Construction of eukaryotic expression plasmid human transforming growth factor beta3 and its transfection into precartilaginous stem cells.

OBJECTIVE: To obtain seed cells for cartilage repair through constructing recombinant human transforming growth factor beta3 vector (hTGF-beta3) and transfecting it into rat's precartilaginous stem cells (PSCs). METHODS: Gene engineering technique was introduced to construct eukaryotic expression plasmid pcDNA3.1 (+)-hTGF-beta3. PSCs of rats were isolated and purified with method of immunomagnetic microbeads. Then PSCs were cotransfected with plasmid hTGF-beta3 and pcDNA3.1 (+)-enhanced green fluorescence protein (EGFP) by liner polyethyleneimine (PEI). And 48 hours later the transient expression of EGFP was observed under a fluorescence microscope, and the expression of hTGF-beta3 was detected with reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). RESULTS: The sequences of the recombinants were consistent with that from Genebank. Cotransfection of EGFP provided fast visual confirmation of successful transduction. The hTGF-beta3 mRNA and protein expression could be detected by RT-PCR and ELISA. CONCLUSIONS: The recombinant plasmid is correctly constructed and successfully transfected into rat's PSCs, which is an important step to treat epiphyseal injury or other osteo-cartilage diseases with transgenic therapy.

Effect of cefazolin loaded bone matrix gelatin on repairing large segmental bone defects and preventing infection / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To explore the possibility of repairing long segmental bone defects and preventing infection with cefazolin loaded bone matrix gelatin (C-BMG).</p><p><b>METHODS</b>C-BMG was made from putting cefazolin into BMG by vacuum absorption and lyophilization techniques. The sustaining period of effective drug concentration in vitro and in vivo was detected. The time of inhibiting bacteria, and the drug concentration in local tissues (bone and muscle) and plasma after implantation of C-BMG were examined by high performance liquid chromatography.</p><p><b>RESULTS</b>The effective inhibition time to staphylococcus aureus of C-BMG was 22 days in vitro; while 14 days in vivo. The cefazolin concentration in local tissues was higher in early stage, and later it kept a stable and low drug release. C-BMG showed an excellent ability to repair segmental long bone defects.</p><p><b>CONCLUSIONS</b>C-BMG can gradually release cefazolin with effective drug concentration and has excellent ability to repair segmental bone defects. It can be used to repair segmental long bone defects and prevent infection after operation.</p>

Effect of rabbit adipose-derived stem cells transfected by adenoviral vector mediated hTGF-?_1 gene on chondrocyte differentiation in vitro / 中华风湿病学杂志

Objective To investigate the proliferation of rabbit adipose-derived stem cell(ADSCs) transfected by adenoviral vector mediated hTGF-?_1 gene and its chondrocyte differentiation potential.Methods The Ad-hTGF-?_1 plasmid vetor which had the hTGF-?_1 gene was developed and transfected the ADSCs.The experimental group was the hTGF-?_1 transfected group.The cells enclosed by alginate were cultured in com- plete chondrogenie medium(CMM).The morphology of the cells were observed,and RT-PCR was used to measure hTGF-?_1 and collagenⅡexpression,at the same time western blot and immunohistochemistry were applied to detect the expression of collagenⅡin ADSCs before and after transfected with hTGF-?_1 gene. Results The hTGF-?_1 transfected ADSCs became the polygon and it proliferated well.The RT-PCR result of hTGF-?_1 on the transfected group was better than the control after transtected for 7 day and 21 day.The dif- ference between the two groups was significant(P