AIM To examine the expression of activin A, amember of the transforming growth factor (TGF-β) superfamily, recently has been reported to beoverexpressed in liver cirrhosis, in the course ofcarbon tetrachloride-induce...AIM To examine the expression of activin A, amember of the transforming growth factor (TGF-β) superfamily, recently has been reported to beoverexpressed in liver cirrhosis, in the course ofcarbon tetrachloride-induced rat hepaticfibrosis.METHODS Hepatic fibrosis was induced in ratsby subcutaneous injections of 40% carbontetrachloride oily solution for a period of 1 to 7weeks. At the end of 1, 2, 3, 4, 5, 6 and 7 weeksafter carbon tetrachloride injections, the ratswere killed in group (6- 10 rats each time) forstudy. The activin A messenger RNA expressionand its protein localization were assessed bysemi-quantitative reverse transcriptionpolymerase chain reaction ( RT-PCR ) andimmunohistochemistry.RESULTS The normal rat liver expressedactivin A mRNA and protein, and its expressionwas transiently decreased and becameundetectable after carbon tetrachlorideinjections for 2 or 3 weeks and then increasedgradually. After injection of carbon tetrachloridefor 6 and 7 weeks, activin A mRNA and proteinexpressions were significantly enchanced in ratliver. Compared with that of the normal ratliver. Activin A mRNA expression levels in ratsreceiving carbon tetrachloride injections for 6and 7 weeks were 1.6 and 2.2 times that of thosein normal rat liver respectively (0.456±0.094 vs0.286± 0.0670, P＜0.01; 0.620± 0.134 vs 0.286±± 0.0670, P＜0.01). Immunohistochemistryshowed that activin A expressed in hepatocytesof normal liver, and its expression wasdecreased in rats receiving carbon tetrachloridefor 2 or 3 weeks. Compared with normal liver,activin A expression distribution mode changedin fibrotic liver, being increased significantly inhepatocytes around fibrotic areas.CONCLUSION Activin A expression wasincreased in latestage of hepatic fibrosis, andthis may be involved in hepatic fibrosisformation in this period.展开更多
The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism...The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism of inhibin by investigating the localization of inhibin and α subunits in the central nervous system （mainly in hypothalamus and pituitary） of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its α subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin α subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and α subunit could be transported through the BBB by the method of “separation and reconstruction”.展开更多
目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA des...目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA desaturase,SCD)抑制剂]组、NC+PluriSIn 1组、sh-INHBA-AS1+PluriSIn 1组、10058-F4(c-Myc抑制剂)组、NC+10058-F4组、sh-INHBA-AS1+10058-F4组、sh-INHBA-AS1+OE-c-Myc组。平板克隆实验检测各组细胞的增殖能力,FCM检测各组细胞的凋亡情况,Transwell小室实验检测各组细胞的侵袭、迁移能力,qPCR法检测各组细胞中INHBA-AS1、c-Myc、SCD和EMT相关基因(N-cadherin、TGF-β、ZEB1)mRNA的表达,WB法检测各组细胞中c-Myc、SCD、EMT相关(N-cadherin、TGF-β、ZEB1)、S-腺苷-甲硫氨酸脱羧酶(SAMDC)和亚精胺/精胺N1-乙酰转移酶(SSAT)蛋白的表达,ELISA检测各组细胞上清液中鸟氨酸脱羧酶(ODC)的含量。结果:敲减INHBA-AS1表达使HeLa细胞的增殖、侵袭和迁移能力显著降低(均P<0.05)而细胞凋亡率显著升高(P<0.05),q PCR、WB法检测结果显示,敲减INHBA-AS1均可显著抑制HeLa细胞中c-Myc、SCD、N-cadherin、TGF-β、ZEB1和SAMDC的表达(均P<0.05),而促进SSAT的表达(P<0.05),并降低HeLa细胞上清液中ODC的含量(P<0.05)。与c-Myc抑制剂和SCD抑制剂单独处理相比,其联合敲减INHBA-AS1后上述作用更加显著(均P<0.05);与sh-INHBA-AS1组相比,进一步过表达c-Myc后HeLa细胞的增殖能力显著升高(P<0.05)、SCD和N-cadherin蛋白表达水平显著升高(P<0.05)、细胞上清液中ODC含量显著升高(P<0.05)。结论:INHBA-AS1可通过c-Myc调控SCD的表达,从而影响HeLa细胞鸟氨酸代谢和EMT进程,进而促进HeLa细胞的增殖、侵袭和迁移能力。展开更多
