锶对hMSCs增殖、分化的影响及含锶脱钙骨对hMSCs的作用

发布时间：2018-05-28 18:34

本文选题：锶 + 间质干细胞　；参考：《第三军医大学》2010年硕士论文

【摘要】： 目的:观察锶(Sr)对人骨髓间充质干细胞(human bone marrow derived mesenchymal stem cells , hMSCs)增殖与成骨分化的影响并探索其最佳作用浓度;制备不同含锶量的脱钙骨基质(decalcified bone matrix,DBM),并与hMSCs复合,成骨诱导培养后观察hMSCs在不同含锶量的脱钙骨中的成骨效果。方法: 1、抽取健康成年志愿者骨髓6ml,体外分离、培养原代hMSCs,流式细胞术鉴定细胞表型。取第4代细胞进行实验,共分为5组,A组为空白对照,培养液中仅加入骨诱导剂(地塞米松10-8mol/L、抗坏血酸50μg/ml、β-甘油磷酸钠10mmol/Lol/L),B、C、D、E组培养液在加入骨诱导剂后分别加入3.75×10-3、3.75×10-2、3.75×10-1、3.75mmol/L的氯化锶(SrCl2)。定期测定各组hMSCs的增殖情况(MTT法)、碱性磷酸酶(alkaline phosphatase,ALP)活性(酶标法)和骨钙素(OCN)含量(放免法),并观察细胞茜素红钙结节染色情况,分析各组钙结节数量及面积。 2、收集临床髋关节置换术后废弃的股骨头(外伤致股骨颈骨折,股骨头无缺血性坏死),切成5mm×5mm×5mm的骨块,以改良Urist法制作脱钙骨基质(DBM)。将Ⅰ型胶原溶液分别与不同浓度的SrCl2溶液混合,使锶-胶原溶液中锶的终浓度分别为:0、3.75×10-3、3.75×10-2、3.75×10-1、3.75mmol/L,分别标记为A’、B’、C’、D’、E’组。将制备的DBM浸入锶-胶原混合溶液中1 h,取出后以真空吸附法使锶-胶原混合物充分吸附于材料表面,将材料取出,环氧乙烷灭菌备用。将培养的第4代hMSCs与各组含锶的DBM复合培养,定期测定各组hMSCs的殖增情况(MTT法)、碱性磷酸酶(alkaline phosphatase,ALP)活性(酶标法)和骨钙素(OCN)含量(放免法)。结果: l、随培养时间增加,各组细胞增殖和成骨分化均明显提高,有显著差异(P0.01);同时间点比较中,第7天的细胞增殖实验、第7、14天的ALP检测、第14、21天的OCN检测以及第21天的钙结节数量、面积定量检测,B~E组均优于A组并有显著差异(P0.05)。第7天的MTT检测及第14天的ALP检测,D组均值最高,与A、B、C组有显著差异( P 0.05),与E组无显著差异( P 0.05);第21天的OCN检测以及钙结节数量、面积定量检测,D组均值最高,与其他4组均有显著性差异( P0.05)。 2、hMSCs与各组含锶DBM复合培养后,随培养时间增加,各组细胞增殖程度明显提高,有显著差异(P0.01);同时间点比较中,MTT:第1、4、7天各组间均无显著性差异( P 0.05);ALP:第7、14天各有显著性差异(P0.01),含锶组活性强于空白组,含锶组间差异不明显( P 0.05);OCN:第7、14、21天,无显著性差异( P 0.05);补充实验证实,不复合细胞的DBM含有不同含量OCN。结论: 1、锶可促进hMSCs的增殖及成骨分化,其最佳作用浓度为3.75×10-1mmol/L。 2、所制作的含锶DBM具有良好的生物相容性,hMSCs与含锶DBM复合培养后能够增殖并成骨分化,含锶组成骨分化优于空白组。因所制作的DBM生物学性状差异较大,不能进一步证明各组间增殖及成骨分化的差异。
[Abstract]:Objective: to observe the effect of strontium on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (bone marrow derived mesenchymal stem cells, hMSCs) and to explore the optimal concentration of strontium, to prepare the decalcified bone matrix with different strontium content and to combine it with hMSCs, and to investigate the effect of strontium on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells (BMSCs). The osteogenic effect of hMSCs in decalcified bone with different strontium content was observed after osteogenic induction. Methods: 1. The bone marrow of healthy adult volunteers was extracted from 6 ml, isolated in vitro, cultured with hMSCs, and identified by flow cytometry. The cells of the 4th passage were divided into 5 groups as blank control group. Only dexamethasone 10-8 mol / L, ascorbic acid 50 渭 g / ml, 尾 -glycerophosphate 10 mmol / L Lolp / L ~ (2 +) C ~ (2 +) C ~ (2 +) C ~ (2 +) -C _ (1) C _ (2) O _ (2) was added to the culture medium of group A (3. 75 脳 10 ~ (-3) C ~ (-3) C ~ (-2) ~ (3) 脳 10 ~ (-2) 脳 10 ~ (-2) 脳 10 ~ (-5) 脳 10 ~ (-1) mol 路L ~ (-1) Sr ~ (-1) O _ (-1) Sr _ (1) O _ (2) chloride. The proliferation of hMSCs, the activity of alkaline phosphatase (ALP) and the content of osteocalcin (OCN) were measured regularly. The staining of alizarin red calcium nodules was observed, and the number and area of calcium nodules in each group were analyzed. 