AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymer...AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.展开更多
目的:建立小鼠腔前卵泡体外海藻酸盐包埋培养的三维培养(3-dimensions in vitro growth,3D-IVG)方法以及成熟卵母细胞体外受精体系。方法:采用机械法分离12日龄雌性昆明小鼠卵巢,获取结构完整的腔前卵泡,腔前卵泡经过包埋后体外立体化培...目的:建立小鼠腔前卵泡体外海藻酸盐包埋培养的三维培养(3-dimensions in vitro growth,3D-IVG)方法以及成熟卵母细胞体外受精体系。方法:采用机械法分离12日龄雌性昆明小鼠卵巢,获取结构完整的腔前卵泡,腔前卵泡经过包埋后体外立体化培养,激素超排,收集黏液化的卵丘-卵母细胞复合体(cumulus-oocyte-complex,COCs),分别计数发泡期(germinal vesicle,GV)卵母细胞、生发泡破裂期(germinal vesicle breakdown,GVBD)卵母细胞和含有第一极体(first polar body,PB)卵母细胞,制备卵母细胞染色体并进行C带染色分析。同时设小鼠体内超排卵母细胞(in vivo convention,IVC)作为对照。再将体外培养成熟的卵母细胞与获能后的小鼠精子受精,观察受精情况。结果:经过3D-IVG,卵泡存活率为82.5%,卵泡发育过程维持完整的三维结构,卵泡直径增长迅速,培养6d后,有91.7%成腔;在激素超排后,有82.6%COCs黏液化。GV、GVBD和含有PB的成熟卵母细胞分别为6.1%,45.4%和48.5%。3D-IVG获得的成熟卵母细胞百分率(48.5%)明显低于体内超排成熟卵母细胞百分率(82.9%),差异有统计学意义(P<0.05)。但3D-IVG所获得的卵母细胞染色体C带分析表明染色体数目为20条,结构未见异常,与体内超排卵母细胞一致。并观察到3D-IVG培养成熟的卵母细胞可以受精成功。结论:建立并完善了小鼠腔前卵泡的三维体外培养体系,为生殖毒性实验研究和胚胎工程研究提供了新的方法。展开更多
基金Supported by National Natural Science Foundation of China,No.30471970National Science and Technology Support Project(the 11th FiveYear Plan)of China,No.2006BAI02A14+1 种基金Scientific Research Special Projects of Health Ministry of China,No.200802011National Data Sharing Project in Human Health,No.2005DKA32403
文摘AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.
