试验旨在对小尾寒羊特异性蛋白1(specificity protein 1,SP1)基因进行克隆和序列分析,并研究SP1基因对前体脂肪细胞分化的影响。选取1月龄小尾寒羊尾部脂肪组织进行前体脂肪细胞的分离、培养和诱导分化;用重叠PCR法克隆SP1基因编码区(co...试验旨在对小尾寒羊特异性蛋白1(specificity protein 1,SP1)基因进行克隆和序列分析,并研究SP1基因对前体脂肪细胞分化的影响。选取1月龄小尾寒羊尾部脂肪组织进行前体脂肪细胞的分离、培养和诱导分化;用重叠PCR法克隆SP1基因编码区(coding DNA sequence,CDS);用生物信息学软件分析SP1基因CDS,并对其编码蛋白的结构、功能结构域、亚细胞定位、翻译后修饰进行预测;用慢病毒包装SP1基因过表达、干扰及对照质粒转染293T细胞后,收取病毒液感染小尾寒羊前体脂肪细胞;用油红O染色检测脂滴沉积能力;用实时荧光定量PCR检测SP1基因和成脂标志基因的mRNA表达量。结果表明,小尾寒羊SP1基因CDS全长2340 bp,编码779个氨基酸;序列相似性分析显示,小尾寒羊SP1基因CDS及其编码序列与绵羊、山羊的相似性最高,其次是牛、马、猪、人、小鼠、大鼠,与鸡的相似性最低,系统进化分析结果与之相符;SP1蛋白定位于细胞核,有109个磷酸化位点,其二级结构由α-螺旋、β-转角、无规则卷曲和延伸链4种结构组成,所占比例分别为16.94%、8.60%、54.17%和20.28%,二级结构和三级结构均存在3个锌指结构域;过表达、干扰及对照质粒转染293T细胞,每组细胞皆出现较强绿色荧光,载体成功转染至293T细胞;病毒液感染小尾寒羊前体脂肪细胞并分化12 d后,过表达组SP1基因表达量极显著高于对照组(P<0.01),干扰组SP1基因表达量极显著低于对照组(P<0.01);过表达SP1基因极显著上调成脂标志基因mRNA的表达量(P<0.01),产生更多脂滴;干扰SP1基因则结果相反。因此,SP1基因对小尾寒羊尾部前体脂肪细胞分化具有正向调控的作用。展开更多
AIM:To explore the expression of Sp1 in gastric carcinoma as well as its association with other clinicopathologic features, and to evaluate the role of Spl as a prognostic indicator of gastric carcinoma. METHODS: By u...AIM:To explore the expression of Sp1 in gastric carcinoma as well as its association with other clinicopathologic features, and to evaluate the role of Spl as a prognostic indicator of gastric carcinoma. METHODS: By using immunohistochemistry, we examined the Spl expression patterns in 65 cases of human gastric cancer, and 40 normal gastric mucosa specimens. Simultaneously, the correlation between Spl expression and clinical outcome or clinicopathologic features was investigated. RESULTS: The percentage of Spl expression was 12.5% (5/40) in normal gastric mucosa, and the Spl protein was mainly expressed in the nuclei of cells located in the mucous neck region. In sharp contrast, strong Spl expression was detected in tumor cells, whereas no or faint Spl staining was detected in stromal cells and normal glandular cells surrounding the tumors. The expression rate of Spl in gastric cancer lesions was 53.85% (35/65). The medium survival duration in patients who had a tumor with negative, weak and strong Spl expressions was 1 700, 1 560 and 1 026 d, respectively (P<0.05). Spl protein expression was closely related to the depth of tumor infiltration (X2 = 13.223, P<0.01) and TNM stage (X2= 11.009, P<0.05), but had no relationship with the number of lymph nodes and Lauren's classification (P>0.05). Cox regression model for multivariate analysis revealed that high Spl expression (P<0.05) and advanced stage (P<0.01) were independent predictors of poor survival. CONCLUSION: Normal and malignant gastric tissues have unique Spl expression patterns. Spl might serve as an independent prognostic factor, by influencing the tumor infiltration and progression.展开更多
Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements withi...Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.展开更多
