ABSTRACT
[ ABSTRACT] AIM:To construct a conditionally replicating adenovirus vector activated by CXCR4 promoter and to evaluate its ability of lysing the lung cancer cells specifically.METHODS:Human CXCR4-E1A gene amplified by PCR was cloned into the shuttle plasmid pDC316-GFP to construct the recombinant shuttle plasmid pDC316-CXCR4-GFP.The recombinat shuttle plasmid and adenovirus genomic plasmid pBHG-lox-E1, 3Cre were transfected into 293 cells to construct the recombinant adenovirus CRAd-CXCR4-GFP.PCR was used to detect the target gene fragments, and the viral titer was determined.A549 cells with the highest mRNA expression of CXCR4 were screened out from 5 kinds of lung cancer cell lines by real-time PCR.CXCR4 promoter activity and adenovirus replication numbers were detected in A549 cells after transfection of CRAd-CXCR4-GFP and Ad-NULL.CRAd-CXCR4-GFP and Ad-NULL were transfected into A549 cells and 16HBE cells, the apoptotic rates were detected by flow cytometry and the viability was analyzed by CCK-8 assay.RE-SULTS:The recombinant plasmid pDC316-CXCR4-GFP was constructed successfully.Green fluorescence was observed in 293 cells under fluorescent microscope after co-transfection of pDC316-CXCR4-GFP and pBHG-lox-E1, 3Cre at 11 d. Green fluorescence was observed in 293 cells after infection of amplified 3rd generational adenovirus.PCR showed that the purpose gene was successfully integrated in recombinant adenovirus genome.The virus in the supernatant reached a titer of 1 ×1013 PFU/L.The mRNA expression of E1A and E4 in the A549 cells after transfection of CRAd-CXCR4-GFP was markedly increased compared with Ad-NULL group.Compared with Ad-NULL group and empty control group, the apoptotic rate and the viability of A549 cells in CRAd-CXCR4-GFP group had no significant difference in the first 4 d, the apoptotic rate increased significantly at 5 d, and the cell viability declined significantly at 5 d, but the apoptotic rate and the viability of 16HBE cells in each group had no significant difference within 5 days.CONCLUSION: The conditionally replicating adenovirus vector CRAd-CXCR4-GFP has been successfully constructed, which has the ability of lysing lung cancer cells specifically.
ABSTRACT
Objective To compare the effect of insulin pump continuous subcutaneous insulin (CSII) and multiple subcutaneous insulin (short-acting insulin before meals + glargine,MSII) for the short duration of type 2 diabetes mellitus (T2DM) patients.Methods Fifty-two newly diagnosed T2DM patients were randomly divided into CSII(n =29) and MSII(n =23) group.Patients in CSII group were given insulin pump continuous subcutaneous plus metformin.And patients in MSII group were given insulin by multiple subcutaneous insulin injection plus metformin.The treating periods was 2 weeks and followed up one month.Results The periods from point of insulin injection to blood glucose back to normal level in CSII group was (4.70 ±2.01) d,shorter than that in MSII group(6.90 ± 1.50) d,and the difference was significant(t =2.056,P ＜0.05).The levels of C peptide in two hours postprandial before and after treatment in CSII group were (4.24 ± 0.25) ng/L,(6.29 ± 0.56) ng/L,and (3.20 ±0.11) ng/L and (7.33 ±0.41) ng/L respectively in MSII group.The levels of C peptide in two hours postprandial after treatment were higher than that of before treatment in two groups (t =2.018,2.436 respectively,P ＜0.05),but there were no significant differences between two groups(t =0.985,P ＞ 0.05).Nineteen cases (65.5％) in CSII group were off insulin treatment in one month,and 9 cases (39.1％) in MSII group.There were significant differences in two groups(x2 =5.11,P ＜0.05).Conclusion The two treatment plans can make the improvement in terms of glucose control.However,CSII plan showed more effective than MSII.