| Esophageal carcinoma is one of malignant tumours which are developed from esophageal mucosa epithelium and glands. It is one of the common digestive tract cancer. Esophageal carcinoma has obvious regionalism and often been discovered in Northwest and North China. Especially the incidence and mortality of esophageal carcinoma in Linzhou City of Henan Province ranks first in our country and the incidence and mortality of esophageal carcinoma in our country also ranks first in the world. Esophageal squamous cell carcinoma is the most common histological types.The lymphatic metastasis of it is one of the important reasons about the disease development or death of esophageal carcinoma patients.MMP-9(matrix metalloproteinase-9) belongs to MMPs.It could degrade the composition of extracellular matrix and basement membrane, and promote the invasiveness and metastasis of tumor cells. TIMP-2(tissue inhibitor of metalloproteinase-2) is one member of TIMPs family,it could specifically restrain the activity of MMP and degradation of the basement membrane,and then restrain the invasiveness and metastasis of tumor cells.The balance of MMPs and TIMPs plays an important role in maintaining the intact state of extracellular matrix. The imbalance of MMPs and TIMPs would become one of important factors about the tumor cells which invade or metastasize.Studies suggest that the expression of MMP-9increases. MMP-9expresses more highly in tumor cells than normal tissues.TIMP-2could restrain all MMPs.The expression of TIMP-2and lymphatic metastasis of esophageal carcinoma appear in a negative correlation. The expression of TIMP-2would be low and negative in the one that has lymphatic metastasis.The lymphatic metastasis of tumor cells is an important approach of tumor spread and metastasis. Tumor lymphatic endothelial cells is an important boundary of lymphatic metastasis concerning tumor. It is a critical step of the interaction between tumor cells and tumor lymphatic endothelial cells. It has not been reported that the research about the correlation of esophageal carcinoma cells and esophageal lymphatic endothelial cells.The subject is the one that using different endothelial cells conditioned mediums culture esophageal carcinoma EC9706cells and esophageal carcinoma KYSE150cells,and then using Immunocytochemistry and Western blot detects the expression of MMP-9protein and TIMP-2protein in each group cells;Using in situ Hybridization and RT-PCR detects the expression of MMP-9mRNA and TIMP-2mRNA in each group cells; Using CCK-8method detects the proliferation ability of each group cells;Using Transwell method detects the invasive ability of each group cells.By constructing the transplanted tumor model of esophageal carcinoma about nude mice,the transplanted tumors and surrounding tissues experimentize immunohistochemical staining of D2-40and LYVE-1, then mark lymphatic vessels and mensurate LMVD. Investigate the interaction between esophageal carcinoma cells of different differentiation degree and esophageal lymphatic endothelial cells, lay the important theoretical foundation about blocking lymphatic metastasis of malignant tumors.Materials and methods1.The esophageal carcinoma EC9706cells are favored by the State Key Laboratory of institute of Oncology in Chinese Academy of Medical Sciences; The esophageal carcinoma KYSE150cells and esophageal lymphatic endothelial cells are purchased from Shanghai Bioleaf biological technology limited company; The lymphatic endothelial cells of Children’s leather are purchased from Sciencell Company; The nude mice are purchased from Xi Puerbikai Experimental Animal Limited Company of Shanghai.2. The esophageal lymphatic endothelial cells and the lymphatic endothelial cells originated from normal children dermis are cultured in the same condition, and when the second generation cells are fused,use serum-free medium culture them overnight and collect the two groups of endothelial cells conditoned medium.3.Use the two groups of endothelial cells conditoned medium culture EC9706cells and KYSE150cells group by group, and simultaneously suppose the blank control group. Culture them after24hours, and then collect each group cells.4.The group of culturing cells:There are three big groups which are classified into experimental group、experimental control group and Blank comtrol group. Experimental group(E group) are classified into El group and E2group. E1group uses the conditioned medium of esophageal lymphatic endothelial cells to culture EC9706cells and E2group uses it to culture KYSE150cells. Experimental control group(C group) are classified into C1group and C2group. C1group uses the conditioned medium of the lymphatic endothelial cells originated from normal children dermis to culture EC9706cells and C2group uses it to culture KYSE150cells. Blank comtrol group(K group) are classified into K1group and K2group.K1group dosn’t change medium and continue to use primary medium to culture them as well as K2group.5.Using immunocytochemistry、Western blot detects the expression of MMP-9protein and TIMP-2protein in each group cells.Using in situ Hybridization、RT-PCR detects the expression of MMP-9mRNA and TIMP-2mRNA in each group cells.6.Using CCK-8method detects the proliferation ability of each group cells.7.Using Transwell method detects the invasive ability of each group cells.8.Construct the transplanted tumor model of esophageal carcinoma about nude mice, the group of nude mice accords with the group of culturing esophageal carcinoma cells which is mentioned above. The nude mice were inoculated tumor cells and were euthanized to take the tumors after five weeks.9.The transplanted tumors and surrounding tissues experimentize immunohistochemical staining of D2-40and LYVE-1, then mark lymphatic vessels and mensurate LMVD.10.Statistical analysis:Using the SPSS17.0statistical software to analyse the data, at an inspection level of a=0.05.Results1. Immunocytochemistry detects the expression of MMP-9protein and TIMP-2protein:The expression of MMP-9in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05);The expression of MMP-9protein in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05); The expression of MMP-9in the E2group of experimental group is higher than E1group, the difference is statistically significant(P<0.05). The expression of TIMP-2protein in experimental group is lower than experimental control group, and it is significantly lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2protein in experimental control group is lower than blank control group, the difference is statistically significant (P<0.05);The expression of TIMP-2protein in the E2group of experimental group is lower than E1group, the difference is statistically significant (P<0.05).2. Situ Hybridization detects the expression of MMP-9mRNA and TIMP-2mRNA:The expression of MMP-9mRNA in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05); The expression of MMP-9mRNA in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05);The expression of MMP-9mRNA in the E2group of experimental group is higher than E1group, the difference is statistically significant (P<0.05). The expression of TIMP-2mRNA in experimental group is lower than experimental control group, and it is significantly lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2 mRNA in experimental control group is lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2mRNA in the E2group of experimental group is lower than E1group, the difference is statistically significant (P<0.05).3.RT-PCR detects the expression of MMP-9mRNA and TIMP-2mRNA:The expression of MMP-9mRNA in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05);The expression of MMP-9mRNA in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05); The expression of MMP-9mRNA in the E2group of experimental group is higher than E1group, the difference is statistically significant (P<0.05). The expression of TIMP-2mRNA in experimental group is lower than experimental control group, and it is significantly lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2mRNA in experimental control group is lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2mRNA in the E2group of experimental group is lower than E1group, the difference was statistically significant (P<0.05).4.Western blot detects the expression of MMP-9protein and TIMP-2protein: The expression of MMP-9in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05); The expression of MMP-9protein in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05); The expression of MMP-9in the E2group of experimental group is higher than E1group, the difference is statistically significant (P<0.05). The expression of TIMP-2protein in experimental group is lower than experimental control group, and it is significantly lower than blank control group, the difference was statistically significant (P<0.05); The expression of TIMP-2protein in experimental control group is lower than blank control group, the difference is statistically significant (P<0.05); The expression of TIMP-2protein in the E2group of experimental group is lower than E1group, the difference is statistically significant (P<0.05).5. CCK-8method detects the proliferation ability of each group cells:The proliferation ability of the cells in experimental group is the strongest. Among them the proliferation ability of the cells in E2group is stronger than El group, and the proliferation ability of the cells in E2group is the strongest in all groups; the proliferation ability of the cells in experimental group is stronger than experimental control group, and it is significantly stronger than blank control group, the difference is statistically significant (P<0.05); The proliferation ability of the cells in experimental control group is stronger than blank control group, the difference is statistically significant (P<0.05).6. Transwell method detects the invasive ability of each group cells:The number of invasion cells of the cells in experimental group is the most. Among them the number of invasion cells of the cells in E2group is more than E1group, and the number of invasion cells of the cells in E2group is the most in all groups, also the invasive ability of it is the strongest. The number of invasion cells of the cells in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05). The number of invasion cells of the cells in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05).7.The transplanted tumors of esophageal carcinoma about nude mice are obvious after inoculating the cells of esophageal carcinoma in nude mice’s subcutaneous tissues. Measure the volume of tumors every two or three days after forming tumors and the nude mice are sacrificed after five weeks.The volume of the tumors in each group:2542.21±.12.65、2872.42±13.12,2264.51±13.95,2334.27±14.03、1437.56±17.37and1643.31±14.09. The volume of transplanted tumors about nude mice in experimental group is the biggest. Among them the volum of E2group is bigger than E1group, and the volum of E2group is the biggest in all groups, the difference is statistically significant (P<0.05).The volume of transplanted tumors about nude mice in experimental group(E group) is bigger than experimental control group(C group), and it is significantly bigger than blank control group(K group), the difference is statistically significant (P<0.05).The experimental control group compared with blank control group has no significant difference, the difference is statistically significant (P<0.05).8.D2-40and LYVE-1mark lymphatic vessels and mensurate LMVD:The LMVD condition of D2-40and LYVE-1of the transplanted tumors and surrounding tissues about nude mice in experimental group is higher than experimental control group, and it is significantly higher than blank control group, the difference is statistically significant (P<0.05); Among them the LMVD condition of D2-40and LYVE-1of E2group is the highest, and it is higher than E1group, the difference is statistically significant (P<0.05); The LMVD condition of D2-40and LYVE-1in experimental control group is higher than blank control group, the difference is statistically significant (P<0.05).Conclusions1.The esophageal lymphatic endothelial cells could promote the proliferation ability and invasive ability of esophageal carcinoma cells in vitro.2.The esophageal lymphatic endothelial cells give different influence on esophageal carcinoma cells of different differentiation degree and they give more obvious influence on poorly differentiated esophageal carcinoma cells; The action perhaps has relation with up-regulating MMP-9expression of esophageal carcinoma cells and down-regulating TIMP-2expression of esophageal carcinoma cells.3.The esophageal lymphatic endothelial cells could promote the growth of transplanted tumor of esophageal carcinoma about nude mice and lymphangion genesis. |
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