丝素肽络合Ca~(2+)含量影响细胞生长的研究
|
摘要
研究丝素肽(SFP)络合Ca~(2+)含量对人正常肝细胞L02生长的影响。采用CaCl_2-乙醇-水三元体系制备丝素蛋白(SF),并获得不同Ca~(2+)含量的SFP后进行相对分子质量分级;采用EDTA滴定法测定SFP中Ca~(2+)含量,细胞增殖率评价其细胞毒性,圆二色谱(CD)检测二级结构。结果表明:随透析时间(12、24、36 h)增加,SFP中Ca~(2+)含量分别为16.00%、9.60%、0.40%,逐渐减小,对L02的生长作用由促进转变为抑制,其中在SFP相对分子质量大于8 kD时尤为明显,L02细胞增殖率从(118.68%±3.65%)减小到(59.78%±1.70%),对应SFP二级结构中3.43%的α螺旋和5.35%的无规则卷曲转变为β折叠。当SFP应用于组织工程支架领域时,络合较高Ca~(2+)含量的SFP更有利于细胞的增殖。
The effects of silk fibroin peptide(SFP) complex Ca~(2+) content on the growth of human normal hepatocyte L02 were studied. The CaCl_2-ethanol-water system was used to prepare silk fibroin(SF), and SFP with different Ca~(2+) content was obtained. Then, the relative molecular mass of SFP was classified. The content of Ca~(2+ )in SFP was measured by EDTA titration. The cytotoxicity of L02 cells was evaluated by the proliferation rate, and circular dichroism(CD) was used to detect the secondary structure of SFP. The results show that as the time of dialysis(12, 24, 36 h) increased, Ca~(2+) content of SFP(16.00%, 9.60%, 0.40%) gradually decreased, and the growth of L02 changed from promotion to inhibition. Especially when the relative molecular mass of SFP was greater than 8 kD, the cell proliferation rate of L02 decreased from(118.68%±3.65%) to(59.78%±1.70%). In the secondary structure of the corresponding SFP, 3.43% of alpha-helix and 5.35% of irregular curl transformed to beta-folding. In the field of tissue engineering scaffolds, SFP with higher content of complex Ca~(2+) is more conducive to cell proliferation.
The effects of silk fibroin peptide(SFP) complex Ca~(2+) content on the growth of human normal hepatocyte L02 were studied. The CaCl_2-ethanol-water system was used to prepare silk fibroin(SF), and SFP with different Ca~(2+) content was obtained. Then, the relative molecular mass of SFP was classified. The content of Ca~(2+ )in SFP was measured by EDTA titration. The cytotoxicity of L02 cells was evaluated by the proliferation rate, and circular dichroism(CD) was used to detect the secondary structure of SFP. The results show that as the time of dialysis(12, 24, 36 h) increased, Ca~(2+) content of SFP(16.00%, 9.60%, 0.40%) gradually decreased, and the growth of L02 changed from promotion to inhibition. Especially when the relative molecular mass of SFP was greater than 8 kD, the cell proliferation rate of L02 decreased from(118.68%±3.65%) to(59.78%±1.70%). In the secondary structure of the corresponding SFP, 3.43% of alpha-helix and 5.35% of irregular curl transformed to beta-folding. In the field of tissue engineering scaffolds, SFP with higher content of complex Ca~(2+) is more conducive to cell proliferation.
引文
[1]MELKE J, MIDHA S, GHOSH S, et al. Silk fibroin as biomaterial for bone tissue engineering [J]. Acta Biomaterialia, 2016, 31:1-16.
[2]VEPARIV C, KAPLAN D L. Silk as a Biomaterial[J]. Progress in Polymer Science, 2007, 32(8): 991-1007.
[3]TEIMOURI A, AZADI M, EMADI R, et al. Preparation, characterization, degradation and biocompatibility of different silk fibroin based composite scaffolds prepared by freeze-drying method for tissue engineering application [J]. Polymer Degradation & Stability, 2015, 121: 18-29.
[4]黄慧明. 蚕丝蛋白肽抗肿瘤与抗氧化作用研究[D]. 泰安: 山东农业大学, 2012.HUANG Huiming. Antitumor and Antioxidation of Silk Protein Peptide[D]. Taian: Shandong Agricultural University, 2012.
[5]YAMADA H, IGARASHI Y, TAKASU Y, et al. Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts [J]. Biomaterials, 2004, 25(3): 467-472.
[6]王富平, 庞亚妮, 陈忠敏. 丝素降解生成的低分子多肽对细胞生长性能的影响[J]. 丝绸, 2017, 54(4):1-4. WANG Fuping, PANG Yani, CHEN Zhongmin. Effect of low-molecular-weight silk fibroin peptides on cell growth property [J]. Journal of Silk, 2017, 54(4): 1-4.
