STAT3-siRNA-前胸腺素α共负载热敏性纳米胶束对肿瘤相关树突状细胞免疫功能调控的影响

发布时间：2018-05-18 19:55

本文选题：胶束 + 温度敏感性　；参考：《苏州大学》2016年硕士论文

【摘要】：目的:将负载前胸腺素(ProTα)多肽片段,具有温度敏感性的聚合物胶束用于负载STAT3-siRNA到达肿瘤相关树突状细胞(TADCs),下调STAT3蛋白的表达,调控肿瘤相关树突状细胞的功能,发挥其体内免疫抗肿瘤效果。方法:通过酰胺键反应合成壳聚糖接枝的泊洛沙姆(PO-CS)载体,采用FT-IR和1H-NMR进行结构验证,并用芘探针测量其形成胶束后的临界胶束浓度。在溶液中形成胶束后,利用马尔文粒径电位测定仪中测量其粒径和电位大小,同时测量15℃,25℃和37℃条件下胶束的粒径变化考察其温度敏感性。本实验还分别测量了泊洛沙姆(PO)、壳聚糖(CS)以及PO-CS载体在不同温度下的粒径变化情况来考察该胶束的温度敏感性来源。将制备好的胶束置于透射电镜(TEM)中观察其表面形态,并通过琼脂糖凝胶电泳验证胶束对siRNA的保护作用。利用MTT的方法考察胶束对树突状细胞(DCs)、脾脏细胞(Spleen Cells)和293T细胞的安全性;并使用流式细胞仪和激光共聚焦显微镜考察PO-CS胶束负载ProTα和siRNA进入DCs的情况及聚合物胶束温度敏感性的考察。利用Western Blot方法检测负载STAT3胶束与TADCs共培养后STAT3蛋白的下调情况,同时使用流式细胞仪检测STAT3-siRNA发挥沉默效果后,ProTα促进TADCs表达CD40,CD86及IL-12的情况。采用IVIS Lumina II小动物成像仪观察胶束负载Cy5-siRNA的体内动态情况,使用红外热成像仪考察胶束体内冷刺激所需的时间。建立B16黑色素瘤肿瘤模型考察负载ProTα和STAT3-siRNA的ProTα-PO-CS载体的体内药效学效果。结果:本实验所构建PO-CS载体容易形成胶束,CMC较低(5.0μg/ml)。TEM结果表明,PO-CS胶束外观圆整,粒径为231.7±4.6nm,电位为17.3±0.3mV,PI值较小,对siRNA的包封率达90%以上,且具有一定的温度敏感性。MTT结果显示,PO-CS胶束的DCs细胞毒性低,并且其负载的siRNA在树突状细胞中的摄取(81%)要比巨噬细胞(25%)高。激光共聚焦显微镜结果表明,ProTα/PO-CS胶束能很好地将siRNA转运到TADCs中,并且具有很好的温度敏感性,能在冷刺激的作用下实现siRNA的释放。Western Blot结果表明,胶束负载的STAT3-siRNA能有效沉默TADCs中STAT3蛋白的表达,并且能在流式细胞仪中观察到ProTα促进TADCs中表面成熟因子CD40,CD86的上调,而未加STAT3-siRNA处理组并无此功能。该结果表明共负载STAT3-siRNA和ProTα处理后,TADCs的功能得到恢复,能向成熟树突状细胞分化。IL-12因子的上调结果也证明经共负载STAT3-siRNA和ProTα胶束治疗后,TADCs恢复正常的功能。体内小动物成像结果表明,胶束负载siRNA后,持续长时间观察到Cy5-siRNA的体内动态情况。红外热成像结果表明,体内达到冷刺激效果温度所需的时间为20-25min。体内肿瘤生长抑制实验结果表明,与PBS组相比,负载ProTα和STAT3-siRNA的ProTα-PO-CS冷刺激胶束组能很好地调控TADCs的功能,发挥免疫抗肿瘤效果,抑制肿瘤的生长,同时释放更多的IL-12,共同抑制肿瘤的生长。结论:ProTα-PO-CS温度敏感性胶束能将siRNA递送到TADCs中,沉默STAT3蛋白的表达,调控TADCs的功能,恢复ProTα促进树突状细胞向成熟树突状细胞分化的功能,发挥联合免疫抗肿瘤的治疗效果。
[Abstract]:Objective: to use the ProT alpha peptide fragment of pre loaded thymosin (ProT alpha) and a temperature sensitive polymer micelle to load STAT3-siRNA to tumor related dendritic cells (TADCs), down regulate the expression of STAT3 protein, regulate the function of tumor related dendritic cells and exert its immune antitumor effect in vivo. The sugar grafted poloxamer (PO-CS) carrier was constructed by FT-IR and 1H-NMR, and the pyrene probe was used to measure the critical micelle concentration after the formation of the micelles. After forming the micelles in the solution, the particle size and potential of the micelles were measured by Malvin particle size potentiometric measuring instrument, and the particle size changes of the micelles at 15, 25 and 37 C were measured. The temperature sensitivity of poloxamer (PO), chitosan (CS) and PO-CS carrier at different temperatures were measured to investigate the temperature sensitivity of the micelles. The surface morphology of the micelles was observed in the transmission electron microscope (TEM), and the micelle to siRN was verified by agarose gel electrophoresis. The protective effect of A. The safety of micelles on dendritic cells (DCs), splenic cells (Spleen Cells) and 293T cells were investigated by using MTT method. The flow cytometry and laser confocal microscopy were used to investigate the condition of PO-CS micelle loading ProT A and siRNA into DCs and the