EphB4特异性抑制剂NVP-BHG712对人成骨肉瘤MG63细胞株影响的体外研究

发布时间：2019-06-13 08:55

【摘要】：正畸治疗是通过适量矫治力的作用使牙齿在牙槽骨中向正确的方向和位置移动的过程。正畸力可通过牙周膜组织传导至牙槽骨，张力侧新骨形成，压力侧破骨吸收，从而实现骨改建。随着牙周膜成纤维细胞和成骨细胞表达的ephrinB2被成功检测，通过EphB4-ephrinB2的作用促进张力侧成骨的观点被国内外学者提出。利用EphB4-ephrinB2特异性结合的特点，通过EphB4特异性抑制剂的应用可以明确在正畸牙移动过程张力侧成骨所涉及的多种信号通路中EphB4-ephrinB2信号所发挥的作用。目的：以人成骨肉瘤MG63细胞株为研究对象，检测EphB4信号特异性抑制剂NVP-BHG712在体外实验中对其增殖活性和碱性磷酸酶活性的影响，从而为进一步应用该抑制剂探究EphB4-ephrinB2信号对正畸牙移动过程中张力侧成骨的作用打下基础。通过对众多已知参与到信号通路中的分子进行调控或进一步探究信号传导的分子基础可以加深对正畸过程中骨改建机制的理解，为改善治疗策略或更为精确合理的控制牙齿移动提供理论依据。方法：冻存的MG63细胞株复苏后培养，以5×103/孔接种于24孔培养板。实验组设置NVP-BHG712浓度分别为6.25ng/ml、12.5ng/ml、25ng/ml、37.5ng/ml及62.5ng/ml，另设不含NVP-BHG712为对照组。利用MTT法检测加入NVP-BHG712后第1、2、3天各组吸光度值，以探求NVP-BHG712对MG63增殖活性的影响。同时利用碱性磷酸酶活性检测结合BCA法蛋白检测计算各组碱性磷酸酶活性值，以探求NVP-BHG712对MG63碱性磷酸酶活性的影响。采用配对t检验方法，，P0.05为差异有统计学意义。结果：MTT结果显示，随着时间的推移，各组细胞数均逐渐增加。3天组当NVP-BHG712浓度为6.25ng/ml时所测OD值高于对照组（P0.05），其余各实验组与对照组相比均无差异。结合碱性磷酸酶活性检测和BCA法蛋白浓度检测，通过计算，结果显示，在第1、3、5、7天各浓度实验组与对照组相比均无统计学差异（P0.05）。结论： 1.浓度为6.25ng/ml的NVP-BHG712对MG63的增殖有促进作用，浓度为12.5ng/ml、25ng/ml、37.5ng/ml及62.5ng/ml时对MG63的增殖活性没有影响。 2.浓度为6.25ng/ml、12.5ng/ml、25ng/ml、37.5ng/ml及62.5ng/ml的NVP-BHG712对MG63的ALP活性没有影响。
[Abstract]:Orthodontic treatment is a process of moving the teeth in the correct direction and position in the alveolar bone by a proper amount of corrective force. The orthodontic force can be transmitted to the alveolar bone, the tension side new bone formation and the pressure side bone-breaking absorption through the periodontal ligament tissue, so that the bone reconstruction can be realized. With the successful detection of the ephrin B2 of the expression of the periodontal ligament fibroblasts and the osteoblast, the viewpoint of promoting the side-to-bone of the tension side by the action of EphB4-ephrin B2 is proposed by the domestic and foreign scholars. The application of EphB4-ephrin to the specific binding of EphB4-ephrin B2 can be used to identify the effect of EphB4-ephrin B2 signal in the various signal paths involved in the tension side of the orthodontic tooth. Objective: To study the proliferation activity and the activity of alkaline phosphatase in human osteosarcoma MG63 cell line. In order to further study the effect of EphB4-ephrin B2 (EphB4-ephrin B2) signal on the tension side-forming of the orthodontic tooth in the process of orthodontic tooth movement, The understanding of the mechanism of bone remodeling in the orthodontic process can be enhanced by regulating or further exploring the molecular basis of a plurality of molecules known to be involved in the signal pathway, providing a theoretical basis for improving the treatment strategy or more precisely and reasonably controlling the movement of the teeth. Methods: The cryopreserved MG63 cell line was recovered and cultured in 5%103/ well for 24 wells. The concentration of NVP-BHG712 in the experimental group was 6.25 ng/ ml, 12.5 ng/ ml,25 ng/ ml, 37.5 ng/ ml and 62.5 ng/ ml, respectively. The absorbance values of group 1,2 and 3 after NVP-BHG712 were detected by MTT method to explore the proliferative activity of NVP-BHG712 on MG63. The activity of alkaline phosphatase in each group was calculated by using alkaline phosphatase activity test and BCA method to explore the activity of NVP-BHG712 on the alkaline phosphatase activity of MG63. The effect of the paired t-test method is that the difference between P0.05 is the difference. Results: The results showed that the number of cells in each group increased gradually over time. The OD value of the three-day group was higher than that of the control group when the concentration of NVP-BHG712 was 6.25 ng/ ml (P0.05). The results showed that there was no statistical difference between the experimental group and the control group at 1,3,5 and 7 days with the detection of the activity of alkaline phosphatase and the detection of the concentration of the BCA protein. 0.0 5). Conclusion:1. The concentration of NVP-BHG712 with a concentration of 6.25 ng/ ml can promote the proliferation of MG63. The concentration is 12.5 ng/ ml,25 ng/ ml, 37.5 ng/ ml and 62.5 ng/ ml. No effect on proliferation activity.2. NVP-BHG712 vs. MG6 with a concentration of 6.25 ng/ ml, 12.5 ng/ ml,25 ng/ ml, 37.5 ng/ ml and 62.5 ng/ ml
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783.5

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