摘要
目的 :探讨肺癌来源的信号素4D(semaphorin 4D,Sema4D)在肺癌骨转移溶骨性骨破坏中的作用及机制。方法 :采用免疫组织化学法检测肺癌骨转移灶及正常骨组织中Sema4D的表达情况。分别采用蛋白质印迹法和实时荧光定量PCR法检测4种肺癌细胞株中Sema4D蛋白及mRNA表达水平。收集4种肺癌细胞条件培养液,采用ELISA法检测其中Sema4D的分泌水平。收集Sema4D高表达(PC9细胞)及低表达(A549细胞)肺癌细胞的条件培养液,与成骨前体细胞MC3T3-E1共培养后,采用碱性磷酸酶染色法检测成骨细胞分化水平,采用茜素红染色法检测成骨细胞矿化能力,采用实时荧光定量PCR法检测成骨细胞分化基因如肝/肾/骨型碱性磷酸酶(alkaline phosphatase, liver/bone/kidney,ALPL)、Oxterix和Ⅰ型胶原α1(collagen typeⅠalpha 1,Col1α1)的表达水平。将Sema4D shRNA转染肺癌PC9细胞后,采用蛋白质印迹法、实时荧光定量PCR及ELISA法分别检测肺癌细胞中Sema4D表达与分泌水平的变化;收集肺癌条件培养液与成骨前体细胞共培养,再次采用碱性磷酸酶染色法、茜素红染色法和实时荧光定量PCR法检测干扰Sema4D表达后肺癌条件培养液对成骨细胞分化的影响。结果:与正常骨组织相比,Sema4D在肺癌溶骨性骨转移灶中高表达。Sema4D在不同肺癌细胞株中呈不同程度的表达与分泌,其中肺癌PC9细胞中表达与分泌水平最高,而肺癌A549细胞中表达与分泌水平最低(P <0.05)。肺癌条件培养液可明显抑制成骨前体细胞在成骨诱导剂诱导下的成骨分化,表现为碱性磷酸酶染色及茜素红染色的相对染色面积减小(P值均<0.05),成骨分化基因ALPL、Oxterix和Col1α1的mRNA表达水平均明显降低(P值均<0.05);并且Sema4D高表达细胞株PC9的条件培养液抑制成骨细胞分化的能力明显强于Sema4D低表达细胞株A549(P <0.05)。shRNA转染可显著降低肺癌细胞中Sema4D的表达与分泌水平(P值均<0.05),并�
Objective:To determine the role of semaphorin 4 D(Sema4 D)in osteolytic bone destruction of bone metastasis in lung cancer,and to explore its mechanism.Methods:Immunohistochemistry was used to detect the expression of Sema4 D in bone metastases of lung cancer and normal bone tissues.The expression levels of Sema4 D protein and mRNA in four kinds of lung cancer cell lines were detected by Western blotting and realtime fluorescent quantitative PCR,respectively.The conditioned medium of lung cancer cells was collected,and ELISA was used to detect the secretion level of Sema4 D in lung cancer conditioned medium.The conditioned medium from lung cancer cells with Sema4 D high expression(PC9 cells)or low expression(A549 cells)was collected and co-cultured with osteoblastic precursor MC3 T3-E1 cells,then the osteoblast differentiation ability was detected by alkaline phosphatase staining,the mineralization ability of osteoblasts was detected by alizarin red staining,the expression levels of osteoblast differentiation genes alkaline phosphatase,liver/bone/kidney(ALPL),Oxterix and collagen typeⅠalpha 1(Col 1α1)were detected by real-time fluorescent quantitative PCR.Sema4 D shRNA was transfected into lung cancer PC9 cells,then the expression and secretion levels of Sema4 D in lung cancer cells were detected by Western blotting,real-time fluorescent quantitative PCR and ELISA,respectively.The conditioned medium of lung cancer PC9 cells transfected with Sema4 D shRNA was collected and co-cultured with preosteoblasts,and the effect of the conditioned medium from lung cancer cells with Sema4 D interference on the osteoblast differentiation was determined by alkaline phosphatase staining,alizarin red staining and real-time fluorescent quantitative PCR,respectively.Results:Compared with the normal bone tissues,Sema4 D was highly expressed in osteolytic bone metastases of lung cancer.Sema4 D was expressed and secreted in different lung cancer cell lines in different degrees.The expression and secretion levels of Sema4 D were th
作者
陈武桂
孙靖
杨思振
张莹
胡旭
李松涛
沈伟伟
廖通权
周跃
初同伟
CHEN Wugui;SUN Jing;YANG Sizhen;ZHANG Ying;HU Xu;LI Songtao;SHEN Weiwei;LIAO Tongquan;ZHOU Yue;CHU Tongwei(Department of Orthopedics,Second Aliated Hospital,Military Medical University,Chongqing 400037,China;Department of Orthopedics,940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army,Lanzhou 730050,Gansu Province,China)
出处
《肿瘤》
CAS
CSCD
北大核心
2019年第9期701-711,共11页
Tumor
基金
国家自然科学基金资助项目(编号:81570800)~~
关键词
肺肿瘤
肿瘤转移
骨
信号素4D
成骨细胞
细胞分化
Lung neoplasms
Neoplasm metastasis,bone
Semaphorin 4D
Osteoblasts
Cell differentiation
