【摘要】：目的：本文以PAMAM為載體，以具有主動靶向功能的HA為被膜材料，構(gòu)建PAMAM/HA用于遞送靶向VEGF mRNA的siRNA，誘導RNAi，通過抗血管生成控制腫瘤生長。由于單一的抗血管生成療法無法快速、完全消除腫瘤及PAMAM/HA無法完全避免體內(nèi)RES吞噬作用的缺陷，在上述復合物的基礎(chǔ)上，將HA進行PEG化后包覆PAMAM用于聯(lián)合遞送靶向VEGF的siRNA和抗腫瘤化療藥物PTX，構(gòu)建兼具靶向及長循環(huán)雙重功能的聯(lián)合載藥系統(tǒng)siRNA/PTX/PAMAM/HA-PEG，以期增強體內(nèi)抗腫瘤作用，實現(xiàn)低毒高效的抗腫瘤效果。 方法：本文構(gòu)建了不同HA包覆修飾量的siRNA/PAMAM/HA（SPH）復合物，瓊脂糖凝膠電泳進行siRNA阻滯實驗，，激光粒度分析儀測定粒徑及電位，MTT法測定細胞毒性，采用熒光顯微鏡和流式細胞儀對復合物的攝取進行考察，從而篩選出最佳的HA包覆量。通過酰胺化反應(yīng)合成HA-PEG，經(jīng)純化后通過1H-NMR對產(chǎn)物結(jié)構(gòu)進行表征。通過靜電吸附及物理包埋法構(gòu)建siRNA和PTX共遞送的復合物載藥系統(tǒng)siRNA/PTX/PAMAM/HA-PEG（SPPG），通過siRNA阻滯實驗、穩(wěn)定性實驗、粒徑、電位、形貌和包封率等對復合物進行表征。MTT法測定細胞毒性，流式細胞儀、HPLC定量考察復合物的攝取情況，RT-PCR測定復合物的RNAi效果，通過流式細胞儀和共聚焦顯微鏡分別考察復合物的內(nèi)吞機制和細胞內(nèi)定位。采用小動物活體熒光成像技術(shù)觀察復合物在體內(nèi)的分布情況。 結(jié)果：激光粒度分析儀測定結(jié)果表明，隨著HA包覆比例的增加，SPH的粒徑增加，zeta電位降低。MTT和細胞攝取實驗表明，HA的包覆使復合物的細胞毒性下降；HA的包覆能增加細胞對該復合物的攝取，增強復合物的主動靶向性，并且當HA的包覆量為25%（電荷比）時，細胞攝取量最高。1H-NMR檢測結(jié)果顯示成功合成HA-PEG。siRNA/PTX/PAMAM（SPP）、siRNA/PTX/PAMAM/HA（SPPH）、siRNA/PTX/PAMAM/HA-PEG（SPPG）的粒徑依次增大，電位降低。細胞實驗表明，HA的包覆使細胞對復合物的攝取最高，RNAi效果最強；而HA-PEG的包覆攝取雖然比未有包覆的復合物低，RNAi效果二者卻未有明顯差異。攝取機理研究結(jié)果表明，三種復合物的細胞攝取均為能量依賴的轉(zhuǎn)運過程，SP經(jīng)網(wǎng)格蛋白途徑入胞，SPH及SPG經(jīng)網(wǎng)格蛋白及小窩蛋白多種途徑入胞。動物實驗表明，SPPH及SPPG均可不斷向腫瘤部位蓄積，具有主動靶向功能；且SPPG能夠在腫瘤部位獲得較長時間的累積，具有一定的長循環(huán)作用。 結(jié)論：本文以PAMAM為載體，通過合成HA-PEG，構(gòu)建了毒性較低的用于siRNA及PTX共遞送的載藥系統(tǒng)SPPG，在保留HA主動靶向的基礎(chǔ)上，兼?zhèn)淞碎L循環(huán)功能，將基因療法與傳統(tǒng)的化療相結(jié)合，有望實現(xiàn)較高的體內(nèi)抗腫瘤作用。
[Abstract]:Aim: in this paper, PAMAM was used as carrier and HA with active targeting function as membrane material to construct siRNA, which was used to deliver targeted VEGF mRNA to induce RNAi, to control tumor growth through anti-angiogenesis. Because the single antiangiogenic therapy can not be rapid, the tumor and PAMAM/HA can not completely avoid the defects of RES phagocytosis in vivo, on the basis of the above complex, In order to enhance the anti-tumor effect in vivo, PEG coated HA was used to deliver the siRNA targeting VEGF and the anti-tumor chemotherapeutic drug PTX, to construct a combined drug delivery system siRNA/PTX/PAMAM/HA-PEG, with both targeting and long circulation functions, so as to enhance the anti-tumor effect in vivo. To achieve low toxicity and high efficiency of anti-tumor effect. Methods: siRNA/PAMAM/HA (SPH) complexes with different amount of HA coating were constructed. Agarose gel electrophoresis was used for siRNA block assay. Particle size and potential were measured by laser particle size analyzer. Cytotoxicity was determined by MTT method. The uptake of the complex was investigated by fluorescence microscope and flow cytometry, and the best HA coating was obtained. HA-PEG, was synthesized by amidation reaction and the structure of the product was characterized by 1H-NMR. By means of electrostatic adsorption and physical entrapping method, a compound drug carrier system of siRNA and PTX was constructed by means of siRNA block experiment, stability test, particle size, potential. The morphology and encapsulation efficiency were used to characterize the complex. MTT assay was used to determine the cytotoxicity, flow cytometry and HPLC were used to quantitatively investigate the uptake of the complex, and RT-PCR was used to determine the RNAi effect of the complex. The endocytosis mechanism and intracellular localization of the complex were investigated by flow cytometry and confocal microscopy, respectively. The distribution of the complex in vivo was observed by in vivo fluorescence imaging of small animals. Results: the results of laser particle size analyzer showed that with the increase of HA coating ratio, the diameter of SPH increased and the zeta potential decreased. The results of MTT and cell uptake tests showed that the coating of HA decreased the cytotoxicity of the complex. The coating of HA can increase the uptake of the complex, enhance the active targeting of the complex, and when the encapsulation amount of HA is 25% (charge ratio), The results of 1H-NMR showed that the particle size of successfully synthesized HA-PEG.siRNA/PTX/PAMAM (SPP), siRNA/PTX/PAMAM/HA (SPPH), siRNA/PTX/PAMAM/HA-PEG (SPPG) increased in turn and the potential decreased. Cell experiments showed that the uptake of complex by HA was the highest and that of RNAi was the strongest, but that of HA-PEG was lower than that of uncoated complex, but there was no significant difference between the two groups. The results of uptake mechanism showed that the uptake of the three complexes was an energy-dependent transport process. SP was transfered through grid protein pathway, SPH and SPG via grid protein and fossa protein. Animal experiments showed that both SPPH and SPPG could accumulate to the tumor site continuously and had active targeting function, and SPPG could accumulate in the tumor site for a long time and had a certain long circulation effect. Conclusion: in this paper, a novel drug delivery system, SPPG, with low toxicity for siRNA and PTX delivery, was constructed using PAMAM as carrier and HA-PEG, as a carrier. The drug delivery system SPPG, could retain the active targeting of HA and possess long cycle function. The combination of gene therapy and traditional chemotherapy is expected to achieve higher anti-tumor effect in vivo.
【學位授予單位】：蘇州大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R94

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