| Objective:Primary bronchial lung cancer,referred to as lung cancer,is a common primary malignant tumor of the lungs.In the past 50 years,with the increase of environmental pollution,especially urban air pollution,the incidence and mortality of lung cancer have increased significantly.Among them,the incidence of lung adenocarcinoma accounts for 40%of the total incidence of lung cancer,making it the most common type of lung cancer.In addition,lung adenocarcinoma generally has no obvious clinical symptoms in the early stage.Most patients are usually diagnosed as diffuse metastatic lung adenocarcinoma at the first diagnosis.Lung adenocarcinoma is currently one of the most aggressive and deadly malignant tumors.Stem cell technology and its application are considered to be the human health project in the 21st century.As the most promising therapy in cell therapy,it has been found that stem cell therapy is expected to treat a variety of clinical chronic and refractory diseases.Stem cell products are rapidly entering the stage of clinical research,most of which are mesenchymal stem cells.Umbilical cord mesenchymal stem cells(UC-MSC)have the advantages of high differentiation potential,easy access to materials,and no ethical controversy.They have been paid more and more attention in the clinical research of mesenchymal stem cells.A number of clinical studies on the treatment of refractory diseases with UC-MSC have been initiated.With the extensive development of clinical research on UC-MSC,its safety has received more and more attention,especially whether it has the effect of promoting tumor metastasis,is still controversial.At present,there is no unified view on the relationship between UC-MSC and tumor growth and metastasis.Studies have shown that UC-MSC has different effects on different tumors,and which effect it plays depends on a series of factors.Such as the type of tumor cells,various cytokines secreted by UC-MSC in vivo or in vitro,and the interaction between tumor cells.The metastasis of UC-MSC to lung adenocarcinoma with high incidence and insidious onset is worthy of our in-depth study.Therefore,this study selected two human lung adenocarcinoma cells:SPC-A-1 and A549.The effects of UC-MSC on the migration and invasion ability of SPC-A-1 and A549 were evaluated by cell migration and cell invasion experiments.And through the detection of cytokine chip,it was found that the expression of human granulocyte chemotactic factor 2(GCP-2)was significantly increased after the mixed culture of UC-MSC and SPC-A-1.Subsequent experiments were conducted to preliminarily explore the mechanisms related to the impact of UC-MSC on the migration and invasion capabilities of SPC-A-1 and A549,And through the detection of cytokines,it was found that the expression of human granulocyte chemotactic factor 2(GCP-2)increased.We continue to explore related mechanisms to provide a theoretical basis for the security of UC-MSC applications.Methods:1.SPC-A-1,A549 cells and UC-MSC required for resuscitation and culture.Flow cytometry to identify the phenotype of UC-MSC and to identify its differentiation potential.2.Cell migration experiments and cell invasion experiments were used to study the effect of UC-MSC on the migration and invasion of human lung adenocarcinoma cells SPC-A-1 and A549.3.The direct co-culture method and Transwell co-culture method were used to collect the supernatant of UC-MSC and SPC-A-1 cells or UC-MSC and A549 cells at three time points of 24 h,48 h and 72 h.ELISA detects the content of GCP-2 in the above-mentioned culture supernatant.4.Transfect the GCP-2 shRNA lentiviral interference vector(Shanghai Jikai Gene)into the cells to knock down the expression of GCP-2 in UC-MSC.Cell migration and invasion experiments were used to detect the effect of GCP-2 knockdown UC-MSC on the migration and invasion ability of SPC-A-1 and A549.5.Increase the dose of GCP-2.Cell migration and invasion experiments were used to detect the effects of the UC-MSC group,GCP-2(10 ng/ml)group,and GCP-2(50 ng/ml)group on the migration and invasion capabilities of SPC-A-1 and A549.6.Different concentrations of GCP-2(GCP-2(10 ng/ml),GCP-2(50 ng/ml),GCP-2(100 ng/ml))interfere with lung adenocarcinoma cells SPC-A-1 and A549,Western Blot detects Vimentin Protein and E-cadherin protein expression.Results:1.The results of UC-MSC morphology and phenotype identification are consistent with the morphological characteristics and cell phenotype of mesenchymal stem cells,and UC-MSC cells have the differentiation potential to differentiate into adipocytes,bone cells,and cartilage.2.UC-MSC can obviously promote the migration and invasion of SPC-A-1 cells,and can obviously promote the invasion of A549 cells,but it has no obvious effect on promoting the migration of A549 cells.3.In the supernatant of UC-MSC and SPC-A-1 co-culture directly and Transwell co-culture,the secretion of GCP-2 was significantly increased,and GCP-2 was secreted by UC-MSC,SPC-A-1 cells did not secrete GCP-2.In the direct co-culture of UC-MSC and A549 cells or in the supernatant of Transwell co-culture,the amount of GCP-2 secreted was significantly increased,and GCP-2 was mainly secreted by UC-MSC,and A549 cells could only secrete a small amount of GCP-2.The GCP-2 in the supernatant of UC-MSC is about 2 ng/ml.4.After knocking down the expression of GCP-2,UC-MSC cells did not significantly promote the migration and invasion of human lung adenocarcinoma cells SPC-A-1 and A549.5.UC-MSC group(GCP-2 content is about 2 ng/ml),GCP-2(10 ng/ml)group,GCP-2(50 ng/ml)group can significantly promote the migration and invasion of SPC-A-1.UC-MSC group(GCP-2 content is about 2 ng/ml),GCP-2(10 ng/ml)group,GCP-2(50 ng/ml)group can significantly promote the invasion ability of A549,but does not significantly promote the migration of A549.As the concentration of GCP-2 increases,the ability to promote the migration of SPC-A-1 also increases.6.Western Blot detection found that after using GCP-2(50 ng/ml)and GCP-2(100 ng/ml)to interfere with SPC-A-1 cells for 24 hours,the expression of Vimentin protein in SPC-A-1 cells of the two groups was higher than that of the control group.At 48 h,the expression of E-cadherin protein in the two groups decreased significantly compared with the control group.After using GCP-2(50 ng/ml)group and GCP-2(100 ng/ml)group to interfere with A549 cells for 48 hours,the Vimentin protein of GCP-2(100 ng/ml)group increased significantly compared with the normal group,while E-The cadherin protein decreased significantly.Conclusions:1.UC-MSC can obviously promote the migration and invasion of SPC-A-1 cells,and can obviously promote the invasion of A549 cells,but it has no obvious effect on promoting the migration of A549 cells.2.In the co-culture supernatant of UC-MSC and SPC-A-1 cells or UC-MSC and A549 cells,the content of GCP-2 was significantly increased,and GCP-2 was mainly secreted by UC-MSC.3.The ability of UC-MSC to promote the migration and invasion of human lung adenocarcinoma cells SPC-A-1 and A549 may be mediated by GCP-2.4.The ability of GCP-2 to promote the migration and invasion of SPC-A-1 and A549 cells may be related to epithelial-mesenchymal transition(EMT). |
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