中华实验和临床病毒学杂志
中華實驗和臨床病毒學雜誌
중화실험화림상병독학잡지
CHINESE JOURNAL OF EXPERIMENTAL AND CLINICAL VIROLOGY
2013年
5期
344-347
,共4页
肝炎病毒,乙型%基因型%细胞凋亡%癌基因蛋白质类,病毒性
肝炎病毒,乙型%基因型%細胞凋亡%癌基因蛋白質類,病毒性
간염병독,을형%기인형%세포조망%암기인단백질류,병독성
Hepatitis B virus%Genotype%Apoptosis%Oncogeme proteins,viral
目的 探讨B、C基因型HBVx蛋白(HBx)对肝癌细胞凋亡的影响 方法 采用PCR法扩增B、C基因型HBV X基因片段,并定向插入绿色荧光蛋白(GFP)真核表达载体构建重组体pGFP-XB及pGFP-XC;使用FugeneHD将载体pEGFP-C1、pGFP-XB及pGFP-XC转染Bel-7402细胞,RT-PCR及Western Blot鉴定HBV X基因的转录与表达,以建立稳定表达细胞株Bel-7402/GFP、Bel-7402/GFP-XB、Bel-7402/GFP-XC,用阿霉素(2.5 μg/ml)分别处理上述细胞,处理后用锥虫蓝染色法和流式细胞术检测各组细胞凋亡率.结果 RT-PCR及Western Blot检测显示在Bel-7402/GFP-XB、Bel-7402/GFP-XC有HBV X基因转录与表达.锥虫蓝染色检测表明阿霉素处理的Bel-7402、Bel-7402/GFP细胞发生了明显的时间依赖性死亡,而在Bel-7402/GFP-XB、Bel-7402/GFP-XC和未加阿霉素处理的空白对照组细胞未见明显细胞死亡；流式细胞术检测显示阿霉素处理48 h后,Bel-7402/GFP-XB、Bel-7402/GFP-XC细胞的凋亡率分别为3.87％、4.01％,两者差异无统计学意义(P＞0.05),且与未处理的空白对照组细胞凋亡率(2.74％、2.91％、3.00％、2.83％)差异无统计学意义(P＞0.05),但明显低于加入阿霉素处理的Bel-7402细胞(27.05％)及Bel-7402/GFP细胞(29.14％)(P＜0.01).结论 成功建立了稳定表达B、C基因型HBV X与GFP融合蛋白的人肝癌细胞系,为进一步研究HBV X的生物学功能提供了基础.在人肝癌细胞,B、C基因型HBV X蛋白均能抑制阿霉素诱导的细胞凋亡,其抑制细胞凋亡的作用无明显差异.
目的 探討B、C基因型HBVx蛋白(HBx)對肝癌細胞凋亡的影響 方法 採用PCR法擴增B、C基因型HBV X基因片段,併定嚮插入綠色熒光蛋白(GFP)真覈錶達載體構建重組體pGFP-XB及pGFP-XC;使用FugeneHD將載體pEGFP-C1、pGFP-XB及pGFP-XC轉染Bel-7402細胞,RT-PCR及Western Blot鑒定HBV X基因的轉錄與錶達,以建立穩定錶達細胞株Bel-7402/GFP、Bel-7402/GFP-XB、Bel-7402/GFP-XC,用阿黴素(2.5 μg/ml)分彆處理上述細胞,處理後用錐蟲藍染色法和流式細胞術檢測各組細胞凋亡率.結果 RT-PCR及Western Blot檢測顯示在Bel-7402/GFP-XB、Bel-7402/GFP-XC有HBV X基因轉錄與錶達.錐蟲藍染色檢測錶明阿黴素處理的Bel-7402、Bel-7402/GFP細胞髮生瞭明顯的時間依賴性死亡,而在Bel-7402/GFP-XB、Bel-7402/GFP-XC和未加阿黴素處理的空白對照組細胞未見明顯細胞死亡；流式細胞術檢測顯示阿黴素處理48 h後,Bel-7402/GFP-XB、Bel-7402/GFP-XC細胞的凋亡率分彆為3.87％、4.01％,兩者差異無統計學意義(P＞0.05),且與未處理的空白對照組細胞凋亡率(2.74％、2.91％、3.00％、2.83％)差異無統計學意義(P＞0.05),但明顯低于加入阿黴素處理的Bel-7402細胞(27.05％)及Bel-7402/GFP細胞(29.14％)(P＜0.01).結論 成功建立瞭穩定錶達B、C基因型HBV X與GFP融閤蛋白的人肝癌細胞繫,為進一步研究HBV X的生物學功能提供瞭基礎.在人肝癌細胞,B、C基因型HBV X蛋白均能抑製阿黴素誘導的細胞凋亡,其抑製細胞凋亡的作用無明顯差異.
