Establishment of Embryonic Stem Cell Lines from C57BL/6J Mice and Generation of Chimeras

Four embryonic stem (ES) cell lines, designated CE1, CE2, CE3 and CE4, were isolated from C57BL/6J blastocysts. The ratio of normal diploid composition of these cell lines is above 70%. To examine the differentiation potential of the ES cells, the CE2 cells were injected subcutaneously into syngenic mice, and many kinds of differentiated cells were observed on the sections of the teratoma derived from this ES cell line. On the other hand, to test the chimeric ability of the ES cells, the CE2 cells were microinjected into the blastocysts of ICR mice, and a chimera was obtained among living pups. These results show that CE2 ES cells are pluripotent stem cells, which can differentiate into many kinds of cell types, and can be used as a cell system for further research.

Stable EGFP Gene Expression in C6 Glioma Cell Line after Transduction with HIV-1-based Lentiviral Vector

Objective： To establish a stable C6/EGFP glioma cell transfected with the human immunodeficiency virus line for studies on glioma. Methods： The C6 glioma cell line was type I （HIV-1） based lentivirus vector containing two enhancer-promoters CMV and EF1α. Enhanced green fluorescent protein （EGFP）-positive C6 cells were sorted out by fluorescence-activated cell sort. Expression of EGFP was observed by fluorescent microscopy. EGFP gene in C6 genome was assessed by Polymerase chain reaction （PCR） and DNA sequencing. Original and transfected cells were compared biologically and cytomorphologically. Results： Lentivirus vector transfection produced up to 40% EGFP-positive cells. After fluorescence-activated cell sort selection, a pure cell line C6/EGFP was established. PCR and DNA sequencing revealed integration of EGFP gene in C6 cell genome. Analysis of cell characteristics revealed no difference between transfected and original cells. Conclusion： A C6/EGFP cell line expressing EGFP as a marker is established, in which the EGFP gene is integrated into the genome. This cell line can be served as a promising tool for further basic research and gene therapy studies.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

小鼠胸腺上皮细胞株MTEC_B自发产生低水平IL－6

本室所建立的小鼠胸腺上皮细胞株ＭＴＥＣＢ以５．０×１０５细胞／ｍｌ的浓度在含１０％小牛血清的ＤＭＥ培养液中，５％ＣＯ２３７℃条件下，培养６ｄ，离心收集上清液。以ＩＬ－６依赖细胞株采用ＭＴＴ法对ＭＴＥＣＢ的上清液进行ＩＬ－６活性检测。结果发现ＭＴＥＣＢ能自发产生低水平的ＩＬ－６，其活性单位为１９．２Ｕ／ｍｌ。ＭＴＥＣＢ的ＩＬ－６产生量低于文献报道的其他胸腺上皮细胞株，说明我们建立的胸腺上皮细胞株有新的特点，反映出胸腺上皮细胞存在高度异质性。

The labeling of C57BL/6j derived embryonic stem cells with enhanced green fluorescent protein

Objective To labele MESPU35, a embryonic stem (ES) cell line derived from C57BL/6j mouse, with enhanced green fluorescent protein (EGFP) for further application.Methods The EGFP gene was controlled by the hybrid CA promoter/enhancer (CMV enhancer/ chicken beta-actin promoter/ beta-actin intron) to construct the vector of the transgene, pCA-EGFP. The vector was transfected into MESPU35 by electroporation.Results We generated EGFP expressing ES cells demonstrating normal properties. The green fluorescence of EGFP expressing cells was maintained in propagation of the ES cells for more than 30 passages as well as in differentiated cells. Cultured in suspension, the 'green' ES cells aggregated, and formed embryoid bodies maintaining the green fluorescence at varying developmental stages. The 'green' embryoid bodies could expand and differentiate into various types of cells, exhibiting ubiquitous green fluorescence. Conclusions The hybrid CA promoter/enhancer used to control the EGFP expressing ES cells, resulted in more intense and ubiquitous activity. The EGFP transfected cells yield bright green fluorescence, which can be visualized in real time and in situ. In addition, the ES cells, MESPU35, are derived from C57BL/6j mice, which are the most widely used in oncology, physiology and genetics. Compared to 129 substrains, C57BL/6j mice avoid a number of potential problems apparent in the other strains.

