骨肉瘤中Axl抑制凋亡及与凋亡蛋白的相关性  

Relationship between receptor tyrosine kinase Axl and apoptosis relative proteins in osteosarcoma

作　　者：蒋念[1] 王雪迪[1] 田锐[1] 谢显彪[2] 赖英荣[1] 彭挺生[1] 

机构地区：[1]中山大学附属第一医院病理科,广州510080 [2]中山大学附属第一医院骨肿瘤科,广州510080

出　　处：《临床与实验病理学杂志》2017年第5期490-496,共7页Chinese Journal of Clinical and Experimental Pathology

基　　金：国家自然科学基金(81302348)

摘　　要：目的探讨受体酪氨酸激酶Axl在骨肉瘤中的抗凋亡作用,分析磷酸化Axl(P-Axl)与抗凋亡蛋白之间的相关性。方法常规培养骨肉瘤细胞株MG63、143B和U2OS,分别设立人重组蛋白Gas6刺激组、Axl siRNA转染组、Gas6刺激+Axl siRNA转染组及各阴性对照组,应用顺铂(DDP)或氨甲喋呤(MTX)致使细胞凋亡后,通过Hoechst 33258染色、MTT实验、Annexin VFIFC等检测法统计分析不同处理组间细胞凋亡率的差异及不同浓度Gas6刺激后骨肉瘤细胞的细胞周期变化;采用免疫组化En Vision两步法检测41例骨肉瘤及18例骨纤维结构不良组织中P-Axl、BCL-2及Bax的表达,分析患者预后与三者的相关性;应用TUNEL染色分析骨肉瘤组织中P-Axl表达水平与细胞凋亡率的相关性。结果 Hoechst 33258、MTT、Annexin V-FIFC等的实验结果一致,均表明骨肉瘤细胞株MG63、143B和U2OS中Gas6通过活化Axl降低DDP或MTX导致的细胞凋亡率(P<0.05),Axl siRNA则引起细胞凋亡率增加(P<0.05),在Axl siRNA干扰前Gas6预刺激有降低凋亡率的趋势。41例骨肉瘤组织中P-Axl、BCL-2、Bax的阳性率分别为85.4%、70.7%及36.6%;18例骨纤维结构不良组织中三者阳性率分别为11.1%、22.2%及11.1%,两组相比骨肉瘤组织中P-Axl、BCL-2、Bax的表达明显增高(P<0.05)。Pearson相关性分析显示BCL-2表达与P-Axl呈显著正相关(r=0.842,P<0.000 1);Cox单因素风险回归分析发现BCL-2和Bax表达是影响患者预后的预测因素;TUNEL结果显示P-Axl高表达的骨肉瘤组织中细胞凋亡率明显降低(P<0.05)。结论骨肉瘤中Gas6/Axl活化后通过调节凋亡蛋白BCL-2,抑制由DDP或MTX诱发的细胞凋亡,从而促进肿瘤的进展。Purpose To identify the role of tyrosine kinase receptor Axl for anti-apoptosis which was induced by cisplatin （DDP） and methotrexate （MTX） chemotherapy and to analyze the relationship between P-Axl and apoptosis-related proteins in osteosarcoma. Methods Osteosarcoma cell lines MG63, 143B and U2OS were used in apoptosis assays, Axl siRNA transfection, cytotoxicity assays, cell cycle analysis, etc. A total of 41 cases of osteosarcom patients were included for immunohistochemistry of EnVision two-step staining and clinico-pathological relative analysis. TUNEL assay was performed in ten cases for apoptosis detection. Results Among the osteosarcoma cell lines, Gas6/Axl could obviously protect tumor cells from apoptosis induced by DDP and MTX （P 〈 0.05 ）. Axl siRNA transfection enhanced cell apoptosis, whereas Gas6 prone to function upon previous knockdown by Axl siRNA. Among the 41 cases, the positive rate of P-Axl, BCL-2, and Bax was 85.4%, 70. 7% , and 36. 6% , respectively. In contrast, the positive rate of them was 22.2% , 11.1% , and 11.1% in osteofibrous dysplasia, respectively. The expression levels of these apoptosisrelated factors were significantly higher in osteosarcoma than in osteofibrous dysplasia （P 〈 0. 05 ）. Through clinico-pathological analysis, there were significant relationships between the survival status and BCL-2 or Bax expression （P 〈 0.05 ）. Pearson correlation analysis demonstrated that BCL-2 was positively correlated to P-Axl with statistical significance （ r = 0. 842, P 〈 0. 000 1 ）. By Cox univariate analysis, BCL-2 or Bax was correlated with the patients＇ prognosis. TUNEL assay also demonstrated that P-Axl high expression inhibited apoptosis in osteosarcoma tissues. Conclusion Gas6/Axl protects osteosarcoma cells from the apoptosis induced by DDP and MTX chemotherapy and inhibits apoptosis in osteosarcoma tissue, possibly through the regulation of apoptosis-related protein BCL-2.

关 键 词：骨肉瘤 AXL BCL-2 细胞凋亡 预后 

分 类 号：R738.3[医药卫生—肿瘤]