CD44剪接变异体在乳腺癌中的表达

目的观察乳腺癌细胞系及原发性乳腺癌组织中CD44剪接变异体种类及其表达情况。方法 RT-PCR法检测乳腺癌细胞系MDA-MB-231、MDA-MB-435和20例原发性乳腺癌组织中CD44变异体表达情况,分析其与临床病理参数之间的关系。结果 RT-PCR分析显示,在已知的8种剪接变异体、乳腺癌细胞系MDA-MB-231、MDA-MB-435和所检测的全部乳腺癌组织标本中,检测到CD44剪接变异体1、2、3、4、5、6、8,其中CD44剪接变异体4、5表达水平较高。CD44剪接变异体与临床病理参数分析显示CD44剪接变异体与患者年龄、TNM分期、淋巴结转移、ER、PR表达无关。CD44剪接变异体1和CD44剪接变异体2 mRNA的高表达与较小的肿瘤直径有关;CD44剪接变异体4的mRNA高表达与组织学高级别有关;CD44剪接变异体2、6的mRNA高表达与Her-2低表达相关;CD44剪接变异体5的mRNA低表达和Her-2高表达相关。结论 MDA-MB-231、MDA-MB-435及所检测的乳腺癌组织标本中,CD44剪接变异体为异质性表达,以标准型CD44表达水平最高。     

CD45分子选择性剪接亚型的功能和调节

白细胞共同抗原CD45分子由一类结构相似、相对分子质量较大的跨膜蛋白组成,其胞浆区段具有蛋白质酪氨酸磷酸酶的作用,因而在细胞的信号传导及功能调节中发挥重要作用。CD45mRNA外显子的不同剪切拼接方式,能编码产生CD45RA、CD45RB、CD45RC、CD45RO等多种蛋白亚型。CD45亚型分子的表达与T淋巴细胞功能相关,且随着细胞的分化、发育和激活,分子亚型可发生转换。近年研究显示,CD45基因DNA的表观遗传学修饰、DNA结合的蛋白以及RNA剪接位点的突变等多种因素可以调控CD45不同外显子的剪接,从而调节淋巴细胞表面CD45分子不同亚型的表达。调控CD45外显子在成熟转录本的保留或者缺失的因素,也可能最终调控淋巴细胞CD45分子亚型的表达以及淋巴细胞的功能,并与临床自身免疫性疾病、血液病、肿瘤等疾病的发病相关。     

5氟尿嘧啶上调干细胞标记物CD133在结肠癌细胞中的表达

目的: 研究通过5氟尿嘧啶(5-FU)对结肠癌干细胞标记物CD133表达的影响,探讨5-FU对结肠癌干细胞的影响。方法: 用流式细胞仪检测CD133在结肠癌细胞株表面的表达, 磁珠细胞分离的方法分离结肠癌细胞株DLD1中CD133阳性和阴性的细胞群,细胞克隆形成实验检测2群细胞的自我更新能力,新型四唑氮盐方法(MTS)检测2群细胞对5-FU敏感性的差异,qPCR方法检测5-FU处理结肠癌细胞后CD133mRNA水平的变化。结果: 结肠癌细胞株DLD1、HT29、SW480、HCT116、Lovo、RKO细胞表面CD133的表达率分别为30.20%、82.00%、0.34%、91.80%、85.30%、0.28%。DLD1细胞中以CD133为标记有2群明显的细胞,MACS方法分离后阳性细胞群中CD133为87.21%±5.33%, 而阴性细胞群中阴性细胞的比例为84.30%±4.65%;CD133阳性的细胞与未分离及CD133阴性细胞相比,克隆形成能力强(46.33%±4.44% vs 31.00%±2.00%,P<0.05),对5-FU的敏感性下降20%,P<0.01。在DLD1和HT29细胞中,5-FU 1 mg/L上调CD133mRNA水平的表达,从1升为1.684±0.012(P<0.01)、HT29细胞从30.702±0.284升为49.379±0.460(P<0.01)。结论: 与CD133阴性细胞相比CD133阳性细胞克隆形成能力强,对5-FU的敏感性下降;5-FU上调干细胞标记物CD133mRNA水平的表达,CD133阳性的结肠癌干细胞在5-FU的治疗过程中被富集。     

