体外构建抗结核性骨组织工程复合体的实验研究

发布时间：2018-06-02 01:52

本文选题：骨结核 + 脂肪干细胞　；参考：《新疆医科大学》2014年硕士论文

【摘要】：目的：基于兔脂肪干细胞(rabbit adipose-derived stem cells, rADSCs)、利福喷丁聚乳酸缓释微球、同种异体部分脱钙骨支架,初步于体外构建抗结核性骨组织工程复合体。方法：分离、培养rADSCs,经检测细胞周期、表面抗原CD44及分化诱导鉴定干细胞特性。用利福喷丁生长液干预rADSCs,按浓度分为0μg/ml、20μg/ml、40μg/ml及60μg/ml四组,分别绘制细胞生长曲线、检测细胞凋亡及碱性磷酸酶(alkaline phosphatase, ALP)含量。制备利福喷丁微球,计算平均直径、载药率及包封率。构建rADSCs-利福喷丁微球二维复合物并成骨诱导。制备骨支架并构建rADSCs-利福喷丁微球-骨支架三维复合物,计算细胞粘附率；扫描电子显微镜及苏木精-伊红(HE)染色观察复合情况；每3d更换生长液,检测体外释药特性。结果：rADSCs呈长梭形生长,G0/G1期占(80.3+3.2)%,表面抗原CD44阳性表达。成骨诱导后ALP、茜素红染色,成脂诱导后油红O染色及成软骨诱导后阿利新蓝染色均为阳性。生长曲线示60μg/ml组第4-12d增值能力较其他三组弱(P0.01)；凋亡检测示60μg/ml组凋亡率高于其他三组(P0.01)；ALP检测示第7、14d60μg/ml组ALP含量较其他三组少(P0.05)；临界浓度为40μg/ml。微球平均直径为(26.4+3.6)μm,载药率为(40.89±0.63)%,包封率为(75.24±0.18)%。二维复合物成骨诱导后茜素红染色阳性。三维复合物细胞粘附率为(96.94±1.24)%；扫描电镜及HE染色观察复合体相容性良好；体外培养第3、6、39d累积释药量分别达总量的8.54%、20.12%及93.66%,每个时间点检测的释药浓度均低于临界浓度,但高于最低抑菌浓度。结论：体外构建的抗结核性骨组织工程复合体具有持续缓慢释放药物的性质,且该释药浓度不影响rADSCs的增值及成骨活性。
[Abstract]:Aim: to construct anti-tuberculous bone tissue engineering complex in vitro based on rabbit adipose stem cells adipose-derived stem cells, rifampicin polylactic acid sustained-release microspheres and allogeneic partial decalcification bone scaffold. Methods: rADSCs were isolated and cultured. Stem cells were identified by cell cycle detection, surface antigen CD44 and differentiation induction. RADSCswere treated with rifapentine growth liquid. The cells were divided into four groups: 0 渭 g / ml, 20 渭 g / ml, 40 渭 g/ml and 60 渭 g/ml. The cell growth curves were plotted, and the cell apoptosis and alkaline phosphatase (ALPaline) content were measured. Rifapentine microspheres were prepared and the mean diameter, drug loading rate and encapsulation efficiency were calculated. Two-dimensional complex of rADSCs- rifapentine microspheres was constructed and induced by osteogenesis. Bone scaffolds were prepared and three dimensional complexes of rADSCsrifopentine microspheres and bone scaffolds were constructed to calculate the cell adhesion rate, scanning electron microscopy and hematoxylin eosin HEhe staining were used to observe the composite, and the growth fluid was changed every 3 days to detect the drug release characteristics in vitro. Results the percentage of CD44 in G _ 0 / G _ 1 phase of G _ 0 / G _ 1 was 80.33.2%, and the expression of surface antigen CD44 was positive. ALP, alizarin red staining, oil red O staining and cartilage induction were all positive after osteogenic induction. The growth curve showed that the increment ability of 60 渭 g/ml group was weaker than that of the other three groups on 4-12 d, and the apoptotic rate of 60 渭 g/ml group was higher than that of the other three groups. The ALP content of 60 渭 g/ml group was lower than that of the other three groups on the 7th day, 14 d, 60 渭 g/ml group, and the critical concentration was 40 渭 g / ml. The average diameter of the microspheres was 26.4.3.6) 渭 m, the drug loading rate was 40.89 卤0.63 and the encapsulation efficiency was 75.24 卤0.18. Alizarin red staining was positive after osteogenic induction of two-dimensional complex. The cell adhesion rate of the three dimensional complex was 96.94 卤1.24%, the compatibility of the complex was well observed by SEM and HE staining, the cumulative release of the complex reached 8.54% and 93.66% of the total drug release at the 639d of culture in vitro, and the drug release concentration detected at each time point was lower than the critical concentration. But above the minimum inhibitory concentration. Conclusion: the anti-tuberculous bone tissue engineering complex constructed in vitro has the properties of sustained slow release of the drug, and the release concentration of the drug does not affect the proliferation and osteogenic activity of rADSCs.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R529.2

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