Expression of articular chondrocytes in rabbits transfected by retroviral vector-mediated transforming growth factor bets 1 gene in vitro
10.3969/j.issn.1673-8225.2010.02.006
- VernacularTitle:反转录病毒载体介导转化生长因子β_1基因体外转染兔膝关节软骨细胞的表达
- Author:
Shuzhong LIN
;
Jun LIU
;
Chuan XIANG
;
Xiaochun WEI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2010;14(2):214-217
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: The functional gene fragments integrate into gene vector, which is then transfacted 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-βl (TGFβ_1) in vitro, and to observe its expression and its effect on biological characters of chondrocytes. METHODS: Rabbit chondrocytas were isolated by use of trypsin digestion method. Vector was PLNCX_2 Hind Ⅲ/Not Ⅰ doubly digested and dephosphorylated, connected with some multiple cloning sites and RFP gene following pDsRed_2 double digestion, to build PLNCX_2-RFP. TGFβ_1 gene was amplified from the PGEMT-TGF and connected with PLNCX_2-RFP following double digestion, to build PLNCX_2-TGFβ_1-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 PLNCX_2 group and transfected PLNCX_2-TGFβ_1-RFP group, continued screening 2 weeks to observe the cellular changes. Cell supematant 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 TGFβ_1 expression in cell culture supernatant. RESULTS AND CONCLUSION: The recombinant gene PLNCX_2-TGFβ_1-RFP was identified correct sequence by the enzyme digestion sequencing TGFβ_1 and RFP, which showed that the eukaryotic expression vector PLNCX_2-TGFβ_1-RFP had been successfully built as expectation. They were then transfected into packaging calls and cultured, the virus titer was defined as 1×10~6 CFU. Following stable transfection of cartilage cells, red fluorescence can be observed, proving successful transfection. After continuous screening 2 weeks, the scattered adherent calls formed positive clones, and gradually diffusely integrated, cell clusters appeared with common dual cores, the calls proliferated actively. NO concentration in the transfected PLNCX_2-TGFβ_1-RFP group was higher than that of transfected PLNCX_2 group (P < 0.05), no difference was significant between control group and transfected PLNCX_2 group. The control group and the group transfected PLNCX_2 showed no TGFβ_1 expression, while TGFβ_1 concentration was (28.08±3.73) ng/L in the transfected PLNCX_2-TGFβ_1-RFP group. PLNCX_2 ratroviral vector-mediated human TGFβ_1 can be effectively transfected into rabbit knee joint cartilage cells and obtain stable expression, while the transfected cartilage calls proliferate actively.
