本文選題：血管內(nèi)皮生長因子 + 萬古霉素��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：研究目的:1.以海藻酸鹽及殼聚糖為載體,研制同時裝載VEGF及萬古霉素的微球型、可塑性骨支架材料,并對其性能進行分析。2.用無菌環(huán)境中制備的雙重載藥多層海藻酸鹽-殼聚糖微球型支架材料完全細(xì)胞培養(yǎng)液浸出液培養(yǎng)第3代HPMSCs,并檢測用支架材料浸出液培養(yǎng)后,HPMSCs增殖活性及成骨活性的變化。研究方法:1.采用組織塊培養(yǎng)法從人足月胎盤組織中分離培養(yǎng)HPMSCs,采用MTT法檢測并繪制細(xì)胞生長曲線,對第3代HPMSCs進行成骨誘導(dǎo)以及成脂誘導(dǎo),驗證其多向分化潛能;2.按照前期實驗在無菌環(huán)境中制備多層載藥緩釋微球的方法,靈活運用海藻酸鹽和殼聚糖交聯(lián)原理,利用海藻酸鹽和殼聚糖交聯(lián)產(chǎn)物的可塑性,將緩釋與支架支撐功能相結(jié)合,選擇微球與支架最優(yōu)配比,探索在無菌環(huán)境中制備雙重載藥多層海藻酸鹽-殼聚糖微球型支架材料;3.將制備好的支架材料與第3代HPMSCs共培養(yǎng),采用CCK-8試劑盒檢測細(xì)胞增殖能力,采用茜素紅染色及ALP試劑盒檢測細(xì)胞成骨能力的變化。研究結(jié)果:1.倒置顯微鏡下觀察到從人足月胎盤組織中分離培養(yǎng)出的細(xì)胞呈成纖維細(xì)胞樣貼壁生長,細(xì)胞生長曲線呈“S”形,并且細(xì)胞經(jīng)過成骨誘導(dǎo)后可以見到胞外有鈣鹽沉積、茜素紅染色陽性,經(jīng)成脂誘導(dǎo)后油紅O染色陽性;2.無菌環(huán)境下制得的雙重載藥多層海藻酸鹽-殼聚糖微球型支架材料形態(tài)可塑,并且兼具緩釋與支架功能;3.與支架材料浸出液共培養(yǎng)后,HPMSCs的增殖能力無顯著變化(P0.05);成骨誘導(dǎo)后,茜素紅染色顯示載藥微球支架組鈣鹽沉積多于空載微球支架組、無支架組和空白對照組,ALP試劑盒測試結(jié)果證實加入載藥微球支架后細(xì)胞內(nèi)堿性磷酸酶含量明顯增高(P0.05),空載微球支架組與無支架組相比也有顯著性差異(P0.05)。研究結(jié)論:1.成功從人足月胎盤組織中分離培養(yǎng)出HPMSCs,對其生物學(xué)特征鑒定后證明其符合干細(xì)胞特性;2.成功制備出形態(tài)可塑的雙重載藥多層海藻酸鹽-殼聚糖微球型支架材料;3.通過雙重載藥多層海藻酸鹽-殼聚糖微球型支架浸出液與HPMSCs共培養(yǎng),證明其對HPMSCs增殖活性無明顯影響,但能顯著改善HPMSCs的成骨活性。
[Abstract]:Objective: 1.Using alginate and chitosan as carriers, microspheres and plastic bone scaffolds loaded with VEGF and vancomycin were prepared and their properties were analyzed.The third generation of HPMSCs was cultured in the third generation of HPMSCs from the multi-layer alginate-chitosan microsphere scaffolds prepared in aseptic environment. The proliferative and osteogenic activities of HPMSCs were detected after the culture of the scaffold extracts.Research method: 1.HPMSCs were isolated and cultured from human full-term placenta by tissue mass culture method. The cell growth curves were measured and plotted by MTT method. The third generation HPMSCs was induced by osteogenesis and adipogenesis to verify its multidirectional differentiation potential.According to the method of preparing multilayer drug loaded sustained-release microspheres in aseptic environment in previous experiments, the principle of alginate and chitosan crosslinking was used flexibly, and the plasticity of alginate and chitosan crosslinking products was used to combine the sustained release with the support function of the scaffold.The optimum ratio of microspheres and scaffolds was chosen to prepare multilayer alginate chitosan microsphere scaffold material in aseptic environment.The prepared scaffolds were co-cultured with the third generation of HPMSCs. The proliferation of the cells was detected by CCK-8 kit, and the osteogenic ability of the cells was detected by alizarin red staining and ALP kit.The result of the study was: 1.The cells isolated from human placenta were observed to be fibroblast-like growth under inverted microscope. The growth curve of the cells was "S", and the extracellular calcium deposits could be seen after osteogenesis induction.Alizarin red staining was positive, and oil red O staining was positive after lipogenesis.The multilayer alginate chitosan microsphere scaffold material prepared under sterile environment has the function of both sustained release and scaffold.The proliferative ability of HPMSCs was not significantly changed after co-culture with scaffold material leaching solution (P 0.05). After osteogenesis induction, alizarin red staining showed that calcium salt deposition in drug-loaded microsphere scaffold group was more than that in no-loaded microsphere scaffold group, while that in drug-loaded microsphere scaffold group was higher than that in non-loaded microsphere scaffold group.The results of ALP kit test in the no stent group and the blank control group confirmed that the content of alkaline phosphatase in the cells increased significantly with the addition of the drug loaded microsphere stent, and there was also a significant difference between the no loaded microsphere stent group and the no stent group (P 0. 05).Conclusion: 1.HPMSCs were isolated and cultured successfully from human full-term placenta. The biological characteristics of HPMSCs were confirmed to be in accordance with stem cell characteristics.The multilayer alginate-chitosan microsphere scaffold material with plastic form was successfully prepared.By co-culture of multi-layer alginate-chitosan microsphere scaffolds with double drug loading with HPMSCs, it was proved that it had no obvious effect on the proliferation of HPMSCs, but could significantly improve the osteogenic activity of HPMSCs.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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