Aquaporin 1 overexpression may enhance glioma tumorigenesis by interacting with the transcriptional regulation networks of Foxo4,Maz,and E2F families

Background The glioblastoma has served as a valuable experimental model system for investigating the growth and invasive properties of glioblastoma.Aquaporin-1(AQP1)in facilitating cell migration and potentially contributing to tumor progression.In this study,we analyzed the role of AQP1 overexpression in glioblastoma and elucidated the main mechanisms involved.Methods AQP1 overexpression recombinant vector was introduced into C6 rat glioma cells to construct an AQP1 overexpression C6 cell line,and its effect on cell viability and migration ability was detected by MTT and Transwell.RNA was extracted by Trizol method for gene sequencing and transcriptomics analysis,and the differentially expressed genes(DEGs)were enriched for up-and downregulated genes by Principal component analysis(PCA),and the molecular mechanism of AQP1 overexpression was analyzed in comparison with the control group using the NCBI GEO database.Statistical analysis was performed using Mann-Whitney paired two tailed t test.Results The cell viability of AQP1-transfected cell lines increased by 23%and the mean distance traveled increased by 67%compared with the control group.Quantitative analysis of gene expression showed that there were 12,121 genes with an average transcripts per million(TPM)value greater than 1.DEGs accounted for 13%of the genes expressed,with the highest correlation with upregulated genes being FOXO4 and MAZ,and the highest with down-regulated genes being E2F TFs.Conclusions AQP1 may be implicated in glioma formation by interacting with the transcriptional regulation networks involving the FOXO4,MAZ,and E2F1/2.These findings shed light on the potential significance of AQP1 in glioma pathogenesis and warrant further investigations to unravel the underlying molecular mechanisms.

乳酸脱氢酶与蚊虫对溴氰菊酯抗性相关性的研究

目的研究乳酸脱氢酶(LDH)与蚊溴氰菊酯抗性之间的关系。方法应用RT-PCR及cDNA末端快速扩增(rapid amplification of cDNA end,RACE)技术扩增白纹伊蚊LDH全长基因;用实时荧光定量PCR检测敏感和抗性蚊C6/36细胞中LDH基因的表达水平;用乳酸脱氢酶活性检测试剂盒检测敏感和抗性蚊细胞中LDH酶活性。结果 LDH基因全长共1831 bp,其中ORF为996 bp,共编码331个氨基酸。该基因与埃及伊蚊、黑腹果蝇、小家鼠和人的乳酸脱氢酶的同源性分别为89%、72%、66%和65%。实时荧光定量PCR结果表明,LDH基因在抗性蚊细胞中高表达,为敏感蚊细胞的1.87倍(P<0.05)。LDH酶活性检测结果显示,LDH在敏感蚊细胞中的酶活性范围为1～95 U/gprot,均值为40 U/gprot;在抗性蚊细胞中的酶活性范围为20～150 U/gprot,均值为75 U/gprot(n=15,P<0.01)。结论抗性蚊细胞中的LDH表达量和酶活性均高于敏感蚊细胞,提示LDH可能与蚊溴氰菊酯抗性相关。

丙型肝炎阳性血清直接感染HepG2细胞的初步研究

目的 :用丙型肝炎 (丙肝 )阳性血清 (抗HCV阳性及HCVRNART PCR阳性 )感染HepG2细胞 ,以期建立稳定的HCV感染的细胞模型。方法 :采用丙肝阳性血清连续感染法感染HepG2细胞 ,用逆转录多聚酶链反应检测传代HepG2细胞内HCVRNA正链和负链 ,电镜观察感染后的细胞内的病毒颗粒 ,免疫组化方法检测感染后的细胞内丙肝病毒蛋白抗原 (HCVNS5、HCVcapsid)的存在。结果 :感染后传代的第一代至第三代细胞内检出HCVRNA正链及负链 ;在感染后传代的细胞内发现球形、密集分布的类丙肝病毒颗粒 ,直径为 30～ 6 0nm ;免疫金银染色发现部分细胞内有聚集的银染颗粒 ;免疫电镜检查在细胞胞浆的囊泡样结构中发现聚集的金颗粒。结论 :HCV可在HepG2细胞内复制 ,可形成病毒颗粒 ,可随细胞传代。

纳秒级陡脉冲电场对荷瘤小鼠乳腺癌的杀伤作用研究

目的以BALB／c荷瘤小鼠为对象，探讨纳秒级陡脉冲电场对在体小鼠乳腺癌的杀伤效应。方法取24只皮下接种小鼠乳腺癌细胞EMT-6的BALB／c荷瘤鼠，分为实验组和对照组，每组12只。在麻醉状态下，采用场强为700V／cm、脉宽为70μs、上升时间为57ns、占空比为50％的陡脉冲电场，利用自制的电极针插入实验组小鼠肿瘤两端进行电刺激处理，脉冲个数为100个，能量为9．75J，有效作用时间仅为7ms。对照组不进行电刺激，其他处理相同。隔天测量动物体质量、肿瘤大小，并在处理后第1、3、5、7天分别取肿瘤组织做病理切片。结果与对照组相比，实验组小鼠肿瘤生长速度较慢，特别在处理后1-3d内肿瘤几乎停止生长。组织病理结果显示电刺激处理后肿瘤组织有明显的凝固性坏死表现，组织水肿明显，处理后第3天肿瘤组织中出现大量空泡样结构，血管损伤在早期即出现。结论在给定电场条件下，陡脉冲电场对肿瘤生长有一定的抑制作用。要想达到更理想的肿瘤治疗效果，需对陡脉冲剂量进行进一步的研究。