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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与突触后致密蛋白PSD95相互作用，进而抑制含NR2B亚基的N-甲基-D-天冬氨酸（N-methyl-D-asparate，NMDA）受体的磷酸化的可行性。方法 ①通过多肽阵列技术的overlapping平台确定Fyn的SH2结构域与突触后致密蛋白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（10 μM, 20 μM, 30 μM）组的穿膜肽内化细胞的效率。③用CCK8法检测不同浓度（10 μM, 20 μM, 100 μM）穿膜肽分别孵育神经细胞(6 h, 24 h, 48 h)的细胞毒性。④将神经细胞分为穿膜肽Fynp-Tat组，乱序穿膜肽mFynp-Tat组和PBS对照组进行孵育，用GST pull-down和Western Blotting验证抑制Fyn与 PSD95相互作用的效率。结果 ①通过overlapping确定的Fyn与 PSD95相互作用的结合基序为（KGAYSL)。②Fynp-Tat组浓度为20 μM时内化效率为（91.5±2.0 %），与10 μM组比较（77.3±1.4 %，P<0.05）有差异；③当Fynp-Tat组浓度为100 μM，作用时间为24 h，才出现细胞活力(75.1±1.5 %)降低,与（100 μM, 6 h）组比较（82.5±3.0 %，P<0.05）有差异；④Fynp-Tat组与mFynp-Tat组、PBS对照组相比，Fynp-Tat组能够抑制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（KGAYSLYGRKKRRQRRR）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。
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Abstract: Objective To examine the potential application of the cell-penetrating peptide based on peptide arrays for the inhibition of interaction between the nonreceptor tyrosine kinase Fyn and post density protein PSD95 in vitro. Methods First, by using peptide array we dentified biding motifs（Fynp）between Fyn SH2 domain and PSD95 PDZ3 domain. Fused Fynp with the transduction domain of the HIV -Tat protein to generate the cell-penetrating peptide Fynp-Tat, and generate a mistake sequence of cell-penetrating peitide mFynp-Tat. Second, we cultured the spinal dorsal horn neurons isolated from the embryonic Sprague-Dawley rats, and investigated the internalization efficiency of Fynp-Tat by immunofluorescence staining in different concentration groups（10μM, 20μM, 30μM）. Third, we calculated the cytotoxicity of Fynp-Tat through CCK8 in different concentrations（10 μM, 20 μM, 100 μM） and different incubateing time groups(6 h, 24 h, 48 h). Four ,we investigated the interruption efficiency of Fynp-Tat on the interaction between Fyn and PSD 95 by using GST pull-down and Western blotting, neurons were incubated by Fynp-Tat(Fynp-Tat group) , mFynp-Tat(mFynp-Tat group ) and PBS(control group). Results Fynp-Tat group (20 μM) interalized neurons（91.5±2.0 %）singnificantly, compared with the cells exposed to 10 μM ( 77.3±1.4 %, P<0.05) . After an incubation of 100 μM Fynp-Tat for 24 h, the cell viability was decreased to 75.1±1.5 %, compared with 82.5±3.0 % of the cells treated for 6 h at 100 μM Fynp-Tat (P<0.05 ). Compared with the PBS control 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). Conclusion Our data suggest that Fynp-Tat can internalize the neurons with little cytotoxicity, and can also inhibit the interaction between Fyn and PSD95 in vitro.
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