上调PTEN基因表达对白血病耐药细胞系K562／ADM细胞化疗增敏作用的实验研究  

Experimental study of up-regulating PTEN gene on increasing the chemosensitivity in K562/ADM cells

作　　者：张弘[1] 杨良春[1] 曹励之[1] 杨明华[1] 谢岷[1] 朱珊[1] 曾佩[1] 俞燕[1] 

机构地区：[1]中南大学湘雅医院儿科,长沙410008

出　　处：《中华血液学杂志》2012年第5期412-416,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金（30973234）

摘　　要：目的探讨上调PTEN表达对白血病耐药细胞化疗增敏的作用及其机制。方法将PTEN真核表达质粒导入耐阿霉素（ADM）的K562细胞（K562／ADM细胞）中，采用Westernblot和RT-PCR方法检测PTEN基因在转染前后细胞的表达，MTF法检测ADM对转染前后K562／ADM细胞存活率的影响，应用流式细胞术检测细胞凋亡百分率，采用重组人半胱天冬酶-3（caspase-3）活性定量试剂盒分析caspase-3的活性，Westernblot检测自噬微管相关蛋白轻链3（LC3）-Ⅰ／Ⅱ、自噬相关蛋白Bec-linl、磷酸化蛋白激酶B（p-Akt）和磷酸化40S核糖体蛋白6s激酶（p-p70S6K）的表达，单丹磺酰戊二胺（MDC）染色和透视电镜观察自噬泡的情况。结果①与未经处理和转染空载体的K562／ADM细胞相比，转染PTEN基因的K562／ADM细胞组PTENmRNA和蛋白表达水平均增高；②转染PTEN基因使K562／ADM细胞对ADM的敏感性增强，与转染空载体细胞比较，其细胞存活率从（94．07±2．60）％降低到（53．83±4．20）％，细胞凋亡率从（11．89±1．70）％增加到（43．69±2．30）％；经caspase-3抑制剂（Z-DEVE-FMK）预处理后，未能完全阻止ADM对细胞的毒性作用；③ADM处理细胞12和24h后，转染PTEN基因的K562／ADM细胞casepase-3活性（2．27±0．13和3．15±0．08）均明显高于转染空载体组（1．19±0．14和1．48±0．06）（P值均〈0．05）；④与转染空载体组比较，转染PTEN基因的K562／ADM细胞LC3-Ⅱ和Beclinl的蛋白表达水平分别增加83％和18％，p-Akt和p-p70S6K的蛋白表达水平分别降低96％和87％；⑤增加PTEN的表达，细胞内自噬泡较转染空载体组明显增多。结论上调PTEN基因表达能明显增加K562／ADM细胞对ADM的敏感性，该作用可能与上调PTEN基因表达、抑制P13K／Ak∥mTOR通路活性从而促进细胞凋亡和自噬的发生有关。Objective To investigate the mechanism of mereasing the K562/ADM cells chemosensitivity by up-regulating expression of PTEN gene. Methods K562/ADM cells were transient transfeeted with pGFP-PTEN or vector. The level of PTEN in K562/ADM cells was assayed by Western blot and RT-PCR. Cell viability on K562/ADM was determined by MTF assay. Cell apoptosis by flow cytometry. Activity of caspase-3 by Caspase Colorimetric Assay Kit. The proteins expression of LC3-Ⅰ/Ⅱ, Beclinl, p-Akt, p- p70S6K by Western blot. The autophagic vacuoles by MDC stain and Electron microscopy. Results ①The mRNA and protein levels of PTEN in K562/ADM cells transfected with pGFP-PTEN were significantly increased compared with the control（ untreated and treasfeeted with empty vector）. ②Enhanced expression of PTEN by gene transfection resulted in a reversal of resistance to ADM. Compared with empty vector group, cell viability decreased from （94.07 ±2.60） % to （53.83 ±4.20） %, the cell apoptotic rate increased from （ 11.89 ± 1.70） % to （43.69 ± 2.30） %, meanwhile, pretreated with caspase-3 inhibitor（Z-DEVE-FMK） didn＇ t completely inhibit the cytotoxicity of ADM to K562/ADM cells. ③After treated with ADM for 12 and 24 h, the activities of casepase-3 in PTEN-transfeeted K562/ADM cells increased compared with those in pG- FP-transfected K562/ADM cells [ （2.27 ± 0.13 ） vs （ 1.19 ±0.14） ] at 12h, [ （ 3.15 ± 0.08） vs （ 1.48 ±0.05）] at 24 h （ P 〈 0.05 ）.④The protein levels of LC3- Ⅱ and Beclinl in K562/ADM cells transfectedwith pGFP-PTEN were increased by 83% and 18% respectively, and the protein levels of p-Akt and p- p70S6K were declined by 96% and 87% respectively, compared with those in K562/ADM cells transfected with pGFP plasmid. ⑤The upregulation of PTEN in K562/ADM cells improved the number of autophagic vac- uoles compared with the empty vector group. Conclusion The upregulation of PTEN expression increases the chemosensitivity of K562/ADM to ADM, which may related with the

关 键 词：基因 PTEN K562/ADM细胞 自噬 凋亡 耐药性 基因转染 

分 类 号：R733.7[医药卫生—肿瘤]