本文選題：外源質(zhì)粒DNA + 胃腸道��； 參考：《江南大學(xué)》2005年博士論文

【摘要】：外源質(zhì)粒DNA通過胃腸道途徑吸收可達(dá)到基因治療、免疫和代謝調(diào)控的目的。外源質(zhì)粒DNA吸收、基因轉(zhuǎn)移可能對機(jī)體造成的影響令人關(guān)注。論文研究了外源質(zhì)粒在胃腸道中的吸收和對機(jī)體免疫功能的影響,以基因芯片為技術(shù)平臺,主要研究外源質(zhì)粒DNA對機(jī)體基因差異表達(dá)的影響。主要研究結(jié)果如下: 1外源質(zhì)粒DNA在小鼠體內(nèi)的組織分布及整合的可能性評價 實(shí)驗(yàn)首先研究外源質(zhì)粒DNA在小鼠體內(nèi)的吸收及對整合的可能性進(jìn)行評價。實(shí)驗(yàn)采用5-6周齡,體重20g左右的健康昆明種小鼠,給每只小鼠灌注pcDNA3s質(zhì)粒溶液200μg,于灌胃后1,3,6,24h,48h及3,6wk分離肺、腎、脾、腸系膜淋巴結(jié)、胸腺、生殖器官、糞便、十二指腸、大腸、血液、肌肉及肝臟等。提取組織總DNA,采用半定量PCR法檢測外源質(zhì)粒DNA在不同組織分布及隨時間的變化情況。凝膠電泳分離宿主基因組DNA與外源質(zhì)粒DNA,以組織基因組DNA為模板,PCR法檢測質(zhì)粒DNA在基因組上的整合率。對照模板中加入不同拷貝數(shù)的pcDNA3s質(zhì)粒,確定PCR反應(yīng)的敏感性。采用氨芐青霉素篩選腸道糞便細(xì)菌中的質(zhì)粒陽性克隆。 灌胃質(zhì)粒pcDNA3s后,可在不同組織、不同時間檢測到質(zhì)粒DNA的存在,至灌胃第6周后,除了腎臟外在其他組織均呈陰性。外源質(zhì)粒DNA在體內(nèi)主要以片段形式存在,在各組織基因組DNA中未檢測到質(zhì)粒DNA的整合。未檢測到腸道糞便細(xì)菌中的質(zhì)粒陽性克隆,說明在腸道中質(zhì)粒DNA不會轉(zhuǎn)化到腸道細(xì)菌中。結(jié)果表明外源質(zhì)粒DNA能被胃腸道吸收,迅速分布于小鼠各個器官中并以碎片的形式在體內(nèi)存留較長的時間。外源質(zhì)粒DNA經(jīng)胃腸道途徑不會整合到宿主染色體基因組上。 2外源質(zhì)粒DNA在小鼠腸道中的吸收機(jī)制研究 實(shí)驗(yàn)采用5-6周齡Balbc/C雄性小鼠6只,隨機(jī)分成2組,一組為實(shí)驗(yàn)對照組,灌胃200μL生理鹽水,另一組給小鼠灌胃1μg/μL質(zhì)粒pcDNA3溶液200μL,灌胃后4h ,分離空腸一段并提取總RNA,RT-PCR逆轉(zhuǎn)錄成cDNA,將雙鏈cDNA進(jìn)行體外轉(zhuǎn)錄合成cRNA, cRNA再次反轉(zhuǎn)錄成cDNA,Cy5-dCTP標(biāo)記實(shí)驗(yàn)組cDNA,Cy3-dCTP標(biāo)記實(shí)驗(yàn)組cDNA,標(biāo)記的DNA與芯片進(jìn)行雜交、洗滌、并進(jìn)行掃描,采用GenePix Pro4.0進(jìn)行數(shù)據(jù)分析。 結(jié)果顯示17667條基因中,一共有4196條基因表達(dá),其中明顯差異表達(dá)的基因有61條,表達(dá)上調(diào)36條,表達(dá)下調(diào)25條。外源質(zhì)粒DNA可誘導(dǎo)腸道陰離子轉(zhuǎn)運(yùn)蛋白的表達(dá),提示腸道中可能存在轉(zhuǎn)運(yùn)蛋白介導(dǎo)外源質(zhì)粒DNA的吸收。外源質(zhì)粒DNA進(jìn)入腸道后能夠引起腸道免疫應(yīng)答基因發(fā)生變化。外源質(zhì)粒DNA上調(diào)微粒體谷胱甘肽硫轉(zhuǎn)移酶和谷胱甘肽過氧化物酶的表達(dá),增強(qiáng)腸道的抗氧化功能。外源質(zhì)粒DNA通過抑制腸道Caspase3基因而抑制腸黏膜細(xì)胞凋亡。腸道絲裂原激活蛋白激酶基因(MAP2K2)發(fā)生上調(diào),說明外源質(zhì)粒DNA可能通過MAPK途徑激活腸黏膜細(xì)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.
【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R346

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