Establishment Of Gastric Carcinoma BGC-823 Cell Line Stably Expressing NM23 Gene Transduced By Adenovirus

Objective: Constructed and identified the recombinant adenovirus vector carrying the NM23 gene,then infected human gastric carcinoma BGC-823 cells,observe its influence on the gastric carcinoma BGC-823 cells,thus to establish the BGC-823 cell line expressing the NM23 gene stably.Methods:(1)We used the established plasmid NM23-PIRES2-EGFP as template and amplified the NM23 gene by PCR,the NM23 gene fragment was cloned into adenovirus vector p CMV-MCS-IRES-EGFP,the recombinant plasmid was digested by restriction enzyme digestion and DNA sequencing.(2)Packaging virus and testing the titer determination to determine the adenovirus infection optimal MOI values of BGC-823 cell;(3)Thehuman gastric carcinoma BGC-823 cells were divi-ded into three groups: the experimental group(NM23-OE group,transfected p CMV-KAI1-IR-ES-EGFP),control group(Ctrl),negative control group(NC group,transfected p CM-V-MCS-IRES-EGFP);(4)Real Time PCR and Western Blot were used to detect the changes of NM23 m RNA and the expression of NM23 protein in three gro-ups of BGC-823 cell respectively;(5)Assessed the apoptosis of carcinom of stomac-h cell line BGC-823 by flow cytometry.Results:(1)The recombinational adenovirus p CMV-NM23-IRES-EGFP was constructed successfully which was tested by enzyme digestion and DNA sequencing.(2)After augmentation,the titer of the recombinantional adenovirus was 4.0x1010 ifu/ml;(3)The expression levels of NM23 m RNA and NM23 protein in the experimental group were increased significantly(P<0.05)compared with the control group and the negative control group by Western Blot and Real Time PCR validation,while the difference between the control group and the negative control group was not statistically significant(P>0.05);(4)The apoptosis of BGC-823 cell in the experimental group was significantly higher than the apoptosis of blank group and the negative control group,while the difference was statistically significant(P<0.05).Conclusion:(1)The adenovirus vector of pCMV-NM23-IRES-EGFP was successfully constructed,which was identified by sequencing;(2)Successfully established the human gastric carcinoma BGC-823 cells which could express the NM23 gene expressed stably;(3)The upregulation of NM23 gene can promote apoptosis of human gastric carcinoma BGC-823 cells.