Recombinant adeno-associated virus-mediated chemokine like factor 1 gene transferring modulates the proliferative activities and osteogenic potentials of human hip ligaments of ankylosing spondylitis patients

Abstract

Objective To investigate the effects of chemokine like factor 1(CKLF1) gene on the proliferative activities and osteogenic potentials of hip ligaments of ankylosing spondylitis (AS) in situ and in vitro. Methods Normal and AS hip ligament specimens were collected from 6 patients with femoral neck fracture and 4 AS patients with severe hip deformities. Ligament specimens were exposed to type Ⅱ colla-genase and obtained a single cell suspension, while phase contrast microscopy and anti-vimentin immuno-fluorescence staining (IFC) were applied to observe the cells. The specimens and fibroblasts were divided and cultured in situ and in vitro respectively, and the recombinant adeno-associated virus (rAAV)-lacZ (E. coli beta-galactosidase gene)and rAAV-hCKLF1 (human CKLF1 cDNA cloned in rAAV-lacZ in place of lacZ) were transduced for 21 days. Cell proliferation (cellularity), secretion of pro-inflammatory cytokines, expression of CKLF1 and CCR4 genes were detected by the water-soluble tetrazolium (WST-1) assay and Hoechst 33258 test (DNA content), enzyme-linked immunosorbent assay (ELISA), IFC test and fluorescent quantitative reverse transcription polymerase chain reaction (RT-PCR), respectively. Statistical analysis significance was conducted using the Student's t test and one-way analysis of variance (ANOVA) (LSD) test where appropriate. Results The second passage of normal and AS cells were positive for anti-vimentin, indicating that the cells were fibroblasts. After transducing with rAAV-hCKLF1 for 21 days, cellularity, WST-1 and Hoechst 33258 assays illustrated that CKLF1 gene transfer promoted cell proliferation (compared with the non-viral transduction and lacZ groups, F=6.98, 64.32, 115.91, P<0.05 or P<0.01). Overexpression of CKLF1 gene enhanced the secretion of pro-inflammatory cytokines (interleukin-6 and tumor necrosis factor-alpha) and the expression of bone- specific extracellular matrix proteins (osteopontin and osteocalcin) (F=34.57, 8.89, P<0.05 or P<0.01). Similar results were observed in fluorescent quantitative RT-PCR test. Conclusion Overexpression of CKLF1 promotes the proliferation of fibroblasts, the secretion of pro-inflammatory cytokines and the expression of osteogenic related target genes, suggesting that CKLF1 might be involved in the pathological ossification of AS. Key words: Spondylitis, ankylosing; Ossification, heterotopic; Fibroblasts; Transfection; Chemokine like factor 1