CAJ | 학술논문

目的研究重组腺病毒介导的野生型p53基因(Ad5wtp53)对人慢性粒细胞白血病(CML)K562细胞系中心体数量和中心体蛋白表达的影响.探讨应用重组腺病毒p53基因对CML进行基因治疗的可行性.方法分别将含有野生型p53基因、突变型p53基因和绿色荧光蛋白基因的重组腺病毒表达载体(AdSwtp53、Ad5mtp53和Ad5GFP)反复扩增,联合多聚季胺共同感染K562细胞.观察感染效率.MTT法确定最适重组腺病毒感染滴度和感染时间.然后用RT-PCR和Western blot检测p53 mRNA和蛋白的表达.最后应用间接免疫荧光染色法对中心体γ-微管蛋白和纺锤体α-微管蛋白同时进行标记,在激光共聚焦显微镜下观察中心体γ-微管蛋白的表达和有丝分裂,并对中心体进行计数.结果多聚季胺可显著提高腺病毒载体对K562细胞的感染效率.多聚季胺的剂量选择为4μg/ml;感染K562细胞的最适腺病毒滴度为20 000 MOI,感染时间为72 h;Ad5wtp53和AdSmtp53转染的K562细胞均可表达p53 mRNA和蛋白.Ad5wtp53感染K562细胞后,中心体数量减少,γ-微管蛋白的表达降低.结论重组腺病毒介导的野生型p53基因能够在白血病K562细胞中持续表达;野生型P53蛋白能够降低K562细胞中心体数量及中心体γ-微管蛋白的表达.
목적 연구중조선병독개도적야생형p53기인(Ad5wtp53)대인만성립세포백혈병(CML)K562세포계중심체수량화중심체단백표체적영향.탐토응용중조선병독p53기인대CML진행기인치료적가행성.방법 분별장함유야생형p53기인、돌변형p53기인화록색형광단백기인적중조선병독표체재체(AdSwtp53、Ad5mtp53화Ad5GFP)반복확증,연합다취계알공동감염K562세포.관찰감염효솔.MTT법학정최괄중조선병독감염적도화감염시간.연후용RT-PCR화Western blot검측p53 mRNA화단백적표체.최후응용간접면역형광염색법대중심체γ-미관단백화방추체α-미관단백동시진행표기,재격광공취초현미경하관찰중심체γ-미관단백적표체화유사분렬,병대중심체진행계수.결과 다취계알가현저제고선병독재체대K562세포적감염효솔.다취계알적제량선택위4μg/ml;감염K562세포적최괄선병독적도위20 000 MOI,감염시간위72 h;Ad5wtp53화AdSmtp53전염적K562세포균가표체p53 mRNA화단백.Ad5wtp53감염K562세포후,중심체수량감소,γ-미관단백적표체강저.결론 중조선병독개도적야생형p53기인능구재백혈병K562세포중지속표체;야생형P53단백능구강저K562세포중심체수량급중심체γ-미관단백적표체.
Objective To observe the effect of recombinant adenovirus-mediated wild-type p53 gene on the number and proteins of eentrosome in K562 cells. To explore the possibility of application of wild-type p53 gene therapy in the treatment of chronic myeloid leukemia. Methods The recombinant adenoviruses car- rying wild-type p53 gene (AdS wtp53}, mutant p53 gene (Ad5 mtp53} or the green fluorescent protein (GFP) gene was repeatedly amplified and co-infected into K562 cells with cation polybrene. The optimal in- fection titer and infection time of the recombinant adenoviruses were determined by MTr assay, p53 mRNA and protein expression were determined by RT-PCR and Western blot respectively. The centrosomal structural proteinγ-tubulin and the spindle protein α-tubulin were marked simultaneously by indirect immunofluores- cenee staining, and the expression of the eentrosomal γ-tubulin protein, the mitosis and the number of centro-some were observed under the laser confoeal microscopy. Results Infection efficiency with recombinant ade-noviruses was facilitated by polybrene in K562 cells, and 4 μ/ml polybrene was chosen. The optimal adeno- virus infection titer was 20 000 MOI and the optimal infection time was 72 hours, p53 mRNA and P53 protein can be expressed in K562 cells by Ad8 wtp53 and Ad5mtp53. Both the expression of the centrosomal γ-tubulin protein and the number of eentrosomes were decreased after AclSwtp53 infection. Conclusion There is sus- tained expression of P53 protein in K562 cells after its infection by Ad5wtp53. Wild-type P53 protein can lead to the down-regulation of the number of centrosomes and the expression of centrosomal γ-tudulin protein in K562 cells.