Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (2): 214-217.doi: 10.3969/j.issn.1673-8225.2010.02.006

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Expression of articular chondrocytes in rabbits transfected by retroviral vector-mediated transforming growth factor beta 1 gene in vitro 

Lin Shu-zhong1, Liu Jun1, Xiang Chuan2, Wei Xiao-chun2   

  1. 1 Department of Orthopedics, Taiyuan Central Hospital, Taiyuan   030009, Shanxi Province, China; 2 Department of Orthopedics, Second Hospital of Shanxi Medical University, Taiyuan   030001, Shanxi Province, China
  • Online:2010-01-08 Published:2010-01-08
  • Contact: Wei Xiao-chun, Professor, Department of Orthopedics, Second Hospital of Shanxi Medical University, Taiyuan 030001, Shanxi Province, China weixiaochun06@yahoo.com.cn
  • About author:Lin Shu-zhong★, Master, Attending physician, Department of Orthopedics, Taiyuan Central Hospital, Taiyuan 030009, Shanxi Province, China dahao116@yahoo.com.cn
  • Supported by:

    the National Natural Science Foundation of China, No.30500515*; Shanxi Province Natural Science Foundation, No.2006021045*

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Abstract:

BACKGROUND: The functional gene fragments integrate into gene vector, which is then transfected into target cells or joint cavity, through the transgenic target cells continue to secrete a large number of functional gene product, local therapeutic concentrations could be maintained within a long period of time, thus repairing articular cartilage injury.
OBJECTIVE: To transfect rabbit articular chondrocytes using recombinant retroviral vector-mediated transforming growth factor-β1 (TGFβ1) in vitro, and to observe its expression and its effect on biological characters of chondrocytes.
METHODS: Rabbit chondrocytes were isolated by use of trypsin digestion method. Vector was PLNCX2 Hind Ⅲ/Not Ⅰ doubly digested and dephosphorylated, connected with some multiple cloning sites and RFP gene following pDsRed2 double digestion, to build PLNCX2-RFP. TGFβ1 gene was amplified from the PGEMT-TGF and connected with PLNCX2-RFP following double digestion, to build PLNCX2-TGFβ1-RFP. Subsequent to packaging retroviral vector, viral supernatant titer was detected. The cultured and transfected chondrocytes in rabbit knee joint were divided into 3 groups: control group (without any transfection), transfected PLNCX2 group and transfected PLNCX2-TGFβ1-RFP group, continued screening 2 weeks to observe the cellular changes. Cell supernatant transfected stably were collected for detecting the effect of gene transfection on the chondrocytes with NO detection kit, ELISA assay was applied to determine human TGFβ1 expression in cell culture supernatant.

RESULTS AND CONCLUSION: The recombinant gene PLNCX2-TGFβ1-RFP was identified correct sequence by the enzyme digestion sequencing TGFβ1 and RFP, which showed that the eukaryotic expression vector PLNCX2-TGFβ1-RFP had been successfully built as expectation. They were then transfected into packaging cells and cultured, the virus titer was defined as 1×106 CFU. Following stable transfection of cartilage cells, red fluorescence can be observed, proving successful transfection. After continuous screening 2 weeks, the scattered adherent cells formed positive clones, and gradually diffusely integrated, cell clusters appeared with common dual cores, the cells proliferated actively. NO concentration in the transfected PLNCX2-TGFβ1-RFP group was higher than that of transfected PLNCX2 group (P < 0.05), no difference was significant between control group and transfected PLNCX2 group. The control group and the group transfected PLNCX2 showed no TGFβ1 expression, while TGFβ1 concentration was (28.08±3.73) ng/L in the transfected PLNCX2-TGFβ1-RFP group. PLNCX2 retroviral vector-mediated human TGFβ1 can be effectively transfected into rabbit knee joint cartilage cells and obtain stable expression, while the transfected cartilage cells proliferate actively.

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