TMP线性二联体多肽的筛选及其对辐射损伤小鼠血小板减少症的救治作用  

Screening for dimmer of thrombopoietin mimetic peptide with potent thrombocytopoietic effect and its effect on radiation-induced thrombocytopenia in mice

作　　者：张舟[1] 陈芳[1] 曾东风[1] 许杨[1] 王崧[1] 申明强[1] 陈默[1] 陈石磊[1] 粟永萍[1] 王军平[1] 

机构地区：[1]第三军医大学军事预防医学院全军复合伤研究所,创伤,烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2013年第19期2005-2009,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金(31071025);全军"十二"后勤科研重点项目(BWS11J009);创伤;烧伤与复合伤国家重点实验室专项基金(SKLZZ201115)~~

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,Western blot分析TMP二联体多肽激活STAT5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5 Gyγ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7 d,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(G8)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STAT5和ERK1/2的磷酸化,该多肽连续作用14 d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)%vs(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(164.8×109/L vs 53.0×109/L),并使血小板水平提前4 d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。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.

关 键 词：TMP二联体 多肽 巨核细胞 血小板 辐射损伤 

分 类 号：R341[医药卫生—基础医学] R558.205