金葡液联合抗坏血酸在骨髓间充质干细胞向成骨细胞分化中的作用研究

发布时间：2018-07-06 15:47

本文选题：抗坏血酸 + 金葡液　；参考：《浙江大学》2008年硕士论文

【摘要】： 研究背景骨髓间充质干细胞(mesenchymal stem cells,MSCs)是一种多潜能成体干细胞,主要存在于骨髓。体外分离培养后,在一定的诱导条件下,MSCs具有向成骨细胞、软骨细胞、神经细胞、脂肪细胞和心肌细胞等多向分化的能力,且分化后各种组织细胞有较强的增殖能力。这种多向分化能力使其成为组织工程、基因治疗等的理想靶细胞。骨折是临床上常见的疾病,在治疗过程中,在骨折断端或新创面处注射金葡素注射液有利于骨折的愈合,这其中发挥作用的是金葡素注射液的主要成分——肠毒素C。朱建民等报道金葡液虽然对成骨细胞的增殖和分化功能无明显作用,但具有明显的促进成骨细胞矿化作用。在动物实验和临床研究均证明了金葡液有加速骨折端血肿吸收、机化和促进骨痂形成、加速骨折愈合的作用;而抗坏血酸可以增强碱性磷酸酶(AKP)的活性、提高成骨细胞的活性。但是金葡液对MSCs是否有作用、是否可以通过金葡液联合抗坏血酸,促进MSCs向成骨细胞分化,达到促进成骨细胞生成、加速骨折愈合的作用,目前尚无研究报道。研究目的本实验旨在研究金葡液联合抗坏血酸对MSCs分化的作用、对成骨细胞的生成及矿化功能的影响等,为临床上金葡液联合抗坏血酸治疗骨折提供理论依据。研究方法将培养第二代的MSCs分组诱导:金葡素注射液诱导组(浓度分别为10ng/L、30 ng/L、100 ng/L);金葡素注射液(浓度分别为10 ng/L、30 ng/L、100ng/L)+抗坏血酸(50μg/ml)诱导组。同时以正常培养液培养的MSCs为对照组。通过碱性磷酸酶活性检测、茜素红染色钙结节显示、扫描电镜、透射电镜观察细胞超微结构等方法,探讨其向成骨细胞分化能力。研究结果经金葡液诱导后,各组碱性磷酸酶(alkaline phosphatase,AKP)活性随金葡液浓度增高,酶活性测定值增加,但无统计学意义;经金葡液联合抗坏血酸诱导后,各组AKP酶活性均显著增高,有统计学意义。各组诱导28 d后,0.1%茜素红-Tris-HCl染色,倒置显微镜下观察,发现较多的红色结节,大小不等,散在或成簇分布。与金葡液组相比,金葡液联合抗坏血酸组钙结节形成数明显增高,结节直径变大。扫描电镜结果显示,与单纯金葡液诱导组相比,该组细胞胞质突起粗大扁平,细胞表面微绒毛丰富,数量明显增多。透射电镜结果显示:MSCs细胞经各诱导组处理后粗面内质网、核糖体、线粒体增多,金葡液联合抗坏血酸组细胞的粗面内质网、核糖体较单用金葡液诱导组更为丰富,线粒体增多且基质密度高。研究结论各组诱导28天后,通过金葡液联合抗坏血酸组诱导的MSCs碱性磷酸酶活性高于金葡组,茜素红染色钙结节增加,并通过电镜观察细胞超微结构,表明其向成骨细胞分化增强,成骨细胞蛋白合成能力增强。因此,金葡液联合抗坏血酸对MSCs向成骨细胞分化有促进作用。
[Abstract]:Background Bone Marrow Mesenchymal Stem cells (mesenchymal stem cells) are multipotent adult stem cells which mainly exist in bone marrow. After isolation and culture in vitro, MSCs have the ability to differentiate into osteoblasts, chondrocytes, nerve cells, adipocytes and cardiomyocytes under certain induction conditions. This multidirectional differentiation makes it an ideal target cell for tissue engineering, gene therapy, etc. Fracture is a common disease in clinic. In the course of treatment, injection of staphylococcus aureus injection at the broken end of fracture or new wound is beneficial to the healing of fracture, and the main component of staphylococcus aureus injection is enterotoxin C. Zhu Jianmin et al reported that staphylococcus aureus had no obvious effect on the proliferation and differentiation of osteoblasts, but it had obvious effect on promoting the mineralization of osteoblasts. Both animal experiments and clinical studies have proved that staphylococcus aureus fluid can accelerate the absorption of hematoma at the fracture end, organize and promote callus formation and accelerate fracture healing, while ascorbic acid can enhance the activity of alkaline phosphatase (AKP) and osteoblast. However, whether the staphylococcus aureus liquid can promote the differentiation of MSCs into osteoblasts and accelerate the fracture healing through the combination of staphylococcus aureus and ascorbic acid has not been reported. Objective to study the effects of staphylococcus aureus combined with ascorbic acid on the differentiation of MSCs and on the formation and mineralization of osteoblasts in order to provide a theoretical basis for the treatment of fracture with staphylococcus aureus combined with ascorbic acid. Methods the cultured MSCs were divided into two groups: staphylococcus aureus injection group (10 ng / L) and staphylococcin injection (10 ng / L ~ (30) ng / L ~ (-1) ascorbic acid (50 渭 g/ml) group. MSCs cultured in normal medium were used as control group. Alkaline phosphatase activity, alizarin red stained calcium nodules, scanning electron microscope and transmission electron microscope were used to observe the ultrastructure of osteoblasts and the ability of differentiation into osteoblasts was studied. Results after induced by staphylococcus aureus, the activity of alkaline phosphatase in each group increased with the concentration of staphylococcus aureus, but there was no significant difference between the two groups, and the activity of alkaline phosphatase was induced by staphylococcus aureus combined with ascorbic acid. The activity of AKP in each group was significantly higher than that in control group. After 28 days of induction, 0.1% alizarin red -Tris-HCl staining was observed and observed under inverted microscope. Compared with the staphylococcus aureus group, the number of calcium nodules and the diameter of the nodules in the staphylococcus aureus combined with ascorbic acid group were significantly increased. The results of scanning electron microscope showed that the cytoplasmic processes of the cells were thicker and flattened, and the number of microvilli on the surface of the cells was more than that of the group induced by staphylococcus aureus. The results of transmission electron microscope showed that the rough endoplasmic reticulum, ribosome and mitochondria were increased after treated with different induction groups, and the rough endoplasmic reticulum and ribosome were more abundant in staphylococcus aureus combined with ascorbic acid group. Mitochondria increased and matrix density was high. Conclusion after 28 days of induction, the alkaline phosphatase activity of MSCs induced by staphylococcus aureus combined with ascorbic acid group was higher than that of staphylococcus aureus group, the calcium nodules stained with alizarin red increased, and the ultrastructure of MSCs was observed by electron microscope. The results showed that the differentiation into osteoblasts was enhanced, and the protein synthesis ability of osteoblasts was enhanced. Therefore, staphylococcus aureus combined with ascorbic acid can promote the differentiation of MSCs into osteoblasts.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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