文摘试验旨在对小尾寒羊特异性蛋白1(specificity protein 1,SP1)基因进行克隆和序列分析,并研究SP1基因对前体脂肪细胞分化的影响。选取1月龄小尾寒羊尾部脂肪组织进行前体脂肪细胞的分离、培养和诱导分化;用重叠PCR法克隆SP1基因编码区(coding DNA sequence,CDS);用生物信息学软件分析SP1基因CDS,并对其编码蛋白的结构、功能结构域、亚细胞定位、翻译后修饰进行预测;用慢病毒包装SP1基因过表达、干扰及对照质粒转染293T细胞后,收取病毒液感染小尾寒羊前体脂肪细胞;用油红O染色检测脂滴沉积能力;用实时荧光定量PCR检测SP1基因和成脂标志基因的mRNA表达量。结果表明,小尾寒羊SP1基因CDS全长2340 bp,编码779个氨基酸;序列相似性分析显示,小尾寒羊SP1基因CDS及其编码序列与绵羊、山羊的相似性最高,其次是牛、马、猪、人、小鼠、大鼠,与鸡的相似性最低,系统进化分析结果与之相符;SP1蛋白定位于细胞核,有109个磷酸化位点,其二级结构由α-螺旋、β-转角、无规则卷曲和延伸链4种结构组成,所占比例分别为16.94%、8.60%、54.17%和20.28%,二级结构和三级结构均存在3个锌指结构域;过表达、干扰及对照质粒转染293T细胞,每组细胞皆出现较强绿色荧光,载体成功转染至293T细胞;病毒液感染小尾寒羊前体脂肪细胞并分化12 d后,过表达组SP1基因表达量极显著高于对照组(P<0.01),干扰组SP1基因表达量极显著低于对照组(P<0.01);过表达SP1基因极显著上调成脂标志基因mRNA的表达量(P<0.01),产生更多脂滴;干扰SP1基因则结果相反。因此,SP1基因对小尾寒羊尾部前体脂肪细胞分化具有正向调控的作用。
基金Supported by the "211" Project sponsored by Ministry of Education,China
文摘AIM:To explore the expression of Sp1 in gastric carcinoma as well as its association with other clinicopathologic features, and to evaluate the role of Spl as a prognostic indicator of gastric carcinoma. METHODS: By using immunohistochemistry, we examined the Spl expression patterns in 65 cases of human gastric cancer, and 40 normal gastric mucosa specimens. Simultaneously, the correlation between Spl expression and clinical outcome or clinicopathologic features was investigated. RESULTS: The percentage of Spl expression was 12.5% (5/40) in normal gastric mucosa, and the Spl protein was mainly expressed in the nuclei of cells located in the mucous neck region. In sharp contrast, strong Spl expression was detected in tumor cells, whereas no or faint Spl staining was detected in stromal cells and normal glandular cells surrounding the tumors. The expression rate of Spl in gastric cancer lesions was 53.85% (35/65). The medium survival duration in patients who had a tumor with negative, weak and strong Spl expressions was 1 700, 1 560 and 1 026 d, respectively (P<0.05). Spl protein expression was closely related to the depth of tumor infiltration (X2 = 13.223, P<0.01) and TNM stage (X2= 11.009, P<0.05), but had no relationship with the number of lymph nodes and Lauren's classification (P>0.05). Cox regression model for multivariate analysis revealed that high Spl expression (P<0.05) and advanced stage (P<0.01) were independent predictors of poor survival. CONCLUSION: Normal and malignant gastric tissues have unique Spl expression patterns. Spl might serve as an independent prognostic factor, by influencing the tumor infiltration and progression.
文摘Nanog gene plays a key role in maintaining pluripotency of ES cells and early embryonic cells. A 5＇ flank sequence of the Nanog gene has been reported to be regulated differentially, and two regulatory elements within the Nanog promoter, namely Oct-4 and Sox-2 binding sites, have been identified to regulate the transcriptional activity ofNanog gene. In this report, we identified the role of two putative Spl binding sites located in the Nanog gene 5＇-flanking region in regulation ofmurine Nanog gene transcription. Mutation studies showed that the two sites were essential for the Nanog promoter activity. Gel shift and supershift analysis showed that both sites specifically bind Spl and Sp3. Furthermore, overexpression of dominant-negative Spl or Sp3 mutants significantly inhibits Nanog promoter activity. These results suggest that the transcription factor Spl and Sp3 are important for Murine Nanog gene expression.
