本文選題：DCN + IL-24��； 參考：《山西醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的：探討核心蛋白聚糖(DCN)基因聯(lián)合白介素-24(IL-24)基因在體外對(duì)PBMC功能的影響。 方法：1.構(gòu)建重組質(zhì)粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24。經(jīng)雙酶切、測(cè)序鑒定基因序列,將重組質(zhì)粒轉(zhuǎn)入大腸桿菌DH-5α中,大量提取pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24重組質(zhì)粒；2.利用脂質(zhì)體2000將重組質(zhì)粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24轉(zhuǎn)入PBMC,鑒定轉(zhuǎn)染效率,并通過RT-PCR檢測(cè)細(xì)胞內(nèi)DCN和IL-24的mRNA表達(dá)；3.測(cè)定重組質(zhì)粒對(duì)PBMC的影響：MTT法檢測(cè)轉(zhuǎn)染后PBMC的增殖、ELISA測(cè)定轉(zhuǎn)染細(xì)胞IFN-r分泌水平、流式細(xì)胞技術(shù)(FCM)檢測(cè)PBMC表面PD-1的表達(dá)。 結(jié)果：1.構(gòu)建的質(zhì)粒經(jīng)EcoRⅠ.NotⅠ雙酶切及PCR鑒定后,所得的基因片段與預(yù)期的基因片段大小相符；經(jīng)DNA測(cè)序證實(shí)：序列與GenBank中IL-24的序列一致；2.通過脂質(zhì)體2000將重組質(zhì)轉(zhuǎn)入PBMC后,熒光顯微鏡下可見細(xì)胞內(nèi)綠色熒光的表達(dá)；RT-PCR檢測(cè)到細(xì)胞內(nèi)有DCN和IL-24的mRNA表達(dá)；3.MTT法檢測(cè)細(xì)胞增殖：轉(zhuǎn)染后96h、120h、144h,DCN與IL-24聯(lián)合組的細(xì)胞增殖率明顯高于DCN組,IL-24組,空質(zhì)粒組,空白對(duì)照組,脂質(zhì)體組；ELISA顯示：DCN與IL-24聯(lián)合組分泌IFN-γ的量顯著高于其他組,有統(tǒng)計(jì)學(xué)差異(P0.05)；流式細(xì)胞技術(shù)(FCM)顯示：PD-1受體的表達(dá)水平,聯(lián)合組高于其它組。 結(jié)論：成功構(gòu)建了pcDNA3.1(+)-IL-24重組質(zhì)粒；利用脂質(zhì)體轉(zhuǎn)染法將DCN和IL-24重組質(zhì)粒轉(zhuǎn)入PBMC,二者能顯著促進(jìn)PBMC的增殖,并增加IFN-γ的分泌及其表面PD-1受體的表達(dá)。提示DCN和IL-24聯(lián)合對(duì)PBMC活性能顯示更強(qiáng)的促進(jìn)作用。
[Abstract]:Objective : To investigate the effect of core protein ( DCN ) gene and interleukin - 24 ( IL - 24 ) gene on the function of PBMC in vitro .



Methods : 1 . The recombinant plasmid pcDNA3.1 ( + ) - DCN and pcDNA3.1 ( + ) - IL - 24 were constructed .
2 . The recombinant plasmid pcDNA3.1 ( + ) - DCN and pcDNA3.1 ( + ) - IL - 24 were transferred into PBMC by liposome 2000 to identify the transfection efficiency , and the mRNA expression of DCN and IL - 24 in the cells was detected by RT - PCR ;
3 . The effect of recombinant plasmid on PBMC was determined : MTT assay was used to detect the proliferation of PBMC , and the level of IFN - r secretion was determined by ELISA , and the expression of PD - 1 was detected by flow cytometry ( FCM ) .



Results : 1 . The constructed plasmid was digested with EcoR鈪，

本文編號(hào)：1922673