达丰度低也是全长克隆困难的原因之一，目前我们还在改良方法进一步尝试全长克隆。虽然未得到此未知片段全长克隆，但我们通过Northern blot（mRNA）分析发现该基因的表达序列全长在1.35Kb左右，去掉mRNA的多聚尾200-300bp估计该基因全长在1000左右，实际上我们已获得片段全长的60%序列，而且其中存在可编码蛋白的开放阅读框架，为先窥视该基因可能存在的功能，对其现有的ORF进行了功能预测和原核、真核表达研究。重组表达质粒pCI-ORF（真核表达质粒pCI与KH基因ORF的重组构<WP=9>建体）热休克法转染至大肠杆菌BL21（DE3）plysS，IPTG诱导30 min后经PAGE电泳，考马斯亮蓝染色发现一个分子量约24kD的蛋白质分子获得表达，1h表达达到峰值。同时采用脂质体转染法将pCI-ORF转染至COS-7细胞，G418筛选后，经RT-PCR结果证实pCI-ORF已成功转染COS-7细胞。进一步通过流式细胞术检测转染pCI-ORF的COS-7细胞、转染pCI-neo vector的COS-7细胞与对照组COS-7细胞经0.2mg/ml的苦参碱处理前后的细胞周期改变，通过瑞氏染色光镜观察细胞形态变化。流式细胞术初步检测结果发现COS-7/pCI-ORF 细胞的G2-M期分布高于COS-7/pCI-neo 细胞与COS-7细胞，经0.2mg/ml的苦参碱作用72h后COS-7更多还原

【Abstract】 The human erythroleukemia K562 cell is a low-differentiation blast cell line with the potentiality of differentiation towards multi-direction. Matrine, one of the components in traditional Chinese herb Sophora flavescens, has the pharmacological effect of anti-tumor. Our previous study showed that a definite concentration of matrine could inhibit proliferation and induce differentiation in K562 cells in vitro. But the direction of differentiation and the molecular mechanism involved are poorly understood. To elucidate the mechanism, present study investigated the changes of cell morphology, cell surface marker-cluster of differentiation (SmCD) and the expression of oncongenes and immediate difference genes treated with matrine at various concentrations and for different time. Focus of the study was to screen some unknown genes involved in differentiation and to understand the function of unknown gene by studying it’s expression product.In the first part of our studies, we observed the effects of matrine on <WP=11>proliferation by trypan blue exclusion and LDH measurement. The effect of matrine on inhibiting proliferation of K562 cells was observed at 0.2mg/ml concentration after the 3rd day. Meanwhile, we found the K562 cells had the morphological tendency of differentiation observed by Wright’s staining under optical-microscopes, and the apoptosis was found on the 9th day. The results indicated that 0.2mg/ml matrine had the effects of inhibiting proliferation and inducing differentiation .In the second part of our studies, the SmCD, such as CD34, CD33, CD14, CD15, CD20, CD27, CD41, HIR2 on K562 cells treated with 0.2mg/ml matrine for 6 days were measured by flow cytometry. The results showed that 0.2mg/ml matrine could induce the differentiation of K562 cells towards erythrocyte and granulocyte. The results were in accordance with the morphology changes.The differentiation of tumor cells is regulated by several kinds of factors, and oncogenes is one of the regulation factors. In the third part, the expression of some proto-oncogenes of K562 cells treated with 0.2mg/ml matrine were measured by RT-PCR assay. The results showed that the changes of c-myc, c-jun and HNF-1αmRNA were dramatic, their relative levels of expression were dropped, and levels of H-ras and p21 mRNA were increased at the early stage (3h). It is suggested that the effect of inducing differentiation of matrine on K562 cells was regulated at the gene level by a network including multiple factors and pathways, and DNA replication, transcription and signal <WP=12>transduction might be involed.In the fourth part, for the purpose of exploring the molecular mechanisms of K562 cells proliferation suppression and differentiation induced by matrine, and discovering unknown genes, a sensitive reverse transcript differential display PCR (DDRT-PCR) was used to screen some important genes with low-level expression in the earlier phase of matrine induced differentiation. After cloning and sequencing of different cDNA by DDRT-PCR, the results were identified through public database of biological information. The results showed that the mRNA levels of several genes coding proteins and proteinase was changed, which may be associated with cell proliferation and differentiation. In addition, we cloned an unknown cDNA fragment (GenBank ID BM077298, dbEST ID 10245338, http://www.ncbi.mlm.nih.gov/GenBank/index.html ,we called KH gene). Then RT-PCR and Northern blot were performed respectively to learn the distribution of unknown cDNA expression in 11 cell lines and 12 human tissues. It was shown that the unknown cDNA distributed mainly in human brain and leukemia cells. Although rapid amplification of cDNA ends (RACE) was used to extend the unknown cDNA fragment to full length ,but we failed to get it. So we cloned, subcloned and reconstructed expressive vector of the open reading frame (ORF) on the unknown cDNA fragment to study the function of the expressive product. A 24kD peptide was obtained in BL21(DE3)plysS by <WP=13>SDS-PAGE. Meanwhile, COS-7 cell更多还原