中国全科医学
中國全科醫學
중국전과의학
CHINESE GENERAL PRACTICE
2015年
24期
2934-2938,2945
,共6页
刘小军%杨琳%潘玉夏%王兴哲%尚银涛%王茜%杨敬慈%罗建民
劉小軍%楊琳%潘玉夏%王興哲%尚銀濤%王茜%楊敬慈%囉建民
류소군%양림%반옥하%왕흥철%상은도%왕천%양경자%라건민
白血病%基因, bcr/abl%RNA, 小分子干扰%细胞增殖%细胞凋亡
白血病%基因, bcr/abl%RNA, 小分子榦擾%細胞增殖%細胞凋亡
백혈병%기인, bcr/abl%RNA, 소분자간우%세포증식%세포조망
Leukemia%Genes,bcr/abl%RNA,small interfering%Cell proliferation%Apoptosis
目的：通过小干扰RNA （ siRNA）沉默K562细胞bcr／abl基因使SH2肌醇磷脂酶（ SHIP）基因表达增加，探讨SHIP基因缺失对K562细胞增殖凋亡的影响及机制。方法构建bcr／abl基因的化学合成siRNA，并转染到K562细胞，分为转染特异性siRNA实验组、转染非特异性siRNA对照组、空白对照组。 Western blotting法检测转染后K562细胞p210表达量，实时荧光定量PCR （ FQ-RT-PCR）检测各组细胞中SHIP mRNA表达量； Western blotting法检测SHIP、磷酸化蛋白激酶B 308（p-Akt308）和磷酸化蛋白激酶B 473（p-Akt473）的表达量； MTT比色法检测K562细胞增殖率；流式细胞仪检测K562细胞凋亡率；酶联免疫吸附试验（ ELISA）法检测核因子（ NF）-κB活性。结果转染特异性siRNA实验组K562细胞p210表达量、 p-Akt308表达量、 p-Akt473表达量较空白对照组和转染非特异性siRNA对照组降低， SHIP mRNA表达量、 SHIP表达量、细胞凋亡率较空白对照组和转染非特异性siRNA对照组升高（P＜0.05）；空白对照组与转染非特异性siRNA对照组K562细胞p210表达量、 SHIP mRNA表达量、 SHIP表达量、 p-Akt308表达量、 p-Akt473表达量、细胞凋亡率比较，差异无统计学意义（P＞0.05）。第1天、第3天、第5天转染特异性siRNA实验组K562细胞增殖率较空白对照组和转染非特异性siRNA对照组降低（P＜0.05）；空白对照组与转染非特异性siRNA对照组K562细胞增殖率比较，差异无统计学意义（ P＞0.05）。第3天、第5天转染特异性siRNA实验组NF-κB活性较空白对照组和转染非特异性siRNA对照组降低（ P＜0.05）；空白对照组与转染非特异性siRNA对照组NF-κB活性比较，差异无统计学意义（ P＞0.05）。结论 SHIP基因表达受bcr／abl基因的调控， SHIP表达缺失可能是K562细胞过度增殖和凋亡减少的重要因素。
目的：通過小榦擾RNA （ siRNA）沉默K562細胞bcr／abl基因使SH2肌醇燐脂酶（ SHIP）基因錶達增加，探討SHIP基因缺失對K562細胞增殖凋亡的影響及機製。方法構建bcr／abl基因的化學閤成siRNA，併轉染到K562細胞，分為轉染特異性siRNA實驗組、轉染非特異性siRNA對照組、空白對照組。 Western blotting法檢測轉染後K562細胞p210錶達量，實時熒光定量PCR （ FQ-RT-PCR）檢測各組細胞中SHIP mRNA錶達量； Western blotting法檢測SHIP、燐痠化蛋白激酶B 308（p-Akt308）和燐痠化蛋白激酶B 473（p-Akt473）的錶達量； MTT比色法檢測K562細胞增殖率；流式細胞儀檢測K562細胞凋亡率；酶聯免疫吸附試驗（ ELISA）法檢測覈因子（ NF）-κB活性。結果轉染特異性siRNA實驗組K562細胞p210錶達量、 p-Akt308錶達量、 p-Akt473錶達量較空白對照組和轉染非特異性siRNA對照組降低， SHIP mRNA錶達量、 SHIP錶達量、細胞凋亡率較空白對照組和轉染非特異性siRNA對照組升高（P＜0.05）；空白對照組與轉染非特異性siRNA對照組K562細胞p210錶達量、 SHIP mRNA錶達量、 SHIP錶達量、 p-Akt308錶達量、 p-Akt473錶達量、細胞凋亡率比較，差異無統計學意義（P＞0.05）。第1天、第3天、第5天轉染特異性siRNA實驗組K562細胞增殖率較空白對照組和轉染非特異性siRNA對照組降低（P＜0.05）；空白對照組與轉染非特異性siRNA對照組K562細胞增殖率比較，差異無統計學意義（ P＞0.05）。第3天、第5天轉染特異性siRNA實驗組NF-κB活性較空白對照組和轉染非特異性siRNA對照組降低（ P＜0.05）；空白對照組與轉染非特異性siRNA對照組NF-κB活性比較，差異無統計學意義（ P＞0.05）。結論 SHIP基因錶達受bcr／abl基因的調控， SHIP錶達缺失可能是K562細胞過度增殖和凋亡減少的重要因素。
