【摘要】：BDNF是神經(jīng)生長因子家族重要成員,研究發(fā)現(xiàn)BDNF及其受體在骨痂中大量表達(dá),神經(jīng)生長因子可能在骨折愈合過程中發(fā)揮重要作用,但其具體機(jī)制尚不明確。骨折延遲愈合、骨折不愈合是創(chuàng)傷骨科臨床常見難題,成骨細(xì)胞是骨折愈合過程中重要的修復(fù)細(xì)胞,研究BDNF對(duì)成骨細(xì)胞調(diào)控作用具有重要意義。研究目的：探求BDNF對(duì)成骨細(xì)胞增殖及凋亡的影響。研究方法：本研究首先在體外培養(yǎng)成骨細(xì)胞,并利用RT-PCR檢測ALP mRNA表達(dá)對(duì)成骨細(xì)胞進(jìn)行鑒定；使用MTT法檢測BDNF對(duì)成骨細(xì)胞增殖的影響；BDNF作用于成骨細(xì)胞,利用annexin V-FITC/PI雙染檢測細(xì)胞凋亡,western blot檢測凋亡相關(guān)蛋白Bcl-2、Bax的表達(dá)。研究結(jié)果：(1)細(xì)胞培養(yǎng)及鑒定：從小鼠顱骨分離培養(yǎng)的成骨細(xì)胞生長旺盛,接種24小時(shí)大部分細(xì)胞已經(jīng)貼壁,原代細(xì)胞7天長滿瓶底,傳代后5天就能長滿瓶底,第三代成骨細(xì)胞仍有較高活性,RT-PCR檢測到成骨細(xì)胞中ALP mRNA高表達(dá),證實(shí)培養(yǎng)細(xì)胞為成骨細(xì)胞。(2)MTT法發(fā)現(xiàn)25ng/ml BDNF處理成骨細(xì)胞未顯示明顯促增殖作用,50、100ng/ml BDNF明顯促進(jìn)成骨細(xì)胞增殖。(3)Annexin V-FITC/PI雙染結(jié)果顯示50、100ng/ml BDNF顯著抑制細(xì)胞凋亡,免疫印跡顯不BDNF促進(jìn)抑凋亡蛋白Bcl-2表達(dá),下調(diào)促凋亡蛋白Bax表達(dá)水平。研究結(jié)論：采用乳小鼠顱骨分離培養(yǎng)的成骨細(xì)胞具有良好活性,50ng/ml以上濃度BDNF處理成骨細(xì)胞能顯著促進(jìn)細(xì)胞增殖,抑制成骨細(xì)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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：R274.1

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