利用表达谱基因芯片筛选溃疡性结肠炎差异表达基因

目的:利用人类全基因组表达谱芯片技术,分析溃疡性结肠炎患者和健康者基因表达谱差异,筛选出溃疡性结肠炎相关基因。方法:采用Trizol法提取8例溃疡性结肠炎患者和8例健康对照者结肠粘膜组织总RNA并纯化,逆转录合成c DNA,利用荧光染料Cy3标记aa UTP,转录合成标记的c RNA,并与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据进行归一化处理,利用倍数差异和t检验计算筛选出相关差异表达基因,采用DAVID在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能,并对部分差异表达基因进行实时荧光定量PCR验证。结果:筛查出溃疡性结肠炎结肠粘膜组织差异表达基因4132个,其中上调基因2004个,下调基因2128个。选取6条差异表达基因进行PCR验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论:溃疡性结肠炎患者与健康对照者基因表达存在明显差异,分析这些差异表达基因有助于我们探索溃疡性结肠炎的发病机制,为疾病的治疗提供理论依据。     

MAPK信号通路基因在小鼠肝再生中的表达谱变化

为了分析丝裂原活化蛋白激酶(Mitogen-Activated Protein Kinases,MAPK)信号通路基因在肝再生中的表达图谱,以及探讨MAPK信号通路在肝再生中的作用,文章利用四氯化碳(Carbon Tetrachloride,CCl4)诱导的小鼠肝损伤再生模型对MAPK信号通路基因的表达进行检测.首先,采用CCl4腹腔注射的方法建立小鼠肝损伤再生模型,通过肝脏切片HE染色和测定血清中谷丙转氨酶活性确认模型的质量,然后,在注射CCl4后的第0、0.5、1.5、4.5、7 d分别采集小鼠肝脏样本,应用Affymetrix公司的小鼠基因表达芯片,检测MAPK信号通路中93个基因的差异表达图谱,并用荧光实时定量PCR法验证芯片检测的结果.结果表明,在芯片检测到的93个MAPK信号通路基因中,有31个在肝再生中有不同程度差异表达,且经荧光实时定量RT-PCR检测的结果与基因芯片的结果相符合.基因表达谱芯片技术可以筛选出肝再生中差异表达的基因,在小鼠肝再生中的第0.5和1.5 d,MAPK信号通路中表达水平上调的基因增多,而在第4.5和7 d,则表达水平下调的基因明显增多.这一结果表明MAPK信号通路对肝再生不同阶段的双重调控作用.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

GMPS,PR,CD40和p21对卵巢癌预后的预测价值(Prognostic values of GMPS,PR CD40,and p21 in ovarian cancer全译)

目的通过一个卵巢癌自发恶性转化模型来识别卵巢癌患者的预后标志物。方法小鼠卵巢表面上皮细胞(MOSECs)在体外培养可以发生自发恶性转化。使用Illumina HiSeq 2000新一代测序平台和生物信息学分析检测在此模型转化过程中的基因表达和信号传导的改变。使用基因表达互作谱分析(http://gepia.cancer-pku.cn/)识别4个差异表达的核心基因,随后使用TCGA数据库与Kaplan-Meier Plotter在线数据库(www.kmplot.com)研究这些核心基因与卵巢癌患者生存期之间的关系。结果数据显示在MOSECs自发恶性转化的初期与晚期差异表达的基因共计263个,其中包括182个上调与81个下调基因。生物信息学数据揭示有4个基因(GMPS、PR、CD40、p21)在卵巢癌发生过程中扮演者重要的角色。此外,使用TCGA数据验证了这4个基因的差异表达及其与卵巢癌患者的预后相关。结论本研究使用卵巢癌进展模型分析差异表达的基因,并鉴定出四种(即GMPS、PR、CD40和p21)基因可以作为卵巢癌患者的预后标志物。未来需要前瞻临床研究来进一步验证这四种基因的特征及其临床实用性。     

