本文選題：膠束 + 溫度敏感性�。� 參考：《蘇州大學》2016年碩士論文

【摘要】：目的:將負載前胸腺素(ProTα)多肽片段,具有溫度敏感性的聚合物膠束用于負載STAT3-siRNA到達腫瘤相關樹突狀細胞(TADCs),下調(diào)STAT3蛋白的表達,調(diào)控腫瘤相關樹突狀細胞的功能,發(fā)揮其體內(nèi)免疫抗腫瘤效果。方法:通過酰胺鍵反應合成殼聚糖接枝的泊洛沙姆(PO-CS)載體,采用FT-IR和1H-NMR進行結(jié)構(gòu)驗證,并用芘探針測量其形成膠束后的臨界膠束濃度。在溶液中形成膠束后,利用馬爾文粒徑電位測定儀中測量其粒徑和電位大小,同時測量15℃,25℃和37℃條件下膠束的粒徑變化考察其溫度敏感性。本實驗還分別測量了泊洛沙姆(PO)、殼聚糖(CS)以及PO-CS載體在不同溫度下的粒徑變化情況來考察該膠束的溫度敏感性來源。將制備好的膠束置于透射電鏡(TEM)中觀察其表面形態(tài),并通過瓊脂糖凝膠電泳驗證膠束對siRNA的保護作用。利用MTT的方法考察膠束對樹突狀細胞(DCs)、脾臟細胞(Spleen Cells)和293T細胞的安全性;并使用流式細胞儀和激光共聚焦顯微鏡考察PO-CS膠束負載ProTα和siRNA進入DCs的情況及聚合物膠束溫度敏感性的考察。利用Western Blot方法檢測負載STAT3膠束與TADCs共培養(yǎng)后STAT3蛋白的下調(diào)情況,同時使用流式細胞儀檢測STAT3-siRNA發(fā)揮沉默效果后,ProTα促進TADCs表達CD40,CD86及IL-12的情況。采用IVIS Lumina II小動物成像儀觀察膠束負載Cy5-siRNA的體內(nèi)動態(tài)情況,使用紅外熱成像儀考察膠束體內(nèi)冷刺激所需的時間。建立B16黑色素瘤腫瘤模型考察負載ProTα和STAT3-siRNA的ProTα-PO-CS載體的體內(nèi)藥效學效果。結(jié)果:本實驗所構(gòu)建PO-CS載體容易形成膠束,CMC較低(5.0μg/ml)。TEM結(jié)果表明,PO-CS膠束外觀圓整,粒徑為231.7±4.6nm,電位為17.3±0.3mV,PI值較小,對siRNA的包封率達90%以上,且具有一定的溫度敏感性。MTT結(jié)果顯示,PO-CS膠束的DCs細胞毒性低,并且其負載的siRNA在樹突狀細胞中的攝取(81%)要比巨噬細胞(25%)高。激光共聚焦顯微鏡結(jié)果表明,ProTα/PO-CS膠束能很好地將siRNA轉(zhuǎn)運到TADCs中,并且具有很好的溫度敏感性,能在冷刺激的作用下實現(xiàn)siRNA的釋放。Western Blot結(jié)果表明,膠束負載的STAT3-siRNA能有效沉默TADCs中STAT3蛋白的表達,并且能在流式細胞儀中觀察到ProTα促進TADCs中表面成熟因子CD40,CD86的上調(diào),而未加STAT3-siRNA處理組并無此功能。該結(jié)果表明共負載STAT3-siRNA和ProTα處理后,TADCs的功能得到恢復,能向成熟樹突狀細胞分化。IL-12因子的上調(diào)結(jié)果也證明經(jīng)共負載STAT3-siRNA和ProTα膠束治療后,TADCs恢復正常的功能。體內(nèi)小動物成像結(jié)果表明,膠束負載siRNA后,持續(xù)長時間觀察到Cy5-siRNA的體內(nèi)動態(tài)情況。紅外熱成像結(jié)果表明,體內(nèi)達到冷刺激效果溫度所需的時間為20-25min。體內(nèi)腫瘤生長抑制實驗結(jié)果表明,與PBS組相比,負載ProTα和STAT3-siRNA的ProTα-PO-CS冷刺激膠束組能很好地調(diào)控TADCs的功能,發(fā)揮免疫抗腫瘤效果,抑制腫瘤的生長,同時釋放更多的IL-12,共同抑制腫瘤的生長。結(jié)論:ProTα-PO-CS溫度敏感性膠束能將siRNA遞送到TADCs中,沉默STAT3蛋白的表達,調(diào)控TADCs的功能,恢復ProTα促進樹突狀細胞向成熟樹突狀細胞分化的功能,發(fā)揮聯(lián)合免疫抗腫瘤的治療效果。
[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.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R943;R96

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