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
This study investigated the contents and distribution of collagen Ⅴ （Col Ⅴ） in skin lesions of the patients with systemic sclerosis （SSc） and its roles in the pathogenesis. The contents and distribution for α1 ...This study investigated the contents and distribution of collagen Ⅴ （Col Ⅴ） in skin lesions of the patients with systemic sclerosis （SSc） and its roles in the pathogenesis. The contents and distribution for α1 chain of collagen type Ⅰ, Ⅲ and V [α1 （Ⅰ）, α1 （Ⅲ） and α1 （Ⅴ）] in skin lesions of 36 patients with SSc （9 cases of mild fibrosis, 14 moderate, and 13 severe） were detected by using im- munohistochemical SP method. Six cases of normal skin tissues served as controls. The results showed that there was diffuse distribution for three kinds of collagens in dermis. The deep staining α1 （Ⅰ） and α1 （Ⅲ） masses or bands were seen in reticular layer, while α1 （Ⅴ） was distributed more ho- mogeneously. From control to weak, moderate and severe fibrosis stages, α1 （Ⅰ）, α1 （Ⅲ） and α1 （V） showed a gradually increased trend in skin lesions （P〈0.05）. α1 （Ⅴ） was obviously elevated in skin lesions at early stage and persisted in whole fibrotic process and risen in greater contents, while α1 （Ⅰ） and α1 （Ⅲ） were to go higher late and were apparently elevated at moderate and late stages. Com- pared with α1 （Ⅰ）, α1 （Ⅴ） took leading increase at early stage in skin lesions （P〈0.01）, and had more elevated contents than α1 （Ⅲ） at moderate and late stages. The fibrotic changes in dermal reticular layer occurred earlier than those in papillary layer, and the abnormalities of α1 （Ⅴ）/α1 （I） ratio ap- peared before α1 （Ⅲ）/α1 （Ⅰ） ratio. It was concluded that a lot of α1 （Ⅴ） began to deposit in greater contents prior to α1 （Ⅰ） and α1 （Ⅲ） at early stage in SSc and persisted in whole fibrotic process. The changes of α1 （Ⅴ） contents in reticular layer occurred earlier than those in papillary layer, and it sug- gested that the fibrosis in reticular layer appeared earlier.展开更多
High myopia(HM)is the primary cause of blindness,with the microstructural organization and composition of collagenous fibers in the cornea and sclera playing a crucial role in the biomechanical behavior of these tissu...High myopia(HM)is the primary cause of blindness,with the microstructural organization and composition of collagenous fibers in the cornea and sclera playing a crucial role in the biomechanical behavior of these tissues.In a previously reported myopic linkage region,MYP5(17q21-22),a potential candidate gene,LRRC46(c.C235T,p.Q79X),was identified in a large Han Chinese pedigree.LRRC46 is expressed in various eye tissues in humans and mice,including the retina,cornea,and sclera.In subsequent cell experiments,the mutation(c.C235T)decreased the expression of LRRC46 protein in human corneal epithelial cells(HCE-T).Further investigation revealed that Lrrc46^(-/-)mice(KO)exhibited a classical myopia phenotype.The thickness of the cornea and sclera in KO mice became thinner and more pronounced with age,the activity of limbal stem cells decreased,and microstructural changes were observed in the fibroblasts of the sclera and cornea.We performed RNA-seq on scleral and corneal tissues of KO and normal control wild-type(WT)mice,which indicated a significant downregulation of the collagen synthesis-related pathway(extracellular matrix,ECM)in KO mice.Subsequent in vitro studies further indicated that LRRC46,a member of the important LRR protein family,primarily affected the formation of collagens.This study suggested that LRRC46 is a novel candidate gene for HM,influencing collagen protein VⅢ(Col8a1)formation in the eye and gradually altering the biomechanical structure of the cornea and sclera,thereby promoting the occurrence and development of HM.展开更多
Collagen Type V(Col.V)plays an essential role in cell behaviors and has attracted increasing attention in recent years.Highpurity Col.V is needed for evaluating its biological properties.In this research,the enzymatic...Collagen Type V(Col.V)plays an essential role in cell behaviors and has attracted increasing attention in recent years.Highpurity Col.V is needed for evaluating its biological properties.In this research,the enzymatic hydrolysis process was combined with ultrafiltration to purify Col.V from the bovine cornea.The purity of Col.V was determined to be above 90%by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and high-performance liquid chromatography methods.The effect of Col.V on cell behaviors was evaluated.The circular dichroism spectroscopy results demonstrated that the extracted Col.V exhibited a complete triple helix structure.SDS-PAGE suggested that the molecular weight of Col.V was 440 kDa.The self-assembly experiment revealed that the proportion of Col.V in the collagen mixture can affect the Col.I fiber diameter.The cell culture results implied that Col.V can inhibit fibroblasts(L929)proliferation.The L929 showed maximum mobility when the addition of Col.V was 30%.Thus,Col.V has the effect of inhibiting L929 proliferation and promoting migration.The high-purity Col.V provides useful information for further understanding its biological implications.展开更多
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.
