【作者】 韩崇旭；

【导师】 张锡然；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2005， 博士

【摘要】 在体外用GM-CSF和IL-4刺激人胚胎骨髓间质干细胞(MSCs)向树突状细胞(DCs)分化的过程中,我们发现了一种新的细胞,经克隆化培养和鉴定,具有肿瘤特性,命名为F6细胞株。本文旨在研究F6肿瘤细胞的发生机制。 我们应用下列方法(1)采用荧光差异显示(FDD)技术寻找MSCs诱导致瘤(F6)的关键基因。(2)采用实时定量PCR(RT-PCR)技术、Western blotting、PI染色流式细胞仪鉴定F6细胞和间质干细胞中相关基因的表达。(3)采用RT-PCR,Western blotting,免疫组化,荧光显微镜,共聚焦显微镜,研究nucleostemin基因在F6细胞和cos7细胞的特性。(4)利用RNAi、Western blotting、流式细胞仪技术,探讨nucleostemin对F6和U937细胞增殖的影响。(5)利用Real-time RT-PCR定量检测F6致裸鼠肿瘤瘤组织及人肺癌患者肿瘤组织的nuleostemin、cyclinⅠ和siva基因的表达特性。研究取得以下结果: (1)荧光差异显示成功地分离了胚胎MSCs和F6肿瘤细胞之间的不同基因片段,TA克隆得到15个EST片段,GeneBank序列比对分析表明,这些片断均与相关基因片断同源,其中C356,G392和G383分别与nucleostemin,cyclin Ⅰ和Siva基因同源(95%,97%,100%)。 (2)从LTEP-a-2细胞中克隆了nucleostemin的全长基因(1650bp,GenBank接受序号:AY825265),并且在大肠杆菌中表达GST-nucleostemin蛋白。观察了它在F6和其它4种人肿瘤细胞株(LTEP-a-2,U937,SW480 and 95D)中的表达特性。同时,用pcDNA3.1(+)-GFP-nucleostemin转染COS7细胞,在4-20周内,其细胞体积和核都增大,细胞呈巨细胞样改变,提示细胞分裂时nucleostemin影响细胞微管和微丝系统。Western blotting表明nucleostemin在F6,LTEP-a-2,U937,SW480 and 95D的细胞核和细胞浆中的表达水平比在MSCs和COS-7中明显增高(<0.01-0.001,n=3)。同免疫组化染色的结果一致。 (3)SCID裸鼠实验表明在注射F6细胞4周后产生肿瘤。实时定量PCR结果更多还原

【Abstract】 A novel tumor cell line, named F6, was acquired during the processes of inducing human embryonic bone marrow mesenchymal stem cells (MSCs) to differentiate into dendritic cells (DCs) by GM-CSF and IL-4 in vitro. The mechanism of tumorigenesis of F6 cells needs to be address .We used the methods as following:(1)Fluorescent differential display (FDD) was applied to identify the difference of key genes expression between F6 cells and MSCs.(2)Quantitative real-time RT-PCR , Western blotting, PI staining flow cytometry were applied to identify the expression of related genes between F6 cells and MSCs.(3)RT-PCR, Western blotting, immunocytochemical staining, fluorescent microscopy and confocal laser scanning microscopy were applied to study the expression characteristics of nucleostemin in F6 and cos7 cells.(4) RNAi, Western blotting, real-time RT-PCR, flow cytometry were used to investigate the effect of nucleostem on proliferation of in F6 and U937 cells.(5) Real-time RT-PCR were applied to study the expression characteristics of nucleostemin, cyclin I and siva genes in F6 tumor tissue and tumor tissue of lung cancer patients.The results were as following:(1) Fifteen cDNA fragments were attained, and three of them (C356, G392 and G383) were highly up-regulated in F6 cells compared with MSCs. By comparing to the GeneBank databases, we found that C356, G392 and G383 fragments were 95%, 97% and 100% homologous to nucleostemin, Cyclin I and siva respectively.(2) The full-length gene (1650bp) of nucleostemin from LTEP-a-2 cells was cloned (GenBank accession number: AY825265), and GST-nucleostemin protein was expressed in E. coli. The expression characteristics of nucleostemin in F6 cell and other human cancer cell lines (LTEP-a-2, U937, SW480 and 95D ) were investigated. The results showed that the transfection with pcDNA3.1(+)-GFP-nucleostemin for certain period of time (4-20weeks) promoted augmentation of cell size and nuclei multiplication, and the cells converted into giant cell-like cells, and suggested that the system of microbule and microfilament was destructed. Western blotting results showed that the expression levels of nucleostemin in the nuclei and cytoplasm of cancer cell lines, e.g., F6, LTEP-a-2, U937, SW480 and 95D, were higher than those in MSCs and COS-7 cells. The levels of nucleostemin in F6 cells were notably high. Similar results were also obtained in immunohistochemical staining nucleostemin.(3) The levels of nucleostemin gene expression were also detected by real-time PCR in F6 tumor tissues which were obtained from SCID nude mice at the 4th, 6th and 7th week after injection of F6 cells, and in lung tissues from five patients with lung cancer. The results showed that the expression of nucleostemin gene increased significantly in F6 tumor tissues and lung cancer tissues(4) FDD results showed that the expression of Cyclin I and Siva genes were highly up-regulated in F6 cells compared with MSCs. In animal model, neoplasia in SCID nude mice was detected four weeks after injection with F6 cells. The results of real-time RT-PCR indicated that in tumour tissues obtained from the nude mice at the 4th, 6th and 7th week after the injection with F6 cells, the expression of Cyclin I and Siva increased significantly with the accreting volume of tumour. In addition, analysis of gene expression levels in lung tissue from five patients with lung cancershowed that Cyclin I and Siva gene expressions were higher in cancer tissue than those in adjacent nonmalignant lung tissue.(5) The RNAi shuttle plasmid (pShuttle-plp2, pShuttle-p3p4) against NS and the recombinated adenoviroplasm (backbone-plP2, backbone-p3P4) with the ability of packing adenovirus were constructed. The expression of NS in transfected F6 and U937 with backbone-plP2, backbone-p3P4 obviously decreased (p<0. 01, n=3) . The expression of NS in transfected U937 cells notably decreased about 2-3times (p<0. 01, n=3)o The result of cell cycle analysis indicated that the number of cells transfected with backbone-plP2, backbone-p3P4 in S state was more than those in control cells transfected with backbone-pshuttle (43. 33, 43. 33% VS 32. 07%) . In addition, analysis of the cell cycle in cells transfected backbone-plP2, backbone-p3P4 showed that d/Go state decreased in control cells transfected by backbone-pshuttle (44.89, 45.00% VS 54.74%) . The number of U937 cells transfected with backbone-PlP2, backbone-P3P4 increased from 1000 per well to 57778 per well and 38333 per well respectively after 6 days. The number were 47% and 32% of those of control cells (backbone-pshuttle transfected cells, 121250 per well) .These findings support the idea that the increase of multiple genes including nucleostemin, cyclin I and siva expression in F6 may play a role in both tumorigenesis and transforming human embryonic bone marrow mesenchymal stem cells into F6 tumor cells. The further work will be done in the future.更多还原