CAJ | 학술논문

目的：：构建人GATA-1全长过表达载体，对其进行过表达，并初步探讨其在人非造血细胞（人脐静脉内皮细胞、人成纤维细胞）中的生物学作用。方法：以人脐带血cDNA为模板，采用高保真PCR获得GATA-1的CDS序列，连接至pGEM-T Easy载体，构建pBPLV-GATA1慢病毒表达质粒，并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定，鉴定正确后，在293T细胞中进行慢病毒包装，浓缩病毒后，对我们原代分离培养的人脐静脉内皮细胞和成纤维细胞进行慢病毒感染，采用流式分选对阳性目的细胞进行纯化，阳性细胞扩增后进行Western印迹鉴定；以人包皮成纤维细胞（HFF）为研究对象，在光学及荧光显微镜下观察细胞形态变化情况，CCK-8法检测细胞增殖情况，流式细胞术检测细胞表面标志表达情况。结果：双酶切和测序结果表明克隆了1242 bp的人GATA-1全长CDS序列，经Western印迹检测，GATA-1在293T细胞中获得瞬时表达；获得了人GATA-1稳转细胞株，对稳转细胞株的检测表明，GATA-1对HFF细胞增殖具有一定的抑制作用，但对HFF细胞形态及细胞表面标志CD90、CD29的表达无明显影响。结论：构建了人GATA-1慢病毒表达载体及其稳转细胞株，GATA-1蛋白对间质细胞的增殖有一定的抑制作用。
목적：：구건인GATA-1전장과표체재체，대기진행과표체，병초보탐토기재인비조혈세포（인제정맥내피세포、인성섬유세포）중적생물학작용。방법：이인제대혈cDNA위모판，채용고보진PCR획득GATA-1적CDS서렬，련접지pGEM-T Easy재체，구건pBPLV-GATA1만병독표체질립，병대상술재체진행NheⅠ、XbaⅠ쌍매절화측서감정，감정정학후，재293T세포중진행만병독포장，농축병독후，대아문원대분리배양적인제정맥내피세포화성섬유세포진행만병독감염，채용류식분선대양성목적세포진행순화，양성세포확증후진행Western인적감정；이인포피성섬유세포（HFF）위연구대상，재광학급형광현미경하관찰세포형태변화정황，CCK-8법검측세포증식정황，류식세포술검측세포표면표지표체정황。결과：쌍매절화측서결과표명극륭료1242 bp적인GATA-1전장CDS서렬，경Western인적검측，GATA-1재293T세포중획득순시표체；획득료인GATA-1은전세포주，대은전세포주적검측표명，GATA-1대HFF세포증식구유일정적억제작용，단대HFF세포형태급세포표면표지CD90、CD29적표체무명현영향。결론：구건료인GATA-1만병독표체재체급기은전세포주，GATA-1단백대간질세포적증식유일정적억제작용。
Objective: To construct human GATA-1 full length expression vector, and overexpress the recombi-nant GATA-1, study its biological effects on human nonhematopoietic cells including human umbilical vein endo-thelial cells(HUVEC) and human foreskin fibroblasts(HFF). Methods: The high fidelity PCR amplication of hu-man GATA-1 CDS sequence was done with human cord blood cDNA as templates. The GATA-1 CDS fragment was linked in pGEM-T Easy vector, and digested into pBPLV-GFP lentivirus vector. The recombinant clones were all identified by double enzyme digestion with NheⅠand XbaⅠ. Lentivirus package was done in 293T cells when the sequencing result of pBPLV-GATA1 was correct. FACS was used to purify the GFP+ cells from the bulk trans-fected cells. Western blot was carried out after the GFP+ cells were expanded. In the study, HFF cells were main-ly studied. Cell morphology was observed under light microscope and fluorescence microscope. CCK-8 assay was used to detect the proliferation of HFF cells. Flow cytometry was used to examine the expression of cell surface protein CD90 and CD29 in HFF cells. Results: Human GATA-1 full length CDS sequence was cloned at a length of 1242 bp. pBPLV-GATA1 lentivirus vector was transiently overexpressed in 293T cells confirmed by Western blot. The stable HFF cells overexpressing human GATA-1 were established. The results suggested that hu-man GATA-1 protein has some inhibiting effects on HFF cell growth but has no effects on the expression of CD90 and CD29. Conclusion: Lentivirus vector encoding human GATA-1 full length and corresponding stable overexpression cells were constructed. Human GATA-1 protein has some inhibiting effects on HFF cell growth.