血管外膜肌成纤维细胞分化的基因表达谱

我们以往的研究表明,TGF-β1可以诱导血管外膜成纤维细胞（adventitial fibroblasts,AFs）向肌成纤维细胞（myofibroblasts,MFs）分化。为寻找可能涉及MF分化的基因,本实验采用寡核苷酸芯片技术动态检测细胞表型转化过程中基因表达的变化,实时定量RT-PCR验证芯片结果。在芯片上的15866条总探针组中,2121个探针组在TGF-β1刺激后至少一个时间点的表达发生2倍以上变化,其中l318个基因表达上调,761个基因表达下调,还有少数基因（42个）在不同的时间点既有上调又有下调表达。在1231个已知功能基因中,分泌磷蛋白l（secreted phosphoprotein1．APP1）、Rhoassociated coiled-coil forming kinase2（ROCK2）的表达趋势与标志基因α-平滑肌肌动蛋白（α-SM-actin）的表达趋势相同,TGF-D1诱导MF分化过程中上调了电压门控性钾通道Shal家族成员2（potassium voltage-gated channel,Shal-related family and member2,KCND2）的表达,这些基因参与了MF的分化;此外,还发现内皮素1（endothelin 1,EDN1）、补体成分、NADPH氧化酶4（NADPH oxidase 4,NOX4）和NAD（P）H dehydrogenase,quinone 1 （NQO1）可能参与了MF分化。本实验用寡核苷酸芯片技术验证了通过其它技术证实的同MF分化相关的基因,并发现了新的涉及该过程的基因,基因表达谱研究有利于鉴定参与细胞分化的基因和通路。     

原位杂交检测microRNA-205在乳腺癌中的表达

目的探讨microRNA-205表达与乳腺恶性病变的关系。方法乳腺疾病及癌组织芯片原位杂交分析microRNA-205的表达;实时定量RT-PCR方法检测正常乳腺细胞株、恶性程度不同的乳腺癌细胞株中microRNA-205的表达。结果原位杂交分析显示,36例正常与良性乳腺病变中,33例(91.67%)表达阳性;36例乳腺癌中,23例(63.89%)表达阳性。microRNA-205的表达在乳腺正常与良性病变中的表达较恶性病变中高且有统计学差异(P=0.011),但与乳腺癌TNM分期、临床分期无关(P0.05)。实时定量RT-PCR结果显示,四个高度恶性乳腺癌细胞株(MDA-MB-231、HS578T、BT549和SUM159PT)中microRNA-205的表达较永生化正常乳腺上皮细胞株MCF10A和四个低度恶性细胞株(MDA-MB-468、T-47D、ZR-75-1和SKBR3)中为低(P0.05)。结论原位杂交适用于microRNA-205的表达分析;组织芯片标本原位杂交与乳腺细胞株实时定量RT-PCR分析结果提示,microRNA-205可能参与了乳腺癌的发生、发展,并随着乳腺癌的演进呈下调趋势。     

肝素酶超表达对HEK293细胞中Arc基因的调节

目的:利用RT-PCR验证基于肝素酶转基因小鼠的大脑皮层组织表达芯片发现的表达上调基因Arc,研究肝素酶基因Hpse对Arc基因表达的影响。方法:在稳定转染小鼠肝素酶基因Hpse的HEK293细胞体系中,采用半定量PCR的方法分析Arc的mRNA表达情况。结果:与前期的小鼠大脑皮层表达芯片结果相反,Hpse下调Arc基因的表达。结论:推断肝素酶可能通过影响Arc基因的表达,从而影响细胞功能。     

