【摘要】：目的:制備載內(nèi)皮抑素殼聚糖納米粒,并對其抗血管和抗腫瘤作用進行研究。方法:利用離子凝膠法制備載內(nèi)皮抑素殼聚糖納米粒(ES-NPs),探究負載內(nèi)皮抑素量的不同對納米粒粒徑、包封率、載藥量的影響,選出最佳的制備方案。利用體外透析法,檢測ES-NPs的釋藥特點。并觀察ES-NPs在不同溫度下血清中的穩(wěn)定性。利用MTT法和Transwell實驗,觀察ES-NPs和游離ES對臍靜脈內(nèi)皮細胞(HUVECs)增殖、遷徙的影響。隨后建立Lewis肺癌移植瘤模型,隨機平均分組,采用PBS、內(nèi)皮抑素注射液、ES-NPs和空納米粒對移植瘤進行治療。每2天測量每只小鼠腫瘤的體積,繪制腫瘤生長曲線。21天觀察結(jié)束后,計算抑瘤率,收集腫瘤組織和血清,免疫組化法檢測各組小鼠移植瘤的微血管密度(MVD),ELISA法檢測各組小鼠血清中內(nèi)皮抑素和VEGF濃度。結(jié)果:我們利用離子凝膠法成功制備出負載不同質(zhì)量內(nèi)皮抑素的ES-NPs,通過對各批次納米粒包封率和載藥量的比較,我們發(fā)現(xiàn),在40 mL殼聚糖溶液(1 mg/mL)中加入500μL ES(11.2mg/mL)制成的ES-NPs在保證較高包封率的同時也具有合適的載藥量和粒徑,能滿足治療的需要,并且通過加入5%海藻糖凍干后能夠穩(wěn)定保存。納米粒外觀呈球形,分布均勻,在體外具有明顯的緩釋性,7d累積釋放量達到(60.22±2.58)%,并且在血清中能夠穩(wěn)定存在至少48h。MTT實驗顯示,ES-NPs對HUVECs的生長有抑制作用。在藥物作用的最初24h,ES-NPs對HUVECs的抑制作用與游離ES相差不大(p=0.577)。但隨著作用時間的延長,ES-NPs的作用效果明顯優(yōu)于游離ES(p0.05)。同樣,在transwell實驗中,ES-NPs對HUVECs的遷徙的抑制作用也要明顯強于游離ES,差異具有統(tǒng)計學差異(p0.05);并且作用時間越長,兩者的作用效果的差距越大。在Lewis肺癌移植瘤模型上,我們觀察到ES-NPs能明顯抑制移植瘤生長。甚至,ES-NPs每7天注射1次的效果都優(yōu)于游離ES連續(xù)注射14天。而ES-NPs連續(xù)注射14天在我們的實驗中取得了最好的治療效果,對移植瘤的抑制率高達63.32%。免疫組化CD31染色結(jié)果顯示,ES-NPs連續(xù)注射14天組腫瘤組織的MVD最低,明顯低于對照組(p0.001),游離ES組(p0.001)。與MVD結(jié)果對應,ES-NPs連續(xù)注射14天組小鼠血清中的內(nèi)皮抑素水平明顯高于其它各組(p值均0.05),而VEGF水平明顯低于其它各組(p值均0.05)。證明了ES-NPs主要通過抑制腫瘤血管生成而起到抗腫瘤作用。結(jié)論:1)殼聚糖是制備內(nèi)皮抑素納米合適的材料,用殼聚糖制備的ES-NPs具有較高的包封率、載藥量和明顯的緩釋性,能滿足治療的需要。2)載內(nèi)皮抑素殼聚糖納米粒在體外能明顯抑制HUVECs的增殖和遷徙,作用效果優(yōu)于游離內(nèi)皮抑素。3)在Lewis肺癌模型上,載內(nèi)皮抑素殼聚糖納米粒能增強內(nèi)皮抑素的治療作用,通過抑制血管生成而明顯抑制腫瘤生長。
[Abstract]:Aim: to prepare endostatin-loaded chitosan nanoparticles and study their anti-vascular and anti-tumor effects. Methods: endostatin loaded chitosan nanoparticles (ES-NPs) were prepared by ionic gel method. The effects of different amount of loaded endostatin on particle size, encapsulation efficiency and drug loading were investigated. The drug release characteristics of ES-NPs were detected by in vitro dialysis. The stability of ES-NPs in serum at different temperature was observed. The effects of ES-NPs and free es on the proliferation and migration of umbilical vein endothelial cells (HUVECs) were observed by MTT and Transwell assay. Then the transplanted tumor model of Lewis lung cancer was established, and the transplanted tumor was treated with PBSs, endostatin injection (ESNPs) and empty nanoparticles. Tumor volume of each mouse was measured every 2 days, tumor growth curve was drawn after 21 days observation, tumor inhibition rate was calculated, tumor tissue and serum were collected. The concentrations of endostatin and VEGF in the serum of mice were detected by immunohistochemical method, and the microvessel density of transplanted tumor in each group was detected by (MVD) Elisa. Results: we successfully prepared ES-NPs loaded with different mass endostatin by ionic gel method. By comparing the encapsulation efficiency and drug loading of different batches of nanoparticles, we found that, The ES-NPs prepared by adding 渭 L ES (11.2mg/mL into 40 mL chitosan solution (1 mg/mL) not only guaranteed high entrapment efficiency, but also had suitable drug loading and particle size, which could meet the need of treatment. The ES-NPs could be stably preserved by adding 5% trehalose into the freeze-dried solution. The nanoparticles were spherical in appearance and uniform in distribution, and the cumulative release amount reached (60.22 鹵2.58) in vitro after 7 days of sustained release. At least 48h.MTT showed that GES NPs could inhibit the growth of HUVECs in serum. The inhibitory effect of ES-NPs on HUVECs was similar to that of free es at the first 24 h of drug action (p0. 577). However, with the prolongation of the action time, the effect of GES NPs was obviously better than that of free es (p0.05). Similarly, in transwell experiment, the inhibitory effect of GES NPs on migration of HUVECs was significantly stronger than that of free ESs, the difference was statistically significant (p0. 05), and the longer the action time, the greater the difference of effect between them. In Lewis lung cancer transplant model, we observed that ES-NPs can significantly inhibit the growth of transplanted tumor. Even the effect of once every 7 days was better than that of free es for 14 days. ES-NPs injection for 14 days achieved the best therapeutic effect in our experiment, and the inhibition rate of transplanted tumor was as high as 63.32. The results of immunohistochemical CD31 staining showed that the MVD of tumor tissue was the lowest in the control group (p0.001) and the free es group (p0.001) after 14 days of continuous injection. The serum levels of endostatin and VEGF were significantly higher in mice of 14-day continuous injection group than those in other groups (p < 0.05), but the levels of VEGF were significantly lower than those in other groups (p < 0.05). It is proved that ES-NPs plays an anti-tumor role mainly by inhibiting tumor angiogenesis. Conclusion: chitosan is a suitable material for preparing endostatin nanoparticles. ES-NPs prepared by chitosan has high encapsulation efficiency, drug loading and obvious slow-release. Endostatin chitosan nanoparticles could significantly inhibit the proliferation and migration of HUVECs in vitro, and the effect was better than that of free endostatin 3) on Lewis lung cancer model. Endostatin-loaded chitosan nanoparticles can enhance the therapeutic effect of endostatin and inhibit tumor growth by inhibiting angiogenesis.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R73-36

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