| Lung cancer is one of the most common malignant tumors in China,more than 80%of which are non-small cell lung cancer(NSCLC).Although the treatment of lung cancer has made great progress in recent years,the overall 5-year survival rate is still only about 15%.70%-80%the patients diagnosed with NSCLC have been in advanced stage,with tumor invasion and metastasis,thus missing the best time for surgical resection,resulting in treatment failure and death.Therefore,we must find more effective treatment.In recent years,lung cancer targeted therapy has become a research hotspot.Some scholars found that umbilical cord mesenchymal stem cells(MSCs)can specifically migrate to the tumor site,have obvious homing effect on glioma,ovarian cancer,breast cancer and other tumors,and can penetrate into the tumor matrix,no damage the normal tissue.At present,MSCs is also regarded as the most promising gene carrier.The use of mesenchymal stem cells of this characteristic,we speculate that in lung cancer targeted therapy,inhibit cancer genes can be transfected into the mesenchymal stem cells by the lentiviral vector,to suppress and even kill the tumor cells,which will provide a new road to clinical lung cancer targeted therapy.In the selection of tumor suppressor genes,we choose Interferon-?(IFN-?)which is a cytokine produced by fibroblasts and have many biological functions.IFN-? can not only be used in the treatment of some infections and autoimmune diseases,but also inhibit the proliferation of many kinds of tumor-derived cells.In the research of glioblastoma,melanoma,ovarian cancer,breast cancer,we found that it can induces apoptosis of tumor cells,inhibits tumor growth.At present,IFN-? gene combined with mesenchymal stem cells is rarely studied in the targeted therapy of lung cancer.Combined with the recent researchs progress at home and abroad,aiming at the problems existing in clinical treatment and gene targeting therapy for lung cancer,we propose using human umbilical cord derived mesenchymal stem cells(UCMSCs)as cell carriers to carry IFN-? gene in the treatment of NSCLC.In this study,IFN-? gene was transfected into UCMSCs(IFN-?-MSCs)by lentivirus infection,and experiments in vivo and in vitro were carried out.The migration ability of IFN-?-MSCs to NSCLC and its effect on the proliferation of NSCLC cells were measured in vitro.IFN-?-MSCs was injected into the model of lung cancer bearing mice by tail vein.The tumor size of the experimental groups and the control group were observed and compared.The effects of IFN-?-MSCs on the biological activity of lung cancer cells were systematically evaluated in vitro and in vivo,and the proliferation,apoptosis,apoptosis-related protein and gene expression of lung cancer cells were detected.Furthermore,the mechanism of UCMSCs infected with IFN-?gene on lung cancer cells was discussed,which provided a new theoretical basis and strategy for gene targeting therapy of lung cancer.[Objectives]1.The effects of IFN-?-MSCs on the proliferation of lung cancer A549 cells and NCI-H2170 cells were confirmed by in vitro experiments.2.The effects of IFN-?-MSCs on hypodermic implantable tumor of lung cancer and on various important organs of nude mice were investigated by in vivo experiments.3.The study on the mechanism of IFN-?-MSCs inhibiting the proliferation and promoting apoptosis of NSCLC cells,which opens a new way for NSCLC targeting therapy,and lays a theoretical foundation.[Methods](?)Vitro experiment1.Extraction of human umbilical cord,isolation,culture and amplification of UCMSCs.2.The third generation surface antigens of UCMSCs were identified by flow cytometry,including surface antigen CD90,CD73,CD105,CD45,CD14,CD19,CD34,HLA-DR.3.The experiment of adipogenic and osteogenic induction of UCMSCs to understand that MSCs has a variety of cell differentiation potential.4.Transfection of IFN-? gene and empty vector of lentivirus both with enhanced Red Fluorescent protein(ERFP)labeled into UCMSCs.Cell morphology and fluorescence infection efficiency was observed under fluorescence microscope.More than 85%of the transfection efficiency can continue the following test.The experiment was divided into:?The control group,?IFN-P group,?IFN?