喉癌发生相关特异基因筛选与验证

 为了检测喉鳞状细胞癌相关的基因表达变化特征,筛选与喉癌发生相关的特异基因. 从3名患者体内分别取正常喉上皮组织和喉鳞状细胞癌组织.应用基因芯片技术进行基因表达差异分析及系统聚类分析,并进行半定量RT PCR验证部分基因芯片结果.本实验基因芯片包括7 26条探针,其中,在3对样本中,表达发生显著差异的基因共有94条,有31条上调,63条下调,并且系统聚类分析将正常和癌组织各分为一类,半定量RT-PCR结果与芯片结果一致.实验表明,细胞的代谢、生长、信号传递相关基因(如RAN、PDCD10、zyxin、TACSTD1等)参与了喉癌的发生、发展,并可能扮演了重要角色     

siRNA介导的PRR11表达抑制导致肺癌细胞系基因表达谱变化的分析

Proline rich 11(PRR11)是本课题组鉴定的一个新的肿瘤相关基因。为研究PRR11介导肺癌发生发展相关的分子机制,本研究分析了PRR11表达被抑制后人肺癌细胞系H1299的全基因组基因表达谱的变化。首先,采用siRNA抑制H1299细胞中PRR11的表达,提取总RNA,采用基因芯片分析全基因组基因表达谱的变化。然后,对呈现差异表达的基因进行GO和Pathway富集分析,并对部分重要的候选基因进行定量RT-PCR验证。基因芯片结果表明,采用siRNA有效抑制H1299细胞中PRR11表达后,共有550个基因的mRNA水平出现明显变化,其中139个基因表达上调,411个基因表达下调。生物信息学分析结果表明,上述差异表达的基因显著富集于细胞周期和MAPK通路。定量RT-PCR验证分析结果表明,PRR11表达抑制后确实可导致多个与细胞周期和肿瘤发生发展密切相关的基因(包括DHRS2、EPB41L3、CCNA1、MAP4K4、RRM1、NFIB)呈现显著的表达变化。这些结果提示,PRR11可能通过上述通路和/或基因的表达变化参与肺癌的发生发展过程。     

RASSF1A对黑色素瘤A375细胞基因网络的影响

RASSF1A(Ras association domain family 1 isoform A)是定位于染色体3p21.3区域的抑瘤基因,编码一个由340个氨基酸残基构成的微管相关蛋白.该基因在包括恶性黑色素瘤在内的多种肿瘤中因启动子高甲基化而表达沉默.本研究建立了RASSF1A稳定表达的恶性黑色素瘤A375细胞系,通过全基因组表达谱基因芯片分析RASSF1A过表达对A375细胞基因表达谱的影响,发现RASSF1A引起184个基因表达上调,26个基因表达下调.通过Realtime RT-PCR对部分差异表达基因进行验证,结果表明与芯片筛选结果一致.RASSF1A影响的差异表达基因功能上归属于细胞生长与增殖、细胞周期、细胞凋亡、细胞间黏附、信号传导等生物过程.采用STRING软件构建了RASSF1A影响的差异表达基因调控网络,结果表明RASSF1A调控的差异表达基因构成一个高连接度的基因网络.其中,炎症细胞因子、转录因子位于网络中央.RASSF1A通过影响炎症细胞因子与转录因子之间的表达,影响A375细胞基因网络,调节黑色素瘤恶性生物学行为.     

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristics of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy.     

Comment to: “Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro” by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957: Spontaneous transformation of mesenchymal stem cells in culture: Facts or fiction?

There is at present a controversy in the literature whether MSCs are susceptible to spontaneous in vitro transformation or not. Several groups have reported spontaneous transformation of MSCs from various species. However, some of these reports were not true transformations and later proven to be due to cross-contaminating cancer cells. To date there is no solid evidence that MSCs can undergo spontaneous transformation in culture. Only two groups used DNA fingerprinting to authenticate their transformed cells, and both groups later showed cross-contamination of cancer cells in their cultures. In this commentary, we address the paper "Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro" by Z. Ren et al. Exp. Cell Res. 317 (2011) 2950-2957. In this article the authors characterize the transformed mesenchymal cells (TMCs) and claim to have verified their origin. We question the authentication of the TMCs made by the authors and we also believe it is in the interest of the scientific community, that a highly controversial finding, such as spontaneous transformation of MSCs, should be properly verified by stringent methods, preferably DNA fingerprinting, in order to validate if an actual transformation event has occurred.     

