Li Juan, Luo Jing, Luo Yi-Qin, Zhou Ming, Zhao Liang, Yao Li-Juan, Dong Hang, Yang Rui-Ning
Department of Laboratory Medicine, The Affiliated Anhui Provincial Hospital of Anhui Medical University, Hefei, China.
Transfusion. 2014 Aug;54(8):2106-17. doi: 10.1111/trf.12617. Epub 2014 Mar 24.
Thrombocytopenia is a common side effect of tumor chemotherapy, the main management approach to which is based on platelet (PLT) transfusion. However, PLTs, containing angiogenesis regulators, play a major role in boosting tumor growth and metastasis. The purpose of the study was to determine whether PLTs have the capacity to overexpress tumstatin by modified megakaryocyte (MK) and PLT precursors using lentivirus-mediated gene transfer, which might lead to alteration in proangiogenic effect of PLTs.
CD34+ hematopoietic stem cells (HSCs) were transduced with recombinant lentivirus carrying tumstatin and induced to produce MKs and PLTs in the culture medium containing a cytokine cocktail. Flow cytometry and aggregation test were used to detect the generation and function of MKs and PLTs. Western blot analysis and confocal microscopy were applied to examine the expression and distribution of tumstatin in transgenic MKs and PLTs. Capillary tube formation of human umbilical vein endothelial cells (HUVECs) was used to evaluate the inhibitory effect of transgenic PLTs.
CD34+ HSCs can be efficiently transduced with lentivirus vectors and successfully differentiated into MKs and PLTs. Large amounts of functional MKs and PLTs could be generated and had correct biologic characteristics. The tests demonstrated the feasibility of tumstatin expression in MKs and PLTs under control of the cytomegalovirus promoter, that thus tumstatin was stored in the α-granules of PLTs, and that the releasate of thrombin or A543 cell-stimulated transgenic PLTs obviously inhibited the growth of capillary tube network structures of HUVECs.
Gene-modified CD34+ HSCs not only successfully differentiated into MKs and PLTs but also expressed tumstatin protein. Release of tumstatin in transgenic PLT granules led to antiangiogenic effect of PLTs.
血小板减少是肿瘤化疗常见的副作用，其主要治疗方法是基于血小板（PLT）输注。然而，含有血管生成调节因子的血小板在促进肿瘤生长和转移中起主要作用。本研究的目的是确定血小板是否有能力通过慢病毒介导的基因转移修饰巨核细胞（MK）和血小板前体来过表达tumstatin，这可能导致血小板促血管生成作用的改变。
用携带tumstatin的重组慢病毒转导CD34+造血干细胞（HSC），并在含有细胞因子混合物的培养基中诱导其产生巨核细胞和血小板。采用流式细胞术和聚集试验检测巨核细胞和血小板的生成及功能。应用蛋白质免疫印迹分析和共聚焦显微镜检查tumstatin在转基因巨核细胞和血小板中的表达及分布。用人脐静脉内皮细胞（HUVEC）的毛细管形成试验评估转基因血小板的抑制作用。
慢病毒载体可有效转导CD34+HSC，并成功分化为巨核细胞和血小板。可产生大量具有正常生物学特性的功能性巨核细胞和血小板。试验证明了在巨细胞病毒启动子控制下tumstatin在巨核细胞和血小板中表达的可行性，tumstatin因此储存在血小板的α颗粒中，并且凝血酶或A543细胞刺激的转基因血小板的释放物明显抑制了HUVEC毛细管网络结构的生长。
基因修饰的CD34+HSC不仅成功分化为巨核细胞和血小板，还表达了tumstatin蛋白。转基因血小板颗粒中tumstatin的释放导致了血小板的抗血管生成作用。
