NANOG基因在急性淋巴细胞白血病细胞的表达及其慢病毒干扰载体的构建

本研究探讨NANOG基因在人急性淋巴细胞白血病（ALL）细胞中的表达并构建特异性干扰NANOG表达的慢病毒载体。利用RT—PCR及Westernblot技术检测MOLT4、CCRF—HSB2、Jurkat等ALL细胞系及在我院住院治疗的15例新诊断的ALL患者骨髓细胞中NANOG的表达情况。通过构建携带NANOG特异性shRNA的慢病毒载体包装病毒颗粒，感染MOLT4细胞后经分选获得稳定表达株，并在基因及蛋白水平检测NANOG干扰效率。结果表明，MOLT-4细胞、CCRF—HSB2细胞以及33．3％的ALL患者中可见NANOG基因mRNA的扩增产物及蛋白表达。测序表明，ALL患者及MOLT-4、CCRF—HSB2细胞主要表达NANOGP8mRNA。构建隧病毒干扰质粒pLB-shNANOG-1、pLB-shNANOG-2及pLB—shcontrol，包装病毒颗粒超速离心后病毒滴度达（1．83—3．12）×10^8IU／ml。病毒感染MOLT-4细胞后经流式细胞仪分选获得GFP＋细胞。实时定量PCR及Westernblot证实，两种shRNA可有效下调NANOG基因及蛋白的表达。结论：MOLT4细胞、CCRF．HSB2细胞以及部分ALL患者骨髓细胞中存在NANOG的表达，其主要为NANOGP8mRNA的转录。成功构建了特异性干扰NANOG表达的慢病毒载体，获得可稳定下调NANOG表达的MOLT4细胞株。

Expression of NANOG Gene in Acute Lymphoblastic Leukemia Cells and Construction of Lentiviral Vector Carrying NANOG Spe- cific shRNA

The aim of this study was to detect the expression of NANOG gene in acute lymphoblastic leukemia （ALL） cells, and to construct the lentiviral vector carrying NANOG specific shRNA. The expression of NANOG was de- tected by RT-PCR and Western blot in MOLT-4, CCRF-HSB2, Jurkat cells and bone marrow cells from 15 patients with ALL in our hospital. The lentiviral vector carrying NANOG specific shRNA was constructed. After infection of MOLT- 4 cells with the lentivirus constructs, GFP ＋ cells were harvested by flow cytometry. The efficiency of RNA interference was detected by real-time quantitative PCR and Western blot. The results showed that the expression of NANOG mRNA and protein was detected in MOLT-4, CCRF-HSB2 cells and 33.3% samples of bone marrow from patients with ALL. The sequencing results demonstrated that the mRNAs amplified from these leukemic cells showed higher homology to NANOGP8 than NANOG1. The lentiviral vector pLB-shNANOG-1, pLB-shNANOG-2 and pLB-shcontrol were construc- ted. The viral particles were harvested and concentrated by ultracentrifugation. The virus titers were （ 1.83 -3.12） × 10^8 IU/ml. After infection of MOLT-4 cells with the lentivirus, flow cytometry detection indicated that the GFP ＋cells were harvested by real-time quantitative PCR and Western blot, the assays showed that the 2 designed shRNA could sig- nificantly down-regulate expression of NANOG gene and protein. It is concluded that NANOGP8 is expressed in various types of ALL cells and in 33.3% of marrow cell samples obtained from ALL patients. After infection with the lentivirus constructs, MOLT-4 cells which stably down-regulat the expression of NANOG mRNA are obtained.