Deletion of RBPJK in Mesenchymal Stem Cells Enhances Osteogenic Activity by Up-Regulation of BMP Signaling

Recently we have demonstrated the importance of RBPjk-dependent Notch signaling in the regulation of mesenchymal stem cell (MSC) differentiation during skeletogenesis both in vivo and in vitro. Here we further performed RBPJK loss-of-function experiments to demonstrate for the first time that RBPJK deficient MSC shows enhanced differentiation and osteogenesis acts via up-regulation of the BMP signaling. In the present study, we first compared the spontaneous and osteogenic differentiation in normal and recombination signal binding protein for immunoglobulin kappa J region (RBPJK) deficient human bone marrow-derived mesenchymal stem cells (MSCs). It was found that RBPJK highly expressed in fresh isolated MSCs and its expression was progressing down-regulated during spontaneous differentiation and even greater in osteogenic media inducted differentiation. Deletion of RBPJK in MSCs not only enhances cell spontaneous differentiation, but also significantly accelerates condition media inducted osteogenic differentiation by showing enhanced alkaline phosphatase (ALP) activity, Alizarin red staining, gene expression of Runx2, Osteopontin (OPN), Type I collagen (COL1a1) in culture. Additionally, BMP signaling responsive reporter activity and phosphor-smad1/5/8 expression were also significantly increased upon removal of RBPJK in MSCs. These data proved that inhibition of Notch signaling in MSCs promotes cell osteogenic differentiation by up-regulation of BMP signaling, and RBPJK deficient MSC maybe a better cell population for cell-based bone tissue engineering.     

Spontaneous transformation of cynomolgus mesenchymal stem cells in vitro: further confirmation by short tandem repeat analysis

It remains a highly debatable issue whether mesenchymal stem cells (MSCs) can undergo spontaneous transformation in culture. Recently, two groups retracted their previous publications due to the finding that the claimed transformed cells are actually contaminating cancer cells, which calls for a more stringent identification of transformed cells in the field. In this study, we continued with our previous finding of spontaneous transformation of cynomolgus MSCs and provided further evidence using short tandem repeat analysis that the transformed mesenchymal stem cells were indeed derived from cynomolgus MSCs.     

Loss of interactions between p53 and survivin gene in mesenchymal stem cells after spontaneous transformation in vitro

Mesenchymal stem cells (MSC) from various animals undergo a spontaneous transformation in long-term culture. The transformed MSCs are highly tumorigenic and are likely to be the tumor-initiating cells of sarcoma. To explain why the transformed MSCs become tumorigenic, the present study investigated the characteristics of rat MSCs before and after spontaneous transformation. It was shown that although the transformed MSCs maintained typical surface markers of MSC, they exhibited some cancer stem cell-like characteristics such as loss of contact inhibition and multi-potency to mesenchymal lineages, and acquirement of ability of anchorage-independent growth. The expression of a key senescence regulator p16 almost disappeared, but the other one, p53 abnormally increased in the transformed MSCs. ChIP assay demonstrated that a normal negative regulation of p53 on survivin gene disappeared in the transformed cells due to a lack of p53 binding to the promoter of survivin gene. DNA sequencing revealed that the p53 gene in transformed MSCs was not a wild-type, but a 942C > T mutant with the mutation located in the sequence coding p53 protein’s DNA-binding domain. These findings indicate that the transformed MSCs express high levels of a p53 mutant that loses the ability to bind survivin gene, leading to an abnormally upregulated expression of survivin, which is a key reason for the cell’s unlimited proliferation.     

