骨折愈合过程中BDNF调控成骨细胞增殖和凋亡的实验研究

发布时间：2019-03-28 17:03

【摘要】：BDNF是神经生长因子家族重要成员,研究发现BDNF及其受体在骨痂中大量表达,神经生长因子可能在骨折愈合过程中发挥重要作用,但其具体机制尚不明确。骨折延迟愈合、骨折不愈合是创伤骨科临床常见难题,成骨细胞是骨折愈合过程中重要的修复细胞,研究BDNF对成骨细胞调控作用具有重要意义。研究目的：探求BDNF对成骨细胞增殖及凋亡的影响。研究方法：本研究首先在体外培养成骨细胞,并利用RT-PCR检测ALP mRNA表达对成骨细胞进行鉴定；使用MTT法检测BDNF对成骨细胞增殖的影响；BDNF作用于成骨细胞,利用annexin V-FITC/PI双染检测细胞凋亡,western blot检测凋亡相关蛋白Bcl-2、Bax的表达。研究结果：(1)细胞培养及鉴定：从小鼠颅骨分离培养的成骨细胞生长旺盛,接种24小时大部分细胞已经贴壁,原代细胞7天长满瓶底,传代后5天就能长满瓶底,第三代成骨细胞仍有较高活性,RT-PCR检测到成骨细胞中ALP mRNA高表达,证实培养细胞为成骨细胞。(2)MTT法发现25ng/ml BDNF处理成骨细胞未显示明显促增殖作用,50、100ng/ml BDNF明显促进成骨细胞增殖。(3)Annexin V-FITC/PI双染结果显示50、100ng/ml BDNF显著抑制细胞凋亡,免疫印迹显不BDNF促进抑凋亡蛋白Bcl-2表达,下调促凋亡蛋白Bax表达水平。研究结论：采用乳小鼠颅骨分离培养的成骨细胞具有良好活性,50ng/ml以上浓度BDNF处理成骨细胞能显著促进细胞增殖,抑制成骨细胞凋亡。
[Abstract]:BDNF is an important member of nerve growth factor family. It has been found that BDNF and its receptor are expressed in callus. Nerve growth factor may play an important role in fracture healing, but its mechanism is still unclear. Delayed union of fracture, nonunion of fracture is a common clinical problem in trauma orthopedics, osteoblast is an important repair cell in the process of fracture healing. It is of great significance to study the role of BDNF in the regulation of osteoblast. Objective: to investigate the effect of BDNF on proliferation and apoptosis of osteoblasts. Methods: firstly, osteoblasts were cultured in vitro, and the expression of ALP mRNA was detected by RT-PCR to identify osteoblasts, and the effect of BDNF on osteoblast proliferation was detected by MTT method. The expression of apoptosis-related protein Bcl-2,Bax in osteoblasts was detected by annexin V-FITC/PI double-staining with BDNF, and the expression of apoptosis-related protein Bcl-2,Bax was detected by, western blot. Results: (1) Cell culture and identification: osteoblasts isolated from mouse skulls grew exuberantly, and most of the cells had adhered to the wall 24 hours after inoculation. The primary cells grew to the bottom of the bottle for 7 days and could grow to the bottom of the bottle 5 days after passage. The third generation osteoblasts still had high activity, and the high expression of ALP mRNA in osteoblasts was detected by RT-PCR, which confirmed that the cultured cells were osteoblasts. (2) MTT assay showed that the osteoblasts treated with 25ng/ml BDNF had no obvious effect of promoting proliferation. 50100ng/ml BDNF significantly promoted the proliferation of osteoblasts. (3) the results of Annexin V-FITC/PI double staining showed that 50100ng/ml BDNF significantly inhibited the apoptosis of osteoblasts. Western blot showed that BDNF did not promote the expression of anti-apoptotic protein Bcl-2 and down-regulated the expression of pro-apoptotic protein Bax. Conclusion: the osteoblasts isolated from the skulls of suckling mice have good activity. BDNF above the concentration of 50ng/ml can significantly promote the proliferation of osteoblasts and inhibit the apoptosis of osteoblasts.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R274.1

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