목적：통과소간우RNA （ siRNA）침묵K562세포bcr／abl기인사SH2기순린지매（ SHIP）기인표체증가，탐토SHIP기인결실대K562세포증식조망적영향급궤제。방법구건bcr／abl기인적화학합성siRNA，병전염도K562세포，분위전염특이성siRNA실험조、전염비특이성siRNA대조조、공백대조조。 Western blotting법검측전염후K562세포p210표체량，실시형광정량PCR （ FQ-RT-PCR）검측각조세포중SHIP mRNA표체량； Western blotting법검측SHIP、린산화단백격매B 308（p-Akt308）화린산화단백격매B 473（p-Akt473）적표체량； MTT비색법검측K562세포증식솔；류식세포의검측K562세포조망솔；매련면역흡부시험（ ELISA）법검측핵인자（ NF）-κB활성。결과전염특이성siRNA실험조K562세포p210표체량、 p-Akt308표체량、 p-Akt473표체량교공백대조조화전염비특이성siRNA대조조강저， SHIP mRNA표체량、 SHIP표체량、세포조망솔교공백대조조화전염비특이성siRNA대조조승고（P＜0.05）；공백대조조여전염비특이성siRNA대조조K562세포p210표체량、 SHIP mRNA표체량、 SHIP표체량、 p-Akt308표체량、 p-Akt473표체량、세포조망솔비교，차이무통계학의의（P＞0.05）。제1천、제3천、제5천전염특이성siRNA실험조K562세포증식솔교공백대조조화전염비특이성siRNA대조조강저（P＜0.05）；공백대조조여전염비특이성siRNA대조조K562세포증식솔비교，차이무통계학의의（ P＞0.05）。제3천、제5천전염특이성siRNA실험조NF-κB활성교공백대조조화전염비특이성siRNA대조조강저（ P＜0.05）；공백대조조여전염비특이성siRNA대조조NF-κB활성비교，차이무통계학의의（ P＞0.05）。결론 SHIP기인표체수bcr／abl기인적조공， SHIP표체결실가능시K562세포과도증식화조망감소적중요인소。
Objective Through silence the expression of bcr/abl with siRNA to increase the expression of SHIP so that we can study the influence of SHIP gene expression to the proliferation and apoptosis of K 562 together with its possible mechanism.Methods Construct synthetic siRNA specific to bcr/abl fusion gene and tranfect it into cell line K 562, non -specific siRNA trasfected group and untreated group were used as control , then Western blotting was employed to detect the expression of p210, SHIP and level of p -Akt (308, 437), SHIP mRNA level was detected through FQ -RT -PCR, proliferation of K562 through MTT, and apoptosis of K562 through flow cytometre, NF-κB activity was examined through ELISA.Results fter transfected with siRNA specific to bcr/abl, the expression of p210, p -Akt308, p -Akt473 was dramatically decreased in the bcr -abl-specific siRNA transfected group compare to the non -specific transfected control and untreated group of K562 cells, while the SHIP mRNA, protein expression, apoptosis rate increased in bcr -abl -specific transfected K562 cells compare to the other two control groups (P<0.05).All these changes were not detected between the non-specific transfected group and untreated group of K 562 cells (P>0.05).The proliferation decreased in the bcr -abl-specific transfected group compare to the other two controls on day 1, day 3 and day 5 respectively (P<0.05), but no significance was found between the two control groups (P>0.05).NF-κB activity decreased in bcr -abl-specific transfected group compare to the other two control groups on day 3 and day 5 ( P<0.05) , but no significance was found between the two control groups ( P>0.05).Conclusion Our results suggested that SHIP expression is controlled by bcr /abl.Reduced expression of SHIP partly responsible for the increased proliferation and decreased apoptosis of bcr /abl positive cells .