-Lentivirus transfection MSCs group(IFN-?-MSCs group),?Lentivirus empty carrier transfection MSCs group(MSCs-lentivirus group)?MSCs group.5.ELISA was used to detect the expression of IFN-? protein in the supernatant of IFN-?-MSCs group,MSCs-lentivirus group and MSCs group after 24h,48h,72h Finally,the OD value of each pore was measured by enzyme standard instrument at the wavelength of 450nm.6.Western blot assay was used to detect the expression of IFN-P protein in IFN-?-MSCs group,MSCs-lentivirus group and MSCs group.P-actin was used as the internal control.The final results are expressed as the ratio of the optical density of the target band to the ?-actin.7.Transwell method was used to detect the migration ability of five groups of A549 cells and NCI-H2170 cells respectively.Finally,Five views(Observed under microscope ×200)were observed in each filter membrane,and the number of cells passing through the microporous membrane in each group was calculated.Migration Inhibition Rate =(the number of transmembrane cells in the control group-the number of transmembrane cells in the test group)/the number of transmembrane cells in the control group x 100%.8.CCK-8 method was used to detect the effect of A549 and NCI-H2170 cell proliferation respectively.The absorbance value(OD)of 450nm wave was measured on the enzyme labelling instrument,the average OD value was calculated.The cell proliferation curve was drawn according to the absorbance values at different time points.(?)Vivo experiments1.Lung cancer A549 cells were used to establish subcutaneous implanted tumor model of lung cancer in nude mice.2.The five groups were divided according to different drugs were injected into the tail vein:The control group(no treatment of lung cancer in nude mice),IFN-? group,IFN-?-MSCs group,MSCs-lentivirus group,MSCs group.All the five groups of nude mice were killed at the 10th week.3.The tumor sizes in the animal models of five groups were compared:The mice were dissected,the tumor organisms were completely removed,the length and the short diameter of the tumors were weighed and measured,and the volumes were calculated and compared;4.The apoptosis of five groups of tumor cells were detected by tunel method.5.The changes of organs in nude mice bearing tumor were observed,and the specimens of organs were stained by HE staining,and the damages of organs in IFN-?group were observed.6.The MSCs group,the IFN-?-MSCs group of mesenchymal stem cells were labeled by ERFP.Fluorescence microscope was used to observe the fluorescence distribution of tumor tissues and organs in the two groups and the control group.The IOD values of each group were calculated and compared.The purpose was to detect whether MSCs have characteristics of the tumor targeted migration.(?)Study on mechanism of tumor Suppressor1.Western blot method was used to detect the prtein levels of Caspase-3,Caspase-8,PKR,VEGF in tumor tissues of the control group,MSCs group,MSCs-lentivirus group,IFN-?-MSCs group and IFN-p group.2.Fluorescence quantitative PCR method was used to detect the mRNA levels of Cas-pase-3,Caspase-8,PKR,VEGF in tumor tissues of the control group,MSCs group,MSCs-lentivirus group,IFN-?-MSCs group and IFN-? group.3.Part of lung cancer nude mice model of IFN-p group were treated with anti-macrophage antibody,macrophages in nude mice were inactivated,and non-inactivated group was set up.Tumor weight and volumes in nude mice model of macrophage inactivation group and non-inactivated group were compared.Further study on the important role of macrophages in the inhibition of lung cancer cell growth by IFN-?.