Effects Of NM23 Gene Transfection On Transplanted Human Gastric Carcinoma Xenografts In Nude Mice

Objective:The adenovirus vector which carrying NM23 metastasis suppressor gene to transfect human poorly differentiated gastric carcinoma cell line BGC-823,to investigate the effect of overexpression of NM23 metastasis suppressor gene on metastasis of transplanted abdominal tumor in nude mice.Method:1.Drawed the growth curve of BGC-823 cells.2.BGC-823 cells were grouped,and blank control group(Ctrl group),and BGC-823 cells infected by pCMV-MCS-IRES-EGFP were selected as negative control group(NC group),and BGC-823 cells infected by pCMV-NM23-IRES-EGFP were selected as experimental group(NM23-OE group)respectively.After 48h incubation,to observed whether the cells carry the enhanced green fluorescence protein EGFP.3.18 female BALB/c-nu nude mice were randomly divided into 3 groups,which 6 in each group;To adjusted cells density were1×10~7cells/ml,and nude mice were inoculated intraperitioneally with 0.2ml single cell suspensions.The nude mice with blank BGC-823 cells for Ctrl group,and the nude mice with BGC-823 cells infected by pCMV-MCS-IRES-EGFP for NC group,and the nude mice with BGC-823 cells infected by pCMV-NM23-IRES-EGFP for NM23-OE group.To observed the living conditions of nude mice at every regular time,and the abdominal circumference of nude mice were measured at every 2 days,and to explored the abdominal cavity in nude mice by application of small animal ultrasound.4.Nude mice were necropsied after 2 weeks of continuous observation,and gross macroscopic observat_ion and microscopic observation were performed respectively.To calculated the number of transplanted tumors disseminated in organs,and calculated the rate of various organs into the tumors,and measured maximum tumor volume,and weighted the tumor weights,and calculated the inhibition rate of NM23-OE group,and completed the exfoliative cytology,and the pathological features of xenograft tumors were observed by HE staining.5.IHC and Western Blot were analyzed the expression of NM23 protein in three groups of nude mice xenografts.Results:1.BGC-823 cells were appeared 1 day of adaptive period after generation.On the secend day,the cells began to multiply.Cells were incubated until day 3,and got to the logarithmic growth phase.After 6 days,the number of cells gradually decreased.2.To observed the infection by using fluorescent microscope.NM23-OE group and NC group have the enhanced green fluorescence protein EGFP,and Ctrl group had no the enhanced green fluorescence protein.3.The xenograft model in nude mice were constructed successfully,the general situation is subpar in Ctrl group and NC group,and the NM23-OE group in good condition.Compared with the Ctrl group and NC group by NM23-OE group,the abdominal circumference growth in nude mice of NM23-OE group obviously lag behind of Ctrl group and NC group,the difference of abdominal circumference in nude mice of NM23-OE group obviously below of Ctrl group and NC group,the difference was statistically significant(p<0.05).Detected the growth of transplanted tumors in abdominal cavity of nude mice by using small animal ultrasonic detector,and description of human gastric carcinoma xenograft model in nude mice were successfully constructed.4.It founded the statistics of tumor formation rates of nude mice,the tumor formation rates of NM23-OE group were lower than Ctrl group and NC group,the difference was statistically significant(p<0.05).The maximum tumor volume in NM23-OE group was significantly lighter than Ctrl group and NC group,the difference was statistically significant(p<0.05).The weight of xenografts in NM23-OE group were significantly lighter than Ctrl group and NC group,the difference was statistically significant(p<0.05).The inhibition rate of NM23-OE group is 75%.There were no significant ascites in NM23-OE group,and Ctrl group and NC group ascites cytology showed:The single free nuclear heterogeneous cells were visible.HE staining showed abnormal gastric carcinoma cells in a mass,with cancer embolus in blood vessels.5.Immunohistochemistry resulted:The expression of NM23 protein in NM23-OE group were higher than in Ctrl group and NC group,the difference was statistically significant(p<0.05).Compared with Ctrl group and NC group,there was no statistical significance(p>0.05).The expression of Ki67 in NM23-OE group were lower than in Ctrl group and NC group,the difference was statistically significant(p<0.05).Compared with Ctrl group and NC group,there was no statistical significance(p>0.05).Spearman rank correlation analysis showed:The positive expression of NM23 and Ki67 in transplanted tumors were negatively correlation,the r was correlation coefficient=-0.87,p<0.05.Western Blot resulted:The expression of NM23protein in NM23-OE group were higher than in Ctrl group and NC group,the difference was statistically significant(p<0.05).However,the expression of NM23 protein in the control group and NC group were lower,and there were no significant difference between the two control groups(p>0.05).Conclusion:1.The adenovirus vector which carrying NM23 metastasis suppressor gene successfully transfect human poorly differentiated gastric carcinoma cell line BGC-823.2.Successfully construct the model of nude mouse abdominal transplantatation tumor.3.BGC-823 cells are infected by the adenovirus vector which carrying the NM23 metastasis suppressor gene,when the NM23 metastasis suppressor gene is overexpressed,the incidence and maximum tumor volume and weight of the tumor in nude mice are decreased,to a certain extent,the NM23 gene has inhibitory effect on the disseminated metastasis of abdominal xenografts in nude mice.