2. The abandoned femoral head (femoral neck fracture caused by trauma, no avascular necrosis of femoral head) was collected after clinical hip arthroplasty. The bone mass of 5mm 脳 5mm 脳 5mm was cut to make decalcified bone matrix (DBM) by modified Urist method. The type I collagen solution was mixed with different concentration of SrCl2 solution. The final concentration of strontium in strontium-collagen solution was 3. 0 0. 75 脳 10-3, 3. 75 脳 10-2, 3. 75 脳 10-1 and 3. 75 mmol / L, respectively. The prepared DBM was immersed in strontium collagen mixed solution for 1 hour, then the strontium collagen mixture was fully adsorbed on the surface of the material by vacuum adsorption method. The material was removed and sterilized by ethylene oxide. The fourth generation of hMSCs was co-cultured with strontium containing DBM. The proliferation of hMSCs in each group was measured by MTT assay. The activity of alkaline phosphatase (ALP) and the content of osteocalcin (hMSCs) were determined by radioimmunoassay. Results: L, with the increase of culture time, the proliferation and osteogenic differentiation of the cells in each group increased significantly, and the difference was significant (P 0.01). In the same time point, the cell proliferation test on the 7th day and the ALP detection on the 7th day were observed at the same time point. The number of calcium nodules and the number of calcium nodules in group E on day 14 and 21 were better than those in group A (P 0.05). The mean values of MTT on day 7 and ALP on day 14 were the highest in group D, and significantly different from those in group C (P 0.05, P 0.05, P 0.05), OCN on day 21 and the number of calcium nodules in group D, and the highest in group D by quantitative area detection. There was significant difference between the four groups (P 0.05). (2) after co-culture of hMSCs with strontium containing DBM, the proliferation of cells in each group increased with the increase of culture time. At the same time, there was no significant difference in MTT between groups on the 7th day (P 0.05) and on the 14th day (P 0.05). The activity of strontium group was stronger than that of blank group, but there was no significant difference between strontium group and strontium group (P 0.05 OCN: day 71421, P < 0.05). There was no significant difference (P 0.05). The supplementation experiment confirmed that the DBM contained different contents of DBM. Conclusion: 1.Strontium could promote the proliferation and osteogenic differentiation of hMSCs, and the optimum concentration was 3.75 脳 10 ~ (-1) mmol 路L ~ (-1) 路L ~ (-1). 2Strontium containing DBM has good biocompatibility and can proliferate and differentiate into osteogenesis after co-culture with strontium containing DBM, and the strontium component bone differentiation is better than that of blank group. The biological characteristics of DBM were different, which could not further prove the difference of proliferation and osteogenic differentiation among groups.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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