TRIP13基因mRNA在慢性淋巴细胞白血病B淋巴细胞的表达及其调控JVM-2细胞增殖和凋亡的分子机制探讨

Study on the expression of TRIP13 mRNA in chronic lymphocytic leukemia B lymphocyte and the molecular mechanism of TRIP13 mediated JVM-2 cell proliferation and apoptosis

【摘要】目的探讨甲状腺激素受体相互作用分子13（TRIP13）基因表达和慢性淋巴细胞白血病（CLL）的相关性，验证TRIP13基因在CLL发生发展中发挥的生物学功能，探讨TRIP13基因调控CLL发生发展的下游分子机制。方法①应用实时荧光定量PCR检测30例CLL患者和12名造血干细胞供者（正常对照组）外周血CD19’B淋巴细胞TRIP13mRNA的表达水平。②使用慢病毒介导的shRNA干扰JVM-2细胞TRIP13mRNA和蛋白表达水平，同时使用随机序列shRNA处理JVM-2细胞作为对照组，分别应用MTT法和流式细胞术检测TRIP13干扰组、对照组JVM．2细胞的增殖和凋亡情况。@Westernblot法检测TRIP13干扰对JVM-2细胞凋亡相关蛋白表达的影响。结果①30例CLL患者外周血B淋巴细胞TRIP13mRNA表达水平（2^△Ct=0．01489）高于正常对照组（2^△Ct=0．00019）（P〈0．001）。②在JVM2细胞中筛选出有效干扰shRNA靶点，下调TRIP13的表达能够有效抑制JVM-2细胞增殖并促进细胞凋亡。③干扰TRIP13后JVM-2细胞Myc和Bcl-2蛋白表达下调（P〈0．001），Bax、caspase3、Bad蛋白表达上调（P〈0．001）。结论CLL患者外周血CD19^＋B淋巴细胞TRIP13mRNA表达上调；TRIP13基因通过影响细胞增殖和凋亡相关蛋白表达而调控JVM2细胞的增殖与凋亡。

Objective To investigate the clinical significance of expression level of thyroid hormone receptor interactors 13 （TRIP13） gene to probe its function and downstream molecular mechanism in chronic lymphocytic leukemia （CLL）. Methods Real-time quantitative PCR method was used to detect the expression levels of TRIP 13 mRNA of CD 19^＋ B lymphocytes in 30 cases of patients with CLL and 12 cases of peripheral blood hematopoietic stem cell donors （normal control group）. Lentivirus mediated shRNA was used to interference the mRNA and TRIP13 protein in CLL cells JVM-2. Scramble sequence was used as control. Methyl thiazolyl tetrazolium colorimetric assay （MTT） and flow cytometry was used to detect the cell proliferation and apoptosis in TRIP13 knocked-down and negative control JVM-2 cells. Results TRIP13 mRNA level was significantly higher in 30 cases of CLL patients （2^△Ct= 0.014 89） compared with 12 healthy donors （2^△Ct= 0.000 19） （P〈0.001）. Validated TRIP13 shRNA target was achieved in JVM2 cell. Compared with the control group, down-regulation of TRIP13 expression could significantly inhibit the proliferation of JVM-2 cells and induce apoptosis. The expressions of Myc and Bcl-2 protein in JVM-2 cells decreased significantly after interference with TRIP 13 （P〈0.001）, and the expressions of Bax, caspase 3 and Bad protein increased significantly （P〈0.001）. Conclusion TRIP 13 mRNA significantly over-expressed in CLL patients CD 19^＋ B lymphocytes. TRIP 13 could influence JVM2 cell proliferation and apoptosis through proliferation- and apoptosis-related proteins.