肿瘤耐药中上皮-间质转化与耐药相关蛋白的相关性研究

肿瘤耐药中上皮-间质转化与耐药相关蛋白的相关性研究[摘要] 目的：通过观察上皮-间质转化（epithelial-mesenchymal transition，EMT）标志物上皮型钙黏蛋白（E-cadherin，E-cad）、神经型钙黏蛋白（N-cadherin，N-cad）和肺耐药相关蛋白（1ung resistance-related protein，LRP）、多药耐药相关蛋白（multidrug resistance-related protein，MRP）、P-糖蛋白（P-glycoprotein，P-gp）蛋白表达及共定位情况，探讨肿瘤耐药中EMT与耐药相关蛋白的相关性。方法：构建TGF-β1空载慢病毒和TGF-β1过表达慢病毒分别转染A549/DDP细胞。将A549/DDP细胞、TGF-β1空载A549/DDP细胞、TGF-β1过表达A549/DDP细胞分别接种到裸鼠腋窝皮下，细胞接种8d后，腹腔注射顺铂（0.0035 g·kg-1），2次/周，细胞接种32d后，处死裸鼠取移植瘤组织。HE染色观察各组肺腺癌荷瘤裸鼠移植瘤组织形态学变化，Western blot检测各组E-cad、N-cad和LRP、MRP、P-gp蛋白表达水平，免疫荧光双染检测各组E-cad、N-cad与LRP、MRP、P-gp 共定位情况。结果：形态学观察可见TGF-β1过表达组细胞松散，连接稀疏，细胞间隙明显增宽，细胞可见大量纺锤形或长梭形形态改变，偶尔可见伪足，形似间质细胞。与空白组、空载组比较，TGF-β1过表达组E-cad蛋白表达显著下调，N-cad和LRP、MRP、P-gp蛋白表达均显著上调（P<0.05）；E-cad荧光表达明显减弱，而LRP、MRP、P-gp荧光表达明显增强，共定位区域较少，E-cad与LRP、MRP、P-gp呈负相关（P<0.05）；N-cad荧光表达明显增强，LRP、MRP、P-gp荧光表达明显增强，共定位区域较多，N-cad与LRP、MRP、P-gp呈正相关（P<0.05）。结论：在肿瘤耐药发生过程中，EMT与耐药相关蛋白的表达呈正相关。肿瘤EMT程度越高，耐药相关蛋白的表达越高，其耐药性越强。

[关键字] 非小细胞肺癌；上皮-间质转化；耐药相关蛋白；耐药；转化生长因子-β1

Correlation reserach between epithelial mesenchymal transition and drug resistance associated proteins in tumor drug resistance

WANG Ying，ZHANG Ying，GAO Yuan，WANG Chun，LIU Chun-ying [ABSTRACT] AIM: To observe the protein expression and co-localization of the markers of epithelial mesenchymal transition (EMT) such as E-cadherin (E-cad) and N-cadherin (n-cad), and drug resistance associated protein such as lung resistance related protein (LRP), multidrug resistance related protein (MRP) and P-glycoprotein (P-gp), and to explore the correlation between EMT and drug resistance related proteins in tumor drug resistance. METHODS: The constructed TGF-β1 empty lentivirus and TGF-β1 overexpression lentivirus were transfected into A549/DDP cells respectively. Eighteen BALB/C nude mice were randomly divided into three groups.

A549/DDP cells were inoculated in the blank group, TGF-β1 empty cells were inoculated in no-load group, and TGF-β1 overexpression A549/DDP cells were inoculated in overexpression group. After eight days of cell inoculation，cisplatin was injected intraperitoneally, 0.0035 g·kg-1, twice a week. After thirty-two days of cell inoculation，nude mice were killed and tumor tissues were taken. The protein expressions of E-cad, N-cad, LRP, MRP and P-gp were detected by Western blot. The co-localization of between E-cad or N-cad and LRP，MRP，P-gp were observed by double immunofluorescence staining，and the correlation was analyzed. RESULTS: The transplanted tumor cells in TGF-β1 overexpression group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers．Compared with the blank group and TGF-β1 empty group, the protein expression of E-cadwas significantly decreased (P<0.05), however those of N-cadherin, LRP, MRP and P-gp were significantly increased in TGF-β1 overexpression group (P<0.05) ; the fluorescence expression of E-cad decreased significantly, LRP, MRP and P-gp increased significantly, co-location areas was fewer，E-cad is negatively correlated with LRP, MRP and P-gp in TGF-β1 overexpression group (P<0.05); the fluorescence expression of N-cad increased significantly, LRP, MRP and P-gp increased significantly,

co-location areas was more，N-cad is positively correlated with LRP, MRP and P-gp in TGF-β1 overexpression group (P<0.05). CONCLUSION: In the process of tumor drug resistance, EMT was positively correlated with the expression of drug resistance related proteins. The higher the degree of EMT, the higher the expression of drug resistance related proteins, the stronger the drug resistance.

[KEY WORDS] Non small cell lung cancer; Epithelial mesenchymal transition; Drug resistance associated protein; Drug resistance; Transforming growth factor - β 1

[基金项目] 国家自然科学基金资助项目（No.81973735）；国家自然科学基金资助项目（No.81774184）

Δ通讯作者Tel：0245-31207092；E-mail：ury@ 肿瘤患者的耐药是化疗过程中的棘手问题，耐药的肿瘤细胞可以不断生长并向远处转移，导致化疗效果差，甚至化疗失败[1]。越来越多的研究证实，上皮间质转化与耐药密切相关[2-3]。上皮间质转化是上皮细胞向间质化转变的生物过程，也是肿瘤细胞发生侵袭和转移的启动环节。上皮间质转化能调节耐药相关蛋白的