Induction of Chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was const...

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Bibliographic Details
Published in:Journal of Huazhong University of Science and Technology. Medical sciences 2013-08, Vol.33 (4), p.536-542
Main Authors: Zheng, Dong, Dan, Yang, Huang, Peng, Xia, Tian, Yang, Shu-hua, Xu, Wei-hua, Yang, Cao, Liu, Guo-hui, Liu, Xian-zhe, Feng, Yong
Format: Article
Language:English
Subjects:
Adipose Tissue - metabolism
Animals
Chondrogenesis - genetics
Female
Male
Medicine
Medicine & Public Health
Rabbits
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Stem Cells - metabolism
Transforming Growth Factor beta3 - genetics
Transforming Growth Factor beta3 - metabolism
免疫组化染色
基因转染
基质金属蛋白酶
干细胞培养
脂肪干细胞
诱导分化
软骨基质
重组融合蛋白
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Summary:Summary： A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells （ADSCs）. The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups： experimental group （MMP group）, negative control group （no MMP） and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen （Col II） was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
ISSN:1672-0733
1993-1352
DOI:10.1007/s11596-013-1155-2