本文選題：樹狀大分子　切入點：基因轉(zhuǎn)染　出處：《上海醫(yī)學(xué)》2017年09期 　論文類型：期刊論文

【摘要】：目的以聚乙二醇化樹狀大分子包裹的納米金顆粒作為非病毒載體,將人鐵蛋白重鏈(FTH1)轉(zhuǎn)染到自然殺傷細(xì)胞92(NK92細(xì)胞)中,并進(jìn)行體外細(xì)胞MRI檢查。方法將第5代聚酰胺-胺樹狀大分子用聚乙二醇(PEG)修飾之后包裹納米金顆粒作為非病毒載體,壓縮FTH1報告基因然后轉(zhuǎn)染到NK92細(xì)胞內(nèi)。應(yīng)用磁共振氫譜、紫外分光光度計和透射電子顯微鏡等對合成的載體材料進(jìn)行表征;采用瓊脂糖凝膠電泳實驗確定載體材料與相應(yīng)基因之間最佳壓縮的氮磷比;應(yīng)用細(xì)胞計數(shù)試劑盒-8(CCK-8)檢測載體材料的毒性;通過測定水動力粒徑來確定不同氮磷比的條件下載體材料和基因形成的復(fù)合物能否順利通過細(xì)胞膜;應(yīng)用熒光顯微鏡和流式細(xì)胞儀測定轉(zhuǎn)染效率;應(yīng)用3.0T的MRI對轉(zhuǎn)染FTH1的NK92細(xì)胞進(jìn)行體外成像。結(jié)果合成的聚乙二醇化的包裹納米金顆粒的樹狀大分子{(Au~0)_(25)-G5.NH_2-mPEG_(17)}DENPs具有較好的生物相容性,材料細(xì)胞毒性較小,在濃度達(dá)到3 000nmol/L時細(xì)胞存活率仍然能達(dá)到60%以上。熒光顯微鏡和流式細(xì)胞儀測定顯示,在氮磷比為1∶1、2∶1、5:1、10:1時,轉(zhuǎn)染效率分別為57.8%、64.0%、80.2%、38.3%。在200和500μmol/L枸櫞酸鐵銨的條件下,MRI檢查發(fā)現(xiàn)T2加權(quán)像(T2WI)的信號均降低,后者更為明顯。結(jié)論聚乙二醇化樹狀大分子包裹的納米金顆粒能夠成功地轉(zhuǎn)染目的基因,MRI檢查能有效地在體外檢測FTH1轉(zhuǎn)染之后的NK92細(xì)胞,為MRI活體示蹤自然殺傷細(xì)胞過繼免疫腫瘤治療奠定了基礎(chǔ)。
[Abstract]:Objective to transfect human ferritin heavy chain FTH _ 1 into natural killer 92(NK92 cells by using nano-gold particles encapsulated by polyethylene glycol dendrimers as non-viral carriers. Methods the 5th generation polyamide-amine dendrimers were modified with polyethylene glycol (PEG) and coated with gold nanoparticles as non-viral carriers, then the FTH1 reporter gene was compressed and transfected into NK92 cells. The synthesized carrier materials were characterized by UV spectrophotometer and transmission electron microscope, and the optimum ratio of nitrogen to phosphorus was determined by agarose gel electrophoresis. The cytotoxicity of carrier material was detected by using cell count kit -8 (CCK-8), and whether the download material with different nitrogen and phosphorus ratio and the complex formed by gene could pass through the cell membrane smoothly, the hydrodynamic particle size was measured to determine whether the download material and the complex formed by gene were able to pass through the cell membrane successfully. The transfection efficiency was determined by fluorescence microscope and flow cytometry, and the NK92 cells transfected with FTH1 were imaged by 3.0T MRI in vitro. Results the synthesized poly (ethylene glycol) coated dendrimer {Austenized gold nanoparticles {{Auroethyleneuryleneglycolated] G5.NH2-mPEGG 17} DENPs had good biocompatibility. The cytotoxicity of the material was relatively small, and the cell survival rate was still over 60% when the concentration reached 3,000 nmol / L. fluorescence microscopy and flow cytometry showed that when the ratio of nitrogen to phosphorus was 1: 1, 2: 2, 1: 5: 1 10: 1, The transfection efficiency was 57.8% and 64.0%, respectively. Under the condition of 200 渭 mol/L and 500 渭 mol/L ammonium citrate, the signal of T2-weighted T2WI was decreased. Conclusion Polyethylene Glycol dendrimers encapsulated nanoparticles can be successfully transfected into NK92 cells after transfection of target gene and can effectively detect NK92 cells after FTH1 transfection in vitro. It lays a foundation for the adoptive immunotherapy of MRI tracer natural killer cells.
【作者單位】： 上海交通大學(xué)附屬第一人民醫(yī)院放射科;東華大學(xué)化學(xué)化工學(xué)院;
【基金】：上海市浦江人才計劃(14PJD028) 上海交通大學(xué)醫(yī)工交叉合作項目(YG2015MS31) 市級醫(yī)院臨床技能與臨床創(chuàng)新三年行動(16CR3091B) 上海市科學(xué)技術(shù)委員會科技發(fā)展項目資助
【分類號】：R445.2;R730.3

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