中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2009年
7期
394-397
,共4页
潘小平%杜维波%盛国平%虞晓鹏%喻成波%吕国良%曹红翠%李君%章益民%陈瑜%李兰娟
潘小平%杜維波%盛國平%虞曉鵬%喻成波%呂國良%曹紅翠%李君%章益民%陳瑜%李蘭娟
반소평%두유파%성국평%우효붕%유성파%려국량%조홍취%리군%장익민%진유%리란연
肝,人工%细胞系%猴病毒40%抗原,病毒,肿瘤%肝细胞%细胞色素P450酶系统
肝,人工%細胞繫%猴病毒40%抗原,病毒,腫瘤%肝細胞%細胞色素P450酶繫統
간,인공%세포계%후병독40%항원,병독,종류%간세포%세포색소P450매계통
Liver,artificial%Cell line%Simian virus 40%Antigens,viral,tumor%Hepatocytes%Cytochrome P-450 enzyme system
目的 建立永生化的人肝细胞系,为生物人工肝、肝细胞移植、体外药物代谢等提供细胞模型.方法 应用胶原酶等四步灌流法分离获得原代人肝细胞,以含有SV40大T抗原(SV40LT)的重组反转录病毒对原代人肝细胞进行永生化,并对永生化人肝细胞进行生物学特性鉴定.结果原代人肝细胞经含有SV40 LT的重组反转录病毒感染3～4周后,获得2株永生化人肝细胞系,分别命名为HepLi2和HepLi3,永生化人肝细胞在相差显微镜下具有典型的肝细胞形态.Western印迹显示有SV40 LT基因的蛋白表达;RT-PCR显示永生化人肝细胞有Alb、谷胱甘肽S转移酶(GSTp)、人凝血因子X(HBCF-X)和β-actin mRNA表达,有细胞色素(CY)P450亚型(CYP3A5、CYP2E1、CYP2C8-19、CYP3A4)mRNA的表达;在HepILi2和HepLi3永生化人肝细胞的培养上清液中可检测到ALT、乳酸脱氢酶(LDH)和Alb.结论 新建立的永生化人肝细胞系具有正常人肝细胞的生物学特性以及完善的肝细胞CYP450酶功能,可进一步用于肝细胞药物代谢研究.
目的 建立永生化的人肝細胞繫,為生物人工肝、肝細胞移植、體外藥物代謝等提供細胞模型.方法 應用膠原酶等四步灌流法分離穫得原代人肝細胞,以含有SV40大T抗原(SV40LT)的重組反轉錄病毒對原代人肝細胞進行永生化,併對永生化人肝細胞進行生物學特性鑒定.結果原代人肝細胞經含有SV40 LT的重組反轉錄病毒感染3～4週後,穫得2株永生化人肝細胞繫,分彆命名為HepLi2和HepLi3,永生化人肝細胞在相差顯微鏡下具有典型的肝細胞形態.Western印跡顯示有SV40 LT基因的蛋白錶達;RT-PCR顯示永生化人肝細胞有Alb、穀胱甘肽S轉移酶(GSTp)、人凝血因子X(HBCF-X)和β-actin mRNA錶達,有細胞色素(CY)P450亞型(CYP3A5、CYP2E1、CYP2C8-19、CYP3A4)mRNA的錶達;在HepILi2和HepLi3永生化人肝細胞的培養上清液中可檢測到ALT、乳痠脫氫酶(LDH)和Alb.結論 新建立的永生化人肝細胞繫具有正常人肝細胞的生物學特性以及完善的肝細胞CYP450酶功能,可進一步用于肝細胞藥物代謝研究.
목적 건립영생화적인간세포계,위생물인공간、간세포이식、체외약물대사등제공세포모형.방법 응용효원매등사보관류법분리획득원대인간세포,이함유SV40대T항원(SV40LT)적중조반전록병독대원대인간세포진행영생화,병대영생화인간세포진행생물학특성감정.결과원대인간세포경함유SV40 LT적중조반전록병독감염3～4주후,획득2주영생화인간세포계,분별명명위HepLi2화HepLi3,영생화인간세포재상차현미경하구유전형적간세포형태.Western인적현시유SV40 LT기인적단백표체;RT-PCR현시영생화인간세포유Alb、곡광감태S전이매(GSTp)、인응혈인자X(HBCF-X)화β-actin mRNA표체,유세포색소(CY)P450아형(CYP3A5、CYP2E1、CYP2C8-19、CYP3A4)mRNA적표체;재HepILi2화HepLi3영생화인간세포적배양상청액중가검측도ALT、유산탈경매(LDH)화Alb.결론 신건립적영생화인간세포계구유정상인간세포적생물학특성이급완선적간세포CYP450매공능,가진일보용우간세포약물대사연구.
Objective To establish immortalized human hepatocyte lines for studies of bioartificial liver,hepatocyte transplantation,and drug metabolism in vitro.Methods Primary human hepatocytes were isolated by 4-step perfusion technique with collagenase and transfected with recombinant retrovirus containing Simian virus 40 large T antigen(SV40 LT).Subsequently,immortalized human hepatocytes were evaluated by analysis of gene expression and functional characteristics in vitro.Results Two immortalized human hepatocyte lines,HepLi2 and HepLi3,were obtained after primary human hepatocytes being infected by SV40 LT containing recombinant retrovirus for 3-4 weeks.The immortalized human hepatocytes showed classical appearance of hepatocyte observed by phase contrast microscope.The protein expression of SV40 LT in HepLi2 and HepLi3 cells were detected by Western blotting.The mRNA expressions of albumin(Alb),glutathione S-transferase(GST-p),human blood coagulation factor X(HBCF-X)and β-actin in HepLi2 and HepLi3 cells were detected by reverse transcription polymerase chain reaction(RT-PCR),and the mRNA expressions of cytochrome(CY)450 subtypes(CYP3A5,CYP2E1,CYP2C8-19 and CYP3A4)in HepLi2 and HepLi3 cells were also observed by RT-PCR.Levels of alanine transaminase (ALT),lactate dehydrogenase(LDH)and Alb were detected in the supernatant of immortalized human hepatoeyte culture.Conclusions The immortalized human hepatocyte lines have the biological characteristics of primary human hepatocytes and have the CYP450 functions of hepatocytes,which may be heIDful for the studies of bioartificial liver,heoatocvte transplantation and drug metabolism in vitro.
