制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合...制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合α1,3Gal的药物GSI-B4联合免疫磁珠筛选,成功分离出自发杂合性缺失(Loss of heterozygosity,LOH)的双等位基因敲除(GGTA1-/-)耳成纤维细胞。单细胞克隆培养与PCR鉴定后,以GGTA1-/-细胞为核供体,体外成熟的猪卵母细胞为核受体构建克隆胚胎并移植。先后将3 122枚重构胚移植到13头受体母猪,其中4头怀孕,产仔12头。采用PCR和Southern blotting进行GGTA1基因型检测,11头为GGTA1-/-猪;流式细胞术分析表明,GGTA1-/-仔猪耳成纤维细胞不表达α1,3Gal。获得能克服异种器官超急性排斥反应(HAR)的GGTA1-/-猪,不仅为异种器官供体的进一步基因修饰提供了平台,也为异种移植临床前研究提供了宝贵资源。展开更多
Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH...Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH of epigenetic microsatellites of PTEN(D10S541,D10S583and D10S1687)was exam-ined in advanced gastric carcinomas(n=56)by PCR-SSCP.The mRNA and protein expressions of PTEN gene were evaluated in normal mucosa(n=56),early(n=11)and advanced carcinomas(n=56)of the stomach using RT?PCR and immunohistochemoistry respectively.PTEN mRNA and protein expressions were compared with clinicopathological staging and lymph node metastasis of tumors.The relationship be-tween PTEN mRNA expression and LOH of microsatellites was discussed,as well as relationship between PTEN mRNA and protein expression.Results.LOH of D10S541,D10S583and D10S1687was found in28.6%(16/56)of advanced gas-tric carcinomas.The positive rates of PTEN expression were80.4%(45/56),45.5%(5/11)and32.1%(18/56)in normal gastric mucosa,early and advanced gastric carcinomas at mRNA level,while78.6%(44/56),36.4%(4/11)and28.6%(16/56)at protein level.PTEN mRNA and protein were less fre-quently expressed in early and advanced gastric carcinomas than normal gastric mucosa(P<0.05).There was negative correlation between PTEN mRNA expression and LOH of microsatellites(P<0.05).PTEN protein expression paralleled to its mRNA expression(P<0.05).The PTEN mRNA and protein expres-sions were negatively correlated with lymph node metastasis of advanced gastric carcinomas(P<0.05).Conclusion.Down?regulated expression of PTEN and frequent LOH of its epigenetic microsatellites might play an important role in gastric carcinogenesis.Reduced PTEN mRNA expression was closely as-sociated with LOH of its epigenetic microsatellites.Altered expression of PTEN might contribute to lymph node metastasis of gastric carcinoma by decreasing cell adhesion and apoptosis,increasing angiogenesis and cell mobility.展开更多
文摘制备α1,3-半乳糖基转移酶(αl,3-galactosyltransferase,GGTA1)基因缺失的五指山小型猪,为我国异种器官移植研究奠定基础。在已经获得的GGTA1单等位基因敲除(GGTA+/-)五指山小型猪基础上,建立GGTA1+/-猪耳成纤维细胞系。利用特异性结合α1,3Gal的药物GSI-B4联合免疫磁珠筛选,成功分离出自发杂合性缺失(Loss of heterozygosity,LOH)的双等位基因敲除(GGTA1-/-)耳成纤维细胞。单细胞克隆培养与PCR鉴定后,以GGTA1-/-细胞为核供体,体外成熟的猪卵母细胞为核受体构建克隆胚胎并移植。先后将3 122枚重构胚移植到13头受体母猪,其中4头怀孕,产仔12头。采用PCR和Southern blotting进行GGTA1基因型检测,11头为GGTA1-/-猪;流式细胞术分析表明,GGTA1-/-仔猪耳成纤维细胞不表达α1,3Gal。获得能克服异种器官超急性排斥反应(HAR)的GGTA1-/-猪,不仅为异种器官供体的进一步基因修饰提供了平台,也为异种移植临床前研究提供了宝贵资源。
文摘Objective.To investigate PTEN expression and loss of heterozygosity(LOH)of its epigenetic microsatel-lites in gastric carcinoma and explore their roles in tumorigenesis and progression of gastric carcinoma.Methods.LOH of epigenetic microsatellites of PTEN(D10S541,D10S583and D10S1687)was exam-ined in advanced gastric carcinomas(n=56)by PCR-SSCP.The mRNA and protein expressions of PTEN gene were evaluated in normal mucosa(n=56),early(n=11)and advanced carcinomas(n=56)of the stomach using RT?PCR and immunohistochemoistry respectively.PTEN mRNA and protein expressions were compared with clinicopathological staging and lymph node metastasis of tumors.The relationship be-tween PTEN mRNA expression and LOH of microsatellites was discussed,as well as relationship between PTEN mRNA and protein expression.Results.LOH of D10S541,D10S583and D10S1687was found in28.6%(16/56)of advanced gas-tric carcinomas.The positive rates of PTEN expression were80.4%(45/56),45.5%(5/11)and32.1%(18/56)in normal gastric mucosa,early and advanced gastric carcinomas at mRNA level,while78.6%(44/56),36.4%(4/11)and28.6%(16/56)at protein level.PTEN mRNA and protein were less fre-quently expressed in early and advanced gastric carcinomas than normal gastric mucosa(P<0.05).There was negative correlation between PTEN mRNA expression and LOH of microsatellites(P<0.05).PTEN protein expression paralleled to its mRNA expression(P<0.05).The PTEN mRNA and protein expres-sions were negatively correlated with lymph node metastasis of advanced gastric carcinomas(P<0.05).Conclusion.Down?regulated expression of PTEN and frequent LOH of its epigenetic microsatellites might play an important role in gastric carcinogenesis.Reduced PTEN mRNA expression was closely as-sociated with LOH of its epigenetic microsatellites.Altered expression of PTEN might contribute to lymph node metastasis of gastric carcinoma by decreasing cell adhesion and apoptosis,increasing angiogenesis and cell mobility.