文摘AIM To examine the expression of activin A, amember of the transforming growth factor (TGF-β) superfamily, recently has been reported to beoverexpressed in liver cirrhosis, in the course ofcarbon tetrachloride-induced rat hepaticfibrosis.METHODS Hepatic fibrosis was induced in ratsby subcutaneous injections of 40% carbontetrachloride oily solution for a period of 1 to 7weeks. At the end of 1, 2, 3, 4, 5, 6 and 7 weeksafter carbon tetrachloride injections, the ratswere killed in group (6- 10 rats each time) forstudy. The activin A messenger RNA expressionand its protein localization were assessed bysemi-quantitative reverse transcriptionpolymerase chain reaction ( RT-PCR ) andimmunohistochemistry.RESULTS The normal rat liver expressedactivin A mRNA and protein, and its expressionwas transiently decreased and becameundetectable after carbon tetrachlorideinjections for 2 or 3 weeks and then increasedgradually. After injection of carbon tetrachloridefor 6 and 7 weeks, activin A mRNA and proteinexpressions were significantly enchanced in ratliver. Compared with that of the normal ratliver. Activin A mRNA expression levels in ratsreceiving carbon tetrachloride injections for 6and 7 weeks were 1.6 and 2.2 times that of thosein normal rat liver respectively (0.456±0.094 vs0.286± 0.0670, P＜0.01; 0.620± 0.134 vs 0.286±± 0.0670, P＜0.01). Immunohistochemistryshowed that activin A expressed in hepatocytesof normal liver, and its expression wasdecreased in rats receiving carbon tetrachloridefor 2 or 3 weeks. Compared with normal liver,activin A expression distribution mode changedin fibrotic liver, being increased significantly inhepatocytes around fibrotic areas.CONCLUSION Activin A expression wasincreased in latestage of hepatic fibrosis, andthis may be involved in hepatic fibrosisformation in this period.
文摘The aims of the study were to determine the distribution of inhibin and its α subunit in some rat tissues by an immunohistochemical method of streptavidin-biotin complex （SABC） and to assess the transport mechanism of inhibin by investigating the localization of inhibin and α subunits in the central nervous system （mainly in hypothalamus and pituitary） of ovariectomized rats. Investigations on the extragonadal tissues of ovariectomized rats showed positive expression of inhibin and its α subunit in heart, kidney, spleen, pancreatic gland cells, but no positive reaction sites were seen in lung, liver, submaxillary gland, and adrenal gland. After injection with inhibin α subunit fragment or inhibin extract, positive reaction sites were observed in hypothalamus and pituitary of ovariectomized rats by SABC. Inhibin and its subunit was present in a wide variety of nonreproductive organs and tissues, and its expression was tissue specific, which indicated that inhibin might play a role in regulating tissue function through autocrine/paracrine mechanisms. Inhibin dimer and α subunit could be transported through the BBB by the method of “separation and reconstruction”.
文摘将鸡催乳素 (PRL)和抑制素 - α亚基 (INB- α)基因编码序列重组为融合基因 ,制备了同时包含这 2种激素基因的融合蛋白。通过 PCR和分子克隆的方法首先将全部粤黄鸡 PRL成熟肽 c DNA克隆到载体 p RSET A的 Bgl 和 Eco R 克隆位点之间 ,获得重组质粒 p PRL- RSET。鸡 INB- α片段经扩增后分别被克隆到质粒 p RSET A和 p PRL- RSET的 Nhe 和 Xho 克隆位点之间 ,获得重组质粒 p INB- RSET和 p INB- PRL。以上重组质粒构建的正确性分别由各特定引物组合扩增的 PCR产物长度、特定限制性内切酶消化各重组质粒所得产物长度以及对各质粒的测序结果得到验证。重组质粒 p PRL- RSET和 p INB- PRL 转化 E.coli BL2 1(DE3)株 ,IPTG诱导后所表达的产物经 SDS- PAGE显示 ,其分别与所预期的重组蛋白分子大小相符。质粒 p PRL - RSET和 p INB- PRL的表达产物和用 Ni- NTA凝胶纯化的 2重组蛋白产物都可与抗鸡 PRL 抗体产生特异的免疫印迹 ,并且表达菌裂解液和相应纯化蛋白的免疫印迹处于同一位置。结果说明 ,试验已成功完成了鸡 PRL、INB-α及 2者融合蛋白的构建。
