中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
Chinese Journal of Immunology
2015年
11期
1476-1485
,共10页
石庆强%左国伟%冯子强%赵绿翠%罗念%游智梅%夏菁%李丹阳%李静%陈地龙
石慶彊%左國偉%馮子彊%趙綠翠%囉唸%遊智梅%夏菁%李丹暘%李靜%陳地龍
석경강%좌국위%풍자강%조록취%라념%유지매%하정%리단양%리정%진지룡
人参皂苷Rh2%HepG2%Gsk-3β%β-catenin
人參皂苷Rh2%HepG2%Gsk-3β%β-catenin
인삼조감Rh2%HepG2%Gsk-3β%β-catenin
Rh2%HepG2,Gsk-3β%β-catenin
目的：研究人参皂苷Rh2对HepG2细胞的作用机制。方法：用pLOV-EF1a-MCS-3FLAG-β-catenin慢病毒感染HepG2细胞，运用荧光显微镜观察细胞荧光强度变化。通过CCK-8法检测细胞增殖反应。采用FCM检测细胞周期及凋亡变化；利用ELISA法检测细胞产生Gsk-3β活性；用PCR法检测细胞Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3基因的表达；用CHIP法检测细胞Bcl2、CyclinD1、Bax、MMP3基因的表达；运用Western blot法检测细胞Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3蛋白的表达。结果：用pLOV-EF1a-MCS-3FLAG-β-catenin慢病毒感染HepG2细胞，被感染的HepG2细胞命名为HepG2-β-catenin；CCK-8分析结果显示，加药组给予（10～160μmol／L） Rh2后HepG2及HepG2-β-catenin细胞的增殖受到抑制，且Rh2对肝癌HepG2-β-catenin和HepG2细胞的生长抑制呈剂量和时间依赖。在HepG2细胞中48、72 h半数抑制率分别为100μmol／L，58.12μmol／L，在HepG2-β-catenin细胞中48、72 h半数抑制率分别是129.2μmol／L，83.33μmol／L，故Rh2作用于HepG2-β-catenin细胞浓度均高于HepG2细胞，与HepG2细胞组比较差异具有统计学意义（P＜0.01）。 FCM检测结果显示，Rh2可诱导HepG2和HepG2-β-catenin细胞周期阻滞在G0／G1期，HepG2＋Rh2组G0／G1期（64.57±0.65），而HepG2-β-catenin＋Rh2组G0／G1期（58.61±2.01）；FCM检测结果显示，Rh2可诱导HepG2和HepG2-β-catenin细胞早期凋亡，HepG2＋Rh2组凋亡率（17.27±2.77），而HepG2-β-catenin＋Rh2组凋亡率（9.02±1.76）。 ELISA结果显示，Rh2作用HepG2细胞12、24、48、72 h后，Gsk-3β的活性随着作用时间延长，逐渐升高，在48 h最高，随后其活性开始降低。 Rh2诱导HepG2和HepG2-β-catenin细胞48 h，与对照组相比，Gsk-3β的活性均增高，而加入Bio后其活性降低，HepG2＋Rh和HepG2-β-catenin＋Rh2组间无明显差异；PCR、CHIP、WB结果显示，人参皂苷Rh2诱导HepG2和HepG2-β-catenin细胞后，Gsk-3β、Bax基因及蛋白表达增加，而β-catenin、CyclinD1、Bcl2、MMP3基因及蛋白表达水平下调。与HepG2-β-catenin＋Rh2组相比，HepG2＋Rh2组除Gsk-3β外各基因及蛋白的表达变化更为显著。结论：过表达β-catenin可削弱人参皂苷Rh2对肝癌HepG2细胞的药理作用。人参皂苷Rh2通过激活Gsk-3β降解β-catenin，影响下游基因的表达，促进肝癌细胞的凋亡及抑制其转移。
目的：研究人參皂苷Rh2對HepG2細胞的作用機製。方法：用pLOV-EF1a-MCS-3FLAG-β-catenin慢病毒感染HepG2細胞，運用熒光顯微鏡觀察細胞熒光彊度變化。通過CCK-8法檢測細胞增殖反應。採用FCM檢測細胞週期及凋亡變化；利用ELISA法檢測細胞產生Gsk-3β活性；用PCR法檢測細胞Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3基因的錶達；用CHIP法檢測細胞Bcl2、CyclinD1、Bax、MMP3基因的錶達；運用Western blot法檢測細胞Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3蛋白的錶達。結果：用pLOV-EF1a-MCS-3FLAG-β-catenin慢病毒感染HepG2細胞，被感染的HepG2細胞命名為HepG2-β-catenin；CCK-8分析結果顯示，加藥組給予（10～160μmol／L） Rh2後HepG2及HepG2-β-catenin細胞的增殖受到抑製，且Rh2對肝癌HepG2-β-catenin和HepG2細胞的生長抑製呈劑量和時間依賴。在HepG2細胞中48、72 h半數抑製率分彆為100μmol／L，58.12μmol／L，在HepG2-β-catenin細胞中48、72 h半數抑製率分彆是129.2μmol／L，83.33μmol／L，故Rh2作用于HepG2-β-catenin細胞濃度均高于HepG2細胞，與HepG2細胞組比較差異具有統計學意義（P＜0.01）。 