摘要目的 探讨基于多肽阵列技术合成的穿膜肽离体条件下抑制非受体酪氨酸激酶Fyn与突触后致密物蛋白(postsynaptic density protein,PSD)95相互作用,进而抑制含N-甲基-D-天冬氨酸受体2B亚基(N-methyl D-aspartate receptor subtype 2B,NR2B)的N-甲基-D-天冬氨酸受体(N-methyl D-aspartate receptor,NMDAR)过度磷酸化,减轻慢性疼痛的可行性.方法 ①通过多肽阵列技术的ovedapping平台确定Fyn的SH2结构域与PSD95的PDZ3结构域相互作用的结合基序,合成含此结合基序的短肽FynP,并与人类免疫缺陷病毒(human immunodeficiency virus,HIV)-Tat蛋白转导结构域连接形成可进入细胞的穿膜肽FynP-Tat,同时合成乱序穿膜肽mFynP-Tat.②原代培养SD大鼠胎鼠脊髓背角神经元,通过免疫荧光检测不同浓度组(10、20、30 μmol/L)多肽内化细胞的效率.③用CCK8法检测不同浓度(10、20、100μmol/L)穿膜肽分别孵育神经细胞(6、24、48 h)的细胞毒性.④将神经细胞分为穿膜肽组(FynP-Tat组)、乱序穿膜肽组(mFynP-Tat组)和PBS组(对照组)进行孵育,用谷胱甘肽转移酶(glutathione S-transferase,GST) pull-down和Western blot验证抑制Fyn与PSD95相互作用的效率. 结果 ①通过overlapping确定Fyn与PSD95相互作用的结合基序为KGAYSL.②与FynP-Tat 10 μmol/L组[(77.3±1.4)％]比较,FynP-Tat 20 μmol/L组[(91.5±2.0)％]和FynP-Tat 30 μmol/L组[(93.4:2.2)％]的内化效率较高,差异有统计学意义(P＜0.05);与mFynP-Tat 10μmol/L组[(75.0±3.6)％]比较,mFynP-Tat 20 μmol/L组[(91.4±1.3)％]和mFynP-Tat 30 μmol/L组[(92.7±2.1)％]的内化效率较高,差异有统计学意义(P＜0.05);③与FynP-Tat(100 μmol/L,6h)组[(82.5±3.0)％]比较,FynP-Tat(100 μmol/L,24 h)组和FynP-Tat(100 μmol/L,48 h)组细胞活力[(75.1±1.5)％和(68.5±1.2)％]降低(P＜0.05);与mFynP-Tat(100 μmol/L,6h)组[(83.3±2.0)％]比较,mFynP-Tat(100 μ mol/L,24 h)组和mFynP-Tat(100 μmol/L,48 h)组细胞活力[(75.6±2.9)％和(68.1±0.7)％]降低(P＜0.05);④与mFynP-Tat组、PBS组比较结果显示,FynP-Tat能够抑制PSD95结合到GST-Fyn融合蛋白上(P＜0.05),但不会抑制PSD95结合到GST-NR2B融合蛋白上(P＞0.05). 结论 离体条件下,基于多肽阵列技术合成的穿膜肽FynP-Tat能够进入神经细胞,细胞毒性低,可有效抑制Fyn与PSD95的相互作用.

abstractsObjective To examine the potential application of the cell-penetrating peptide based on peptide array for the inhibition of interaction between the nonreceptor tyrosine kinase Fyn and post density protein postsynaptic density protein (PSD) 95 in vitro.Methods First,by using peptide array we identified binding motif (FynP) between Fyn SH2 domain and PSD95 PDZ3 domain.We fused Fynp with the transduction domain of the human immunodeficiency virus (HIV)-Tat protein to generate the cellpenetrating peptide (FynP-Tat),and generate a mistake sequence of cell-penetrating peptide (mFynP-Tat).Second,we cultured the spinal dorsal horn neurons isolated from the embryonic Sprague-Dawley rats,and investigated the internalization efficiency of peptides by immunofluorescence staining in different concentration groups(10,20,30 μmol/L).Third,we calculated the cytotoxicity of the cell-penetrating peptides through CCK8 in different concentrations(10,20,100 μmol/L) and different incubating time groups(6,24,48 h).Fourth,we investigated the interruption efficiency of FynP-Tat on the interaction between Fyn and PSD95 by using GST pulldown and Western blotting,and neurons were incubated by FynP-Tat(Fynp-Tat group),mFynP-Tat(mFynP-Tat group) and PBS(control group).Results ① Peptide array showed that the binding motif between Fyn and PSD95 was KGAYSL.② FynP-Tat 20 μmol/L group [(91.5±2.0)％] and FynP-Tat 30 μmol/L group [(93.4±2.2)％] internalized neurons significantly,compared with the cells exposed to FynP-Tat 10 μmol/L group[(77.3±1.4)％](P＜0.05),mFynP-Tat 20 μmol/L group[(91.4± 1.3)％] and mFynP-Tat 30 μmol/L group[(92.7±2.1)％] internalized neurons singnificantly,compared with the cells exposed to mFynP-Tat 10 μmol/L group[(75.0±3.6)％] (P＜0.05).③ After an incubation of 100 μmol/L FynP-Tat for 24 h and 48 h,the cell viability was decreased to [(75.1±1.5)％ and (68.5±1.2)％],compared with[(82.5±3.0) ％] of the cells treated for 6 h at 100 μmol/L FynP-Tat (P＜0.05).After an incubation of 100 μmol/L mFynP-Tat for 24 h and 48 h,the cell viability was decreased to [(75.6±2.9)％ and (68.1 ±0.7)％],compared with (83.3±2.0)％ of the cells treated for 6 h at 100 μmol/L mFynP-Tat (P＜0.05).④ Compared with the control group and the mFynP-Tat group,the FynP-Tat group inhibited PSD95 binding to GST-Fyn fused protein markedly (P＜0.05),but did not inhibit PSD95 binding to GST-NR2B fused protein (P＞0.05).Conclusions The data suggest that FynP-Tat can internalize the neurons with little cytotoxicity,and can also inhibit the interaction between Fyn and PSD95 in vitro.