Infantis Bifidobacterium/Recombinant Human Decorin Tumor Targeted Gene Therapy System And Its Effection On K562Cell Effect

Objective To construct human decorin (hDCN) prokaryotic expression vector PGEX-4T-1-DCN.Transform the recombinant plasmid into bifidobacterium of infantis, until fully expressed, take supernatant to observe the effect of bifidobacterium in K562cell proliferation, apoptosis and cell cycle.Method1.Use Double-Restriction enzyme method to amplify and recovery of DCN gene fragments, use molecular recombination technology to construction of prokaryotic expression vector PGEX-4T-1-DCN and transform the recombinant plasmid transformation into Bifidobacterium by means of electric transformation method, then double enzyme digest and DNA sequencing EcoRI and NotI2. Extracted the supernatant, then put them in the corresponding experimental groups and observe suppression, morphological changes and apoptosis of K562cell under inverted microscope.3. Detect the24h,48h and72h apoptosis of K562cell in the experimental group using Flow Cytometry, and analysis cell cycle arrest.Results1. Recombinant plasmid by double enzyme, the size of product gene fragment recovered match with the target gene segment.Compare with Genebank sequence (NT029419) after sequencing,it was corresponding to the comparison DCN gene sequence exactly.There was no mutation.2. Observed under inverted microscope to DCN plasmid transformation of Bifidobacterium supernatant group, empty plasmid transformation of Bifidobacterium supernatant group, simple group of Bifidobacterium supernatant cell showed apoptotic morphological changes. The conversion of DCN Bifidobacterium group under the effect of cell shape changes are the most obvious, visible cell shape is not regular, and nuclear fragmentation, apoptosis, cell proliferation was significantly slowed down.3. Flow cytometry found that,simple bifidobacterium supernatant group and transforming empty carrier bifidobacterium supernatant group can induce K562cells appearing apoptosis and early apoptotic rate were not much difference(11.26±1.54%and13.85±1.33%, respectively).Transforming the target vector supernatant group was up to23.79±2.48(%), had a significantly different with the blank control group and the other experimental groups (P<0.01).3. Cell cycle analysis results show that, compared with the control group (5.04±0.78%), the percentage of cells in G2/M phase in the pure bifidobacteria supernatant group (10.56± 0.81%), dead body of Bifidobacterium supernatant group (12.98±1.66%) DCN and transformation of Bifidobacterium supernatant group (13.37±0.74%) significantly increased (P<0.01), statistical meaning:pure bifidobacteria supernatant group and empty vector supernatant group G0/G1of bifidobacteria were realized as (65.59±1.54%,63.89±1.24%), compared to the blank group (62.23±0.72%) was not statistically significant (P>0.05), and DCN transformation of double branch bacilli from group GO/Glwas (72.24±0.54%), and other the three groups were compared, the difference was statistically significant (P<0.01).Conclusion1. Construct human decorin prokaryotic expression vector, hDCN PGEX-4T-1-DCN successfully.2. Transform the recombinant plasmid transformation into Bifidobacterium by means of electric transformation method successfully.3. The expression product of exogenous genes DCN and bifidobacterium infantis connection inhibited K562cells proliferation and induces apoptosis and can produce synergistic stronger inhibition of K562cell growth and induce their apoptosis than separate application.4. Bifidobacterium infantis can make K562cell G2/M arrest, while DCN into bifidobacteria, can result in K562cell and G2/M and GO/Glblock, cell growth in the two node also blocked.