新型高分子脂质体优化转染VEGF siRNA对视网膜新生血管抑制的实验研究

发布时间：2018-06-25 20:47

本文选题：高分子脂质体 + 基因转染　；参考：《天津医科大学》2012年博士论文

【摘要】：目的：对载质粒DNA的高分子脂质体进行性能分析,优化其转染条件；观察其细胞毒性及转染不同细胞的转染效率；研究高分子脂质体作为基因载体在氧诱导视网膜病变动物模型中对视网膜新生血管的抑制作用,体现其独特的穿膜能力和缓释功能,为将来制备靶向性药物/基因共载多功能给药系统提供实验基础和理论依据。方法： 1.课题组自行合成羧甲基壳聚糖十八烷基季铵盐(OQLCS/chol)纳米粒子,将穿膜肽(Tat)与其表面的功能基团连接制备出兼具跨膜、长循环功能的高分子脂质体,并对其表征进行检测。研究该高分子脂质体对质粒DNA在不同质量比、不同作用时间及血清存在与否条件下的包载效能,制定最优的转染条件；采用高速离心法进行体外释放动力学实验,绘制体外释放曲线；利用CCK-8法检测不同浓度高分子脂质体在不同时间分别对视网膜色素上皮(RPE)细胞的毒性,找到适于细胞转染的安全浓度；在优化条件下转染RPE细胞,同时以商品化的Lipofectamine2000做对照,比较两者的转染效率。 2.建立缺氧条件下RPE细胞模型,转染48时后收集细胞上清通过ELISA法检测各组VEGF量的变化,细胞爬片后行免疫组织化学染色分析各组VEGF蛋白的表达,采用Real-time PCR检测各组RPE细胞内VEGF mRNA的变化。 3.采用改良的Smith's法建立氧诱导视网膜病变C57BL/6J小鼠模型,共95只,另有27只小鼠于正常氧环境中饲养。P11天时行小鼠玻璃体腔内注药,不同时间点,于正常打药组中随机选取两只小鼠行视网膜铺片,荧光显微镜下观察GFP的表达情况；各组中抽取两只小鼠FITC-dextran心脏灌注后行视网膜铺片观察视网膜血管形态的变化；分别于各组随机抽取两只小鼠,行HE染色,统计突破视网膜内界膜的血管内皮细胞核数；于各组随机抽取的两只小鼠做5gm冰冻切片,DAPI核染后荧光显微镜下观察,以反映高分子脂质体携带质粒DNA的穿膜能力。结果： 1.纳米粒平均粒度为134nm,Zeta电位为+39.64mV,包载质粒后为236nm。透射电镜下纳米粒呈大小均匀的圆形粒子。体外释放实验显示载质粒DNA的高分子脂质体在体外最初5天为突释相,约有70%的质粒释放；此后至第14天呈稳态缓释。该高分子脂质体毒性较低,最高安全浓度为20ug/mL。 2.通过电泳阻滞实验,筛选出高分子脂质体与质粒DNA质量比≥2：1时、混合时间为30-40min、无血清存在的培养液为最优转染条件；并以此条件转染RPE、Hela及RF/16,对RPE的转染率约为69%,对Hela细胞的转染效率约为52%,对RF/16转染效率约为80%。 3.对缺氧后的RPE转染后观察,高分子脂质体组和Lipo组均能有效地将质粒DNA转染入细胞并表达。48小时后收集细胞上清,行ELISA法检测各组VEGF量,高分脂质体组和Lipo组中VEGF含量较模型组明显降低,差异均有显著性(P0.01),其中Lipo组抑制程度略较高分脂质体组大,但差异不具有显著性(P=0.10)。72小时后行细胞免疫组化显示细胞内VEGF的变化,Lipo组和高分脂质体组对VEGF表达产生明显的抑制作用,胞浆内棕色着染明显淡薄,OD值相比高分子脂质体组略低于Lipo组(P=0.443)。Real-time PCR结果显示转染后48小时,高分脂质体组和Lipo组VEGF mRNA的变化与ELISA结果相似。 4.成功建立了氧诱导视网膜病变小鼠模型。玻璃体腔内注射后可见注药后第1天视网膜内即有GFP表达,至第6天达到高峰,第11天时在高分子脂质体组仍能看到GFP表达,Lipo组不明显。FITC(?)心脏灌注后显示视网膜无灌注区及新生血管范围在高分子脂质体组及Lipo组明显改善,效果相当。HE染色后行突破视网膜内界膜的血管内皮细胞核数,结果表明P17天时高分子脂质体组及Lipo组均能有效抑制新生血管生成,P22天时,高分子脂质体效果仍能显现,新生血管内皮细胞核数较Lipo组少,差异具有显著性(P0.01)。冰冻切片显示,注射后第1天,在高分子脂质体组及Lipo组均可以观察到玻璃体腔内有GFP的表达,高分子脂质体组有部分于视网膜表面表达；注射后第6天,两组的表达量均较高,且GFP表达位于RPE层附近；注射后第11天,在高分子脂质体组切片中仍能找到GFP的表达,但在Lipo组已很难找出。行VEGF的Western Blot检测,玻璃体腔内注射后6天,高分子脂质体组与Lipo组抑制效果相当(P=0.092)；P22时高分子脂质体组较Lipo组抑制明显,差异有显著性(P0.05)。Real-timePCR结果与western blot结果相似。结论： 1.自行制备的Tat化的高分子脂质体O-羧甲基壳聚糖十八烷基季铵盐/胆固醇具有粒径均匀、分散性好、Zeta电位较高、细胞毒性较小的特点,体外释放能在较短时间达到治疗浓度,后续可缓慢平稳释放。 2.该高分子脂质体能携带基因很好的转染RPE细胞、Hela细胞及RF/16细胞,转染效率与商品化的Lipofectamine2000相当。 3.该高分子脂质体携带治疗基因可以减少视网膜新生血管的生成,且具有显著的穿膜能力及缓释功能,在体内可维持较长时间的治疗效果,具有临床应用的开发前景。
[Abstract]:Objective:
