人Nanog基因的克隆及其在COS-7L细胞中的表达

目的 :克隆人Nanog基因 ,构建其真核表达载体 ,并观察其在哺乳动物细胞COS 7L中的表达。方法 :利用HE2 93细胞的人基因组DNA为模板 ,以LA PCR技术 ,扩增Nango的基因 ,定向克隆到带Flag标签的pCMV载体中 ,测序后 ,挑选序列正确的真核表达质粒pFlag Nanog转染COS 7L细胞。用抗Flag标签的抗体 ,进行Westernblot和间接免疫荧光染色法检测Nango蛋白的表达。结果 :从人基因组DNA中克隆到序列正确的Nanog全长编码序列。所构建的Nanog质粒在COS 7L细胞中获得高效表达。结论 :人Nanog基因的克隆、真核表达载体的构建及在COS 7L中的表达均获得成功 ,为进一步研究其功能 ,尤其是探讨其在神经干细胞中的作用奠定了基础。     

胚胎干细胞关键因子Nanog在胶质瘤中的表达及意义

目的 探讨胚胎干细胞关键因子Nanog在人胶质瘤中的表达及意义。方法 通过免疫荧光双标技术,分析40例胶质瘤组织内胚胎干细胞关键因子Nanog与肿瘤干细胞相关基因Nestin、CD133及胚胎干细胞全能性相关基因Oct4 的共表达情况,并通过RT-PCR、Western blotting技术分析胶质瘤组织内Nanog与脑胶质瘤恶性程度之间的关系。结果 Nanog在胶质瘤组织中的表达随着胶质瘤病理级别增高而升高,而且超过50%的Nanog阳性细胞同时表达Nestin和CD133。95%以上的Nanog阳性细胞都同时表达胚胎干细胞全能性相关基因Oct4。结论 胶质瘤组织中Nanog多表达在肿瘤干细胞中,与胶质瘤的恶性程度呈正相关,在胶质瘤的发生、发展过程中起着重要作用,为研究胶质瘤的起源及胶质瘤的诊断和预后判断提供帮助。     

八聚体结合转录因子4和胚胎干细胞转录因子对胰腺癌干细胞体内干性特征的影响

目的 观察八聚体结合转录因子4(Oct4)和胚胎干细胞转录因子(Nanog)对胰腺癌干细胞(PCSCs)在体内干性特征的影响.方法 利用流式细胞分选技术分选人胰腺癌细胞-1(PANC-1)中CD44+ CD24+上皮特异性抗原(ESA)+胰腺癌干细胞,通过慢病毒载体介导特异性短发卡RNA (shRNA)沉默胰腺癌干细胞中的Oct4、Nanog基因,并通过Western blot检测干扰效率;将Oct4、Nanog沉默的胰腺癌干细胞、未沉默的胰腺癌干细胞及胰腺癌PANC-1细胞株分别接种于BALB/c裸鼠皮下及腹腔,建立裸鼠异位移植瘤模型,观察沉默Oct4、Nanog对胰腺癌干细胞体内致瘤性、侵袭性及耐药性的影响.结果 PANC-1细胞株中CD44+ CD24+ ESA+胰腺癌干细胞占细胞总数的0.1％ ～0.8％;shRNA沉默Oct4和Nanog的效率分别是(46.00 ±0.08)％和(78.00±0.12)％;体内实验结果显示,沉默Oct4、Nanog基因后,裸鼠的致瘤性和肿瘤转移性显著下降,对吉西他滨耐药性减弱.结论 沉默Oct4、Nanog表达可抑制裸鼠体内胰腺癌干细胞的干性特征.     

