[Exogenous gene expression in vitro and in vivo in bone marrow mesenchymal stem cells modified by hPDGF-A and hBD(2)].
The objective of this study was to investigate the expression of exogenous hPDGF-A and hBD(2) in gene-modified bone marrow mesenchymal stem cells (BM-MSCs) in vitro and in vivo. Recombinant adenovirus vector expressing hPDGF-A/hBD(2) genes was constructed and packaged into virion. Primary isolated and cultured BM-MSCs were transfected by using hPDGF-A hBD(2), then the expressions of exogenous hPDGF-A/hBD(2) were detected by immunocytochemical staining in vitro. The conditioned medium (serum-free cultured supernatant of BM-MSCs transfected with recombinant adenovirus) collected from gene-modified BM-MSCs was applied to scratch wound on monolayer cells of multipotential cell line 10T1/2 in order to confirm the stimulative effect of hPDGF-A on cell migration. Gene-modified BM-MSCs were topically transplanted on wound of rats with radiation and skin excision combined injury. The distribution of BM-MSCs and expression of hPDGF-A/hBD(2) on the wound was observed by fluorescent microscopy and immunohistochemical staining respectively. The results indicated that the rat BM-MSCs transfected with recombinant adenovirus could express the EGFP in vitro. The immunofluorescent cytochemistry assay showed that the gene-modified BM-MSCs expressed the hPDGF-A and hBD(2). The scratch test confirmed that the percentage of healing area of wound in cultured supernatant group of gene-modified BM-MSCs was significant higher than that in control group on 8, 12, 24 and 48 hours (p < 0.05). The fluorescence microscopy of exogenous gene-modified BM-MSCs transplanted on wound revealed that the gene-modified BM-MSCs could higher express exogenous genes of EGFP at least within 2 weeks. The immunohistochemistry staining of wound indicated that the expression of exogenous genes began from day 3, reached to peak on day 7, and still visible on day 21 even though the expression became weak because of the possible dilution of the exogenous genes during cell division. It is concluded that efficient expression of exogenous hPDGF-A/hBD(2) in gene-modified BM-MSCs are demonstrated both in vitro and in vivo, which suggests that the molecular mechanism underlying chronic wound-healing accelerated by the strategy combining cell therapy with gene therapy.
Hao
Lei
L
Department of Radiation Medicine, Third Military Medical University, Chongqing, China.
Sun
Hui-Qin
HQ
Guo
Xiao Shu
XS
Yan
Guo-He
GH
Luo
Cheng-Ji
CJ
Cheng
Tian-Min
TM
Su
Yong-Ping
YP
Zou
Zhong-Min
ZM
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D023083:beta-Defensins / Q000235:genetics*
D010982:Platelet-Derived Growth Factor; D023083:beta-Defensins