[7]LUO Q, CHEN Z, HAO X, et al. Preparation and properties of nanometer silk fibroin peptide/polyvinyl alcohol blend films for cell growth [J]. International Journal of Biological Macromolecules, 2013, 61(10): 135-141.
[8]武明扬, 吴悦, 杨高强, 等.家蚕丝素重链非重复区肽段在大肠埃希菌中的优化表达[J]. 蚕业科学, 2017, 43(2): 238-244. WU Mingyang, WU Yue, YANG Gaoqiang, et al. Expression optimization of non-repetitive polypeptide derived from silk fibroin heavy chain in escherichia coli[J]. Science of Sericulture, 2017, 43(2): 238-244.
[9]钟一鸣, 杨宇红, 陈新, 等. 铜离子对水溶液中再生丝素蛋白构象转变的诱导作用[J]. 高分子学报,2009(10): 1056-1061. ZHONG Yiming, YANG Yuhong, CHEN Xin, et al. CU(Ⅱ)-Induced conformation transition of regenerated silk fibroin in aqueous solutions [J]. Acta Polymerica Sinica, 2009(10): 1056-1061.
[10]SHEN T, WANG T, CHENG G, et al. Dissolution behavior of silk fibroin in a low concentration CaCl2-methanol solvent: from morphology to nanostructure[J]. International Journal of Biological Macromolecules, 2018, 113:458-463.
[11]ZHOU P, XIE X, KNIGHT D P, et al. Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR [J]. Biochemistry, 2004, 43(35):11302.
[12]AJISAWA A. Studies on the dissolution of silk fibroin(Ⅱ): the dissolution of silk fibroin by CaCl2-E2O-R.OH ternary system solution [J]. Fiber, 1969, 24(2):61-64.
[13]刘玉强, 刘鹏翔, 田保中. 碳酸钙-丝素粉体材料结构性能的探究[J]. 丝绸, 2012, 49(6): 1-4.LIU Yuqiang, LIU Pengxiang, TIAN Baozhong. Exploration on the structure and properties of calcium carbonate-silk fibroin powder material[J]. Journal of Silk, 2012,49(6): 1-4.
[14]刘海林, 王磊. 肝脏组织工程种子细胞来源研究进展[J]. 国际消化病杂志, 2003, 23(S1): 20-21.LIU Hailin, WANG Lei. Research progress of seed cells in liver tissue engineering [J]. International Journal of Gastroenterology, 2003, 23(S1): 20-21.
[15]陈忠敏, 郝雪菲. 一种纳米蚕丝丝素蛋白粉的制备方法: 200710092969.6[P]. 2011-07-13.CHEN Zhongmin, HAO Xuefei. Method for preparing nano silk fibroin protein powder: 200710092969.6 [P]. 2011-07-13.
[16]LI M, TAO W, KUGA S, et al. Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment [J]. Polymers for Advanced Technologies, 2010, 14(10): 694-698.
[17]中华人民共和国国家质量监督检验检疫总局. GBT 16886.5—2003 医疗器械生物学评价第5部分: 体外细胞毒性试验[S]. 2003.People’s Republic of China General Administration of Quality Supervision, Inspection and Quarantine. GBT 16886.5-2003 Biological assessment of medical devices The fifth part: in vitro cytotoxicity test [S]. 2003.
[18]ZHU Z H, ZHU L J, XUAN L U. Research on the technology of degradation and desalination of silk fibroin[J]. Amino Acids & Biotic Resources, 2003, 25(3): 37-40.
[19]于海洋, 王昉, 刘其春, 等. 新型丝素蛋白膜的结构和热分解动力学机理[J]. 物理化学学报, 2017, 33(2): 344-355. YU Haiyang, WANG Fang, LIU Qichun, et al. Structure and kinetics of thermal decomposition mechanism of novel silk fibroin films [J]. Acta Physico-Chimica Sinica, 2017, 33(2): 344-355.
[20]HE H, DONG W, HUANG F. Anti-amyloidogenic and anti-apoptotic role of melatonin in alzheimer disease [J]. Current Neuropharmacology, 2010, 8(3): 211-217.
[2]VEPARIV C, KAPLAN D L. Silk as a Biomaterial[J]. Progress in Polymer Science, 2007, 32(8): 991-1007.
[3]TEIMOURI A, AZADI M, EMADI R, et al. Preparation, characterization, degradation and biocompatibility of different silk fibroin based composite scaffolds prepared by freeze-drying method for tissue engineering application [J]. Polymer Degradation & Stability, 2015, 121: 18-29.
[4]黄慧明. 蚕丝蛋白肽抗肿瘤与抗氧化作用研究[D]. 泰安: 山东农业大学, 2012.HUANG Huiming. Antitumor and Antioxidation of Silk Protein Peptide[D]. Taian: Shandong Agricultural University, 2012.
