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
The human cornea is exposed directly to particulate matter （PM） in polluted air. This exposure can cause eye discomfort and corneal injury. Ultrafine PM （diameter ~100 nm） is thought to be particularly harmful to ...The human cornea is exposed directly to particulate matter （PM） in polluted air. This exposure can cause eye discomfort and corneal injury. Ultrafine PM （diameter ~100 nm） is thought to be particularly harmful to health, but there is limited research investigating its toxicity to the eye. In this study, we evaluated toxiciW differences among 30-, 40-, 100- and 150-nm silicon dioxide nanoparticles （Si02 NPs） on the cornea. A 24-hour in vitro exposure of primary human corneal epithelial cells （hCECs） to ultrafine （30 and 40 nm） SiO2 NPs produced toxicity, as evidenced by cell membrane damage, reduced cell viability, increased cell death and mitochondrial dysfunction. In vivo exposure to the same nanoparticles produced observable corneal injury. These effects were more severe with ultrafine than with fine （100 and 150 nm） Si02 NPs. Common antioxidant compounds, e.g., glutathione, did not protect the cornea from SiO2 NP-induced damage. However, foetal bovine serum （FBS） did significantly reduce toxicity, likely by forming a protective protein corona around the nanoparticles. This finding suggests that FBS （or its derivatives） may be a useful clinical therapy for corneal toxicity caused by ultrafine particulates.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.
基金supported by the National Key R&D program of China(2018YFA0107302,2016YFA0201600)the National Natural Science Foundation of China(81570890)the Foundation of Southwest Hospital(SWH2016LHYS-03)
文摘The human cornea is exposed directly to particulate matter （PM） in polluted air. This exposure can cause eye discomfort and corneal injury. Ultrafine PM （diameter ~100 nm） is thought to be particularly harmful to health, but there is limited research investigating its toxicity to the eye. In this study, we evaluated toxiciW differences among 30-, 40-, 100- and 150-nm silicon dioxide nanoparticles （Si02 NPs） on the cornea. A 24-hour in vitro exposure of primary human corneal epithelial cells （hCECs） to ultrafine （30 and 40 nm） SiO2 NPs produced toxicity, as evidenced by cell membrane damage, reduced cell viability, increased cell death and mitochondrial dysfunction. In vivo exposure to the same nanoparticles produced observable corneal injury. These effects were more severe with ultrafine than with fine （100 and 150 nm） Si02 NPs. Common antioxidant compounds, e.g., glutathione, did not protect the cornea from SiO2 NP-induced damage. However, foetal bovine serum （FBS） did significantly reduce toxicity, likely by forming a protective protein corona around the nanoparticles. This finding suggests that FBS （or its derivatives） may be a useful clinical therapy for corneal toxicity caused by ultrafine particulates.
