18 24 2014 06 11 Chinese Journal of Tissue Engineering Research June 11, 2014 Vol.18, No.24 ISSN 2095-4344 CN 21-1581/R CODEN: ZLKHAH 3821 www.CRTER.org 1987 510630 doi:10.3969/j.issn.2095-4344. 2014.24.009 [http://www.crter.org] :R318 :A :2095-4344 (2014)24-03821-08 2014-05-16 Yu Hao-tao, Master, Department of Spinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China Corresponding author: Xu Yi-chun, M.D., Department of Spiny Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China Accepted: 2014-05-16 7 PI3K/Akt ( 510630) 1 7 7 7 2 3 7 PI3K/Akt LY294002 Akt 7 PI3K/Akt BAD-Caspase 9 7 PI3K/Akt p-Akt BAD Caspase 9 7 (2009B030801095 2007A0300005) 7 7 PI3K/Akt 7 Pfirrmann 12 15% 48 h 7 PI3K/Akt LY294002 p-Akt Akt p-Akt BAD Caspase 9 7 LY294002 (P < 0.05) p-Akt 7 p-Akt BAD Caspase 9 (P < 0.05) Akt LY294002 p-Akt (P < 0.05) 7 PI3K/Akt BAD-Caspase 9 . 7 PI3K/Akt [J]. 2014 18(24):3821-3828. Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway Yu Hao-tao, Xu Yi-chun, Wang Qi-you, Ou Ding-qiang, Zhou Wei (Department of Spinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China) Abstract BACKGROUND: Bone morphogenetic protein 7 (BMP-7) can promote extracellular matrix synthesis of human nucleus pulposus cells, and delay 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 induction 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 herniated nucleus pulposus tissue. The collected nucleus pulposus cells were divided into four groups. Blank group: cells were
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中国组织工程研究 第 18卷 第 24期 2014–06–11出版 Chinese Journal of Tissue Engineering Research June 11, 2014 Vol.18, No.24
Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway
Yu Hao-tao, Xu Yi-chun, Wang Qi-you, Ou Ding-qiang, Zhou Wei (Department of Spinal Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou 510630, Guangdong Province, China)
Abstract BACKGROUND: Bone morphogenetic protein 7 (BMP-7) can promote extracellular matrix synthesis of human nucleus pulposus cells, and delay 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 induction 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 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; 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 apoptosis 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 pulposus 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 positive 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 expression 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. Subject headings: apoptosis; bone morphogenetic protein-7; protein kinase; serum Funding: Science and Technology Program of Guangdong Province, No. 2009B030801095, 2007A0300005 Yu HT, Xu YC, Wang QY, Ou DQ, Zhou W. Bone morphogenetic protein-7 inhibits apoptosis of human nucleus pulposus cells through activation of PI3K/Akt pathway. Zhongguo Zuzhi Gongcheng Yanjiu. 2014;18(24):3821-3828.
Quad % Gated % Total X Mean Y MeanUL 2.03 1.51 9.60 61.57UR 5.03 3.73 211.13 107.38LL 91.83 68.05 3.06 4.81LR 1.11 0.82 91.10 10.60
Quad % Gated % Total X Mean Y MeanUL 0.88 0.21 9.00 65.52UR 52.21 12.14 250.33 106.07LL 37.29 8.67 6.27 7.39LR 9.62 2.24 176.97 18.89
Quad % Gated % Total X Mean Y MeanUL 0.65 0.19 10.02 63.87UR 21.85 6.25 264.93 87.80LL 70.15 20.07 5.55 6.27LR 7.35 2.10 177.25 17.03
Quad % Gated % Total X Mean Y MeanUL 0.11 0.02 13.46 34.29UR 31.61 6.38 486.82 76.72LL 45.63 9.22 7.27 6.91LR 22.65 4.57 387.21 13.15
Quad % Gated % Total X Mean Y MeanUL 1.27 0.37 10.78 71.93UR 35.98 10.39 135.79 89.55LL 54.26 15.67 5.50 7.45LR 8.49 2.45 112.96 19.60
Quad % Gated % Total X Mean Y MeanUL 0.80 0.26 9.95 65.25UR 32.09 10.31 209.01 94.42LL 59.33 19.07 5.97 6.18LR 7.78 2.50 127.15 15.87
A B C
D E F
图 3 流式细胞术凋亡率检测无血清诱导凋亡处理下骨形态发生蛋白 7对人髓核细胞的影响 Figure 3 Flow cytometry results of the anti-apoptosis effect of bone morphogenetic protein-7 in serum-free induced human nucleus pulposus cells图注:流式结果图以 PI表达为纵坐标,FITC表达为横坐标。早期凋亡细胞(PI-)及晚期凋亡细胞(PI+)分别位于右上象限(UR)和右下象限(LR)。凋亡率的统计结果为早期凋亡百分比+晚期凋亡百分比。图中 A-F分别为空白对照组,阳性对照组,低、中、高剂量骨形态发生蛋白 7组(50,100,200 μg/L)和 LY294002拮抗组细胞凋亡情况,上述 6组细胞凋亡率均差异有显著性意义。
图 4 免疫荧光检测骨形态发生蛋白 7处理无血清诱导人髓核细胞凋亡后 p-Akt表达(×200) Figure 4 Immunofluorescence results of p-Akt expression in bone morphogenetic protein-7 treated apoptotic human nucleus pulposus cells induced under serum-free condition (×200) 图注:图中 A为 DAPI染核;B为 p-Akt荧光;C为 DAPI和 p-Akt叠加荧光染色。实验使用兔抗人多克隆 p-Akt及羊抗兔 IgG-FITC进行 p-Akt绿色荧光标记,DAPI染核(蓝染)明确细胞位置。相较于空白对照组、阳性对照组以及拮抗组,加入骨形态发生蛋白 7的处理组中细胞内 p-Akt蛋白表达明显(红色箭头所示)。
图 5 无血清诱导人髓核细胞凋亡各组蛋白印迹法检测各组相关凋亡蛋白结果 Figure 5 Apoptotic relative protein expressions in serum-free induced human nucleus pulposus cells detected by western blot assay 图注:图中 A 为无血清诱导人髓核细胞凋亡各组蛋白印迹法检测各组相关凋亡蛋白结果;图中 B-D 为各实验组 p-Akt/BAD/Caspase 9 蛋白印
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