CAJ | 학술논문

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-mCMV-ZsGreen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738 bp 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tum-statin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。
목적이만병독위기인재체,장tumstatin cDNA도입CD34+조혈간세포,재체외유도생성tumstatin기인수식적거핵세포( MKs)화혈소판,검측산생적혈소판대혈관내피세포관상결구형성적작용。방법구건 pLVX-tumstatin-mCMV-ZsGreen중조재체후전염293T세포진행병독포장。용밀도제도리심법결합면역자주분리법부집제혈중CD34+조혈간세포。용만병독감염CD34+조혈간세포,재세포인자조합배양액중유도MKs생성,류식세포의화형태학검측MKs적생성급산혈소판정황。응용RT-PCR법화Western blot법검측tumstatin기인적표체,통과인제정맥혈관내피세포관상결구형성억제시험연구혈소판내용물생물학활성。결과선택최가감염복수(MOI)30：1감염간세포시효솔최고；류식세포술검측결과현시,세포재유도과정중,전염조여미전염조세포도유MKs여혈소판적생성,차생장속도화분화추세기본상동。재전염적MKs기인조리,RT-PCR법검측도738 bp tumstatin기인편단。 Western blot법검측도tumstatin재전기인세포래원적혈소판중은정표체,혈소판가명현억제인제정맥내피세포관상결구형성。결론기인수식적CD34+조혈간세포재체외성공유도분화위MKs화혈소판병표체tum-statin단백,차저충혈소판재체외현저억제인제정맥혈관내피세포적관상결구형성。
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.