Objective To construct recombinant adenovirus vector containing human transforming growth factor beta 3 (TGF-β3), which was transfected into marrow mesenchymal stem cells(MSCs) and to observe its expression. Methods The cDNA TGF-β3 was intergraded into the shuttle vector of pAdTrack-CMV and recombinated with adenovirus skeleton vector pAdEasy-1 by homologous recombination. Then the product was transfected into package cell HEK293 by lipofedtamine and the recombinant adenovirus expressing the TGF-β3genewas generated. The rabbit’s MSCs were isolated, cultivated, purified, and then transfected with recombinant adenovirus containing the TGF-β3 gene. The green fluorescence protein expression was observed after 10 days, and the TGF-β3 expression was observed in MSCs transfected by recombinated adenovirus with TGF-β3 gene after 4 days. Results PCR showed that TGF-β3 cDNA was inserted into the recombinantadenoviral plasmid. The recombinant virus vectors with TGF-β3 gene were collected by the packaging HEK293 cells. The fusion rate of MSCs was 70%-80% with an intensive adhesion and uninform shape after the cultured 10th day. Fluorescent microscopy and immunocytochemistry demonstrated that TGF-β3 was expressed in MSCs. Conclusion Successful construction of human TGF-β3 recombinant adenovirus and its expression in MSCs provide a basis of research for the gene therapy of wound healing.
【Abstract】 Objective To investigate the effects on forming of hypertrophic scar after BMSCs infected with adenovirus carrying TGF-β3c2s2 were transplanted into the wound of animal scar model. Methods The third passage of rabbit’ s BMSCs were infected with 150 mutiple infection, and were cultured 24 hours. The concentration of the BMSCs infected with recombinant adenovirus containing the TGF-β3c2s2 gene was 1×105cell/mL. The purified and evaporated recombinant adenovirus grains containing the TGF-β3c2s2 gene were diluted by DMEM/F12 (without FBS) to 1×108 pfu/mL. The animal scar model of the standard Japanese big ear rabbit was establ ished. Eighty wounds were generated on the gastroside of ear and were randomized to 4 groups in each rabbit, which were divided into 3 control groups (A: control, B: Ad-TGF-β3c2s2, C: BMSCs) and 1 experimental group (D: BMSCs/Ad-TGF-β3c2s2). Then the wounds were tranplanted with cells. On 45 days and 90 days after wounded, thicknessand hardness of scars were measured with color ultrasound diagnostic unit and especial measurement for skin and scar hardness. On 21, 45 and 90 days, three specimens were harvested respectively for further histological study. Results The wound of groups A, B, C gradually formed the different degree scars after epithel ial ization. The hyperplasty of scars reached peak on 45 days after wounded and lasted about 90 days. There was no prominent scar formed in group D during the whole observed procedure. Thickness and hardness of scar of group D and group E were approximate on 45 days and 90 days. Thickness and hardness of scar of groups A, B and C were lower than those of group D (P lt; 0.01), and group B showed more lower than group A and group C (P lt; 0.01). Disorder structure and overlapping arrangement, enlargement collagen fibers were showed in the HE histological sections of the scars of groups A, C. The structure of the scars of groups B, C were similar to Group E. The constitutionsof groups A, B, C, D on 90 days resembled to each one on 45 days. In section of immunohistochemistry after wounded on21 days and 45 days, positive expressions of BrdU in nucleus of Groups C, D were observed. Negative expressions of BrdU in Groups A, B, E were showed. Conclusion BMSCs with Ad-TGF-β3c2s2 gene transplanted into wound could inhibit the forming of hypertrophic scar.