To analyze the performance of the high molecular liposomes carrying plasmid DNA, optimize the transfection conditions, observe the cytotoxicity and transfection efficiency of different cells, and study the inhibition effect of polymer liposome as gene carrier on retinal neovascularization in the oxygen induced retinopathy animal model, reflecting its unique membrane ability and delay. It provides experimental basis and theoretical basis for the preparation of targeted drug / gene co loading multifunctional drug delivery system in the future.
Method:
1. the 1. subject group syntheses carboxymethyl chitosan eighteen alkyl quaternary ammonium salt (OQLCS/chol) nanoparticles, and connects the membrane peptide (Tat) with its surface functional groups to prepare polymer liposomes with cross membrane and long circulation function, and detect its characterization. The high sub liposomes have different effects on the plasmid DNA in different mass ratio. The optimal transfection conditions were established under the conditions of whether there was or not in the presence of serum and serum, and the release curves were plotted in vitro by high speed centrifugation, and the CCK-8 method was used to detect the toxicity of different concentration of polymer liposomes to retinal pigment epithelium (RPE) cells at different time. The transfection efficiency of RPE cells was compared with that of commercial Lipofectamine2000.
2. the RPE cell model was established under the condition of hypoxia, and the cell supernatant was collected by ELISA to detect the changes of VEGF in each group after 48 time transfection. The expression of VEGF protein in each group was analyzed by immunohistochemical staining, and the changes of VEGF mRNA in each group of RPE cells were detected by Real-time PCR.
3. the modified Smith's method was used to establish the C57BL/6J mice model of oxygen induced retinopathy. There were 95 mice in the normal oxygen environment and 27 mice were fed in the glass cavity of the normal oxygen environment. At different time points, two mice were randomly selected in the normal drug group, and the expression of GFP was observed under the fluorescence microscope. In each group, two mice were selected to observe the changes of retinal vascular morphology after FITC-dextran heart perfusion. Two mice were randomly selected in each group. HE staining was performed to break through the number of vascular endothelial nuclei of the inner boundary membrane of the retina; two mice were randomly selected from each group for 5gm frozen section, and the DAPI nucleus was stained with fluorescence. Microscopic observation was carried out to reflect the penetrating ability of plasmid DNA carrying polymer liposomes.
