壳聚糖力学性能与胆固醇-g-壳聚糖对细胞成骨分化的影响研究

发布时间：2018-05-01 23:59

本文选题：壳聚糖 + 胆固醇　；参考：《暨南大学》2017年硕士论文

【摘要】：壳聚糖具有良好的生物相容性、可降解性和生物安全性,且来源广泛,但壳聚糖力学性能差、降解速度与矿化速率慢等缺点限制了其应用,故壳聚糖的改性已经成为目前国内外研究的热点。胆固醇是人体内的重要生物大分子,对细胞膜具有热力学亲和作用,是影响细胞生存及功能的重要因素,对细胞的信号传导、细胞粘附和细胞迁移都很有意义。本研究在前期研究基础上,进一步考察胆固醇琥珀酸-g-壳聚糖(CHCS)对骨髓间充质干细胞(HMSCs)分化的影响机制以及不同力学强度的壳聚糖对于细胞增殖分化的影响机制。本研究首先以壳聚糖为基材,采用天然的生物交联剂京尼平,制备具有不同力学强度的壳聚糖膜。探讨不同力学强度与细胞行为之间的相互关系。结果表明:随着壳聚糖力学强度的增加,MC3T3-E1s细胞增殖率不断增大,但力学强度到一定值后,增殖率开始减小;而细胞的成骨分化能力则随着力学强度的增强而增大。其次,为了探索胆固醇-g-壳聚糖及其力学性能对HMSCs成骨分化的影响,本研究将HMSCs种植在改性后的CHCS膜上进行培养。研究表明:CHCS膜上比CS膜上的细胞增殖率要大,且诱导组细胞比非诱导组增殖明显,ALP活性增大,表明CHCS比CS更有利于促进HMSCs的增殖与成骨分化。钙结节染色结果表明:诱导后的细胞染色明显,且肉眼可见,未诱导的细胞则无法看到红色,且CHCS组细胞染色程度大于CS材料组。CLSM结果表明:CHCS膜上的细胞肌动蛋白相比纯CS膜上的细胞更为定向排列。RT-PCR结果表明:RUNX2、OCN、ALP、COL-I等四组基因的表达随培养时间的延长有所增大,且诱导组的基因表达大于未诱导组,而CHCS基因表达比CS更明显。Western blot结果表明:CHCS中HMSCs成骨标志蛋白的表达水平高于CS。粘附性能测试发现细胞在CHCS上粘附性能优于在CS上。最后,通过RT-PCR和Western blot技术探究胆固醇对Wnt信号通路里的wif1和cdh26基因蛋白的表达量的影响,通过环糊精包被胆固醇来控制胆固醇存在的条件,结果发现:胆固醇的存在能够促进wif1和cdh26蛋白的高表达,从而激活Wnt信号通路,影响HMSCs的成骨分化。
[Abstract]:Chitosan has good biocompatibility, biodegradability and biosafety, and has a wide range of sources. However, the poor mechanical properties of chitosan and slow degradation rate and slow mineralization have limited its application. Therefore, the modification of chitosan has become a hot topic at home and abroad. The effect of thermodynamic affinity is an important factor affecting the survival and function of cells. The signal transduction, cell adhesion and cell migration of cells are very significant. On the basis of previous studies, this study further investigated the mechanism of the effect of cholesterol succinic acid -g- chitosan (CHCS) on the differentiation of bone marrow mesenchymal stem cells (HMSCs) and different mechanics. The effects of Chitosan on cell proliferation and differentiation. First, chitosan was used as the base material and a natural biological cross-linking agent genipin was used to prepare chitosan membranes with different mechanical strength. The relationship between different mechanical strength and cell behavior was discussed. The results showed that with the increase of the mechanical strength of chitosan, MC3T3-E The proliferation rate of 1s cells increased, but the proliferation rate began to decrease with the mechanical strength to a certain value, and the osteogenic differentiation capacity of the cells increased with the strength of the mechanical strength. Secondly, in order to explore the effect of cholesterol -g- chitosan and its mechanical properties on the osteogenesis of HMSCs, this study planted HMSCs on the modified CHCS membrane for culture. The study showed that the proliferation rate of cells on the CHCS membrane was larger than that on the CS membrane, and the induced cell proliferation was obvious than that in the non induction group, and the activity of ALP increased, indicating that CHCS was more beneficial to the proliferation and osteogenic differentiation of HMSCs than CS. The results of calcium nodule staining showed that the induced cells were stained clearly, and the naked eyes were visible, and the uninduced cells could not see red. Color, and the staining degree of cells in group CHCS was greater than that of the CS group.CLSM. The results showed that the cell actin on the CHCS membrane was more directed to.RT-PCR than those on the pure CS membrane. The results showed that the expression of four groups of genes of RUNX2, OCN, ALP, COL-I were increased with the prolongation of the culture time, and the gene expression of the induced group was larger than that of the uninduced group, and the CHCS base was found. The expression of.Western blot was more obvious than that of CS showed that the expression level of HMSCs osteogenic protein in CHCS was higher than that of CS. adhesion test. The adhesion performance of the cells on CHCS was better than that on CS. Finally, the effect of cholesterol on the expression of the protein in the Wnt signal pathway was explored through RT-PCR and Western blot technology. It is found that the presence of cholesterol can promote the high expression of Wif1 and cdh26 proteins, thus activating the Wnt signaling pathway and affecting the osteogenic differentiation of HMSCs.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O636.1;R318.08

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