恶性肿瘤干细胞标记物CD34在鼻咽癌细胞系的表达

BACKGROUND:Previous research have confirmed that CD34 is closely related to oncogenesis, progress, recurrence, metastasis and drug-resistance of various cancers, but its role in nasopharyngeal carcinoma remains unclear. OBJECTIVE:To sort cells positive and negative for CD34 in nasopharyngeal carcinoma cell lines and to detect cell proliferation and migration. METHODS:Expressions of CD34 in nasopharyngeal carcinoma cell lines 5-8F, 6-10B, CNE1 and CNE2 were detected by flow cytometry. And CD34+ and CD34^- cells were sorted based on cell surface markers for purity identification. Afterwards, proliferation and migration of CD34⁺ and CD34^- cells were detected by MTT assay, colony-formation assay and scratch assay. RESULTS AND CONCLUSION:All four nasopharyngeal carcinoma cell lines expressed CD34 in 0.1%-0.2%, and the level of CD34 was closely related to the cell growth density. The purity of CD34⁺ cell was more than 98% in the sorted CD34⁺ cell populations, but no CD34⁺ cells were found in the sorted CD34^- cell populations. At 1, 3, 5 and 7 days the proliferation rate of CD34⁺ cell, populations was significantly higher than that of CD34^- cells (P < 0.05). Consistently, the colony-formation efficiency of CD34⁺ cell was significantly higher than that of CD34^- cells (P < 0.05). Moreover, CD34⁺ cells migrated significantly faster than CD34^- cells by scratch assay (P < 0.05). In conclusion, CD34⁺ cells cultured in vitro display higher proliferation and migration capacities, indicating that CD34⁺ cells have the potential of nasopharyngeal carcinoma stem cells.     

选择性剪接在低氧应答中的调控作用

细胞为适应低氧环境,其相关基因的表达方式发生了改变,其中选择性剪接在低氧应答调控过程中起到了重要的作用.低氧诱导因子介导的低氧应答信号通路在机体适应低氧环境过程中起到了十分重要的作用,低氧诱导因子剪接体通过此通路调控红细胞生成、血管生成、糖酵解等过程.而抑制性PAS蛋白质、脯氨酸羟化酶、促血管生长因子、芳香羟受体核转运蛋白剪接体则通过其它通路进行调控.选择性剪接不仅在低氧应答中起重要作用,而且与阿尔茨海默病、动脉粥样硬化、癌症等常见人类疾病相关.     

CD44V6在不同转移能力人大肠癌细胞系中的表达

目的：探讨大肠癌细胞ＣＤ４４Ｖ６的表达与其转移潜能之间的关系。方法：应用原位分子杂交、免疫组化、免疫荧光定量分析技术，从蛋白水平到分子水平、从定性到定量，分析不同转移能力大小肠癌细胞ＣＤ４４Ｖ６的表达。结果：人大肠癌ＨＴ２９、ＬｏＶｏ细胞系均有ＣＤ４４Ｖ６变异体ｍＲＮＡ和蛋白表达。在ｍＲＮＡ和蛋白水平，高转移能力的ＬｏＶｏ细胞表达强度均高于低转移能力的ＨＴ２９细胞。实验还发现：ＬｏＶｏ细胞系内不同     

7株小鼠胸腺基质细胞系诱导裸鼠骨髓干细胞表达CD4及CD8分子

为研究胸腺微环境在Ｔ细胞发育中的作用，我室在体外建立了７株小鼠胸腺基质细胞系，命名为ＭＴＥＣＩ～ＭＴＥＣ７。通过初步分离得到裸鼠骨髓富含干细胞的细胞群，表面ＣＤ４及ＣＤ８分子均为阴性。将分离的骨髓干细胞与胸腺基质细胞共育３天后，经双色荧光抗体染色，ＦＡＣＳ分析发现胸腺基质细胞可诱导裸鼠骨髓干细胞表达ＣＤ４ＣＤ８分子。ＭＴＳＣ－ＳＮ及ＭＴＳＣ主要诱导的是ＣＤ４＋ＣＤ８－细胞，部分ＣＤ４＋ＣＤ８＋细胞，而ＣＤ４－ＣＤ８＋细胞极少，这种诱导特点可能和基质细胞系的类型有关。     

不同选择剪接形式的小鼠Era在大肠杆菌中的表达及纯化

目的:对不同剪接形式的小鼠era基因(mera)进行克隆、原核表达,并进行纯化,检测抗人Era蛋白抗体对于两种剪接形式鼠Era蛋白的特异性,为以后鼠Era蛋白的研究奠定基础。方法:采用两种剪接形式era基因的MBP融合表达载体(pMAL-meraW,pMAL-meraS),在大肠杆菌中进行表达,对其进行纯化,并应用Westernblot鉴定了抗人Era蛋白抗体的特异性。结果:原核表达的MBP-mEraW、MBP-mEraS融合蛋白经过薄层扫描后发现其分别占菌体总蛋白的17%、19%;纯化后的融合蛋白纯度为67%和61%;用抗人Era蛋白抗体进行Westernblot发现抗体特异性较好,适合两种剪接形式的鼠Era蛋白的检测。结论:利用原核系统高效表达了不同剪切形式的鼠era基因,并检测了兔抗人Era蛋白抗体对不同剪接形式鼠Era蛋白的特异性,为以后对鼠era基因的研究奠定了基础。     