Result:
The average particle size of 1. nanoparticles was 134nm, the Zeta potential was +39.64mV, and the loaded plasmid was 236nm. transmission electron microscope with uniform round particles. The release experiment in vitro showed that the high molecular liposomes containing plasmid DNA were released in the first 5 days in vitro, about 70% of the plasmids released. The toxicity of plastids was low, and the highest safety concentration was 20ug/mL.
2. through the electrophoretic block test, the mixing time was 30-40min when the mass ratio of high molecular liposomes and plasmid DNA was more than 2:1, and the culture solution without serum was the optimal transfection condition, and RPE, Hela and RF/16 were transfected on this condition, the transfection rate to RPE was about 69%, the transfection efficiency of Hela cells was about 52%, and the efficiency of RF/16 transfection was about 80%..
3. after transfection of RPE after hypoxia, both high molecular liposome group and Lipo group could effectively transfect plasmid DNA into cells and express cell supernatant after.48 hours. ELISA method was used to detect VEGF quantity in each group. The content of VEGF in the liposome group and Lipo group was significantly lower than that in the model group. The difference was significant (P0.01), in which the Lipo group was inhibited. The difference was not significant (P=0.10), but the difference was not significant (P=0.10).72 hours after cell immuno histochemistry showed the changes of intracellular VEGF, Lipo and high fat liposomes group had obvious inhibitory effect on VEGF expression, and the brown staining in the cytoplasm was obviously weak, and the OD value was slightly lower than the Lipo group (P=0.443).Real-time compared with the polymer liposome group. PCR results showed that the changes of VEGF mRNA in high liposomes and Lipo groups were similar to those of ELISA at 48 hours after transfection.
4. a mouse model of oxygen induced retinopathy was successfully established. After intravitreal injection, GFP expression was found in the retina first days after injection, and reached the peak at sixth days. At eleventh days, the expression of GFP could still be seen in the polymer liposome group. The Lipo group was not significantly.FITC (?) after heart perfusion, the area of retinal perfusion and the range of neovascularization were shown. The effect of high molecular liposome group and Lipo group was obviously improved. The result was that after.HE staining, the number of vascular endothelial nuclei was broken through the inner boundary membrane of the retina. The results showed that both the high molecular liposomes group and the Lipo group could effectively inhibit the formation of the neovascularization at P17 days, and the effect of the polymer liposomes still appeared at P22 days, and the number of the nucleus of the neovascular endothelial cells was more than that of the Lipo group. The difference was significant (P0.01). The frozen section showed that the expression of GFP in the vitreous body could be observed in the high molecular liposomes group and the Lipo group first days after the injection. The high molecular liposome group was partly expressed on the retina surface, and the expression of the two groups was higher on the sixth day after injection, and the expression of GFP was near the RPE layer; after the injection, the expression was in the RPE layer. On the 11 day, the expression of GFP could still be found in the high molecular liposomes group, but it was difficult to find out in the Lipo group. Western Blot detection of VEGF and the 6 days after intravitreal injection, the inhibition effect of the polymer liposome group and the Lipo group was equal (P=0.092), and the high molecular liposome group was significantly more inhibited than the Lipo group when P22, and the difference was significant (P0.05).Real-timePC. The results of R were similar to that of Western blot.
Conclusion:
1. the Tat carboxymethyl chitosan eighteen alkyl quaternary ammonium salt / cholesterol prepared by ourselves has the characteristics of homogeneous particle size, good dispersibility, high Zeta potential and less cytotoxicity. In vitro release can reach the treatment concentration in a short time, and the subsequent release can be slowly and smoothly released.
2. the liposome can carry well transfected RPE cells. The transfection efficiency of Hela cells and RF/16 cells is comparable to that of commercialized Lipofectamine2000 cells.
3. the polymer liposomes carrying the therapeutic gene can reduce the formation of retinal neovascularization, and have significant membrane ability and sustained release function. It can maintain the therapeutic effect for a long time in the body, and has the prospect of clinical application.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R774.1

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