AIM To develop methods of articular cartilage preparation for X-ray-electron probe microanalysis and to study its elements content in experimental osteoarthrosis.METHODS Twenty dogs aged 2-8 years were divided in rese...AIM To develop methods of articular cartilage preparation for X-ray-electron probe microanalysis and to study its elements content in experimental osteoarthrosis.METHODS Twenty dogs aged 2-8 years were divided in research(aged 2 years, induction of osteoarthrosis-IOA) and intact group. Intact group included three subgroups(aged 2, 5 and 8 years). Samples of cartilage after araldite saturation and pouring were partially cut into semithin sections stained with methylene blue and with methylene blue-basic fuchsin. Their smooth surfaces were investigated by X-ray-electron probe microanalysis. Spatial distribution of sulfur, calcium and phosphorus and their concentrations(weight %) were investigated.RESULTS X-ray electron probe microanalysis revealed non-uniformsulfur distribution in cartilage of intact animals: Its content increases from superficial zone to deep one, this regularity was preserved in animals with IOA. Differences of IOA with spontaneous chondropathy were revealed. Spontaneous aging was characterized by calcium and phosphorus storage in deep and calcified zones and compensatory increase of sulfated glycosaminoglycans in intermediate and deep cartilage zones as evidenced by the metachromatic reaction and microanalysis data. Unlike spontaneous chondropathy connected with aging in experimentally stimulated osteoarthrosis more intensive storage of calcium but minor phosphorus in intermediate zone were marked. In IOA the calcified cartilage thinning and osteoclastic resorption are apparent with few changes of elements composition; the only difference from control is minority phosphorus content.CONCLUSION The obtained results demonstrate specific tricks of X-ray electron probe microanalysis and its possibility in the research of mechanisms of articular cartilage alterations in osteoarthrosis.展开更多
OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients wer...OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.展开更多
基金Supported by The RF Ministry of Health within government-mandated program for FSBI Russian Ilizarov Scientific Center "Restorative Traumatology and Orthopaedics"(RISC"RTO") for Scientific Research,No.01201155770
文摘AIM To develop methods of articular cartilage preparation for X-ray-electron probe microanalysis and to study its elements content in experimental osteoarthrosis.METHODS Twenty dogs aged 2-8 years were divided in research(aged 2 years, induction of osteoarthrosis-IOA) and intact group. Intact group included three subgroups(aged 2, 5 and 8 years). Samples of cartilage after araldite saturation and pouring were partially cut into semithin sections stained with methylene blue and with methylene blue-basic fuchsin. Their smooth surfaces were investigated by X-ray-electron probe microanalysis. Spatial distribution of sulfur, calcium and phosphorus and their concentrations(weight %) were investigated.RESULTS X-ray electron probe microanalysis revealed non-uniformsulfur distribution in cartilage of intact animals: Its content increases from superficial zone to deep one, this regularity was preserved in animals with IOA. Differences of IOA with spontaneous chondropathy were revealed. Spontaneous aging was characterized by calcium and phosphorus storage in deep and calcified zones and compensatory increase of sulfated glycosaminoglycans in intermediate and deep cartilage zones as evidenced by the metachromatic reaction and microanalysis data. Unlike spontaneous chondropathy connected with aging in experimentally stimulated osteoarthrosis more intensive storage of calcium but minor phosphorus in intermediate zone were marked. In IOA the calcified cartilage thinning and osteoclastic resorption are apparent with few changes of elements composition; the only difference from control is minority phosphorus content.CONCLUSION The obtained results demonstrate specific tricks of X-ray electron probe microanalysis and its possibility in the research of mechanisms of articular cartilage alterations in osteoarthrosis.
文摘OBJECTIVE: To observe the distribution of copper in the subcellular structure for the understanding of primary pathogenesis of hepatolenticular degeneration (HLD). METHODS: Skin fibroblasts taken from HLD patients were cultured as an in vitro model of HLD, and the control cells taken from healthy volunteers were clutured in the same way. The distribution of copper inside and outside of lysosomes in fibroblasts was detected by quantitative electron probe X-ray microanalysis. The relationship between the subcellular location of copper and the genotype of the patients, and relationship between the distribution of copper and the course of the disease were analyzed. RESULTS: The content of Cu^(2+) inside lysosomes of HLD cells (14.6±2.1 mmol/kg) and of heterozygote cells (11.6±0.6 mmol/kg) was higher than that of normal cells (4.5±1.2 mmol/kg) (P<0.01). The content of Cu^(2+) outside lysosomes of HLD cells (17.5±4.2 mmol/kg) and of heterozygote cells (12.0±0.9 mmol/kg) was higher than that of normal cells (4.7±1.2 mmol/kg) (P<0.01). The distribution of copper in the subcellular structure was correlated with disease courses of HLD patients. With the progression of the disease, more copper was deposited in lysosomes (r=0.85, P<0.01). The content of copper in the diffused cytoplasmic compartment in HLD cells was correlated with that of sulfur (r=0.86, P<0.05), but not in heterozygote and normal cells. CONCLUSIONS: In the early stage of HLD, copper is accumulated outside lysosome, which is paralleled with increase of metallothionein-like proteins (copper and sulfur-binding proteins). With the development of the disease, more copper is deposited inside lysosome than outside lysosome. We conclude that the up-regulation expression of copper and sulfur-binding proteins and copper accumulation in lysosomes may play an important role in lowering the ATP7B gene mutation-induced toxic effects of free copper on the cell.
