This study sought to elucidate the effect of mechanical strain on the differentiation of mesenchymal stem cells into osteoblasts. Under the conditons of inducing osteoblasts, Immunohistochemical methods and RT-PCR technology were applied in osteogenic supplements medium to detect: (1) the expression of Alkaline phosphatase (ALP), Type I collagen (COL I ), Osterx (Osx) and Osteocalcin (OCN) mRNA, with cyclic strain (3%, 0.5 Hz) applied for 15 min, 30 min, 1 h, 2 h, 4 h, 3 d, 7 d, 14 d; (2) the expression of Osx mRNA and OCN mRNA with 3% strain for 1 h. The results showed: (1) ALP mRNA expression was higher at 7 days; COL I mRNA expression was greater obviously at 7 days and 14 days than that at 3 days and that of the unstrained cells; (2) the expression of Osx mRNA was up-regulated after 15min by strain stimulation,which was significantly increased at 30 min and 1 h in the unstrained cells. The expression of OCN mRNA was not affected in the unstrained cells at 15 min, whereas strain could promote the expression of OCN mRNA at this period. The expression of OCN mRNA was more obviously upregulated in the strained cells at 30 min and 1 h when compared with that in the unstrained cells; (3) the strain (1% and 3%) significantly promoted the expression of Osx mRNA; 10% strain had a little effect on Osx mRNA expression. The expression of OCN mRNA was up-regulated by 3% strain, whereas it had little effect at 1% and 10% strain. In summary, mechanical strain can promote the differentiation of mesenchymal stem cells into osteoblasts.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cells, Cultured ; Mechanoreceptors ; physiology ; Mechanotransduction, Cellular ; physiology ; Mesenchymal Stromal Cells ; cytology ; Mice ; Osteoblasts ; cytology ; Osteocalcin ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Sp7 Transcription Factor ; Stress, Mechanical ; Transcription Factors ; genetics ; metabolism

OBJECTIVETo construct a recombinant adenovirus (pAdxsi-GFP-HIF) encoding human hypoxia inducible factor 1 α gene (HIF-1 α) and to express it in endothelial cells.

METHODSHIF-1 α gene was obtained from human lung cancer cell line A549, which was cultured in hypoxia condition, by RT-PCR. The HIF-1 α gene was subcloned into shuttle vector p Shuttle-CMV-EGFP at KpnI and BamHI sites. After identified with restriction enzymes, plasmid p Shuttle-GFP-HIF was linearized by digestion with restriction endonuclease I-CeuI and I-SceI, and subsequently cotransformed into E.coli DH5a with adenoviral backbone plasmid pAdxsi to make homologous recombination. After linearized by PacI, the homologous recombinant adenovirus plasmid was transfected into 293 cells to package and amplify. The recombinant adenovirus was infected with human umbilical vein endothelial cells (ECV304), and the expression level of HIF-1 α protein was evaluated by ELISA.

RESULTSThe recombinant adenovirus vector containing HIF-1 α gene (pAdxsi-GFP-HIF) was successfully constructed and amplified with titer of 3.38 X 10(10) pfu/mL. The green fluorescence protein was detected under fluorescent microscope in ECV304 at 24h after transfection and with a stronger degree after 48h. The concentration of HIF-1 protein was (48.93 ±3.86)ng/mL in supernatant at 48 h after transfection.

CONCLUSIONA recombinant adenovirus vector pAdxsi-GFP-HIF, encoding human hypoxia inducible factor 1 α gene, has been constructed in vitro and expressed successfully in ECV304 cells.

Adenoviridae ; genetics ; Cells, Cultured ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; Plasmids ; genetics

The present study developed an oral hepatocyte growth factor (HGF) gene therapy strategy for gastric ulcers treatment. An attenuated Salmonella typhimurium that stably expressed high HGF (named as TPH) was constructed, and the antiulcerogenic effect of TPH was evaluated in a rat model of gastric ulcers that created by acetic acid subserosal injection. From day 5 after injection, TPH (1 × 10⁹ cfu), vehicle (TP, 1 × 10⁹ cfu), or sodium bicarbonate (model control) was administered orally every alternate day for three times. Then ulcer size was measured at day 21 after ulcer induction. The ulcer area in TPH-treated group was 10.56 ± 3.30 mm², which was smaller when compared with those in the TP-treated and model control groups (43.47 ± 4.18 and 56.25 ± 6.38 mm², respectively). A higher level of reepithelialization was found in TPH-treated group and the crawling length of gastric epithelial cells was significantly longer than in the other two groups (P < 0.05). The microvessel density in the ulcer granulation tissues of the TPH-treated rats was 39.9 vessels/mm², which was greater than in the TP-treated and model control rats, with a significant statistical difference. These results suggest that TPH treatment significantly accelerates the healing of gastric ulcers via stimulating proliferation of gastric epithelial cells and enhancing angiogenesis on gastric ulcer site.
Acetic Acid ; Animals ; Epithelial Cells ; Genetic Therapy ; Granulation Tissue ; Hepatocyte Growth Factor* ; Hepatocytes* ; Microvessels ; Models, Animal ; Rats* ; Salmonella typhimurium ; Salmonella* ; Sodium Bicarbonate ; Stomach Ulcer* ; Ulcer