[Results](?)Vitro experiment1.Human umbilical cord-derived mesenchymal stem cells were easy to separate,easy to be cultured.After 14 days,under the microscope,MSCs could be seen to swim out.The shape of MSC is like a comma,and continue to be cultivated to see the cell becoming longer,thinner and more numerous.The third generation of cell formation and colony can be dense into radiate and vortex-like,with larger volume and uniform shape.2.Flow cytometry was used to identify the surface antigen of the third generation of UCMSCs.The positive expression rates of CD90,CD73,CD105:96.3%,97.5%,96.9%.The negative expression rates of CD45,CD 14,CD 19,CD34 and HLA-DR:2.8%,3.5%,4.2%,2.2%,2.6%.Therefore,the purity of UCMSCs isolated and cultured in this experiment is very high.3.The cells became oval,round,and a large number of lipid droplets were observed in the cytoplasm of UCMSCs on the 20th day.The oil red " O " staining showed that the lipid droplets were red.After induction of osteogenic differentiation medium and alizarin red staining,small calcified nodules appeared on the surface of the cells,and the nodules increased with the time of culture.This is consistent with the characteristics of UCMSCs differentiation into fat and osteocytes in vitro.4.In the group of IFN-?-MSCs,MSCs-Lentivirus group was observed by fluorescence microscope:The efficiency of fluorescence infection was above 85%and the cells grew well.The results showed that the target plasmid transfection was normal and the target plasmid fluorescent marker gene expression was normal.5.The level of IFN-? protein in the supernatant of IFN-?-MSCs group was significantly higher than that in MSCs-lentivirus group and MSCs group after 24h,48h,72h(P<0.05).There was no statistical difference between MSCs-lentivirus group and MSCs group(P>0.05).6.The expression of IFN-P protein in IFN-?-MSCs group was significantly higher than that in MSCs-lentivirus group and MSCs group(P<0.01).7.Effects of five groups on migration ability of A549 cell line and NCI-H2170 cell ine:The inhibition rate of in IFN-?-MSCs group was significantly higher than that in control group(P<0.01),and the migration ability in IFN-?-MSCs group was significantly lower than that in IFN-? group(P<0.01).8.Effects of five groups on proliferation of A549 cell line and NCI-H2170 cell line:The absorbance of IFN-? group,IFN-? MSCs group,MSCs-lentivirus group and MSCs group were all lower than those in the control group(P<0.05).The absorbance of IFN-?-MSCs group was lower than that of IFN-? group,MSCs-lentivirus group and MSCs group(P<0.05).The IFN-? group was lower than MSCs-lentivirus group and MSCs group(P<0.05).There was no statistical difference between mscs-lentivirus group and MSCs group(P>0.05).(?)Vivo experiment1.Success rate of the nude mice model with lung cancer:98%.One of the nude mice died of being thin,eating little and intolerable.In IFN-? group,one nude mice was injected with IFN-? for three weeks,and then died.The cause of death was nude mice weight loss,poor appetite,poor mental state,and could not tolerate the side effects of drugs.All nude mice in IFN-? group had diarrhea,loss of appetite and mental retardation.Five groups of nude mice were killed at the 10th week after intravenous injection:nine in the control group,nine in IFN-? group,ten in IFN-?-MSCs group,ten in MSCs-lentivirus,ten in MSCs group.2.The tumor tissue sections were stained with HE.The cancer nests were found in all five groups,and the tumor area was bleeding and necrotic in varying degrees and the morphology and structure of the cells disappeared.3.Tumors of IFN-? group and IFN-?-MSCs group grew more slowly than those of the control group,MSCs-lentivirus group and MSC group(P<0.01).Compared with control group,the tumor weight and volume of MSCs-lentivirus group and MSC group increased,but there was no significant difference(P>0.05).4.The apoptosis rate of tumor cells was significantly increased in the IFN-? group and IFN-?-MSCs group compared with the control group,MSCs-lentivirus group and MSCs group(P<0.01).There was no statistically significant difference between IFN-? group and IFN-?-MSCs group(P>0.05).There was no statistically significant difference between the control group,MSCs-lentivirus group and MSCs group(P>00.05).5.There were no obvious pathological changes of lung,kidney,liver,spleen in the control group,IFN-?