中文摘要：

背景与目的：WT1基因编码一个锌指转录因子，在白血病细胞中高表达并与预后呈负相关。WT1基因mRNA通过2个可变性的剪切位点产生4种不同的拼接异构体，4种异构体在表达WT1的不同组织中各以其相对稳定的比例表达，白血病细胞中WT1不同异构体表达的意义尚不清楚。本研究拟用WT1基因异构体WTA转导急性早幼粒细胞性白血病细胞株NB4，以探讨WT1基因异构体对NB4细胞增殖的影响及其分子机制。方法：用电穿孔的方法将携带WT1基因异构体WTA（-17AA／-KTS）全长cDNA的重组真核表达载体及其对照质粒pCB6＋转导NB4细胞，建立WT1基因异构体表达比例改变的细胞株并用有限稀释法进行单克隆化。采用台盼蓝拒染法、MTT比色法、甲基纤维素集落形成实验及细胞周期动力学检测，了解WT1基因异构体表达比例改变对NB4细胞生长的影响。用半定量RT—PCR检测NB4细胞中p53、p21、Cyclin E、Cyclin D1、Cyclin A1基因表达的改变。结果：WT1基因异构体能够通过电穿孔方法成功转染NB4细胞，外源基因在NB4细胞中稳定整合及表达，并通过有限稀释法将永久表达细胞株单克隆化。用台盼蓝拒染法绘制细胞生长曲线，提示转染WT1基因异构体的细胞NB4／WTA生长明显慢于对照组NB4／CMV细胞及未转染的NB4细胞，NB4／WTA细胞生长曲线平缓，在第3天达平台期（78．7±18．0）×10^4／ml，对照组NB4／CMV细胞及NB4细胞在第4天出现生长平台期达（146．0±21．0）×10^4/ml。MTT法检测细胞增殖活性，结果显示，NB4／WTA细胞在各个时间段的抑制率均高于对照组，P〈0．005。甲基纤维素集落形成试验中NB4／WTA克隆形成率明显下降，集落形成抑制率为46．9％。在As2O3（终浓度0．2μmol·L^-1）作用后集落形成抑制更加明显。细胞周期动力学检测提示，NB4／WTA组S期细胞比例升高。RT—PCR检查提示，NB4／WTA细胞p21、Cycli

英文摘要：

BACKGROUND ＆ OBJECTIVE. The Wilms＇ tumor gene, WT1, encodes a zinc finger transcription factor, and is highly expressed in leukemia cells and negatively correlated with the prognosis of leukemia. WT1 mRNA undergoes alternative splicing at 2 sites resulting in 4 isoforms. The mRNA splice isoforms are constantly expressed in all tissues expressing WT1 with fixed proportions. The functional significance of WT1 isoforms expression in leukemia is unclear. This study was designed to explore the potential effects of exogenous WT1 gene isoforms on proliferation of leukemia cell line NB4 and its possible molecular mechanisms. METHODS. The recombinant eukaryotic expression vector pCB6＋/WTA containing full-length human WT1 isoform WTA （-17AA/-KTS） cDNA, and pCB6＋ vector with neomycin resistance gene were transfected into NB4 cells by electroperforation. The positive cell clones （NB4/WTA） were selected with G418. The transfected cells were monocloned by limited dilution. The expressing of WTA mRNA and protein in transfected cells was detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. The proliferating ability of leukemia cells was measured by trypan blue exclusion assay, MTT assay, colony formation assay and cell cycle analysis. The expression of p53, p21, Cyclin E, Cyclin D1 and Cyclin A1 was detected by semi-quantitative RTPCR. RESULTS： WT1 gene isoforms were transfected into NB4 cells,and the cell line stably expressing exogenous WT1 gene isoforms was established successfully. The monoclonal cells overexpressing WT1 were obtained. The growth of NB4/WTA cells was slower than that of untransfected NB4 cells and NB4 cells transfected with pCB6＋ vector （NB4/CMV）： the growth of NB4/ WTA cells reached plate phase [（78.7±18.0）×10^4/ml] at the 3rd day, while that of control cells reached plate phase [（146.0±21.0）×10^4/ml] at the 4th day. The inhibitory rates of NB4/WTA cells at different time points were significantly higher than those of control NB4 ce