Xian Huaguang Bio engineering Company

Fengcheng, China

Xian Huaguang Bio engineering Company

Fengcheng, China
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Xiaojiang T.,First Affiliated Hospital | Jinsong Z.,Histology and Embryology | Jiansheng W.,First Affiliated Hospital | Chengen P.,Xi'an Jiaotong University | And 2 more authors.
Cancer Investigation | Year: 2010

To further enhance anticancer effect of Shepherdin and overcome limitation of peptide therapy, recombinant adeno-associated virus (rAAV) was constructed with following strategies: therapeutic peptide secretory expression and adeno-associated virus gene transfer system. MTT assay and flow cytometric analysis revealed that rAAV harboring fusion gene NT4-Ant-Shepherdin significantly suppressed A549 cell growth in a time-dependent manner and induced apoptosis. In the infected A549 cells, survivin expression level decreased strongly while Caspase-3/7 activities increased significantly. These results indicated that rAAV harboring fusion gene NT4-Ant-Shepherdin may be a novel strategy in lung cancer peptide therapy. Copyright © 2010 Informa Healthcare USA, Inc.

Sun L.,PLA Fourth Military Medical University | Hao Y.,PLA Fourth Military Medical University | Nie X.,PLA Fourth Military Medical University | Zhang X.,PLA Fourth Military Medical University | And 2 more authors.
Experimental and Therapeutic Medicine | Year: 2012

The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the immunohistochemical method and results showed that the expression was positive in the experimental group. Myocardium perfusion MRI displayed that the infracted area significantly decreased under the action of pSS-HRE-CMV-NT4-PR39-PolyA-AAV. The artificial minimal HRE in CRL-1730 cells effectively and rapidly regulates the expression of the downstream gene NT4-TAT-His-PR39 of the CMV promoter. Recombinant pSS-HRE-CMV-NT4-PR39-PolyA-AAV promotes neoangiogenesis in the ischemic area, reduces the area of infarction and improves heart function.

Zhu A.,Tianjin University | Ren Y.,Xi'an Jiaotong University | Wang N.,Tianjin University | Jin Q.,Tianjin University | And 3 more authors.
Gene | Year: 2015

Gene therapy, a significantly crucial strategy for treatment of malignancies, has been gradually accepted in recent years. However, this therapeutic approach has being facing great challenges concerning problems which include complicated development of cancer with multiple gene control, effective target shortage, low efficiency of gene transferring and safety of the vector delivery system. Shepherdin, a novel peptidomimetic molecule designed from Lys-79 to Leu-87 of survivin, has been identified as a tumor suppressor with the function that can not only competitively interfere with the interaction between survivin and Hsp90 (heat shock protein-90) leading to the degradation of survivin to anti-tumor, but also competitively target the ATP-dependent binding pocket of Hsp90 resulting in the dysfunction of Hsp90 chaperone to cell apoptosis via a mitochondrial dependent or independent pathway. In the present study, we designed and constructed a recombinant Adeno-associated virus (rAAV) loading fusion gene NT4-TAT-6His-Shepherdin. The expression of Shepherdin in gallbladder carcinoma (GBC) cells was detected and its strong inhibitory effects against GBC growth were evaluated after AAV mediated gene transfer of Shepherdin into GBC cells and xenograft tumors. MTT assay and flow cytometric analysis demonstrated that rAAV containing Shepherdin gene could significantly inhibit the growth of GBC and this effect was closely associated with apoptosis. These results indicated that rAAV-NT4-TAT-6His-Shepherdin may be considered a novel therapeutic strategy in the gene therapy for gallbladder carcinoma. © 2015 Elsevier B.V..

Qin T.-J.,Xi'an Jiaotong University | Ma W.,Xi'an Jiaotong University | Liu S.-X.,Xi'an Jiaotong University | Yang G.-X.,Xian Huaguang Bio engineering Co. | And 2 more authors.
Molecular Medicine Reports | Year: 2010

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo.

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