Osteogenic Activity of MSC Infected by Recombinant Adenovirus Vector Ad-LMP-1 / 中山大学学报(医学科学版)

[Objective]This study was designed to construct a recombinant adenovirus vector contains LMP-1 gene,and investigate the osteoinductive activity of MSC which were transfected recombinated adenoviral vector carrying LMP-1 gene.[Methods]Total RNA was extracted from mt osteoblast and the LMP-1 gene was acquired by RT-PCR,the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology,then the entry clone and the expression vector were used to create the expression clone throush the LR recombination reaction.The adenovirus expression clone was linearized by PacI and transfected to the 293A cell line to harvest a high titer.Ad-LMP-1 was infected into the 3rd passage MSC,the expression of LMP-1 was detected by Western blot.The osteogenic activity of MSC was evaluated by the expression of collagen Ⅰ,ALP,osteocalcin and the formation of bone nodule.[Result]The LMP-1 gene was successfully acquired and confirmed,the entry clone and the expression clone were both verified by enzymes digestion,and the expression clone was further confirmed by sequenced.The expression of LMP-1 was detected successfully in MSC.The increasing expression of collagen Ⅰ,osteocalcin.ALP and bone nodule were observed by comparing to the control group.[Conclusion]Gateway technology not only make construction of the pAd-LMP-1 recombination adenovirus vector simple and fast,but also get a high transfection efficacy in MSC.LMP-1 gene can induce the osteoblast differention of MSCs,and improve its osteogenic activity.The adenovirus vector is reliable to be used in further gene therapy research.

Transfection and Expression of eNOS Gene on Canine Endothelial Cells / 上海第二医科大学学报

Objective To study endothelial nitric oxide synthase (eNOS) gene transfecting endothelial cells (ECs). Methods eNOS gene was transfected into the steady ECs system by cationic liposomal transduction and check the transfection effect by RT-PCR and immunohistochemistry assay. To test the concentrations of NOS, nitric oxide (NO) and von willebrand factor (vWF) in culture media by colorimetry and ELISA, respectively,and transfected EC function was observed. Results The effect of transfection was satisfactory with RT-PCR products electrophoresis, sequence and immunohistochemistry. The concentrations of NOS and NO in transfected EC culture media increased with time (P

Construction and target gene expression of recombinant adenovirus vectors containing vascular endothelial growth factor 165 and angiopoietin-1 / 中国矫形外科杂志

[Objective]To construct the recombinant adenovirus vector co-expressing human vascular endothelial growth factor 165(VEGF165) and angiogenin-1(Ang-1),and to observe the expression of target gene after transfection.[Method]Molecular biologic techniques were used to clone the genes of VEGF165 and Ang-1,and PCR was used to amplify the DNA sequence of IRES(internal ribozyme entry site) from pIRES2-EGFP.The genes of VEGF165,IRES and Ang-1 were subcloned to pTrack-CMV one by one to get the plasmid named pTrack-CMV-VIA,which contained all the three genes.The linearized shuttle plasmid was cotransformed into BJ5183 bacteria with backbone vector AdEasy-1.Further the recombinant plasmid was packaged and amplified in QBI-293A cells after Pac I digestion to get adenovirus vector pAd-VIA.The expression of the gene of interests was evaluated by fluorescence microscopic analysis of GFP expression and enzyme linked immunosorbent assay(ELISA).[Result]The recombinant adenovirus plasmid was consistent with that shown by sequencing and restriction endonucleases digestion.The recombinant pAd-VIA vector was packaged and amplified successfully in QBI-293A cells.The titer was 2?1010 PFU/ml after amplifying.High positive GFP was expressed in the field through fluorescence microscopy after being transfected by recombinant pAd-VIA.ELISA indicated the expression of the target genes.The difference between the tansfected and non-transfected group was significant(P

Study on the toxicity and transfection efficiency of PEI/MYOC gene complexes in vivo / 眼科

Objective To investigate the toxicity and transfection efficiency of PEI-mediated plasmid DNA encoding myocilin (MYOC) gene transfected into trabecular meshwork.Design Experimental study.Participants SD rats.Methods PEI and plasmid encoding MYOC gene complex was prepared and 20?l PEI/DNA complex was directly injected into anterior chamber of SD rat by microinjection. The eyes were extracted at day 1,3,7,and 14 after injection for the structure analysis of trabecular meshwork by HE staining and transmission electron microscope.Expression of the transferred myocilin gene was monitored by fluorescence microscopy. Main Outcome Measures HE staining,immunohistochemistry and electron microscope.Results No significant toxicity or inflammation was detected under the HE staining and electron microscope.PEI/DNA complex were seen in cytoplasm of trabecular meshwork cells. The fluorescence intensity of myocilin expression in the trabecular meshwork cells was increasing with time and peaked on clays 3 and declined afterward.Conclusion Plasmid DNA mediated by PEI could be efficiently transfected into trabecular meshwork cells by directly injecting into anterior chamber without any toxicity.

Inhibitory effect of mouse angiostatin cDNA gene transduction on human liver cancer cell / 中华消化杂志

Objective To study the effect of the mouse angiostatin cDNA gene transfecting into human liver cancer cell line HCC7721 on cell growth, cell cycle phase distribution, cell morphology in vitro and tumorigenesis in vivo , and the mechanisms. Methods The gene fragment of mouse angiostatin cDNA was directly cloned into an eukaryotic expression plasmid pcDNA3.1(+) of the promoter CMV between the multicloning sites HindⅢ and XbaⅠ and confirmed the correct recombinant plasmid pcDNA3.1(+) angio through enzymatic digestion and gene sequencing. Then it was transfected into human liver cancer cell line HCC7721 with liposome, pcDNA3.1(+) as vecter control and liposome as mock control. After 30 day selection by neomycin G418, angiostatin expression at the levels of mRNA and protein in vitro were tested by RNA dot blot and FACS respectively. Cell morphology under was observed light microscope, made growth curves were made, and cell cycle distribution checked in FACS. Animal model was set up to study tumorigenesis in vivo of those cells transplanted subcutaneously in the right hind legs of nude mice BALBc. Microvessel density (MVD) was analyzed and angiostatin expression in the tumor tissues by in situ immunohistochemistry. Results The recombinant eukaryotic expression plasmid pcDNA3.1(+) angio was constructed Auccessfully. After selection of G418 for about 4 weeks there were macroscopic cell clones in the experimental group and vector control group, but no survival cells in the mock control. In vitro angiostatin were expressed in the experimental group, but not in the vector control. There weren't significant changes in cell morphology, cell growth curves and cell cycle distribution between the experimental and the control group. However, the nude mice experiment showed that tumorigenic capability of the experimental cells had been reduced greatly. Immunochemistry study showed that there were much less microvessels and conversely stronger angiostatin positive staining in the tumor tissues than in the control group. Conclusions Mouse angiostatin has no direct inhibition on human liver cancer cell line HCC7721 in vitro , but it inhibits effectively the tumorigenesis of HCC7721 in vivo , which is probably due to its inhibition on tumor angiogenesis in a paracrine path way.