[5]YAMADA H, IGARASHI Y, TAKASU Y, et al. Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts [J]. Biomaterials, 2004, 25(3): 467-472.
[6]王富平, 庞亚妮, 陈忠敏. 丝素降解生成的低分子多肽对细胞生长性能的影响[J]. 丝绸, 2017, 54(4):1-4. WANG Fuping, PANG Yani, CHEN Zhongmin. Effect of low-molecular-weight silk fibroin peptides on cell growth property [J]. Journal of Silk, 2017, 54(4): 1-4.
[7]LUO Q, CHEN Z, HAO X, et al. Preparation and properties of nanometer silk fibroin peptide/polyvinyl alcohol blend films for cell growth [J]. International Journal of Biological Macromolecules, 2013, 61(10): 135-141.
[8]武明扬, 吴悦, 杨高强, 等.家蚕丝素重链非重复区肽段在大肠埃希菌中的优化表达[J]. 蚕业科学, 2017, 43(2): 238-244. WU Mingyang, WU Yue, YANG Gaoqiang, et al. Expression optimization of non-repetitive polypeptide derived from silk fibroin heavy chain in escherichia coli[J]. Science of Sericulture, 2017, 43(2): 238-244.
[9]钟一鸣, 杨宇红, 陈新, 等. 铜离子对水溶液中再生丝素蛋白构象转变的诱导作用[J]. 高分子学报,2009(10): 1056-1061. ZHONG Yiming, YANG Yuhong, CHEN Xin, et al. CU(Ⅱ)-Induced conformation transition of regenerated silk fibroin in aqueous solutions [J]. Acta Polymerica Sinica, 2009(10): 1056-1061.
[10]SHEN T, WANG T, CHENG G, et al. Dissolution behavior of silk fibroin in a low concentration CaCl2-methanol solvent: from morphology to nanostructure[J]. International Journal of Biological Macromolecules, 2018, 113:458-463.
[11]ZHOU P, XIE X, KNIGHT D P, et al. Effects of pH and calcium ions on the conformational transitions in silk fibroin using 2D Raman correlation spectroscopy and 13C solid-state NMR [J]. Biochemistry, 2004, 43(35):11302.
[12]AJISAWA A. Studies on the dissolution of silk fibroin(Ⅱ): the dissolution of silk fibroin by CaCl2-E2O-R.OH ternary system solution [J]. Fiber, 1969, 24(2):61-64.
[13]刘玉强, 刘鹏翔, 田保中. 碳酸钙-丝素粉体材料结构性能的探究[J]. 丝绸, 2012, 49(6): 1-4.LIU Yuqiang, LIU Pengxiang, TIAN Baozhong. Exploration on the structure and properties of calcium carbonate-silk fibroin powder material[J]. Journal of Silk, 2012,49(6): 1-4.
[14]刘海林, 王磊. 肝脏组织工程种子细胞来源研究进展[J]. 国际消化病杂志, 2003, 23(S1): 20-21.LIU Hailin, WANG Lei. Research progress of seed cells in liver tissue engineering [J]. International Journal of Gastroenterology, 2003, 23(S1): 20-21.
[15]陈忠敏, 郝雪菲. 一种纳米蚕丝丝素蛋白粉的制备方法: 200710092969.6[P]. 2011-07-13.CHEN Zhongmin, HAO Xuefei. Method for preparing nano silk fibroin protein powder: 200710092969.6 [P]. 2011-07-13.
[16]LI M, TAO W, KUGA S, et al. Controlling molecular conformation of regenerated wild silk fibroin by aqueous ethanol treatment [J]. Polymers for Advanced Technologies, 2010, 14(10): 694-698.
[17]中华人民共和国国家质量监督检验检疫总局. GBT 16886.5—2003 医疗器械生物学评价第5部分: 体外细胞毒性试验[S]. 2003.People’s Republic of China General Administration of Quality Supervision, Inspection and Quarantine. GBT 16886.5-2003 Biological assessment of medical devices The fifth part: in vitro cytotoxicity test [S]. 2003.
[18]ZHU Z H, ZHU L J, XUAN L U. Research on the technology of degradation and desalination of silk fibroin[J]. Amino Acids & Biotic Resources, 2003, 25(3): 37-40.
[19]于海洋, 王昉, 刘其春, 等. 新型丝素蛋白膜的结构和热分解动力学机理[J]. 物理化学学报, 2017, 33(2): 344-355. YU Haiyang, WANG Fang, LIU Qichun, et al. Structure and kinetics of thermal decomposition mechanism of novel silk fibroin films [J]. Acta Physico-Chimica Sinica, 2017, 33(2): 344-355.
[20]HE H, DONG W, HUANG F. Anti-amyloidogenic and anti-apoptotic role of melatonin in alzheimer disease [J]. Current Neuropharmacology, 2010, 8(3): 211-217.