Preparation Of HVEGF₁₂₁/EGFP-Expressing Mesenchymal Stem Cells And The Effect Of Hypoxia On Its Expression

Objective: To construct human vascular endothelial growth factor 121eukaryotic expression plasmid that contains green fluorescent protein report gene coding region and transfect bone marrow mesenchymal stem cells with the recombinants. Determine the VEGF expression level of hVEGF₁₂₁/EGFP-expressing bone marrow mesenchymal stem cells and blank MSCs that both after different time course of hypoxic culture, then approach the differences and meanings.Method: 1. We designed the total length amplification primer of hVEGF₁₂₁ which with restriction endonuclease cutting cites. Xho I cutting cite was added to the 5'end of sense primer and EcoR I cutting cite added to the 5'end of anti-sense primer. This pair of primer was used to amplify the coding fragment of hVEGF₁₂₁ from pCD2-hVEGF₁₂₁ plasmid by PCR. The products of PCR—the hVEGF₁₂₁ coding fragments with restriction endonuclease cutting cites , and vector—pEGFP-N₂ were digested by Xho I and EcoR I. The hVEGF₁₂₁ coding fragment was ligated directly into MCS of pEGFP-N₂ by sticky ends ligating clone method. Then, the recombinant plasmid- pEGFP-N₂-hVEGF₁₂₁ was constructed. 2. Recombinant was identified by XhoI,EcoR I and HincII digestion, PCR method , and DNA sequencing technology. 3. BM-MSCs were isolated by adhering-cell culture method, then passaged several times. We identified cells according to morphology and surface markers analysis. 4. BM-MSCs were transfected with pEGFP-N₂-hVEGF₁₂₁ by positive ionic liposome transfection method. 5. Green fluorescent light was observed under fluorescent microscope to detect the expression of hVEGF₁₂₁/EGFP fusion protein. 6. hVEGF₁₂₁/EGFP-expressing BM-MSCs were cultured under hypoxic condition that time course was separately set in 6, 12, 24 hours. Control group were blank BM-MSCs and BM-MSCs transfected with blank vector- pEGFP-N₂ under the same hypoxic intervention. The VEGF mRNA expression level of each experimental group was detected by RT-PCR method and results were compared.Results: 1. The construction of pEGFP-N₂-hVEGF₁₂₁: The PCR products of the designed total length amplification primer of hVEGF₁₂₁ with restriction endonuclease cutting cites length in 459bp, it's consistent with the length of hVEGF₁₂₁ coding sequences. The products of PCR - the hVEGF₁₂₁ coding fragments with restriction endonuclease cutting cites and vector– pEGFP-N₂ were digested correctly by Xho I and EcoR I, so we obtained an insert fragment (453bp) and a vector fragment (4.6kbp), then, we ligated them successfully and constructed a recombinant. Digested the recombinant plasmid with Xhoâ… ,EcoRâ… , we got a 453bp fragment which consistent with the length of hVEGF₁₂₁ coding sequences; PCR with hVEGF₁₂₁ specific partly amplification primer we got correct products (317bp) from recombinant. DNA sequencing completely confirmed that The hVEGF₁₂₁ coding fragment was inserted into MCS of pEGFP-N₂ directly and correctly and demonstrated we constructed the plasmid pEGFP-N₂- hVEGF 121 successfully.2. The BM-MSCs which transfected with pEGFP-N₂-hVEGF₁₂₁ was observed under the fluorescent microscope, we saw obvious green fluorescent light. It hinted the expression of hVEGF₁₂₁/EGFP fusion protein. 3. After 6, 12, 24 hours'hypoxic culture which mimic the microenvironment of myocardial anoxia in vitro, results of RT-PCR shows: Recombinant transfected BM-MSCs expressed higher level of VEGF mRNA than control groups(p<0.01) after each time course of hypoxic culture, and there was no statistic differences among each time course. The VEGF mRNA expression level of blank BM-MSCs group was upgrading along with the prolongation of the hypoxic culture time. The VEGF mRNA expression level of blank BM-MSCs group versus blank vector transfected BM-MSCs group shows no statistic differences (p>0.05).Conclusion: This study successfully constructed human vascular endothelial growth factor 121eukaryotic expression plasmid that contains green fluorescent protein report gene coding region, and expressed pEGFP-N₂-hVEGF₁₂₁ in BM-MSCs. Confirmed that hVEGF₁₂₁/EGFP fusion protein-expressing BM-MSCs can endure adequate time of hypoxic environment, and sustain stronger and more steadily VEGF expression. It provided a good basis for further research on combine of VEGF gene therapy and stem cells transplantation for ischemial cardiovascular disease, and also, offered feasibility evidence for transplant VEGF-expressing BM-MSCs into acute ischemial or infarct myocardium.