CD24 and Nanog identify stem cells signature of ovarian epithelium and cysts that may develop to ovarian cancer

Ovarian cancer is the most lethal gynecological cancer. There is a general debate whether ovarian cancer is an intrinsic or an imported disease. We investigated whether in normal morphological appearance and in early stages of ovarian tumorgenesis typical cancer cell markers such as CD24 and Nanog are expressed. In 25% of normal appearing ovaries of post-menopausal women there was co-localization of CD24 and Nanog in the walls of the ovarian cysts, leaving the epithelial cells on the surface of these ovaries free of Nanog or CD24 expression. In benign ovarian tumors 37% of specimens were positive to CD24 and Nanog labeling while 26% of them were localized in the cyst walls. In contrast, in serous borderline tumors 79% specimens were labeled with CD24, 42% of them were localized in cysts and in 32% of them showed co-localization with CD24 and Nanog was evident: the rest were labeled in the ovarian epithelial cells. In serous ovarian carcinomas 81% specimens were labeled with CD24 antibodies. In 45% of them co-localization with Nanog was evident in the bulk of the cancerous tissue. In mucinous carcinomas no labeling with CD24 or Nanog was evident. In view of the synergistic effect of CD24 and Nanog expressed in malignant cancer development in other systems, it is suggested that such an analysis can be valuable for early detection of ovarian cancer. Moreover, the abundance of these markers in cysts in the development of ovarian cancer may suggest that they present an intrinsic source of the development of the highly malignant disease. Finally, since CD24 is exposed on the surface of the cancer cells, it may be highly beneficial to target these cells with antibodies to CD24 conjugated to cytotoxic drugs for more efficient treatment of this malignant disease.     

胶质瘤组织中Nanog基因的表达及其意义

目的探讨Nanog基因在不同病理级别胶质瘤组织中的表达及其意义。方法采用免疫组织化学染色法和逆转录酶-聚合酶链反应（RT-PCR）方法从蛋白和mRNA水平检测50例不同病理级别胶质瘤组织和7例正常脑组织标本中Nanog的表达。结果 Nanog蛋白在胶质瘤组织WHOⅡ级、Ⅲ级、Ⅳ级的阳性细胞率分别为18.3%±9.6%、37.9%±19.0%、53.4%±19.8%,差异有统计学意义（F=14.918,P=0.000）;NanogmRNA的相对含量在胶质瘤组织WHOⅡ级、Ⅲ级、Ⅳ级中分别为0.1824±0.0310、0.3730±0.0961、0.4594±0.0453,差异有统计学意义（F=65.901,P=0.000）,Nanog蛋白和mRNA表达强度随着病理级别增高而升高,而在正常脑组织中均未见表达。结论 Nanog在胶质瘤组织中高表达,其表达强度与病理级别密切相关,为进一步研究其与肿瘤干细胞关系及其在胶质瘤的生物学行为中所发挥的作用奠定基础。     

胚胎干细胞关键因子Nanog在膀胱癌中的表达及与肿瘤干细胞的关系

目的:检测胚胎干细胞(Embnyonie stem cell,ES)自我更新的关键调节因子Nanog在人膀胱癌组织中的表达,探讨Nanog的表达在膀胱癌发生发展中的意义及与肿瘤干细胞的关系.方法:采用半定量RT-PCR检察32例膀胱癌Nanog的表达,32例癌旁非癌膀胱组织,用免疫组织化学方法在蛋白质水平检测Nanog的表达.结果:Nanog在膀胱癌中的表达率为84.38%(27/32).而在非癌膀胱组织低水平(P<0.01)表达(5/32),免疫组织化学方法分析结果显示:Nanog主要的集中在肿瘤组织的细胞胞核,在非癌组织中没有或低免疫反应性,Nanog表达的阳性细胞率及强度与膀胱癌的分级、复发和转移无明显相关性(P>0.05)>.结论:膀胱癌中有少量细胞表达伞能干细胞标志物Nanog,这些细胞很可能是膀胱癌干细胞,这将为揭示膀胱肿瘤干细胞的存在并分离和鉴定膀胱肿瘤干细胞提供依据,Nanog在膀胱癌中的表达表明胚胎基因与肿瘤的发生发展关系密切.