外源质粒DNA经胃肠道吸收对小鼠基因表达谱影响的初探

发布时间：2018-04-28 12:34

本文选题：外源质粒DNA + 胃肠道　；参考：《江南大学》2005年博士论文

【摘要】：外源质粒DNA通过胃肠道途径吸收可达到基因治疗、免疫和代谢调控的目的。外源质粒DNA吸收、基因转移可能对机体造成的影响令人关注。论文研究了外源质粒在胃肠道中的吸收和对机体免疫功能的影响,以基因芯片为技术平台,主要研究外源质粒DNA对机体基因差异表达的影响。主要研究结果如下: 1外源质粒DNA在小鼠体内的组织分布及整合的可能性评价实验首先研究外源质粒DNA在小鼠体内的吸收及对整合的可能性进行评价。实验采用5-6周龄,体重20g左右的健康昆明种小鼠,给每只小鼠灌注pcDNA3s质粒溶液200μg,于灌胃后1,3,6,24h,48h及3,6wk分离肺、肾、脾、肠系膜淋巴结、胸腺、生殖器官、粪便、十二指肠、大肠、血液、肌肉及肝脏等。提取组织总DNA,采用半定量PCR法检测外源质粒DNA在不同组织分布及随时间的变化情况。凝胶电泳分离宿主基因组DNA与外源质粒DNA,以组织基因组DNA为模板,PCR法检测质粒DNA在基因组上的整合率。对照模板中加入不同拷贝数的pcDNA3s质粒,确定PCR反应的敏感性。采用氨苄青霉素筛选肠道粪便细菌中的质粒阳性克隆。灌胃质粒pcDNA3s后,可在不同组织、不同时间检测到质粒DNA的存在,至灌胃第6周后,除了肾脏外在其他组织均呈阴性。外源质粒DNA在体内主要以片段形式存在,在各组织基因组DNA中未检测到质粒DNA的整合。未检测到肠道粪便细菌中的质粒阳性克隆,说明在肠道中质粒DNA不会转化到肠道细菌中。结果表明外源质粒DNA能被胃肠道吸收,迅速分布于小鼠各个器官中并以碎片的形式在体内存留较长的时间。外源质粒DNA经胃肠道途径不会整合到宿主染色体基因组上。 2外源质粒DNA在小鼠肠道中的吸收机制研究实验采用5-6周龄Balbc/C雄性小鼠6只,随机分成2组,一组为实验对照组,灌胃200μL生理盐水,另一组给小鼠灌胃1μg/μL质粒pcDNA3溶液200μL,灌胃后4h ,分离空肠一段并提取总RNA,RT-PCR逆转录成cDNA,将双链cDNA进行体外转录合成cRNA, cRNA再次反转录成cDNA,Cy5-dCTP标记实验组cDNA,Cy3-dCTP标记实验组cDNA,标记的DNA与芯片进行杂交、洗涤、并进行扫描,采用GenePix Pro4.0进行数据分析。结果显示17667条基因中,一共有4196条基因表达,其中明显差异表达的基因有61条,表达上调36条,表达下调25条。外源质粒DNA可诱导肠道阴离子转运蛋白的表达,提示肠道中可能存在转运蛋白介导外源质粒DNA的吸收。外源质粒DNA进入肠道后能够引起肠道免疫应答基因发生变化。外源质粒DNA上调微粒体谷胱甘肽硫转移酶和谷胱甘肽过氧化物酶的表达,增强肠道的抗氧化功能。外源质粒DNA通过抑制肠道Caspase3基因而抑制肠黏膜细胞凋亡。肠道丝裂原激活蛋白激酶基因(MAP2K2)发生上调,说明外源质粒DNA可能通过MAPK途径激活肠黏膜细胞。
[Abstract]:Exogenous plasmid DNA can be absorbed through gastrointestinal tract to achieve the purpose of gene therapy, immune and metabolic regulation. The possible effects of exogenous plasmid DNA absorption and gene transfer on the body are of concern. In this paper, the effects of exogenous plasmids on gastrointestinal tract absorption and immune function were studied. The effects of exogenous plasmids DNA on gene differential expression were studied on the platform of gene microarray. The main findings are as follows: 1 the possibility of tissue distribution and integration of exogenous plasmid DNA in mice In this experiment, the absorption of exogenous plasmid DNA in mice and the possibility of integration were studied. Healthy Kunming mice (5-6 weeks old, weight about 20g) were injected with 200 渭 g pcDNA3s plasmid solution. Lung, kidney, spleen, mesenteric lymph nodes, thymus, genital organs, feces, duodenum, large intestine were isolated from each mouse for 48 h and 6 wk after gastric perfusion, and the mice were divided into two groups: lung, kidney, spleen, mesenteric lymph node, thymus, genital organs, feces, duodenum and large intestine. Blood, muscle, liver, etc. The total tissue DNA was extracted and the distribution of exogenous plasmid DNA in different tissues and the changes with time were detected by semi-quantitative PCR method. The host genome DNA and exogenous plasmid DNA were separated by gel electrophoresis, and the integration rate of plasmid DNA on the genome was determined by polymerase chain reaction (PCR) with tissue genomic DNA as template. PcDNA3s plasmids with different copy numbers were added to the control template to determine the sensitivity of PCR reaction. Ampicillin was used to screen plasmid positive clones from intestinal fecal bacteria. The existence of plasmid DNA was detected in different tissues and at different time after administration of plasmid pcDNA3s. After 6 weeks of gastric administration, all the tissues except kidney were negative. Exogenous plasmid DNA existed mainly in the form of fragments in vivo, and no integration of plasmid DNA was detected in the genomic DNA of various tissues. No plasmid positive clones were detected in fecal bacteria, indicating that plasmid DNA could not be transformed into intestinal bacteria. The results showed that exogenous plasmid DNA could be absorbed by gastrointestinal tract and distributed rapidly in various organs of mice and remained in vivo for a long time in the form of fragments. Exogenous plasmid DNA is not integrated into the host chromosomal genome via gastrointestinal pathway. Study on the absorption Mechanism of exogenous plasmid DNA in Mouse intestine Six male Balbc/C mice aged 5-6 weeks were randomly divided into two groups: one group was treated with 200 渭 L normal saline, the other group was given 1 渭 g / 渭 L plasmid pcDNA3 solution (200 渭 L) for 4 h after gavage. A segment of jejunum was isolated and total RNAs were extracted by reverse transcription (RT-PCR) to cDNA. the double-stranded cDNA was transcribed and synthesized into cRNAs in vitro. CRNA was then reversed to cDNA-Cy5-dCTP-labeled experimental group cDNAs. The labeled DNA was hybridized with microarray, washed and scanned. The data were analyzed by GenePix Pro4.0. The results showed that there were 4196 genes expressed in 17667 genes, 61 of which were differentially expressed, 36 were up-regulated and 25 were down-regulated. Exogenous plasmid DNA could induce the expression of intestinal anion transporter, suggesting that there might be transporter mediated the absorption of exogenous plasmid DNA. Exogenous plasmid DNA can cause changes in intestinal immune response genes when it enters the intestine. Exogenous plasmid DNA upregulated the expression of microsomal glutathione S-transferase and glutathione peroxidase and enhanced the antioxidant function of intestinal tract. Exogenous plasmid DNA inhibits intestinal mucosal cell apoptosis by inhibiting intestinal Caspase3 gene. Mitogen-activated protein kinase gene MAP2K2) was up-regulated, suggesting that exogenous plasmid DNA might activate intestinal mucosal cells through MAPK pathway.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R346

【参考文献】