本文選題：DCN + IL-24　； 參考：《山西醫(yī)科大學》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PBMC功能的影響。 方法：1.構建重組質粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24。經雙酶切、測序鑒定基因序列,將重組質粒轉入大腸桿菌DH-5α中,大量提取pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24重組質粒；2.利用脂質體2000將重組質粒pcDNA3.1(+)-DCN和pcDNA3.1(+)-IL-24轉入PBMC,鑒定轉染效率,并通過RT-PCR檢測細胞內DCN和IL-24的mRNA表達；3.測定重組質粒對PBMC的影響：MTT法檢測轉染后PBMC的增殖、ELISA測定轉染細胞IFN-r分泌水平、流式細胞技術(FCM)檢測PBMC表面PD-1的表達。 結果：1.構建的質粒經EcoRⅠ.NotⅠ雙酶切及PCR鑒定后,所得的基因片段與預期的基因片段大小相符；經DNA測序證實：序列與GenBank中IL-24的序列一致；2.通過脂質體2000將重組質轉入PBMC后,熒光顯微鏡下可見細胞內綠色熒光的表達；RT-PCR檢測到細胞內有DCN和IL-24的mRNA表達；3.MTT法檢測細胞增殖：轉染后96h、120h、144h,DCN與IL-24聯(lián)合組的細胞增殖率明顯高于DCN組,IL-24組,空質粒組,空白對照組,脂質體組；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)計學差異(P0.05)；流式細胞技術(FCM)顯示：PD-1受體的表達水平,聯(lián)合組高于其它組。 結論：成功構建了pcDNA3.1(+)-IL-24重組質粒；利用脂質體轉染法將DCN和IL-24重組質粒轉入PBMC,二者能顯著促進PBMC的增殖,并增加IFN-γ的分泌及其表面PD-1受體的表達。提示DCN和IL-24聯(lián)合對PBMC活性能顯示更強的促進作用。
[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：1922673