目的:构建人Cdc25C基因的克隆载体和原核表达载体,并诱导其在大肠杆菌中表达.方法:从人肝癌细胞株Bel-7404中提取总R N A,经RT-P C R法扩增人C d c25C c D N A后,将其正确插入克隆载体pMD18-T和表达载体pET-32a(+),并转化至BL21(DE3)、B...目的:构建人Cdc25C基因的克隆载体和原核表达载体,并诱导其在大肠杆菌中表达.方法:从人肝癌细胞株Bel-7404中提取总R N A,经RT-P C R法扩增人C d c25C c D N A后,将其正确插入克隆载体pMD18-T和表达载体pET-32a(+),并转化至BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中,分别采用0.25 mmol/L IPTG和ArtMediaTM Protein Expression自动诱导表达培养基诱导表达,并对纯化的融合蛋白进行考马斯亮蓝染色和质谱分析鉴定.结果:成功扩增了Cdc25C基因,并获得p M D18-T-C d c25C克隆载体和p E T-32a(+)Cdc25C表达载体;重组质粒在BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中均诱导表达出TRx-His-Cdc25C融合蛋白;考马斯亮蓝染色和蛋白质谱分析结果显示基因重组蛋白与目的蛋白相符.结论:获得肿瘤相关抗原Cdc25C重组蛋白,为后续研究奠定基础.展开更多
Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy.To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma trea...Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy.To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery,we evaluated the expression of cell cycle checkpoint-related proteins Chk2,Cdc25C,and Cyclin D1.A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery.Pretreatment tumor biopsy specimens were analyzed for Chk2,Cdc25C,and Cyclin D1 expression by immunohistochemistry.High expression of Chk2,Cyclin D1,and Cdc25C was observed in 44(78.6%),15(26.8%),and 27(48.2%) patients,respectively.The median survival was 16 months(range,3-154 months),with a 5-year overall survival rate of 19.6%.Overexpression of Chk2 was associated with smoking(P = 0.021),overexpression of Cdc25C was associated with patient age(P = 0.033) and tumor length(P = 0.001),and overexpression of Cdc25C was associated with pathologic complete response(P = 0.038).Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival.In multivariate analysis,Cdc25C was the most significant independent predictor of better survival(P = 0.014) for patients treated with radiotherapy followed by surgery.Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery.These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients.Thus,immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.展开更多
文摘目的:构建人Cdc25C基因的克隆载体和原核表达载体,并诱导其在大肠杆菌中表达.方法:从人肝癌细胞株Bel-7404中提取总R N A,经RT-P C R法扩增人C d c25C c D N A后,将其正确插入克隆载体pMD18-T和表达载体pET-32a(+),并转化至BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中,分别采用0.25 mmol/L IPTG和ArtMediaTM Protein Expression自动诱导表达培养基诱导表达,并对纯化的融合蛋白进行考马斯亮蓝染色和质谱分析鉴定.结果:成功扩增了Cdc25C基因,并获得p M D18-T-C d c25C克隆载体和p E T-32a(+)Cdc25C表达载体;重组质粒在BL21(DE3)、BL21(DE3)pLysS和Transetta(DE3)三种感受态大肠杆菌中均诱导表达出TRx-His-Cdc25C融合蛋白;考马斯亮蓝染色和蛋白质谱分析结果显示基因重组蛋白与目的蛋白相符.结论:获得肿瘤相关抗原Cdc25C重组蛋白,为后续研究奠定基础.
基金supported by grants from the National High Technology Research and Development Program of China(863 Program)(No.2006AA020707 and No.2006AA02A403)
文摘Biomarker identification is crucial for the selection of patients who might benefit from radiotherapy.To explore potential markers for response and prognosis in patients with locally advanced esophageal carcinoma treated with radiotherapy followed by surgery,we evaluated the expression of cell cycle checkpoint-related proteins Chk2,Cdc25C,and Cyclin D1.A total of 56 patients with locally advanced esophageal squamous cell carcinoma were treated with radiotherapy followed by surgery.Pretreatment tumor biopsy specimens were analyzed for Chk2,Cdc25C,and Cyclin D1 expression by immunohistochemistry.High expression of Chk2,Cyclin D1,and Cdc25C was observed in 44(78.6%),15(26.8%),and 27(48.2%) patients,respectively.The median survival was 16 months(range,3-154 months),with a 5-year overall survival rate of 19.6%.Overexpression of Chk2 was associated with smoking(P = 0.021),overexpression of Cdc25C was associated with patient age(P = 0.033) and tumor length(P = 0.001),and overexpression of Cdc25C was associated with pathologic complete response(P = 0.038).Univariate analysis demonstrated that overexpression of Cdc25C and pathologic complete response was associated with better survival.In multivariate analysis,Cdc25C was the most significant independent predictor of better survival(P = 0.014) for patients treated with radiotherapy followed by surgery.Overexpression of Cdc25C was significantly associated with pathologic complete response and better survival of patients with locally advanced esophageal cancer treated with radiotherapy followed by surgery.These results suggest that Cdc25C may be a biomarker of treatment response and good prognosis for esophageal carcinoma patients.Thus,immunohistochemical staining of Cdc25C in a pretreatment specimen may be a useful method of identifying optimal treatment for patients with esophageal carcinoma.