temperature sensitivity of polymer micelles. The Western Blot method was used to examine the temperature sensitivity of the micelles. After measuring the downregulation of STAT3 protein after co culture of STAT3 micelles and TADCs, and using flow cytometry to detect the silencing effect of STAT3-siRNA, ProT alpha promoted TADCs to express CD40, CD86 and IL-12. The dynamic state of the micellar load in vivo was observed by IVIS Lumina II small animal imaging instrument, and the infrared thermal imaging instrument was used. A B16 melanoma tumor model was established to examine the pharmacodynamic effects of the B16 melanoma tumor model to investigate the pharmacodynamic effects of the ProT alpha -PO-CS carrier loaded with ProT A and STAT3-siRNA. Results: the PO-CS vector constructed in this experiment was easy to form micelles, and the low CMC (5 u g/ml).TEM results showed that the PO-CS micelle was round, the particle size was 231.7 + 4.6nm, and the potential was 1. 7.3 + 0.3mV, PI value is smaller, the encapsulation efficiency of siRNA is over 90%, and a certain temperature sensitivity.MTT results show that PO-CS micelle DCs cell toxicity is low, and the uptake of siRNA in dendritic cells (81%) is higher than macrophage (25%). The results of laser confocal microscopy show that ProT a /PO-CS micelle can be a good siRNA. It is transported to TADCs, and has good temperature sensitivity. The release of siRNA under the effect of cold stimulation,.Western Blot results show that the STAT3-siRNA loaded by micelles can effectively silence the expression of STAT3 protein in TADCs, and can be observed in the flow cytometry to promote the increase of the surface maturity factor CD40 and CD86 up of TADCs in TADCs. No STAT3-siRNA treatment group did not have this function. The results showed that the function of TADCs was restored after the co loading of STAT3-siRNA and ProT alpha, and the up-regulated result of.IL-12 factor to mature dendritic cells also proved that TADCs restored normal function after the co loaded STAT3-siRNA and ProT alpha micellar treatment. The dynamic state of Cy5-siRNA in vivo was observed for a long time after the micelles were loaded with siRNA. The infrared thermal imaging results showed that the time required for the cold stimulation temperature in the body was 20-25min. tumor growth inhibition test. Compared with the PBS group, the ProT alpha -PO-CS cold stimulation micelle loaded with ProT alpha and STAT3-siRNA could be very good. Regulate the function of TADCs, play an immune anti-tumor effect, inhibit the growth of tumor, and release more IL-12, and jointly inhibit the growth of the tumor. Conclusion: ProT alpha -PO-CS sensitive micelle can deliver siRNA to TADCs, silence the expression of STAT3 protein, regulate the power of TADCs, and restore the ProT a to promote dendritic cells to mature dendritic cells The function of cell differentiation is to play a combined immunological effect against tumor.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R943;R96

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