中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2013年
6期
438-442
,共5页
谢斌辉%王小农%刘凤恩%何晓%谢元康%周奇
謝斌輝%王小農%劉鳳恩%何曉%謝元康%週奇
사빈휘%왕소농%류봉은%하효%사원강%주기
RNA,小分子干扰%受体,白细胞介素8B%肝肿瘤,实验性%基因沉默
RNA,小分子榦擾%受體,白細胞介素8B%肝腫瘤,實驗性%基因沉默
RNA,소분자간우%수체,백세포개소8B%간종류,실험성%기인침묵
RNA,small interfering%Receptors,interleukin-8B%Liver neoplasms,experimental%Gene silencing
目的 研究CXCR2-小干扰RNA(siRNA)基因体外抑制肝癌细胞增殖的作用.方法 将CXCR2-siRNA以脂质体转染法转染肝癌细胞hepG2,24h后荧光显微镜观察细胞荧光含量.采用RT-PCR及免疫印迹法分别检测正常肝Chang细胞、未转染肝癌hepG2细胞以及转染CXCR2-siRNA基因后hepG2细胞在转染24、48、72h后的CXCR2 mRNA和蛋白表达水平.以未转染的hepG2细胞为对照,CCK8法检测转染CXCR2-siRNA的hepG-2细胞在转染24、48、72 h后的增殖.以未转染的hepG2细胞为对照,检测转染CXCR2-siRNA 24 h后hepG2细胞与Transwell小室孵育24h时的侵袭能力.结果 CXCR2-siRNA转染hepG2 24 h后镜下可见大量绿色荧光,转染率为80％.hepG2细胞CXCR2mRNA和蛋白表达水平高于Chang细胞(mRNA:1.69±0.22比0.63±0.31,蛋白:1.93±0.25比0.84±0.29,均P＜0.05).转染CXCR2-siRNA24和48 h后hepG2细胞CXCR2mRNA表达量低于未转染的hepG2细胞(0.75±0.24、0.83±0.21比1.78±0.25,均P＜0.05),72 h后CXCR2-siRNA虽然仍能抑制hepG2细胞CXCR2mRNA的表达,但与未转染的hepG2细胞相比差异并无统计学意义(1.35±0.18比1.78±0.25,P＞0.05).转染CXCR2-siRNA24和48 h后的hepG2细胞CXCR2蛋白表达低于未转染的hepG2细胞(0.91±0.25、1.16±0.23比1.98±0.31,均P＜0.05),转染72 h后蛋白水平有所上升,与对照组相比差异无统计学意义(1.71±0.18比1.98±0.31,P＞0.05).转染CXCR2-siRNA 24、48、72 h后hepG2细胞mRNA和蛋白表达差异均无统计学意义.与未转染的hepG2细胞相比,转染CXCR2-siRNA 24、48、72 h后hepG2细胞增殖受到抑制(均P＜0.05).转染CXCR2-siRNA的hepG2细胞对Transwell小室的侵袭能力明显低于未转染的hepG2细胞[(36±5)个腐倍视野(×200)比(526±9)个府倍视野(×200),P＜0.05].结论 siRNA沉默CXCR2基因体外可有效抑制肝癌细胞的增殖.
目的 研究CXCR2-小榦擾RNA(siRNA)基因體外抑製肝癌細胞增殖的作用.方法 將CXCR2-siRNA以脂質體轉染法轉染肝癌細胞hepG2,24h後熒光顯微鏡觀察細胞熒光含量.採用RT-PCR及免疫印跡法分彆檢測正常肝Chang細胞、未轉染肝癌hepG2細胞以及轉染CXCR2-siRNA基因後hepG2細胞在轉染24、48、72h後的CXCR2 mRNA和蛋白錶達水平.以未轉染的hepG2細胞為對照,CCK8法檢測轉染CXCR2-siRNA的hepG-2細胞在轉染24、48、72 h後的增殖.以未轉染的hepG2細胞為對照,檢測轉染CXCR2-siRNA 24 h後hepG2細胞與Transwell小室孵育24h時的侵襲能力.結果 CXCR2-siRNA轉染hepG2 24 h後鏡下可見大量綠色熒光,轉染率為80％.hepG2細胞CXCR2mRNA和蛋白錶達水平高于Chang細胞(mRNA:1.69±0.22比0.63±0.31,蛋白:1.93±0.25比0.84±0.29,均P＜0.05).轉染CXCR2-siRNA24和48 h後hepG2細胞CXCR2mRNA錶達量低于未轉染的hepG2細胞(0.75±0.24、0.83±0.21比1.78±0.25,均P＜0.05),72 h後CXCR2-siRNA雖然仍能抑製hepG2細胞CXCR2mRNA的錶達,但與未轉染的hepG2細胞相比差異併無統計學意義(1.35±0.18比1.78±0.25,P＞0.05).轉染CXCR2-siRNA24和48 h後的hepG2細胞CXCR2蛋白錶達低于未轉染的hepG2細胞(0.91±0.25、1.16±0.23比1.98±0.31,均P＜0.05),轉染72 h後蛋白水平有所上升,與對照組相比差異無統計學意義(1.71±0.18比1.98±0.31,P＞0.05).轉染CXCR2-siRNA 24、48、72 h後hepG2細胞mRNA和蛋白錶達差異均無統計學意義.與未轉染的hepG2細胞相比,轉染CXCR2-siRNA 24、48、72 h後hepG2細胞增殖受到抑製(均P＜0.05).轉染CXCR2-siRNA的hepG2細胞對Transwell小室的侵襲能力明顯低于未轉染的hepG2細胞[(36±5)箇腐倍視野(×200)比(526±9)箇府倍視野(×200),P＜0.05].結論 siRNA沉默CXCR2基因體外可有效抑製肝癌細胞的增殖.
