To investigate the effect of excessive fluoride ingestion (EFI) on the differential expression of collagen phenotypes and chondrocyte differentiation in cartilage, 60 Sprague-Dawley rats, 4-6 weeks old, were divided into three groups and exposed to O (control), 50, and 100 ppm fluoride (from NaF) in drinking water for 3 to 5 months. Correlating with the increased F in the serum and bone in the two groups exposed to EFI, chondrocytes in the proliferative and hypertrophic zones of growth plate cartilage (GPC) and rib cartilage (RC) were retained and formed a mixture tissue of cartilage peninsulas or islands with ossified islands in the deep zone of GPC and RC. Staining reactions for types II and X collagen were increased in the over-thickened region of GPC and were decreased in RC. Type X collagen staining reactions were also increased in the upper and middle zones in articular cartilage (AC) of tibia in the higher fluoride (EFI-II) group. Abnormal collagen expression in rats of the EFI groups resulted from abnormal differentiation of chondrocytes in proliferative and hypertrophic zones in GPC and RC and from degenerative changes in AC of rats in the EFI groups. Four types of abnormal chondrocyte differentiation in hyaline cartilage of rats with skeletal fluorosis (SF) suggested two pathogenic mechanisms for early stage of SF: osteomalacia-like changes in GPC and RC coupled with delayed mineralization and degenerative changes in AC.

The purpose of the current study was to investigate the abnormal expression of Col X, PTHrP, TGF-β, bFGF, and VEGF in cartilage from patients with Kashin-Beck disease (KBD) to understand the pathogenesis of chondronecrosis in KBD. Articular cartilage and growth plate cartilage collected were divided into four groups: control children (8 samples, 5 cases), KBD children (19 samples, 9 cases), control adults (8 samples, 6 cases), and KBD adults (16 samples, 15 cases). The presence of PTHrP, TGF-β1, bFGF, VEGF, and collagen X in articular cartilage and in growth plate cartilage was analyzed by immunohistochemistry. Articular cartilage and growth plate were each divided in three zones, and the rate of positive cells was counted by light microscope for cytoplasmic and pericellular staining. Results showed that (1) in KBD children, Col X expression was lower in the deep zone of growth plate cartilage than in normal children; in articular cartilage of KBD adults, however, collagen X expression was higher in the middle zone compared to the controls; (2) staining for bFGF, PTHrP, TGF-β1, and VEGF in KBD adult patients was prominent in the chondrocyte clusters and the eroded surface of articular cartilage, and the percentage of chondrocyte staining was significantly higher than in control samples (t=3.64-10.34, df=12 for children and 19 for adults, P=0.002-0.0001); and (3) the enhanced PTHrP, TGF-β1, and VEGF staining in the deep and middle zone of KBD articular cartilage correlated with the high incidence of chondronecrosis in the middle zone (48.5% ± 10.2%) and deep zone (70.6%±27.0%) of adult KBD cartilage. In conclusion, Col X expression was reduced in areas of chondrocyte necrosis in the deep zone of KBD articular cartilage, indicating changes in terminal chondrocyte differentiation. PTHrP, TGF-β1, and VEGF expression was significantly altered and indicated degenerative changes in KBD cartilage, which initially resemble those occurring in osteoarthritis, but lead eventually to chondronecrosis, an event not observed in osteoarthritis.

目的 探讨利用荧光激活细胞分选技术获得有效重组逆转录病毒包装细胞系的方法。方法 用脂质体介导pLEGFP转染PA317细胞用荧光显微镜观察转染结果：依据EGFP荧光利用流式细胞仪的分选技术获得多克隆和单克隆源性的包装细胞。并利用PCR、RT-PCR对其鉴定；以NIH3T3为靶细胞。对其滴度进行测定。结果 荧光显微镜显示用脂质体介导的方法成功的转染PA317细胞在4次连续流式细胞仪分选后得到了稳定表达的多克隆和单克隆源性的包装细胞。PCR和RT-PCR分别从多克隆和单克隆源性的包装细胞细胞基因组DNA以及多克隆和部分（6/8）单克隆源性的包装细胞培养上清中的重组逆转录病毒RNA中扩增出了插入的EGFP片段。滴度测定显示得到的包装细胞能够产生有效的病毒滴度。结论 荧光激活细胞分选技术是获得有效病毒滴度的包装细胞的有效方法。