中国癌症杂志
中國癌癥雜誌
중국암증잡지
CHINA ONCOLOGY
2013年
9期
713-720
,共8页
白血病%RNA干扰%Apollon基因%多药耐药性%细胞凋亡%K562细胞
白血病%RNA榦擾%Apollon基因%多藥耐藥性%細胞凋亡%K562細胞
백혈병%RNA간우%Apollon기인%다약내약성%세포조망%K562세포
Leukemia%RNA interference%Apollon genes%Multiple drug resistance%Apoptosis%K562 cells
背景与目的：Apollon基因在白血病等多种肿瘤内高表达。本试验通过构建高效干扰Apollon基因的短发夹RNA(short hairpin RNA，shRNA)真核表达载体，探讨RNA干扰技术能否逆转人髓系白血病K562细胞多药耐药。方法：构建靶向Apollon基因真核表达载体pGPHI-GFP-Neo-Apollon，应用LipofectamineTM2000转染K562细胞，G418稳定筛选。采用反转录-聚合酶链反应(RT-PCR)法、细胞免疫荧光法分别检测重组载体稳定转染K562细胞后Apollon mRNA及蛋白的表达情况；四甲基偶氮唑盐(MTT)、流式细胞术检测细胞转染前后对长春新碱(leurocristine，VCR)、足叶乙苷(etoposide，VP16)的敏感性及凋亡率的变化。结果：成功构建了pGPHI-GFP-Neo-Apollon载体并在K562细胞内稳定表达的细胞克隆，经G418筛选后，重组载体能有效沉默Apollon的表达，Apollon mRNA及蛋白表达水平明显下降。MTT结果显示，基因干扰组细胞对VCR、VP16的敏感性明显增强，其半数抑制浓度(half-inhibitory，IC50)值分别为(0.144±0.018)mg/L、(17.336±3.571) mg/L，明显低于细胞对照组(P<0.05)。流式细胞术检测结果表明，基因干扰组细胞联合化疗药物后细胞凋亡率显著升高(P<0.05)，而转染阴性对照组细胞凋亡率与正常对照组比较差异无统计学意义(P>0.05)。结论：pGPHI-GFP-Neo-Apollon载体能显著增强VCR和VP16对白血病K562细胞的诱导凋亡作用，提高K562细胞对化疗药物的敏感性，提示RNA干扰Apollon基因表达能一定程度逆转白血病细胞的多药耐药。
揹景與目的：Apollon基因在白血病等多種腫瘤內高錶達。本試驗通過構建高效榦擾Apollon基因的短髮夾RNA(short hairpin RNA，shRNA)真覈錶達載體，探討RNA榦擾技術能否逆轉人髓繫白血病K562細胞多藥耐藥。方法：構建靶嚮Apollon基因真覈錶達載體pGPHI-GFP-Neo-Apollon，應用LipofectamineTM2000轉染K562細胞，G418穩定篩選。採用反轉錄-聚閤酶鏈反應(RT-PCR)法、細胞免疫熒光法分彆檢測重組載體穩定轉染K562細胞後Apollon mRNA及蛋白的錶達情況；四甲基偶氮唑鹽(MTT)、流式細胞術檢測細胞轉染前後對長春新堿(leurocristine，VCR)、足葉乙苷(etoposide，VP16)的敏感性及凋亡率的變化。結果：成功構建瞭pGPHI-GFP-Neo-Apollon載體併在K562細胞內穩定錶達的細胞剋隆，經G418篩選後，重組載體能有效沉默Apollon的錶達，Apollon mRNA及蛋白錶達水平明顯下降。MTT結果顯示，基因榦擾組細胞對VCR、VP16的敏感性明顯增彊，其半數抑製濃度(half-inhibitory，IC50)值分彆為(0.144±0.018)mg/L、(17.336±3.571) mg/L，明顯低于細胞對照組(P<0.05)。流式細胞術檢測結果錶明，基因榦擾組細胞聯閤化療藥物後細胞凋亡率顯著升高(P<0.05)，而轉染陰性對照組細胞凋亡率與正常對照組比較差異無統計學意義(P>0.05)。結論：pGPHI-GFP-Neo-Apollon載體能顯著增彊VCR和VP16對白血病K562細胞的誘導凋亡作用，提高K562細胞對化療藥物的敏感性，提示RNA榦擾Apollon基因錶達能一定程度逆轉白血病細胞的多藥耐藥。
배경여목적：Apollon기인재백혈병등다충종류내고표체。본시험통과구건고효간우Apollon기인적단발협RNA(short hairpin RNA，shRNA)진핵표체재체，탐토RNA간우기술능부역전인수계백혈병K562세포다약내약。방법：구건파향Apollon기인진핵표체재체pGPHI-GFP-Neo-Apollon，응용LipofectamineTM2000전염K562세포，G418은정사선。채용반전록-취합매련반응(RT-PCR)법、세포면역형광법분별검측중조재체은정전염K562세포후Apollon mRNA급단백적표체정황；사갑기우담서염(MTT)、류식세포술검측세포전염전후대장춘신감(leurocristine，VCR)、족협을감(etoposide，VP16)적민감성급조망솔적변화。결과：성공구건료pGPHI-GFP-Neo-Apollon재체병재K562세포내은정표체적세포극륭，경G418사선후，중조재체능유효침묵Apollon적표체，Apollon mRNA급단백표체수평명현하강。MTT결과현시，기인간우조세포대VCR、VP16적민감성명현증강，기반수억제농도(half-inhibitory，IC50)치분별위(0.144±0.018)mg/L、(17.336±3.571) mg/L，명현저우세포대조조(P<0.05)。류식세포술검측결과표명，기인간우조세포연합화료약물후세포조망솔현저승고(P<0.05)，이전염음성대조조세포조망솔여정상대조조비교차이무통계학의의(P>0.05)。결론：pGPHI-GFP-Neo-Apollon재체능현저증강VCR화VP16대백혈병K562세포적유도조망작용，제고K562세포대화료약물적민감성，제시RNA간우Apollon기인표체능일정정도역전백혈병세포적다약내약。
