【摘要】 目的本文旨在构建STAT6-ORF表达载体,包装成慢病毒颗粒。方法应用嵌套PCR法从生长状态良好的293FT细胞的c DNA中扩增STAT6的完整读码框片段(2544 bp),根据需要在引物的5’端分别引入Cla I和Mlu I酶切位点,将PCR产物克隆到p MD20T载体上,得到p MD20T-STAT6-ORF克隆;测序验证后,用Cla I和Mlu I双酶切p MD20T-STAT6克隆,回收酶切产物得到的STAT6-ORF片段,通过T4 DNA连接酶连接到p LVTHm表达载体上,经双酶切和测序验证正确,获得p LVTHm-STAT6-ORF表达载体。结果构建带有增强绿色荧光蛋白EGFP(enhanced green fluorescence protein)标记的STAT6-ORF表达载体,包装成慢病毒颗粒,结果表明成功构建及包装带STAT6-ORF目的片段的慢病毒表达载体。结论 p LVTHm-STAT6过表达载体构建成功,可以进行慢病毒包装与鉴定。更多还原

【Abstract】 Objective To observe the effect of Nesfatin-1 on secretion of gastric acid and pepsin in rats gastric mucosa,and explore its possible mechanism. Methods 18 male SD rats after intracerebroventricular catheter were divided into,high dose group,low dose group and contral group,each rat was injected with Nesfatin-1 0. 05 g,0. 5 g and 5 μL equal volume sterile water by the lateral ventricle injection respectively. Gastric juice were collected by pylorus ligation,after 3h the rats were executed to measure changes of their gastric acid and pepsin,and H+-K+-ATPase expression levels in gastric tissue was detected by RT-PCR,protein expression levels of histidine decarboxylase(Histidine decarboxylase,HDC) in gastric mucosa was detected by Western blot,histamine content in gastric mucosa was detected by ELISA. Results After 3h of rats with Nesfatin-1 injection,compared with control group with equal volume of sterile water for injection,the secretion of gastric acid and pepsin was significantly reduced,there was a significant difference(P < 0. 01). The difference of pepsin secretion level between the two groups was statistical significance(P < 0. 05). Histamine secretion in rat gastric mucosa declined,the difference was statistically significant(P < 0. 05). Compared with the control group,after treatment,two groups of rats gastric H+-K+-ATP enzyme mRNA expression was significantly reduced,the difference was statistically significant(P < 0. 01). Meanwhile HDC protein expression in rat gastric mucosa in high dose group,low dose group decreased significantly compared with the control group,the presence of differences,with statistical significance(P < 0. 05). Conclusion Intracerebroventricular injection Nesfatin-1,could inhibit the secretion of gastric acid and pepsin,and its mechanism may inhibit gastric acid secretion by affecting the central nervous system,H+-K+-ATP enzyme mRNA expression levels,expression of key enzymes. in histamine synthesis pathway.更多还原