목적 탐토B、C기인형HBVx단백(HBx)대간암세포조망적영향 방법 채용PCR법확증B、C기인형HBV X기인편단,병정향삽입록색형광단백(GFP)진핵표체재체구건중조체pGFP-XB급pGFP-XC;사용FugeneHD장재체pEGFP-C1、pGFP-XB급pGFP-XC전염Bel-7402세포,RT-PCR급Western Blot감정HBV X기인적전록여표체,이건립은정표체세포주Bel-7402/GFP、Bel-7402/GFP-XB、Bel-7402/GFP-XC,용아매소(2.5 μg/ml)분별처리상술세포,처리후용추충람염색법화류식세포술검측각조세포조망솔.결과 RT-PCR급Western Blot검측현시재Bel-7402/GFP-XB、Bel-7402/GFP-XC유HBV X기인전록여표체.추충람염색검측표명아매소처리적Bel-7402、Bel-7402/GFP세포발생료명현적시간의뢰성사망,이재Bel-7402/GFP-XB、Bel-7402/GFP-XC화미가아매소처리적공백대조조세포미견명현세포사망；류식세포술검측현시아매소처리48 h후,Bel-7402/GFP-XB、Bel-7402/GFP-XC세포적조망솔분별위3.87％、4.01％,량자차이무통계학의의(P＞0.05),차여미처리적공백대조조세포조망솔(2.74％、2.91％、3.00％、2.83％)차이무통계학의의(P＞0.05),단명현저우가입아매소처리적Bel-7402세포(27.05％)급Bel-7402/GFP세포(29.14％)(P＜0.01).결론 성공건립료은정표체B、C기인형HBV X여GFP융합단백적인간암세포계,위진일보연구HBV X적생물학공능제공료기출.재인간암세포,B、C기인형HBV X단백균능억제아매소유도적세포조망,기억제세포조망적작용무명현차이.
Objective To investigate the apoptosis regulation on hepatoma cells by HBx between genotype B and C.Methods Genotype B and C HBx gene fragments were amplified and inserted into green fluorescent protein(GFP) eukaryotic expression vector pEGFP-C1 to construct recombinant pGFP-XB and pGFP-XC.The pEGFP-C1,pGFP-XB and pGFP-XC were introduced into Bel-7402 cells by Fugene HD to obtain Bel-7402 cells expressing GFP.The transcription and expression of HBx gene were demonstrated by RT-PCR and Western Blot analysis.Bel-7402,Bel-7402/GFP,Bel-7402/GFP-XB,Bel-7402/GFP-XC cells were treated with adriamycin(2.5 μg/ml),and the apoptosis of the cells was determined by trypan blue exclusion,and flow cytometry analysis.Results RT-PCR and Western Blot analysis showed that HBx genes of genotypes B and C were transcribed and expressed in Bel-7402/GFP-XB,Bel-7402/GFP-XC cells.Trypan blue exclusion showed adriamycin induced time-dependent cell death in Bel-7402,Bel-7402/GFP cells while no significant cell death was observed in Bel-7402/GFP-XB,Bel-7402/GFP-XC cells.Flow cytometry analysis indicated that no significant differences of apoptosis rates of Bel-7402/GFP-XB (3.87％) and of Bel7402/GFP-XC(4.01％) were observed (P ＞ 0.05),moreover,no significant differences of Bel-7402/GFP-XB(3.87％),Bel7402/GFP-XC (4.01％)and of the untreated cells.Apoptosis rates in Bel-7402/GFP-XB (3.87％),Bel-7402/GFP-XC (4.01％)cells were significantly lower than those in Bel-7402 (27.05％) and Bel-7402/GFP (29.14％) cells at 48 hours after the adriamycin treatment (P ＜ 0.01).Conclusions Bel-7402 cell lines expressing GFP,GFP-XB and GFP-XC fusion proteins were successfully established.HBV X protein blocks adriamycin-induced apoptosis of Bel-7402 cells.There is no difference between HBx of genotype B and C in inhibiting apoptosis induced by adriamycin.
