本文選題：量子點 + 熒光納米顆粒�。� 參考：《南京醫(yī)科大學(xué)學(xué)報(自然科學(xué)版)》2017年10期

【摘要】：目的:構(gòu)建一種可通過熒光成像進(jìn)行體內(nèi)外示蹤的納米基因載體,并對其生物相容性進(jìn)行初步評價。方法:在量子點Qdots表面修飾殼聚糖后形成熒光納米顆粒CS-Qdots,檢測其電鏡形態(tài)、光學(xué)特征、表面電荷和傅里葉轉(zhuǎn)換近紅外光譜(FTIR),并將其注射入裸鼠移植瘤內(nèi)觀察體內(nèi)成像信號;以凝膠阻滯電泳檢測CS-Qdots攜帶質(zhì)粒DNA的能力,并利用激光共聚焦顯微鏡觀察其轉(zhuǎn)染報告基因綠色熒光蛋白在細(xì)胞內(nèi)的表達(dá)情況。通過MTT試驗檢測細(xì)胞相對增殖率(RGR)、測定溶血率和小鼠急性毒性試驗評價CS-Qdots的生物相容性。結(jié)果:電鏡觀察顯示CS-Qdots納米顆粒粒徑為20~30 nm,zeta電位分析其表面電位為(28.02±1.15)m V,FTIR圖譜顯示出殼聚糖的特征譜帶,發(fā)射光譜分析CS-Qdots最大發(fā)射峰值在630 nm。凝膠阻滯電泳顯示納米顆粒和DNA的比例大于10∶1混合以后不再向正極泳動,激光共聚焦觀察CS-Qdots能攜帶質(zhì)粒p EGFP-C1在Hep G2細(xì)胞內(nèi)表達(dá)綠色熒光蛋白,小鼠活體成像中CS-Qdots在裸鼠移植瘤內(nèi)有較強(qiáng)熒光成像信號。MTT試驗顯示,50、100、200和400μg/m L的CS-Qdots共孵育的細(xì)胞RGR分別為1.000、1.000、0.917和0.875,而相應(yīng)濃度的Qdots孵育細(xì)胞RGR為1.000、0.850、0.621和0.326;濃度在100μg/m L以上的Qdots量子點溶血率均大于5%,而CS-Qdots納米顆粒在400μg/m L以內(nèi)溶血率均小于5%。小鼠尾靜脈注射CS-Qdots 72 h急性毒性試驗顯示,與生理鹽水對照組相比,沒有明顯的臟器病理損傷,肝腎功能正常,血細(xì)胞計數(shù)正常。結(jié)論:成功構(gòu)建了熒光納米顆粒CS-Qdots,它能有效轉(zhuǎn)染基因在細(xì)胞內(nèi)表達(dá),具有較高的生物相容性,并可在體內(nèi)外進(jìn)行熒光成像,是可示蹤基因的運(yùn)送納米載體。
[Abstract]:Aim: to construct a novel in vivo and in vitro tracer gene vector and evaluate its biocompatibility. Methods: the fluorescent nanoparticles CS-Qdotswere formed on the surface of Qdots. The morphology, optical characteristics, surface charge and Fourier transform near infrared spectroscopy (FT-NIR) of CS-Qdotswere measured, and then injected into nude mice to observe the imaging signals in vivo. The ability of CS-Qdots to carry plasmid DNA was detected by gel retardation electrophoresis, and the expression of green fluorescent protein (GFP) was observed by confocal laser microscopy. The relative cell proliferation rate was measured by MTT assay, and the biocompatibility of CS-Qdots was evaluated by hemolysis rate and acute toxicity test in mice. Results: the surface potential of CS-Qdots nanoparticles was determined to be 28.02 鹵1.15m ~ (-1) m ~ (-1) V ~ (-1). The characteristic band of chitosan was found in FTIR spectra. The maximum emission peak value of CS-Qdots was 630nm ~ (-1) nm. Gel block electrophoresis showed that the ratio of nanoparticles to DNA was more than 10:1, and the ratio of nanoparticles to DNA did not move towards the positive electrode after mixing. Laser confocal observation showed that CS-Qdots could carry plasmid p EGFP-C1 to express green fluorescent protein in Hep G2 cells. In vivo imaging of mice, CS-Qdots showed strong fluorescence imaging signal in nude mice xenografts. MTT test showed that the RGR of cells incubated with CS-Qdots of 50100200 and 400 渭 g / mL were 1.0000.0000.000,0.917 and 0.875respectively, while the corresponding concentrations of Qdots incubated cells RGR were 1.000,0.8500.0.621 and 0.326, respectively, and the concentrations were 100 渭 g / mL. The hemolysis rates of Qdots QDs on the above QDs are all greater than 5, while the hemolysis rates of CS-Qdots nanoparticles are less than 5 within 400 渭 g / mL. The acute toxicity test of 72 h CS-Qdots injection in mice showed that there was no obvious organ pathological injury, normal liver and kidney function and normal blood cell count compared with normal saline control group. Conclusion: the fluorescent nanoparticles CS-Qdotswere successfully constructed, which can effectively transfect gene expression in cells, have high biocompatibility, and be able to carry out fluorescence imaging in vivo and in vitro, which can be used as a tracer gene delivery nano-carrier.
【作者單位】： 東南大學(xué)附屬中大醫(yī)院檢驗科;
【基金】：國家自然科學(xué)基金青年基金項目(81501525)
【分類號】：R450

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