中文摘要：

目的筛选具有显著促进巨核细胞增殖、分化和血小板生成活性的血小板生成素模拟肽（TMP）二联体线性多肽，并观察其对血小板减少症的救治作用。方法设计合成系列TMP二联体线性多肽，其中两个头尾串联的TMP之间设置不同长度的连接肽，CCK-8法检测并筛选促巨核细胞增殖活性最强的TMP二联体多肽，Westernblot分析TMP二联体多肽激活STATS和ERK1／2磷酸化情况，流式细胞术分析小鼠骨髓来源Sca＋-I细胞经TMP二联体多肽处理后巨核细胞标志分子CD41、CD61的表达变化。BALB／c小鼠用5Gy1射线全身一次性照射后皮下注射TMP二联体多肽，每天1次，连续用药7d，分析外周血血小板水平变化和骨髓造血组织病理改变。结果连接肽长度和氨基酸组成可显著影响TMP二联体多肽的促巨核细胞增殖活性，其中由1个脯氨酸（P）和8个甘氨酸（G。）连接的TMP二联体活性较为突出。TMP-PG8-TMP多肽处理能够显著促进巨核细胞中STATS和ERK1／2的磷酸化，该多肽连续作用14d能够较IL-3对照组显著上调Sca＋-I细胞CD41、CD61的双阳性表达率[（83．26±5．38）％郴（15．70±2．12）％，P〈0．01]。此外，体内应用该多肽能够较辐照对照显著提高急性辐射损伤小鼠外周血血小板最低值水平（164．8×10^9／Lvs53．0×10^9／L），并使血小板水平提前4d恢复。结论通过筛选连接肽最终获得具有显著促巨核细胞增殖和血小板生成活性的TMP二联体线性多肽。

英文摘要：

Objective To screen the dimmer of thrombopoietin mimetic peptide （dTMP） with potent thrombocytopoietic effect on the promotion of thrombopoiesis and the proliferation and differentiation of megakaryocyte. Methods A series of linear peptides containing two TMP joined by peptide linkers at different lengthes were designed and synthesized. The promoting activity of the dTMP on megakaryocyte proliferation was determined by cell counting kit-8 （CCK-8） assay. The ability of dTMP to phosphorylate ERK1/2 and STAT5 signal pathways was measured by Western blotting. The expression level of CD41/CD61 was analyzed by flow cytometry to reveal the ability of dTMP peptide to promote murine marrow Sea ＋- I cells to differentiate into megakaryocytes. Finally, dTMP was administered to BALB/c mice with thrombocytopenia induced by 5 Gy total body irradiation through subcutaneous injection, in order to investigate the effect of dTMP on platelet recovery in vivo. Results The length of peptide linkers significantly influenced the promoting activity of dTMP peptides on megakaryocyte proliferation. The dTMP peptide with a linker composed by a proline and eight glycine （TMP- PGs-TMP） displayed a remarkable effect on megakaryocyte proliferation. Western blot assay demonstrated that TMP-PGs-TMP had the ability to activate the phosphorylation of ERK1/2 and STAT5. The expression level of CD41/CD61 in the cells derived from Sca＋- I marrow cells treated with TMP-PGs-TMP was much higher than that of IL-3 control group [ （83.26 ±5.38 ） % vs （ 15.70 ± 2.12） %, P 〈 0.01 ]. Meanwhile, dTMP-treated mice were about 4 d earlier in the platelet recovery than that of saline control, demonstrating that this peptidewas able to be used for the treatment of thrombocytopenia. Conclusion A dTMP peptide with potent thrombo- cytopoietic activity is finally obtained by peptide linker screening.