FCM檢測結果顯示，Rh2可誘導HepG2和HepG2-β-catenin細胞週期阻滯在G0／G1期，HepG2＋Rh2組G0／G1期（64.57±0.65），而HepG2-β-catenin＋Rh2組G0／G1期（58.61±2.01）；FCM檢測結果顯示，Rh2可誘導HepG2和HepG2-β-catenin細胞早期凋亡，HepG2＋Rh2組凋亡率（17.27±2.77），而HepG2-β-catenin＋Rh2組凋亡率（9.02±1.76）。 ELISA結果顯示，Rh2作用HepG2細胞12、24、48、72 h後，Gsk-3β的活性隨著作用時間延長，逐漸升高，在48 h最高，隨後其活性開始降低。 Rh2誘導HepG2和HepG2-β-catenin細胞48 h，與對照組相比，Gsk-3β的活性均增高，而加入Bio後其活性降低，HepG2＋Rh和HepG2-β-catenin＋Rh2組間無明顯差異；PCR、CHIP、WB結果顯示，人參皂苷Rh2誘導HepG2和HepG2-β-catenin細胞後，Gsk-3β、Bax基因及蛋白錶達增加，而β-catenin、CyclinD1、Bcl2、MMP3基因及蛋白錶達水平下調。與HepG2-β-catenin＋Rh2組相比，HepG2＋Rh2組除Gsk-3β外各基因及蛋白的錶達變化更為顯著。結論：過錶達β-catenin可削弱人參皂苷Rh2對肝癌HepG2細胞的藥理作用。人參皂苷Rh2通過激活Gsk-3β降解β-catenin，影響下遊基因的錶達，促進肝癌細胞的凋亡及抑製其轉移。
목적：연구인삼조감Rh2대HepG2세포적작용궤제。방법：용pLOV-EF1a-MCS-3FLAG-β-catenin만병독감염HepG2세포，운용형광현미경관찰세포형광강도변화。통과CCK-8법검측세포증식반응。채용FCM검측세포주기급조망변화；이용ELISA법검측세포산생Gsk-3β활성；용PCR법검측세포Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3기인적표체；용CHIP법검측세포Bcl2、CyclinD1、Bax、MMP3기인적표체；운용Western blot법검측세포Gsk-3β、β-catenin、Bcl2、CyclinD1、Bax、MMP3단백적표체。결과：용pLOV-EF1a-MCS-3FLAG-β-catenin만병독감염HepG2세포，피감염적HepG2세포명명위HepG2-β-catenin；CCK-8분석결과현시，가약조급여（10～160μmol／L） Rh2후HepG2급HepG2-β-catenin세포적증식수도억제，차Rh2대간암HepG2-β-catenin화HepG2세포적생장억제정제량화시간의뢰。재HepG2세포중48、72 h반수억제솔분별위100μmol／L，58.12μmol／L，재HepG2-β-catenin세포중48、72 h반수억제솔분별시129.2μmol／L，83.33μmol／L，고Rh2작용우HepG2-β-catenin세포농도균고우HepG2세포，여HepG2세포조비교차이구유통계학의의（P＜0.01）。 FCM검측결과현시，Rh2가유도HepG2화HepG2-β-catenin세포주기조체재G0／G1기，HepG2＋Rh2조G0／G1기（64.57±0.65），이HepG2-β-catenin＋Rh2조G0／G1기（58.61±2.01）；FCM검측결과현시，Rh2가유도HepG2화HepG2-β-catenin세포조기조망，HepG2＋Rh2조조망솔（17.27±2.77），이HepG2-β-catenin＋Rh2조조망솔（9.02±1.76）。 ELISA결과현시，Rh2작용HepG2세포12、24、48、72 h후，Gsk-3β적활성수착작용시간연장，축점승고，재48 h최고，수후기활성개시강저。 Rh2유도HepG2화HepG2-β-catenin세포48 h，여대조조상비，Gsk-3β적활성균증고，이가입Bio후기활성강저，HepG2＋Rh화HepG2-β-catenin＋Rh2조간무명현차이；PCR、CHIP、WB결과현시，인삼조감Rh2유도HepG2화HepG2-β-catenin세포후，Gsk-3β、Bax기인급단백표체증가，이β-catenin、CyclinD1、Bcl2、MMP3기인급단백표체수평하조。여HepG2-β-catenin＋Rh2조상비，HepG2＋Rh2조제Gsk-3β외각기인급단백적표체변화경위현저。결론：과표체β-catenin가삭약인삼조감Rh2대간암HepG2세포적약리작용。인삼조감Rh2통과격활Gsk-3β강해β-catenin，영향하유기인적표체，촉진간암세포적조망급억제기전이。
Objective: To investigate the inhibitory effect of Rh2 on HepG2 cells and explore the underlying mechanism.Methods: We used lentivirus carrying β-catenin to infect HepG2 cell, and detected expression of β-catenin using fluorescence microscopy.The effect of Rh2 on proliferation of HepG2-β-catenin and HepG2 cells was measured by CKK-8 assay,and flow cytometry was used to detect cell cycle and apoptosis.The activity of Gsk-3βwas checked by ELISA kit.The expression