Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund’ s adjuvant present fibrosis and vasculitis of organs usually affected by systemic sclerosis. In this way, we studied ...Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund’ s adjuvant present fibrosis and vasculitis of organs usually affected by systemic sclerosis. In this way, we studied the fibrillogenesis process to identify possible factors involved in altered remodeling observed in this scleroderma-like model. Additionally, we have done a very preliminary comparison with human skins obtained from scleroderma patients (n = 3). Female New Zealand rabbits (n = 10) were immunized subcutaneously with two doses of 1 mg collagen V (COL V) plus complete Freund’ s adjuvant for a 30-day interval, followed by two additional intramuscular booster immunizations in incomplete Freund’ s adjuvant for a 15-day interval. Animals from control group (n = 10), were only inoculated with complete and incomplete Freund’ s adjuvant given at same conditions of COL V. Histological analysis of skins from animals and patients were done by Masson’ s trichrome staining, and immunofluorescence method to detect collagen fibers and interactions of types I, III and V collagen in the remodeling process. The analysis of animal skins showed collagen fibril deposits in the dermis after 7 days of sensibilization and an increase in these deposits after 75 and 120 days, respectively. Skin thickness and atrophy of sebaceous and sweat glands were progressively more intense in late sacrificed animals and correlated with increased amount of collagen deposition. Surprisingly, type V collagen was overexpressed both in animals and patients, forming dense and atypical collagen fibers in the dermis. We suggest that this anomalous expression of morphologically different type V collagen could justify the remodeling observed in scleroderma plaque.展开更多
We used bovine cornea as starting material, pepsin treatment in acetic acid solution to extract the mixture of type I and V collagens, and salt precipitation and dialysis to purify and isolate each type of the collage...We used bovine cornea as starting material, pepsin treatment in acetic acid solution to extract the mixture of type I and V collagens, and salt precipitation and dialysis to purify and isolate each type of the collagens. The preparation was analyzed using sodium dodecyl sulphate polyacrylamide gel electrophoresis. 2-mercaptoethanol used as reducing agent cut off the disulfide bonds, which was utilized to analyze the structure of disulfide bonds involved between α chains in some types of collagens. At the same time, we discovered that the structure of disulfide bonds among α chains potentially existed in the type V collagen prepared from the pepsin-treatment extraction at 4℃. Through quantitative analysis, we obtained that, compared with those pepsin-treated at 4℃, the relative molecular weights of α1 （V） and α 2 （V） subunits pepsin-treated at room temperature decreased by 4.6% and 6.0%, respectively. It is concluded that type V collagen can be prepared from bovine coruea by use of pepsin treatment, salt precipitation and dialysis. The interchain and/or intermolecular disulfide bonds potentially lie near the edges of termini of type V collagen molecules existing in extracellular matrix, and there are few of the intermolecular and/or intramolecular crosslinks formed by lysine or hydroxylysine or histidine residues in type V collagen.展开更多
文摘Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund’ s adjuvant present fibrosis and vasculitis of organs usually affected by systemic sclerosis. In this way, we studied the fibrillogenesis process to identify possible factors involved in altered remodeling observed in this scleroderma-like model. Additionally, we have done a very preliminary comparison with human skins obtained from scleroderma patients (n = 3). Female New Zealand rabbits (n = 10) were immunized subcutaneously with two doses of 1 mg collagen V (COL V) plus complete Freund’ s adjuvant for a 30-day interval, followed by two additional intramuscular booster immunizations in incomplete Freund’ s adjuvant for a 15-day interval. Animals from control group (n = 10), were only inoculated with complete and incomplete Freund’ s adjuvant given at same conditions of COL V. Histological analysis of skins from animals and patients were done by Masson’ s trichrome staining, and immunofluorescence method to detect collagen fibers and interactions of types I, III and V collagen in the remodeling process. The analysis of animal skins showed collagen fibril deposits in the dermis after 7 days of sensibilization and an increase in these deposits after 75 and 120 days, respectively. Skin thickness and atrophy of sebaceous and sweat glands were progressively more intense in late sacrificed animals and correlated with increased amount of collagen deposition. Surprisingly, type V collagen was overexpressed both in animals and patients, forming dense and atypical collagen fibers in the dermis. We suggest that this anomalous expression of morphologically different type V collagen could justify the remodeling observed in scleroderma plaque.
文摘We used bovine cornea as starting material, pepsin treatment in acetic acid solution to extract the mixture of type I and V collagens, and salt precipitation and dialysis to purify and isolate each type of the collagens. The preparation was analyzed using sodium dodecyl sulphate polyacrylamide gel electrophoresis. 2-mercaptoethanol used as reducing agent cut off the disulfide bonds, which was utilized to analyze the structure of disulfide bonds involved between α chains in some types of collagens. At the same time, we discovered that the structure of disulfide bonds among α chains potentially existed in the type V collagen prepared from the pepsin-treatment extraction at 4℃. Through quantitative analysis, we obtained that, compared with those pepsin-treated at 4℃, the relative molecular weights of α1 （V） and α 2 （V） subunits pepsin-treated at room temperature decreased by 4.6% and 6.0%, respectively. It is concluded that type V collagen can be prepared from bovine coruea by use of pepsin treatment, salt precipitation and dialysis. The interchain and/or intermolecular disulfide bonds potentially lie near the edges of termini of type V collagen molecules existing in extracellular matrix, and there are few of the intermolecular and/or intramolecular crosslinks formed by lysine or hydroxylysine or histidine residues in type V collagen.
