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 / 中华风湿病学杂志

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.