生长抑制DNA损伤基因45与p53对卵巢癌细胞凋亡作用的研究

目的：研究生长抑制ＤＮＡ损伤基因（ｇｒｏｗｔｈａｒｅｓｔａｎｄＤＮＡｄａｍａｇｅ，ＧＡＤＤ）对卵巢癌细胞的作用及与ｐ５３蛋白表达的关系。方法：利用ＲＴ－ＰＣＲ鉴定卵巢癌ＳＫＯＶ３和３ＡＯ细胞株ＧＡＤＤ４５的表达；采用ＲＴ－ＰＣＲ和ＷｅｓｔｅｒｎＢｌｏｔ鉴定ＳＫＯＶ３和３ＡＯ细胞株ｐ５３蛋白的表达。通过载体构建将目的基因ＧＡＤＤ４５重组于ｐｃＤＮＡ－Ⅲ真核质粒内。利用脂质体（ＤＯＴＡＰ）法转染ＳＫＯＶ３和３ＡＯ细胞株；Ｇ４１８抗性筛选，ＲＴ－ＰＣＲ进行表达鉴定。采用流式细胞仪分析（ＦＣＭ）以及ＤＮＡ梯度法对细胞凋亡现象进行检测。结果：卵巢癌ＳＫＯＶ３和３ＡＯ细胞株ＧＡＤＤ４５表达阴性。ＳＫＯＶ３细胞株ｐ５３蛋白表达阳性；３ＡＯ细胞株ｐ５３蛋白表达阴性。转染ＧＡＤＤ４５基因的ＳＫＯＶ３细胞株经化学药物足叶已甙（ＶＰ１６）和紫外线诱导后，可以使该细胞产生凋亡现象，而对转染ＧＡＤＤ４５基因的３ＡＯ细胞株经诱导后，未见细胞凋亡现象。结论：转染ＧＡＤＤ４５基因，对ｐ５３蛋白表达阳性的细胞株ＳＫＯＶ３经诱导后，可使细胞进入凋亡状态。对ｐ５３蛋白表达阴性的细胞株３ＡＯ转染ＧＡＤＤ４５基因后，经诱导细胞无凋亡现象，证实ＧＡＤ?

Study of the effects of GADD45 and p53 protein on apoptosis of ovarian carcinoma cell lines

Objective:To study the effects of growth arrest and DNA damage(GADD)gene 45 on ovarian cancer cell lines and the relationship between the effect and the expression of p53 protein.Methods:Expressions of GADD45 and p53 protein in ovarian cancer cell liines SKOV3 and 3AO were identified respectively using RT PCR and Western Blot A eukaryotic expressing vector encoding GADD45 named pcDNA GADD45 was constructed and transfected into SKOV3 and 3AO cell lines with lipofectin(DOTAP),then selected by anti G418 The expression of GADD45 was identified using RT PCR.Apoptosis was analyzed using FCM and DNA ladder.Results:The expression of GADD45 in ovarian cancer cell lines SKOV3 and 3AO was not detected.There was positive expression of p53 protein in SKOV3 whereas negative expression in 3AO.It was in the SKOV3 transferred GADD45 gene after induction by VP16 and UV irradiation that the apoptosis was found,but no apoptosis in the 3AO transferred the same gene.Conclusions:The cell line,which has positive expression of p53 protein transferred GADD45 gene shows the apoptosis after induction by VP16 and UV irradiation.The cell line,however,which has negative expression of p53 protein,couldn