文摘This study investigated the contents and distribution of collagen Ⅴ （Col Ⅴ） in skin lesions of the patients with systemic sclerosis （SSc） and its roles in the pathogenesis. The contents and distribution for α1 chain of collagen type Ⅰ, Ⅲ and V [α1 （Ⅰ）, α1 （Ⅲ） and α1 （Ⅴ）] in skin lesions of 36 patients with SSc （9 cases of mild fibrosis, 14 moderate, and 13 severe） were detected by using im- munohistochemical SP method. Six cases of normal skin tissues served as controls. The results showed that there was diffuse distribution for three kinds of collagens in dermis. The deep staining α1 （Ⅰ） and α1 （Ⅲ） masses or bands were seen in reticular layer, while α1 （Ⅴ） was distributed more ho- mogeneously. From control to weak, moderate and severe fibrosis stages, α1 （Ⅰ）, α1 （Ⅲ） and α1 （V） showed a gradually increased trend in skin lesions （P〈0.05）. α1 （Ⅴ） was obviously elevated in skin lesions at early stage and persisted in whole fibrotic process and risen in greater contents, while α1 （Ⅰ） and α1 （Ⅲ） were to go higher late and were apparently elevated at moderate and late stages. Com- pared with α1 （Ⅰ）, α1 （Ⅴ） took leading increase at early stage in skin lesions （P〈0.01）, and had more elevated contents than α1 （Ⅲ） at moderate and late stages. The fibrotic changes in dermal reticular layer occurred earlier than those in papillary layer, and the abnormalities of α1 （Ⅴ）/α1 （I） ratio ap- peared before α1 （Ⅲ）/α1 （Ⅰ） ratio. It was concluded that a lot of α1 （Ⅴ） began to deposit in greater contents prior to α1 （Ⅰ） and α1 （Ⅲ） at early stage in SSc and persisted in whole fibrotic process. The changes of α1 （Ⅴ） contents in reticular layer occurred earlier than those in papillary layer, and it sug- gested that the fibrosis in reticular layer appeared earlier.
基金supported by the National Natural Science Foundation of China(82330030,82271120,82121003,82201234)the CAMS Innovation Fund for Medical Sciences(2019-12 M-5-032,2021LY06)Sichuan Science and Technology Program(2021YFS0369,2022ZYD0131,24YSZH0012,23ZYZYTS0271,TB2023093,2023ZY0059)。
文摘High myopia(HM)is the primary cause of blindness,with the microstructural organization and composition of collagenous fibers in the cornea and sclera playing a crucial role in the biomechanical behavior of these tissues.In a previously reported myopic linkage region,MYP5(17q21-22),a potential candidate gene,LRRC46(c.C235T,p.Q79X),was identified in a large Han Chinese pedigree.LRRC46 is expressed in various eye tissues in humans and mice,including the retina,cornea,and sclera.In subsequent cell experiments,the mutation(c.C235T)decreased the expression of LRRC46 protein in human corneal epithelial cells(HCE-T).Further investigation revealed that Lrrc46^(-/-)mice(KO)exhibited a classical myopia phenotype.The thickness of the cornea and sclera in KO mice became thinner and more pronounced with age,the activity of limbal stem cells decreased,and microstructural changes were observed in the fibroblasts of the sclera and cornea.We performed RNA-seq on scleral and corneal tissues of KO and normal control wild-type(WT)mice,which indicated a significant downregulation of the collagen synthesis-related pathway(extracellular matrix,ECM)in KO mice.Subsequent in vitro studies further indicated that LRRC46,a member of the important LRR protein family,primarily affected the formation of collagens.This study suggested that LRRC46 is a novel candidate gene for HM,influencing collagen protein VⅢ(Col8a1)formation in the eye and gradually altering the biomechanical structure of the cornea and sclera,thereby promoting the occurrence and development of HM.
基金supported by the National Key Technology R&D Programs of China(2021YFC2400800)Science and Technology Program of Guangzhou,China(201803010086)+1 种基金Open Funding Project of the State Key Laboratory of Biochemical Engineering(2021KF-04)Independent Research Project of the State Key Laboratory of Biochemical Engineering(2021ZZ-03).
文摘Collagen Type V(Col.V)plays an essential role in cell behaviors and has attracted increasing attention in recent years.Highpurity Col.V is needed for evaluating its biological properties.In this research,the enzymatic hydrolysis process was combined with ultrafiltration to purify Col.V from the bovine cornea.The purity of Col.V was determined to be above 90%by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and high-performance liquid chromatography methods.The effect of Col.V on cell behaviors was evaluated.The circular dichroism spectroscopy results demonstrated that the extracted Col.V exhibited a complete triple helix structure.SDS-PAGE suggested that the molecular weight of Col.V was 440 kDa.The self-assembly experiment revealed that the proportion of Col.V in the collagen mixture can affect the Col.I fiber diameter.The cell culture results implied that Col.V can inhibit fibroblasts(L929)proliferation.The L929 showed maximum mobility when the addition of Col.V was 30%.Thus,Col.V has the effect of inhibiting L929 proliferation and promoting migration.The high-purity Col.V provides useful information for further understanding its biological implications.
