Preparation and functional study of an adenovirus vector expressing human plasminogen kringle 5 gene / 生物工程学报

Tumor angiogenesis plays a pivotal role in the progress of tumor. Among the various endogenous angiogenic inhibitors discovered, the human plasminogen kringle 5 (K5) has been demonstrated to be a potential inhibitor of the proliferation and migration of vascular endothelial cells in vitro. The replication-incompetent adenovirus (Ad) vector Adeno-X-CMV-K5 (Ad-K5) (where CMV is cytomegalovirus) was constructed and its antiangiogenic effect was tested on vascular endothelial cell and tumor cell. For the construction, the K5 cDNA was fused in-frame with human plasminogen signal sequence and inserted into the eukaryotic expression vector pcDNA3 to form pcDNA3K5. The recombinant plasmid was subcloned into the shuttle plasmid pShuttle under the control of the constitutive CMV immediate-early promoter. The plasmid carrying the cDNA for K5 (pShuttleKS) was then recombined with the Adeno-X viral DNA and transformed into E. coli DH5alpha. The resultant recombinant plasmid pAd-K5 was transfected into human embryonic kidney (HEK) 293 cells with liposome. The adenovirus expressing human plasminogen kringle 5 (Ad-K5) was successfully packaged and propagated in 293 cells, as detected by the cytopathic effect (CPE) on the cells, and the viral titer in the supernatant was 5 x 10(8) pfu/mL by plaque assay. Both human umbilical vein endothelial cell line ECV304 and human breast carcinoma cell line MDA-MB-231 were infected with Ad-K5 and Ad-LacZ, which was used the negative control, and assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Compared with uninfected control and Ad-LacZ infected control, Ad-K5 infected group at 80 MOI (multiplicity of infection) significantly inhibited ECV304 proliferation; the difference between uninfected control and Ad-LacZ infected control was not significant. In contrast, there was no significant difference in the proliferation of MDA-MB-231 among all the treatments. In addition, the Ad-K5 at 100 MOI inhibited the differentiation and tube formation of ECV304 on ECMatrix gel. These results suggested that the recombinant replication-defective Adenovirus expressing human plasminogen kringle 5 inhibited the proliferation, differentiation and tube formation of ECV304 and had no effect on the proliferation of MDA-MB-231. Adenovirus mediated human plasminogen kringle 5 gene therapy may be a potential treatment of cancer through angiogenesis inhibition.

Preliminary study of ALK3 downstream genes related to ventricular septum defect / 生物工程学报

To investigate the function of ALK3 gene, the gene regulation and the signaling pathway related to ventricular septum defect during heart development. The model mice with ALK3 gene knock-out via alpha-MHC-Cre/lox P system were bred. The mRNA expression level of control group was compared with that of experiment group and ALK3 downstream genes were screened using PCR-select cDNA subtraction microarray. The mRNA of control group was extracted from E11.5 normal mouse hearts, and that of experiment group, from E11.5 hearts of mice with alpha-MHC Cre(+/-) ALK3(F/+) genotype. It was found that the mice with ALK3 gene knock-out produced heart defects involving the interventricular septum. The platelet-activating factors acetylhydrolase and the transcription factor Pax-8 and so on, were down-regulated. However, the Protein Tyrosine Kinase (PTK) of Focal Adhesion Kinase (FAK) subfamily and beta subtype protein 14-3-3 were up-regulated in the alpha-MHC Cre(+/-) ALK3(F/-) mice. These data provide support that ALK3 gene played an important role during heart development. The platelet-activating factors acetylhydrolase and Pax-8 genes could be important ALK3 downstream genes in the BMP signaling pathway during interventricular septum development. PTK and beta subtype protein 14-3-3 might be regulatory factors in this pathway.

A study on myocardial Pax-8 gene / 中华儿科杂志

<p><b>OBJECTIVE</b>Conventional deletion of ALK3, also termed as bone morphogenetic protein (BMP) receptor IA, in mice might result in early embryonic lethality. To investigate the function of ALK3 in cardiac development, the cardiac-specific deletion of ALK3 in mice was made by Dr. Schneider, using Cre recombinase driven by the alpha-MHC promoter that Dr. Fukushipe worked out. Such specific deletion of ALK3 caused death in mid-gestation with defects in the trabeculae, interventricular septum, and endocardial cushion. Since ALK3 is not a cardiac-specific gene, it is extremely important to identify ALK3 downstream genes.</p><p><b>METHODS</b>Alpha-MHC Cre+/-, ALK3 F/- and alpha-MHC Cre+/-, ALK3 F/+ embryos were obtained after 20 alpha-MHC Cre+/-, ALK3 +/- mice and 20 ALK3 F/F mice were mating. The ALK3 downstream genes were screened using microarray made in Germany that could identify 25000 genes in mouse. Two populations of mRNA, one derived from the embryonic heart (11.5 days) of alpha-MHC Cre+/-, ALK3 F/- mice, and the other derived from the alpha-MHC Cre+/-, ALK3 F/+ mice, were compared. Cardiac-specific ALK3 downstream genes were identified using real time quantitative RT-PCR and in situ hybridization.</p><p><b>RESULTS</b>The expression of 12 genes, such as Pax-8 and Hox-3.5 were down-regulated in alpha-MHC Cre+/-, ALK3 F/- mouse heart. The expression of 16 genes including Ras-related protein Rab-5b and EPS-8 protein was up-regulated in the group of alpha-MHC Cre+/-, ALK3 F/-. It was found that the Box protein Pax-8 gene was down-regulated by 7.1 fold (P < 0.001) in the alpha-MHC Cre+/-, ALK3 F/- mice by real time quantitative RT-PCR. It was also revealed that the Box protein Pax-8 gene was expressed stronger in alpha-MHC Cre+/-, ALK3 F/+ than alpha-MHC Cre+/-, ALK3 F/- E11.5 days mouse heart by means of in situ hybridization.</p><p><b>CONCLUSION</b>The Box protein Pax-8 gene is an important and cardiac-specific ALK3 downstream gene in the BMP signaling pathway during inter-ventricular septum development.</p>