Background and purpose:Apollon gene is highly expressed in leukemia and other tumors. The study aimed to discuss whether RNAi technology can reverse multidrug resistance of chronic myeloid leukemia cell line K562 through constructing a eukaryotic vector of short hairpin RNA (shRNA) targeting at Apollon gene. Methods:The eukaryotic vector pGPHI-GFP-Neo-Apollon with shRNA targeting at Apollon gene was constructed and then transfected into K562 cells by LipofectamineTM2000, and G418 pressure selection. Reverse transcription-polymerase chain reaction (RT-PCR) and immunolfuorescence were used to detect the expression of Apollon mRNA and protein after Apollon was transfected stably in K562 cells. The changes of sensitivity of K562 cells to leurocristine (VCR) and etoposide (VP16) after transfection with shRNA-Apollon were detected by MTT method, and the apoptosis rate was detected by flow cytometry. Results: pGPHI-GFP-Neo-Apollon carrier was constructed successfully and expressed stably in K562 cells, and after G418 screening, it silenced Apollon mRNA and protein expression effectively. According to the result of MTT, the sensitivity of K562 cells to VCR and VP16 increased significantly in the group of gene interference, with half of its inhibition concentration (half-inhibitory, IC50) value signiifcantly lower than the control group (P<0.05);Flow cytometry showed that the cell apoptosis rate was increased signiifcantly (P<0.05), but there was no statistically signiifcant difference in the apoptosis rate between shRNA negative control group and normal control group (P>0.05). Conclusion:pGPHI-GFP-Neo-Apollon carrier can enhance the abilities of VCR and VP16 to induce the apoptosis of K562 cells, namely an increase of sensitivity to these chemotherapeutics in K562 cells, it is hinted that RNA interference targeting Apollon gene may reverse the multidrug resistance of leukemia cells in some degree.