목적 연구CXCR2-소간우RNA(siRNA)기인체외억제간암세포증식적작용.방법 장CXCR2-siRNA이지질체전염법전염간암세포hepG2,24h후형광현미경관찰세포형광함량.채용RT-PCR급면역인적법분별검측정상간Chang세포、미전염간암hepG2세포이급전염CXCR2-siRNA기인후hepG2세포재전염24、48、72h후적CXCR2 mRNA화단백표체수평.이미전염적hepG2세포위대조,CCK8법검측전염CXCR2-siRNA적hepG-2세포재전염24、48、72 h후적증식.이미전염적hepG2세포위대조,검측전염CXCR2-siRNA 24 h후hepG2세포여Transwell소실부육24h시적침습능력.결과 CXCR2-siRNA전염hepG2 24 h후경하가견대량록색형광,전염솔위80％.hepG2세포CXCR2mRNA화단백표체수평고우Chang세포(mRNA:1.69±0.22비0.63±0.31,단백:1.93±0.25비0.84±0.29,균P＜0.05).전염CXCR2-siRNA24화48 h후hepG2세포CXCR2mRNA표체량저우미전염적hepG2세포(0.75±0.24、0.83±0.21비1.78±0.25,균P＜0.05),72 h후CXCR2-siRNA수연잉능억제hepG2세포CXCR2mRNA적표체,단여미전염적hepG2세포상비차이병무통계학의의(1.35±0.18비1.78±0.25,P＞0.05).전염CXCR2-siRNA24화48 h후적hepG2세포CXCR2단백표체저우미전염적hepG2세포(0.91±0.25、1.16±0.23비1.98±0.31,균P＜0.05),전염72 h후단백수평유소상승,여대조조상비차이무통계학의의(1.71±0.18비1.98±0.31,P＞0.05).전염CXCR2-siRNA 24、48、72 h후hepG2세포mRNA화단백표체차이균무통계학의의.여미전염적hepG2세포상비,전염CXCR2-siRNA 24、48、72 h후hepG2세포증식수도억제(균P＜0.05).전염CXCR2-siRNA적hepG2세포대Transwell소실적침습능력명현저우미전염적hepG2세포[(36±5)개부배시야(×200)비(526±9)개부배시야(×200),P＜0.05].결론 siRNA침묵CXCR2기인체외가유효억제간암세포적증식.
Objective To investigate the inhibitory effect of siRNA-CXCR2 on in vitro proliferation of hepatocellular carcinoma.Methods CXCR2-siRNA plasmid was transfected by using the liposome technique into hepG2,the hepatocellular carcinoma cells,for the determination of fluorescence under fluorescence microscope at hour 24.The expression of CXCR2 mRNA and protein in the Chang cells derived from normal liver tissues,untransfected hepG2 cells and the hepG2 cells transfected with siRNA-CXCR2 was detected by reverse transcriptase polymerase chain reaction and Western blotting at hours 24,48 and 72,respectively.The CCK8 technique was,by using the untransfected hepG2 cells as control,employed to examine the proliferation of hepG2 cells transfected with CXCR2-siRNA at hours 24,48 and 72.The invasive capacity of the transfected hepG2 cells at hour 24 following transfection and following incubation in a Transwell chamber was assessed respectively compared with hepG2 cells.Results Following transfection,there was a preponderance of green fluorescence (80％) in hepG2 cells at hour 24 under fluorescent microscope and the transfected rate was 80％.Compared with Chang cells,the transfected hepG2 cells yielded a higher levels of CXCR2 mRNA and protein expression (1.69±0.22 vs 0.63 ±0.31 for mRNA and 1.93±0.25 vs 0.84±0.29 for protein,both P＜0.05).Compared with untransfected hepG2 cells,the transfected cells were associated with a reduced level of CXCR2 mRNA expression at hours 24 and 48 (0.75±0.24 and 0.83±0.21 vs 1.78±0.25,both P＜0.05).This held true even at hour 72,however,the level of which was not statistically different from that of untransfected cells (1.35 ±0.18 vs 1.78 ±0.25,P＞0.05).The transfected cells were,compared with untransfected cells,linked to lower level of CXCR2 protein expression at hours 24 and 48 (0.91±0.25 and 1.16±0.23 vs 1.98±0.31,P＜0.05).Despite that there was an increase in CXCR2 protein expression at hour 72,which was not markedly different from that of untransfected cells (1.71 ±0.18 vs 1.98±0.31,P＞0.05).Furthermore,there was no statistically significant difference in the mRNA and protein expression in the transfected hepG2 cells at hours 24,48 and 72.However,transfection with CXCR2-siRNA resulted in inhibition of hepG2 cell proliferation at hours 24,48 and 72 compared with untransfected cells (all P＜0.05).In addition,compared with untransfected cells,the transfeeted cells yielded a markedly attenuated capacity of invasion to the Transwell chamber [(36±5)/HP(×200) vs (526±9)/HP(×200),P＜ 0.05].Conclusion Silencing of CXCR2 genes with siRNA effectively inhibits proliferation of hepatocellular carcinoma cells.
