tumstatin基因修饰的CD34^+造血干细胞生成抗血管生成活性的血小板  

Tumstatin gene-modified CD34^+ hematopoietic stem cells produce anti-angiogenic platelets

作　　者：李娟[1] 罗以勤[1] 丁邦胜[1] 贺学姣[1] 周明[2] 赵亮[1] 姚丽娟[1] 

机构地区：[1]安徽医科大学附属省立医院检验科,合肥230001 [2]安徽医科大学附属省立医院输血科,合肥230001

出　　处：《安徽医科大学学报》2014年第5期576-581,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:11040606M208);安徽省高等学校省级自然科学研究项目(编号:KJ2011A163)

摘　　要：目的以慢病毒为基因载体,将tumstatin cDNA导入CD34+造血干细胞,在体外诱导生成tumstatin基因修饰的巨核细胞(MKs)和血小板,检测产生的血小板对血管内皮细胞管状结构形成的作用。方法构建pLVX-tumstatin-mCMVZsGreen重组载体后转染293T细胞进行病毒包装。用密度梯度离心法结合免疫磁珠分离法富集脐血中CD34+造血干细胞。用慢病毒感染CD34+造血干细胞,在细胞因子组合培养液中诱导MKs生成,流式细胞仪和形态学检测MKs的生成及产血小板情况。应用RT-PCR法和Western blot法检测tumstatin基因的表达,通过人脐静脉血管内皮细胞管状结构形成抑制试验研究血小板内容物生物学活性。结果选择最佳感染复数(MOI)30∶1感染干细胞时效率最高;流式细胞术检测结果显示,细胞在诱导过程中,转染组与未转染组细胞都有MKs与血小板的生成,且生长速度和分化趋势基本相同。在转染的MKs基因组里,RT-PCR法检测到738bp tumstatin基因片段。Western blot法检测到tumstatin在转基因细胞来源的血小板中稳定表达,血小板可明显抑制人脐静脉内皮细胞管状结构形成。结论基因修饰的CD34+造血干细胞在体外成功诱导分化为MKs和血小板并表达tumstatin蛋白,且这种血小板在体外显著抑制人脐静脉血管内皮细胞的管状结构形成。Objective CD34 ＋ hematopoietic stem cells transfected with recombinant lentivirus vector carrying tum-statin cDNA were in vitro induced to produce megakaryocytes （ MKs） and platelets. The inhibitory effect of the platelets on the growth of capillary tube structures of HUVEC were detected. Methods Constructed pLVX-tumsta-tin-mCMV-ZsGreen recombinant vector was transfected into 293T cells for virus packaging. Cord blood CD34 ＋hematopoietic stem cells enriched by immunomagnetic separation were transfected with recombinant lentivirus and induced to produce megakaryocytes in the culture medium combinations of cytokines. Flow cytometry and morpho-logical observation were used to detect the generation of megakaryocytes and platelets. RT-PCR and Western blot a-nalysis were applied to examine the expression of tumstatin. Capillary tube structures assay of HUVEC was used to evaluate the inhibitory effect of the platelets in vitro. Results CD34 ＋ hematopoietic stem cells transfected with re-combinant lentivirus at the best multiplicity of infection （ MOI ） being 30 ： 1 , ZsGreen positive rate of which was highest. The transfected and untransfected cells generated megakaryocyte and platelets, the growth rate and differ-entiation trend of which were substantially identical by flow cytometry analysis. RT-PCR detected a 738 bp tumsta-tin cDNA in transfected MKs. Western blot confirmed the expression of tumstatin protein in gene-modified megakaryocyte and platelets. Tumstatin gene-modified platelets inhibited the capillary tube structures of HUVEC. Conclusion Gene-modified CD34 ＋ hematopoietic stem cells not only successfully differentiate into megakaryocyte and platelets but also express tumstatin protein, and this transgenic platelets significantly inhibit the capillary tube structures of HUVEC in vitro.

关 键 词：TUMSTATIN 慢病毒载体 CD34+造血干细胞 血管内皮细胞 巨核细胞 血小板 

分 类 号：R349.63[医药卫生—基础医学] R331.2