1、骨形态发生蛋白 7 通过 PI3K/Akt 通路可抑制人髓核细胞的凋亡余灏涛，徐义春，王其友，欧定强，周 葳中山大学附属第三医院脊柱外科，广东省广州市 510630Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathwayYu Hao-tao, Xu Yi-chun, Wang Qi-you, Ou Ding-qiang, Zhou WeiDepartment of Spinal Surgery, the Third
2、Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China摘要 背景：骨形态发生蛋白 7 可促进人髓核细胞的细胞外基质合成，减缓椎间盘退变。近年来，有学者提出其可能通过对抗髓核细胞凋亡从而发挥上述作用，但其进一步的分子机制一直未被详细阐明。目的：观察骨形态发生蛋白 7 对无血清诱导下发生凋亡的人髓核细胞产生的作用及其对 PI3K/Akt 通路的影响，分析并探讨骨形态发生蛋白 7 抑制人髓核细胞凋亡的分子机制。方法：通过改良 Pfirrmann 分级及相关条件选取 12 例患
3、者获取椎间盘组织，采用酶消化法获取人髓核细胞后分组实验，以含体积分数 15%胎牛血清的培养基正常培养的髓核细胞设为空白组；使用无血清培养基培养 48 h 诱导凋亡作为阳性对照组；在无血清条件下，通过加入不同剂量的骨形态发生蛋白 7 以及同时添加 PI3K/Akt 通路拮抗剂 LY294002 形成处理组和拮抗组。使用流式细胞术检测各组细胞凋亡率；免疫荧光观察 p-Akt 表达；蛋白印迹法检测 Akt，p-Akt，BAD 和 Caspase 9 等通路蛋白的表达变化。结果与结论：无血清凋亡诱导下，流式细胞术结果显示，随着骨形态发生蛋白 7 处理浓度上升，髓核细胞凋亡率明显下降，加入 LY2940
4、02 共同作用后细胞凋亡率再次升高( P 0.05)。p-Akt 免疫荧光和蛋白印迹法检测结果进一步表明，与凋亡阳性对照组相比，加入骨形态发生蛋白 7 的实验组中，p-Akt 表达明显增加，其下游凋亡相关蛋白 BAD、Caspase 9 蛋白表达减少(P 0.05)，而在同时加入 Akt 通路拮抗剂 LY294002 后，p-Akt 蛋白表达下降而凋亡相关蛋白的表达又恢复到相对较高的水平(P 0.05)。结果证明，骨形态发生蛋白 7 在无血清诱导的人类髓核细胞凋亡中通过激活 PI3K/Akt 通路，拮抗了BAD-Caspase 9 相关的细胞凋亡过程，抑制了髓核细胞的退变。中国组织工程研究杂志
5、出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：关键词 ： 组织构建； 骨组织工程； 组织工程基础实验； 骨形态发生蛋白 7； PI3K/Akt 通路； 人髓核细胞； 凋亡； 无血清；p-Akt； BAD 蛋白； Caspase 9； 流式细胞术； 蛋白印迹法； Abstract：BACKGROUND: Bone morphogenetic protein 7 (BMP-7) can promote extracellular matrix synthesis of human nucleus pulposus cells, and d
6、elay intervertebral disk degeneration. Recently scholars proposed that the above effects can be achieved through anti-apoaptosis, but further molecular mechanism remains poorly understood.OBJECTIVE: To observe the effects of BMP-7 on the apoptotic human nucleus pulposus cells under serum-free induct
7、ion and the PI3K/Akt pathway, investigate the molecular mechanism underlying the anti-apoptosis effect of BMP-7. METHODS: Human nucleus pulposus tissue were harvested from 12 patients according to the modified Pfirrmann classification, and human nucleus pulposus cells were obtained from digestion of
8、 herniated nucleus pulposus tissue. The collected nucleus pulposus cells were divided into four groups. Blank group: cells were cultured in normal culture medium containing 15% feral bovine serum; positive control group: cells were induced to apoptosis in the serum-free culture medium for 48 hours;
9、treated group: cells were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7; antagonism group: cells were induced to apoptosis in the serum-free culture medium containing different dosages of BMP-7 and LY294002, an PI3K/Akt pathway inhibitor. Cellular apopto
10、sis rate was quantified by flow cytometry. Immunoflurorescence was performed to observe p-Akt expressions. Changes of Akt, p-Akt, BAD, and Caspase 9 expression were detected with western blot analysis. RESULTS AND CONCLUSION: Results from flow cytometry showed that, the apoptosis rate of nucleus pul
11、posus cells was significantly decreased as the increase of BMP-7 concentration, under the serum-free induction; after the cells were induced with LY294002, the apoptosis rate was increased again (P 0.05). The p-Akt immunoflurorescence and western blot analysis demonstrated that, compared with positi
12、ve control group, the p-Akt expression was significantly increased, while the expression of downstream apoptosis-related proteins such as BAD and Caspase 9, was significantly decreased in the cells treated with BMP-7 (P 0.05). Furthermore, in the group treated with BMP-7 and LY294002, the p-Akt expr
13、ession was decreased, while the expression of apoptosis-related proteins was recovered to high levels (P 0.05). BMP-7 activates the PI3K/Akt pathway, suppresses the downstream BAD and Caspase 9 signaling, and inhibits the apoptosis of human nucleus pulposus cells induced by serum-free situation.中国组织
14、工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程全文链接：Key words： apoptosis; bone morphogenetic protein-7; protein kinase; serum;中图分类号: R318 基金资助: 广东省科技攻关项目(2009B030801095，2007A0300005)通讯作者: 徐义春，博士。中山大学附属第三医院脊柱外科，广东省广州市 510630 作者简介: 余灏涛，男，1987 年生，广东省广州市人，汉族，中山大学附属第三医院毕业，硕士，目前从事椎间盘退变相关研究。引用本文: 余灏
15、涛,徐义春 ,王其友等 . 骨形态发生蛋白 7 通过 PI3K/Akt 通路可抑制人髓核细胞的凋亡 J. 中国组织工程研究, 2014, 18(24):3821-3828.Yu Hao-tao,Xu Yi-chun,Wang Qi-you et al. Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathwayJ. Chinese Journal of Tissue Engineering Research, 2014, 18(24): 3821-3828.