-MSCs group,MSCs-lentivirus group,MSCs group.In IFN-?group,inflammatory exudation was observed in liver,lung and kidney.HE staining showed a large number of inflammatory cells infiltrating the lungs,filling the entire alveoli.Liver cells showed partial necrosis of liver cells,cytoplasmic hyperchromia,nucleation,and degeneration of hepatocyte,a small amount of vacuoles appeared in the cytoplasm.The kidney can be seen with inflammatory cells,and the glomeruli is mildly hardened.6.Under fluorescence microscope,clear red fluorescent dye was observed in tumor tissue of IFN-?-MSCs group and MSCs group(P<0.01),but there was no red fluorescence expression in liver,lung,kidney and spleen,and there was no red fluorescence expression in tumor tissues and organs in the control group(P>0.05).(?)Study on mechanism of tumor Suppressor1.Expression proteins of Caspase-3,Caspase-8,PKR,VEGF in five groups:The expression levels of Caspase-3 protein and Caspase-8 protein in IFN-?-MSCs group and IFN-? group were significantly higher than those of other groups(P<0.01).The expression levels of those in MSCs group and MSCs-lentivirus group were significantly lower than those in the control group(P<0.01).The expression levels of PKR protein in IFN-? group and IFN-?-MSCs group were significantly higher than in other groups(P<0.01).There was no significant difference among the control group,MSCs group and MSCs-lentivirus group(P>0.05).The expression levels of VEGF protein in IFN-? group and IFN-?-MSCs group were significantly lower than those of other groups(P<0.01).The expression levels of VEGF protein in MSCs group and MSCs-lentivirus were significantly higher than that in the control group(P<0.01).There was no significant difference between IFN-?-MSCs group and IFN-? group(P>0.05)and no significant difference between MSCs group and MSCs-lentivirus group(P>0.05).2.Expression mRNA of Caspase-3,Caspase-8,PKR,VEGF in five groups:The expression levels of Caspase-3 and Caspase-8 were the same in MSCs group and MSCs-lentivirus group(P>0.05),but the expression levels were decreased compared with the control group(P<0.01).The expression levels of Caspase-3 caspase-8 in IFN-?-MSCs group and IFN-? group were almost the same(P>0.05),but those compared with the control group and MSCs group,MSCs-lentivirus group were significantly increased(P<0.01).The expression levels of PKR were the same in IFN-?-MSCs and IFN-? groups(P>0.05),but the expression levels were significantly higher than the other three groups(P<0.01).In the control group,MSCs group and MSCS-lentivirus group,the expression levels were the same(P>0.05).The expression levels of VEGF in MSCs group and MSCS-lentivirus group were higher than that in the control group(P>0.05),but the expression levels in IFN-?-MSCs group and IFN-? group were lower than that in the control group(P<0.01).The expression levels of VEGF in MSCs group and MSCS-lentivirus group were the same(P>0.05).The expression levels of VEGF in IFN-?-MSCs group and IFN-? group were the same(P>0.05).3.The weight and volume of tumor in the experimental group injected with anti-macrophage antibody were significantly increased than that in the control group(P<0.01).[Conclusion]1.Vitro experiments showed that UCMSCs was easy to be isolated and cultured,and had the potential to differentiate into fat cell and osteocytes.IFN-?-MSCs,IFN-? and MSCs can inhibit the migration of lung cancer A549 cell line and NCI-H2170 cell line.2.It was found from nude mice model of lung cancer that the growth of subcutaneous implanted tumor could be inhibited by IFN-?-MSCs and IFN-?,but IFN-? had obvious damage to organs of nude mice.IFN-?-MSCs can direct to tumor cells and play the role of targeted therapy.MSCs showed a slight promotion of tumor growth,but MSCs infected with IFN-? gene could effectively avoid the effect of MSCs.3.The mechanism of IFN-? inhibits the growth of lung canceris is related to PKR/Caspase mediated apoptosis pathways,and inhibits VEGF so as to block the blood supply of the tumor,and the activation of macrophages.4.Therefore,Vitro and vivo studies have demonstrated that the study of IFN-?-MSCs as targeted therapy for NSCLC is of great significance. |
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