活性多肽IKVAV纳米纤维凝胶的自组装及其生物相容性检测

Self-assembly of IKVAV peptide-AmpHipHile and pilot study of biocompatibility

目的:合成神经活性多肽IKVAV(isoleucine-lysine-valine-alanine-valine,异亮氨酸-赖氨酸-丙氨酸-缬氨酸)多肽两亲性分子,在体外进行自组装成为纳米纤维,并且初步检测其生物相容性。方法:实验于2005-12/2006-03在华中科技大学附属协和医院骨科实验室完成。①IKVAV多肽两亲性分子委托上海波泰生物科技公司合成,并用高效液相色谱仪和质谱仪进行纯化和分析。②IKVAV纳米纤维凝胶自组装:分别取200μL10,5和2.5g/LIKVAV两亲性分子(IKVAVPeptide-AmpHipHile,IKVAV-PA)溶液置入小玻璃瓶中,分别加入等体积的含有钙(200mg/L)、镁(97.67mg/L)二价阳离子的DMEM细胞培养基溶液,自组装成为5,2.5和1.25g/L的材料。通过HCl溶液和蒸汽使IKVAV-PA自组装。③透射电镜检测:用场发射能量过滤型透射电镜对自组装后材料进行检测。④生物相容性初步检测:培养L929细胞系,加入不同浓度的IKVAV自组装材料,各组IKVAV-PA的最终浓度分别为0,20,60,160,500mg/L,用CCK-8试剂盒检测IKVAV自组装材料对L929细胞存活和活性的影响。结果:①IKVAV-PA合成后检测结果:成功合成了IKVAV多肽两亲性分子,相对分子质量为1351,纯度为98%。②IKVAV-PA自组装结果:加入含钙、镁二价阳离子的细胞培养基或者降低pH值均可引发其自组装。当凝胶中IKVAV-PA浓度为5g/L时,自组装形成的凝胶完整,有足够的支撑力维持凝胶的形状。但是当IKVAV-PA浓度为2.5g/L和1.25g/L时,自组装形成的凝胶不完整,只在局部形成凝胶颗粒。③透射电子显微镜检测结果:分子自组装形成直径为7.0～8.0nm的纳米纤维。④生物相容性和生物活性初步检测结果:当IKVAV自组装材料生物相容性良好,并且当IKVAV的浓度为60或160mg/L时,能增强L929细胞的活性。结论:IKVAV多肽两亲性分子能自组装成为纳米纤维凝胶,并且具有很好的生物相容性和生物活性。

AIM： To synthesize bioactive IKVAV （isoleucine-lysine-valine-alaninev-aline） Peptide-AmpHipHile （IKVAV-PA） and self-assemble it in vitro, so as to make a pilot study of its biocompatibility. METHODS： The experiment was conducted at the laboratory of Department of Orthopedics, Huazhong University of Science and Technology between December 2005 and March 2006. ①IKVAV-PA was synthesized by the Shanghai Botai Biological Technology Company, and purified and tested with high-performance liquid chromatography and mass spectrometry.②Self-assembly of IKVAV nanofiber gel： 200 μL, 10, 5 and 2.5 g/L IKVAV-PA solution were put into the small glass bottle, and DMEM cell culture media with divalent cation of calcium （Ca 200 mg/L） and magnesium （Mg 97.67 mg/L） was added to induce into 5, 2.5 and 1.25 g/L assembled material by HCl solution and steam. ③Transmission Electron Microscope （TEM） test： The assembled material was detected with TEM of field energy filtering type. ④Pilot study of biocompatibility： L929 cells were cultured and added different concentrations of IKVAV-PA assembled material with the teminal concentrations of 0, 20, 60, 160 and 500 mg/L. CCK-8 kit was used to test the effect of IKVAV assembled material on survival and bioactivity of L929 cells. RESULTS： ①Detection of IKVAV-PA： The IKVAV-PA was synthesized successfully. The relative molecular mass was 1 351 with the purity quotient of 98%. （②IKVAV-PA self-assembly： When cell adding culture media containing Ca^2＋ and Mg^2＋ or decreasing the pH value, IKVAV-PA could self-assemble to produce a gel containing 0.5 g/L peptide, which could maintain the appearance of gel. But IKVAV-PA with concentrations of 2.5 and 1.25 g/L, the self-assembled gel was not complete with only gel granules in local. ③TEM results： The assembled material was a kind of nanofiber with diameter of 7.0-8.0 nm. ④Pilot study of biocompatibility and bioactivity： IKVAV nanofibers with concentrations of 60 μg/mL or 160 μg/mL could enhance the bioactivity of L929 cells. CONCLUSION： IKVAV-PA can self-assemble to nanofiber gel, which has good biocompatibility and bioactivity.