of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 genes were measured by qRT-PCR.In order to checked the relationship between β-catenin and TCF4,CHIP assay kit was used,the expression of Bax,Bcl2,CyclinD1,MMP3 genes were measured by PCR.The expressions of Gsk-3β,β-catenin,Bax,Bcl2,CyclinD1,MMP3 proteins were examined by Western blot.Results:HepG2 cells were successfully infected by pLOV-EF1a-MCS-3FLAG-β-catenin lentivirus,named HepG2-β-catenin.CCK-8 showed that ginsenoside Rh2 could effectively inhibit the proliferation of HepG2 and HepG2-β-catenin cells in vitro,which exhibits a dose-dependent manner at range of 10-160 μmol/L Rh2.The IC50 of Rh2 exposure on HepG2 cell for 48,72 h were 100 μmol/L and 58.12 μmol/L,but the IC50 of Rh2 exposure on HepG2-β-catenin for 48,72 h were 129.2 μmol/L,83.33 μmol/L,respectively.The IC50 of Rh2 exposure on HepG2-β-catenin cell was higher than HepG2 cell, compared with HepG2 group the differences was statistically significant ( P<0.01 ).Flow cytometry indicated that Rh2 could arrest HepG2 and HepG2-β-catenin cells in G0/G1 phase;the cell population in G0/G1 phase of HepG2+Rh2 group was(64.57±0.65)%,HepG2-β-catenin+Rh2 group was(58.61±2.01)%.Flow cytometry indicated that Rh2 could induced early apoptosis in HepG2 and HepG2-β-catenin cells.The apoptosis rate of HepG2 +Rh2 group was (17.27 ±2.77)%,HepG2-β-catenin +Rh2 group(9.02 ±1.76)%.The ELISA results indicated that HepG2 cells was induced by Rh2 for 12,24,48,72 h,the activity of Gsk-3βgradually increased,peak in 48 h,then decreased.Compared with control group,Rh2 induced HepG2 and HepG2-β-catenin cells for 48 hours, Gsk-3βactivity were increased, and their activity reduced after adding Bio, there were no significant differences between HepG2+Rh2 and HepG2-β-catenin+Rh2 groups.The PCR,CHIP and WB results showed that the expression of Gsk-3β,Bax gene and proteins increased,while theβ-catenin,CyclinD1,Bcl2,MMP3 gene and proteins down-regulation in HepG2 and HepG2-β-catenin cell induced by Rh2.Compared with HepG2-β-catenin +Rh2 group, the expression of other gene and proteins changed significantly,however,Gsk-3βwas no significant difference.Conclusion:Over-expression of β-catenin may weaken the phar-macological effects of ginsenoside Rh2 on HepG2 cells.The activity of Gsk-3βwas increased by ginsenoside Rh2 to degradeβ-catenin, affecting the expression of downstream genes,promoting apoptosis of liver cancer cells and inhibiting metastasis.