OCT4异构体在人胚胎干细胞和间充质干细胞中的表达

目的比较OCT4异构体(OCT4A、OCT4B、OCT4B1)及其调控因子在人胚胎干细胞(hESC)和人间充质干细胞(hMSC)中的表达。方法利用RT-PCR、免疫荧光染色、流式细胞分析及体内/外分化实验,鉴定hESC及hMSC的生物学特性;应用real-time PCR、Western blot和流式细胞分析比较OCT4异构体及其转录因子NANOG,SOX2和mRNA结合蛋白LIN28在hESC及hMSC中的表达水平。结果 OCT4异构体mRNA在hESC和hMSC中均有表达,在hESC中的表达显著高于在hMSC中,并以OCT4A的差别最为显著(P0.01);在蛋白水平,hESC表达OCT4A和OCT4B-256aa,hMSC不表达OCT4异构体蛋白。hESC高表达OCT4的调控因子NANOG、SOX2和LIN28;hMSC低表达SOX2,不表达NANOG和LIN28。结论 NANOG、SOX2和LIN28调控OCT4的表达,OCT4异构体在hESC和hMSC中的表达差异提示其可能是不同发育阶段干细胞自我更新和分化潜能等方面差别的主要因素之一。     

Hyaluronate binding assay study of transfected CD44 V4–V7 isoforms into the human gastric carcinoma cell line SC-M1

The potential human metastasis molecule CD44 and its isoforms V5 and V6 are overexpressed in human gastric carcinoma. Among the numerous extracellular matrix components, hyaluronate, a CD44 ligand, is of increasing interest in relation to its role in cancer cell development and invasion. By using the dynabead separation method, the SC-M1 cell line was separated into V5 and V6 isoform-positive and -negative populations. The V5 and V6 isoform-negative populations exhibited significantly higher hyaluronate binding activity than the corresponding positive cells. The hyaluronate binding activity of V5 and V6-positive cells could be restored by pretreatment with anti-CD44 V5 and V6 monoclonal antibodies (MAbs). In addition, transfection of an expression vector containing CD44 V5 and V6 into V5 and V6-negative cells decreased their hyaluronate binding activity to the levels of CD44 V5 and V6-positive cells. Cells transfected with V5 and V6 recovered their hyaluronate binding activity after pretreatment with MAbs against V5 and V6. These data suggest that cell adhesion involving hyaluronate can be regulated by multiple mechanisms, one of which involves alternative splicing of CD44 isoforms. © 1998 John Wiley & Sons, Ltd.     

Isolation and characterization of CD105+/CD90+ subpopulation in breast cancer MDA-MB-231 cell line

Background: The epithelial-mesenchymal transition (EMT) generates cells with properties of stem cells, if that happened, the stem cell should be with mesenchymal property. This study aimed to identify a group of cells with mesenchymal stem cell (MSC)-like characteristics in breast cancer bone metastatic cell line MDA-MB-231, moreover, the relevance between breast cancer stem cells and the EMT was observed. CD105 and CD90, identified as the standards of MSCs, were used for the identification. Methods: The CD105⁺/CD90⁺ and CD105^-/CD90^- subpopulation of MDA-MB-231 cells were detected and sorted by flow cytometry. MSC-like characteristics in cell proliferation, migration and cell cycle were investigated here by MTT asaay, transwell migration assay, and PI staining respectively. The expression profiles of some stem cell-associated genes were also observed by quantitative real time PCR. Results: Around 0.99% and 90.77% of parental cells were identified as CD105⁺/CD90⁺ and CD105^-/CD90^- cell subpopulations respectively. The CD105⁺/CD90⁺ cells exhibited stronger migratory capacity as compared to parental and CD105^-/CD90^- cells, while less CD105⁺/CD90⁺ cells were arrested in the S phase. Besides, pluripotent stem cell factors, like Oct-4, Nanog, Klf4 and Sox-2, were all upregulated in CD105⁺/CD90⁺ cells, with also proliferation increase, as compared with other two populations. Conclusion: The CD105⁺/CD90⁺ subpopulation from breast cancer MDA-MB-231 cells was proven to possess “mesenchymal stem cell-like” characteristics, and its high migratory ability might be associated with EMT. Moreover, using the surface markers of CD105 and CD90 for the identification of MSCs might provide new theoretical basis for the recurrence and metastasis of breast cancer.     

Prognostic value of cancer stem cell marker CD133 expression in non-small cell lung cancer: a systematic review

Objective: To investigate the correlation between CD133-positive non-small cell lung cancer (NSCLC) and clinicopathological features and its impact on survival. Methods: A search in the Pubmed, Embase and Wanfang databases (up to July 15, 2013) was performed. Only articles in which CD133 antigen was detected in situ localization by immunohistochemical staining were included. This meta-analysis was done using RevMan 5.2 software. Outcomes included overall survival and various clinicopathological features. Results: A total of 1004 NSCLC patients from 11 studies were included. Meta-analysis showed that CD133 expression patients had a significant worse 5-year overall survival compared to the low expression ones (RR = 3.19, 95% CI: 2.05-4.98, P<0.0001 fixed random). With respect to clinicopathological features, CD133 expression by IHC method was closely correlated with tumor T stage (OR = 0.91, 95% CI: 0.59-1.39, P = 0.67 fixed-effect) and tumor grade (OR = 1.20, 95% CI: 0.80-1.79, P = 0.37 fixed-effect). Conclusion: CD133-positive NSCLC patients had worse prognosis, and was associated with common clinicopathological poor prognostic factors.