中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
Chinese Journal of Experimental Surgery
2015年
10期
2338-2341
,共4页
熊志勇%姚志成%李明亮%颜见%胡昆鹏%范伟明%许瑞云%邓美海
熊誌勇%姚誌成%李明亮%顏見%鬍昆鵬%範偉明%許瑞雲%鄧美海
웅지용%요지성%리명량%안견%호곤붕%범위명%허서운%산미해
乙型肝炎病毒X蛋白%癌,肝细胞%化疗耐药%联合位点突变
乙型肝炎病毒X蛋白%癌,肝細胞%化療耐藥%聯閤位點突變
을형간염병독X단백%암,간세포%화료내약%연합위점돌변
Hepatitis B virus X protein%Carcinoma,hepatocellular%Chemotherapy resistance%Combo mutation
目的 观察联合位点突变的乙型肝炎病毒x蛋白对肝癌耐药性的影响,探讨乙型肝炎病毒在肝癌耐药机制中的作用.方法 通过基因合成A1762T/G1764A/T1753A/T1768A位点联合突变后的乙型肝炎病毒x蛋白(HBx),慢病毒感染肝癌细胞株Huh7并构建稳定表达HBx蛋白的肝癌细胞,细胞计数试剂盒(CCK-8)法检测转染后肝癌细胞对不同浓度化疗药物顺铂(15、30、60 mg/L)的耐药性,实时荧光定量聚合酶链反应(FQ-PCR)及Western blot法分别检测转染后肝癌细胞多重耐药基因-1(MDR-1) mRNA及蛋白的表达.结果 经嘌呤毒素药物筛选后,可构建出稳定表达株,转染率达90％以上;在不同浓度顺铂(15、30、60 mg/L)作用下,转染后肝癌细胞存活率为84.6％、76.7％、98.6％,明显高于对照组(57.7％、72.8％、77.3％)和空载组(55.0％、70.6％、78.9％),差异有统计学意义(P＜0.05),而对照组细胞存活率与空载组比较,差异无统计学差异(P＞0.05).转染后细胞内MDR-1 mRNA表达明显高于对照组和空载组,差异有统计学意义(P＜0.05),MDR-1蛋白表达明显增高.结论 联合位点突变的乙型肝炎病毒x蛋白可通过上调肝癌细胞内MDR-1的表达提高其化疗抵抗性.
目的 觀察聯閤位點突變的乙型肝炎病毒x蛋白對肝癌耐藥性的影響,探討乙型肝炎病毒在肝癌耐藥機製中的作用.方法 通過基因閤成A1762T/G1764A/T1753A/T1768A位點聯閤突變後的乙型肝炎病毒x蛋白(HBx),慢病毒感染肝癌細胞株Huh7併構建穩定錶達HBx蛋白的肝癌細胞,細胞計數試劑盒(CCK-8)法檢測轉染後肝癌細胞對不同濃度化療藥物順鉑(15、30、60 mg/L)的耐藥性,實時熒光定量聚閤酶鏈反應(FQ-PCR)及Western blot法分彆檢測轉染後肝癌細胞多重耐藥基因-1(MDR-1) mRNA及蛋白的錶達.結果 經嘌呤毒素藥物篩選後,可構建齣穩定錶達株,轉染率達90％以上;在不同濃度順鉑(15、30、60 mg/L)作用下,轉染後肝癌細胞存活率為84.6％、76.7％、98.6％,明顯高于對照組(57.7％、72.8％、77.3％)和空載組(55.0％、70.6％、78.9％),差異有統計學意義(P＜0.05),而對照組細胞存活率與空載組比較,差異無統計學差異(P＞0.05).轉染後細胞內MDR-1 mRNA錶達明顯高于對照組和空載組,差異有統計學意義(P＜0.05),MDR-1蛋白錶達明顯增高.結論 聯閤位點突變的乙型肝炎病毒x蛋白可通過上調肝癌細胞內MDR-1的錶達提高其化療牴抗性.
목적 관찰연합위점돌변적을형간염병독x단백대간암내약성적영향,탐토을형간염병독재간암내약궤제중적작용.방법 통과기인합성A1762T/G1764A/T1753A/T1768A위점연합돌변후적을형간염병독x단백(HBx),만병독감염간암세포주Huh7병구건은정표체HBx단백적간암세포,세포계수시제합(CCK-8)법검측전염후간암세포대불동농도화료약물순박(15、30、60 mg/L)적내약성,실시형광정량취합매련반응(FQ-PCR)급Western blot법분별검측전염후간암세포다중내약기인-1(MDR-1) mRNA급단백적표체.결과 경표령독소약물사선후,가구건출은정표체주,전염솔체90％이상;재불동농도순박(15、30、60 mg/L)작용하,전염후간암세포존활솔위84.6％、76.7％、98.6％,명현고우대조조(57.7％、72.8％、77.3％)화공재조(55.0％、70.6％、78.9％),차이유통계학의의(P＜0.05),이대조조세포존활솔여공재조비교,차이무통계학차이(P＞0.05).전염후세포내MDR-1 mRNA표체명현고우대조조화공재조,차이유통계학의의(P＜0.05),MDR-1단백표체명현증고.결론 연합위점돌변적을형간염병독x단백가통과상조간암세포내MDR-1적표체제고기화료저항성.
Objective To observed the effecof combo mutation of hepatitiviruX protein on drug resistance.MethodWe synthesized combo mutation of hepatitiviruX protein with A1762T/G1764A/T1753A/T1768gene locumutation, then transfected iinto hepatocellulacarcinom(HCC) cell line (Huh7) so ato construchepatocellulacarcinomcell line which stably expressed HBx protein.Cell counting kit-8 (CCK-8) method waapplied to assay HCcellto differendensity chemotherapeutiresistance (15, 30, and 60 mg/L).Real-time fluorescenquantitative polymerase chain reaction (FQ-PCR) and Western blotting were used to detecthe expression of multidrug resistance gene-1 (MDR-1) mRNand protein transfected in hepatomcellrespectively.ResultThe transfection rate of Huh7 cellwhich stably expressed HBx protein wamore than 90％.When Huh7 cellwere exposed in differenconcentrationof cisplatin (15, 30 and 60 mg/L), the survival rate of transfected hepatomcellwa84.6％, 76.7％, and 98.6％ respectively, which wasignificantly highethan in control group (57.7％, 72.8％, and 77.3％ respectively) and empty vectogroup (55.0％, 70.6％, and 78.9％ respectively) (P ＜ 0.05), buthere wano significandifference between the control group and empty vectogroup (P ＞ 0.05).The expression of MDR-1 mRNand protein waincreased significantly in the transfected hepatomcellcompared to the control group and empty vectogroup cells.Conclusion The combo mutation of hepatitiviruX protein can increase the chemotherapy resistance by increasing the expression of MDR-1 in Huh7 cells.
