摘要
目的 探讨p5 3基因诱导白血病细胞凋亡过程中内源性TGF β1 、TNF α、端粒酶活性、Bcl 2表达的改变及其意义。方法 利用脂质体将温敏型p5 3基因 [pN5 3cG(Val135 ) ]导入p5 3基因缺失的白血病细胞系HL 6 0、K5 6 2细胞 ,经G4 18筛选 ,获得稳定表达P5 3蛋白的抗性克隆细胞HL 6 0pN5 3cG ,K5 6 2 pN5 3cG细胞。采用缺口末端标记法、片段化DNA分析、RT PCR、定量PCR、ELISA、PCR ELISA、流式细胞术等方法检测外源性p5 3基因诱导白血病细胞凋亡过程中TGF β1 、TNF α、bcl 2、端粒酶反转录酶 (hTERT)mRNA表达变化、细胞上清TGF β1 水平和端粒酶活性及TGF β1 、TNF α反义PS ODNS对白血病细胞凋亡和Bcl 2蛋白表达的影响。结果 ①外源性野生型p5 3基因诱导细胞凋亡过程中内源性TGF β1 和TNF αmRNA表达上调 ,bcl 2及hTERTmRNA表达下调 ,细胞培养上清TGF β1 蛋白水平明显升高 ,端粒酶活性水平下降 ;②TGF β1 、TNF α反义PS ODNS能够明显抑制野生型p5 3基因诱导细胞凋亡的发生 ,并使细胞bcl 2mRNA及蛋白表达恢复到处理前的水平。结论 p5 3基因诱导白血病细胞凋亡可通过上调内源性TGF β1 和TNF α水平 ,下调hTERTmRNA表达及端粒酶活性 ,抑制bcl 2基因表达而发挥作用。
Objective To investigate the expression changes of intrinsic cytokines TGF-β 1 and TNF-α,telomerase activity and bcl-2 during ongoing apoptosis of HL-60 and K562 cells induced by p53. Methods pN53cG(Val135), a temperature sensitive p53 mutant, which behaved like wild type p53(wt-p53) at 32.5 ℃, were introduced into p53-null HL-60 and K562 cells respectively by lipofectin . In the presence of G418, HL-60-pN53cG and K562-pN53cG clones expressing p53 protein were selected. The ongoing expression of intrinsic cytokines (TGF-β 1 and TNF-α), bcl-2 oncogene and hTERT mRNA during the apoptosis of HL-60 and K562 cells induced by p53 and the effects of exogenous p53 gene, TGF-β 1 and TNF-α antisense PS-ODNS on the apoptosis of HL-60 and K562 cells and the expression of bcl-2 were studied by RT-PCR,quantitative RT-PCR, DNA fragmentation, TdT-mediated dUTP nick end labeling(TUNEL) and flow cytometery. The levels of secreted TGF-β 1 and telomerase activity were detected by ELISA and PCR-ELISA, respectively. Results ①The expressions of intrinsic TGF-β 1 and TNF-α mRNA were up-regulated, while that of bcl-2 and hTERT down-regulated. The levels of TGF-β 1 in the supernatant of HL-60 and K562 cells were increased, and the level of telomerase activity decreased.②Antisense PS-ODNS of TGF-β 1 and TNF-α could obviously inhibit the p53 inducing cell apoptosis, and restore bcl-2 mRNA and protein to pre-treated level. Conclusions Exogenous p53 induces leukemia cell apoptosis via up-regulating the expression of intrinsic TGF-β 1 and TNF-α and down-regulating the expression of hTERT and bcl-2.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2003年第12期640-643,共4页
Chinese Journal of Hematology
基金
国家教委优秀年轻教师基金资助项目 ( 95 5 0 3 )
福建省教委科研基金资助项目 (K95 0 3 3 )
关键词
P53基因
诱导
白血病
细胞凋亡
Gene,p53
Cell line,HL-60
Cell line,K562
Cell apoptosis
Hematopoietic cell growth factor
