医学分子生物学杂志
醫學分子生物學雜誌
의학분자생물학잡지
FOREIGN MEDICAL SCIENCES
2010年
2期
99-103
,共5页
高涵%邹朝霞%刘文杰%张宏宇%高旭
高涵%鄒朝霞%劉文傑%張宏宇%高旭
고함%추조하%류문걸%장굉우%고욱
血红素加氧酶-1%肝癌%细胞周期%细胞周期调控因子
血紅素加氧酶-1%肝癌%細胞週期%細胞週期調控因子
혈홍소가양매-1%간암%세포주기%세포주기조공인자
heme oxygenase-1%hepatoma%cell cycle%cell cycle regulators
目的 观察血红素加氧酶-1(heme oxygenase 1,HO-1)对人肝癌细胞HepG2细胞周期调控因子的影响.方法 构建含有野生型和突变型HO-1基因的重组载体pcDNA3.1(+)-wtHO-1和pcDNA3.1(+)-mHO-1G143H.利用脂质体介导的方法将构建好的重组载体转染肝癌细胞系HepG2,以空载体转染作为对照组.通过G418筛选建立稳定表达野生型和突变型HO-1的HepG2肝癌细胞系.经半定量RT-PCR、Western印迹检测转染细胞系中HO-1 mRNA和蛋白的表达水平.在HO-1表达改变的稳转细胞系中,利用Western 印迹检测转染细胞系中P21、P27蛋白表达水平.结果 成功实现了野生型和突变型HO-1在HepG2细胞中的过表达;野生型和突变型HO-1过表达均能诱导抑癌基因p21和p27的表达.结论 HO-1过表达诱导抑癌基因p21和p27的表达与血红素分解产物无关.HO-1可能通过其它机制调节p21和p27的表达.
目的 觀察血紅素加氧酶-1(heme oxygenase 1,HO-1)對人肝癌細胞HepG2細胞週期調控因子的影響.方法 構建含有野生型和突變型HO-1基因的重組載體pcDNA3.1(+)-wtHO-1和pcDNA3.1(+)-mHO-1G143H.利用脂質體介導的方法將構建好的重組載體轉染肝癌細胞繫HepG2,以空載體轉染作為對照組.通過G418篩選建立穩定錶達野生型和突變型HO-1的HepG2肝癌細胞繫.經半定量RT-PCR、Western印跡檢測轉染細胞繫中HO-1 mRNA和蛋白的錶達水平.在HO-1錶達改變的穩轉細胞繫中,利用Western 印跡檢測轉染細胞繫中P21、P27蛋白錶達水平.結果 成功實現瞭野生型和突變型HO-1在HepG2細胞中的過錶達;野生型和突變型HO-1過錶達均能誘導抑癌基因p21和p27的錶達.結論 HO-1過錶達誘導抑癌基因p21和p27的錶達與血紅素分解產物無關.HO-1可能通過其它機製調節p21和p27的錶達.
목적 관찰혈홍소가양매-1(heme oxygenase 1,HO-1)대인간암세포HepG2세포주기조공인자적영향.방법 구건함유야생형화돌변형HO-1기인적중조재체pcDNA3.1(+)-wtHO-1화pcDNA3.1(+)-mHO-1G143H.이용지질체개도적방법장구건호적중조재체전염간암세포계HepG2,이공재체전염작위대조조.통과G418사선건립은정표체야생형화돌변형HO-1적HepG2간암세포계.경반정량RT-PCR、Western인적검측전염세포계중HO-1 mRNA화단백적표체수평.재HO-1표체개변적은전세포계중,이용Western 인적검측전염세포계중P21、P27단백표체수평.결과 성공실현료야생형화돌변형HO-1재HepG2세포중적과표체;야생형화돌변형HO-1과표체균능유도억암기인p21화p27적표체.결론 HO-1과표체유도억암기인p21화p27적표체여혈홍소분해산물무관.HO-1가능통과기타궤제조절p21화p27적표체.
Objective To elucidate the effect of heme oxygenase-1 on cell cycle regulator in HepG2 cell line.Methods To construct eukaryotic expression vectors expressing wild-type HO-1 and G143H mutant HO-1,named pcDNA3.1 (+) -wtHO-1 and pcDNA3.1 (+) - mHO-1G143H. HepG2 cells were transfected with wtHO-1 and mHO-1 using lipofectamine 2000. Stable transfected cells were selected by G418. Expression of HO-1 mRNA and protein were detected using RT-PCR and Western blot respectively. In addition,expression of p21 and p27,the major cyclin-dependent kinase(Cdk)inhibitors in the two cell lines were examined.Results HepG2 cell lines with wild type and mutant HO-1 overexpression were established successfully. Overexpression of both wild type and mutant HO-1 enhanced the expression of tumor suppressor P21 and P27.Conclusion HO-1 overexpression upregulated the level of P21 and P27. This regulation is not dependent of HO-1 catalytic activity,but probably other mechanisms.
