胶原蛋白作为细胞外基质的主要成分与细胞的增殖、分化等密切相关。为探究制备具有促进皮肤成纤维细胞增殖功能的马鹿茸胶原蛋白肽的适宜工艺条件,本研究以马鹿茸为原料,采用Alcalase和胰蛋白酶进行酶解,以水解度、多肽得率和正常人皮...胶原蛋白作为细胞外基质的主要成分与细胞的增殖、分化等密切相关。为探究制备具有促进皮肤成纤维细胞增殖功能的马鹿茸胶原蛋白肽的适宜工艺条件,本研究以马鹿茸为原料,采用Alcalase和胰蛋白酶进行酶解,以水解度、多肽得率和正常人皮肤成纤维细胞(NHDF细胞)增殖率为目标值进行单因素实验和正交实验,确定适宜的工艺条件。实验结果表明,马鹿茸胶原蛋白肽适宜制备条件为:先在底物浓度1%,酶底比2%,p H 9.0,温度60℃条件下采用Alcalase进行酶解;再在底物浓度1%,酶底比6%,p H 8.0,温度42℃条件下采用胰蛋白酶酶解,酶解产物最终水解度为21.7%,多肽得率是86%,细胞增殖率为22.75%。Alcalase-胰蛋白酶双酶分步水解马鹿茸胶原蛋白可获得促进正常人皮肤成纤维细胞增殖率较高的胶原蛋白肽。展开更多
目的检测新疆5个不同产地塔里木亚种马鹿鹿茸质量性状、多肽(42,26,15 k Da)含量及其对小鼠前成骨细胞增殖和活性的影响。方法对比分析不同产地鹿茸性状(直径、干鲜比、茸长、眉枝、水分、总灰分和浸出物);采用BCA蛋白定量试剂盒测定鹿...目的检测新疆5个不同产地塔里木亚种马鹿鹿茸质量性状、多肽(42,26,15 k Da)含量及其对小鼠前成骨细胞增殖和活性的影响。方法对比分析不同产地鹿茸性状(直径、干鲜比、茸长、眉枝、水分、总灰分和浸出物);采用BCA蛋白定量试剂盒测定鹿茸多肽中的蛋白含量并作聚类分析,采用SDS-PAGE电泳法鉴别鹿茸多肽类型;建立小鼠成骨细胞模型,采用MTT及ELISA法分析其对细胞增殖和功能的作用。结果不同产地塔里木鹿茸药材中鹿茸质量性状存在差异,塔里木河沙雅县鹿茸质量性状较好,鹿茸多肽含量高,显著提高了小鼠成骨细胞增殖率40.74%及碱性磷酸酶活性。结论新疆沙雅地区塔里木亚种马鹿鹿茸活性较好。本实验为建立塔里木马鹿亚种鹿茸质量标准的提升提供了数据支撑。展开更多
Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and re- versed-phase high-performance liquid chromatography（RP-HPLC） were used to isolate and purify the natural poly- pept...Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and re- versed-phase high-performance liquid chromatography（RP-HPLC） were used to isolate and purify the natural poly- peptide from velvet antler（nVAP） of Cervus elaphus（C, elaphus）, which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4--50 gg/mL, nVAP promotes mitosis in epidermal ceils, chondrocytes and NIH3T3 fibroblasts pri- marily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide（sVAP） according to its primary struc- ture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectro- metry（MALDI-TOF-MS） was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAE Bioactivity test shows that at a dose of 5--40 μg/mL, sVAP promotes the pro- liferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon（C, nippon） and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus, sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler pol展开更多
文摘胶原蛋白作为细胞外基质的主要成分与细胞的增殖、分化等密切相关。为探究制备具有促进皮肤成纤维细胞增殖功能的马鹿茸胶原蛋白肽的适宜工艺条件,本研究以马鹿茸为原料,采用Alcalase和胰蛋白酶进行酶解,以水解度、多肽得率和正常人皮肤成纤维细胞(NHDF细胞)增殖率为目标值进行单因素实验和正交实验,确定适宜的工艺条件。实验结果表明,马鹿茸胶原蛋白肽适宜制备条件为:先在底物浓度1%,酶底比2%,p H 9.0,温度60℃条件下采用Alcalase进行酶解;再在底物浓度1%,酶底比6%,p H 8.0,温度42℃条件下采用胰蛋白酶酶解,酶解产物最终水解度为21.7%,多肽得率是86%,细胞增殖率为22.75%。Alcalase-胰蛋白酶双酶分步水解马鹿茸胶原蛋白可获得促进正常人皮肤成纤维细胞增殖率较高的胶原蛋白肽。
文摘目的检测新疆5个不同产地塔里木亚种马鹿鹿茸质量性状、多肽(42,26,15 k Da)含量及其对小鼠前成骨细胞增殖和活性的影响。方法对比分析不同产地鹿茸性状(直径、干鲜比、茸长、眉枝、水分、总灰分和浸出物);采用BCA蛋白定量试剂盒测定鹿茸多肽中的蛋白含量并作聚类分析,采用SDS-PAGE电泳法鉴别鹿茸多肽类型;建立小鼠成骨细胞模型,采用MTT及ELISA法分析其对细胞增殖和功能的作用。结果不同产地塔里木鹿茸药材中鹿茸质量性状存在差异,塔里木河沙雅县鹿茸质量性状较好,鹿茸多肽含量高,显著提高了小鼠成骨细胞增殖率40.74%及碱性磷酸酶活性。结论新疆沙雅地区塔里木亚种马鹿鹿茸活性较好。本实验为建立塔里木马鹿亚种鹿茸质量标准的提升提供了数据支撑。
基金Supported by the Natural Science Foundation of Jilin Provincal Science ＆ Technology Department, China（No.201215244） and the Fundamental Research Foundation of Jilin University, China（No.201103232）
文摘Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and re- versed-phase high-performance liquid chromatography（RP-HPLC） were used to isolate and purify the natural poly- peptide from velvet antler（nVAP） of Cervus elaphus（C, elaphus）, which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4--50 gg/mL, nVAP promotes mitosis in epidermal ceils, chondrocytes and NIH3T3 fibroblasts pri- marily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide（sVAP） according to its primary struc- ture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectro- metry（MALDI-TOF-MS） was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAE Bioactivity test shows that at a dose of 5--40 μg/mL, sVAP promotes the pro- liferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon（C, nippon） and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus, sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler pol