Spontaneous transformation and immortalization of mesenchymal stem cells in vitro

Mesenchymal stem cells (MSCs) possess plasticity and unlimited proliferative activity in vitro, which makes them an attractive object for studies focused on new resources for regenerative medicine. MSC application is effective for treating patients with degenerative and traumatic diseases of different tissues; however, the biological basis for the therapeutic efficacy of MSCs is still obscure. We found that the long-term culture of MSCs that expressed transgenic green fluorescence protein (GFP) led to an increase in their proliferative activity and reduced adhesion, loss of differentiation, and GFP production. At the first passages, MSCs showed karyotypic features of transformation, which were complicated at the later passages by the appearance of tumorigenic properties that were detected after transplantation into syngenic recipients. Tumor cells originated from MSCs explanted in vitro did not express GFP and could not be induced to differentiate. However, in contrast to the parent cells, they showed decreased clonogenic and proliferative activity. We assume that even the short-term cultivation of MSCs in vitro may result in their spontaneous transformation. We hypothesize that immortality and unlimited MSC expansion in vitro are consequences of their transformation rather than intrinsic stem-cell properties.     

Improved isolation and expansion of bone marrow mesenchymal stromal cells using a novel marrow filter device

Background aimsMesenchymal stromal cells (MSCs) have been studied as cell therapy to treat a vast array of diseases. In clinical MSC production, the isolated cells must undergo extensive ex vivo expansion to obtain a sufficient dose of MSCs for the investigational treatment. However, extended tissue culture is fraught with potential hazards, including contamination and malignant transformation. Changes of gene expression with prolonged culture may alter the therapeutic potential of the cells. Increasing the recovery of MSCs from the freshly harvested bone marrow allowing for less ex vivo expansion would represent a major advance in MSC therapy.MethodsHuman bone marrow cells from eight healthy donors were processed using a marrow filter device and, in parallel, using buoyant density centrifugation by two independent investigators. The initial nucleated cell recovery and the final yield, immunophenotype and trilineage differentiation potential of second-passage MSCs were examined.ResultsThe marrow filter device generated significantly greater initial cell recovery requiring less investigator time and resulted in approximately 2.5-fold more MSCs after the second passage. The immunophenotype and differentiation potential of MSCs isolated using the two methods were equivalent and consistent with the defining criteria. The two independent investigators generated comparable results.ConclusionsThis novel filter device is a fast, efficient and reliable system to isolate MSCs and should greatly expedite pre-clinical and clinical investigations of MSC therapy.     

Alteration of Histone Acetylation Pattern during Long-Term Serum-Free Culture Conditions of Human Fetal Placental Mesenchymal Stem Cells

Increasing evidence suggests that the mesenchymal stem cells (MSCs) derived from placenta of fetal origin (fPMSCs) are superior to MSCs of other sources for cell therapy. Since the initial number of isolated MSCs is limited, in vitro propagation is often required to reach sufficient numbers of cells for therapeutic applications, during which MSCs may undergo genetic and/or epigenetic alterations that subsequently increase the probability of spontaneous malignant transformation. Thus, factors that influence genomic and epigenetic stability of MSCs following long-term expansions need to be clarified before cultured MSCs are employed for clinical settings. To date, the genetic and epigenetic stability of fPMSCs after long-term in vitro expansion has not been fully investigated. In this report, alterations to histone acetylation and consequence on the expression pattern of fPMSCs following in vitro propagation under serum-free conditions were explored. The results show that fPMSCs maintain their MSC characteristics before they reached a senescent state. Furthermore, acetylation modification patterns were changed in fPMSCs along with gradually increased global histone deacetylase (HDAC) activity and expression of HDAC subtypes HDAC4, HDAC5 and HDAC6, as well as a down-regulated global histone H3/H4 acetylation during in vitro culturing. In line with the acetylation alterations, the expression of oncogenes Oct4, Sox2 and TERT were significantly decreased over the propagation period. Of note, the down-regulation of Oct4 was strongly associated with changes in acetylation. Intriguingly, telomere length in fPMSCs did not significantly change during the propagating process. These findings suggest that human fPMSCs may be a safe and reliable resource of MSCs and can be propagated under serum-free conditions with less risk of spontaneous malignancy, and warrants further validation in clinical settings.     

Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function

MSCs are kind of cultured cells that reside in different tissues as inducers or regulators of physiological and pathological processes. Here, we derived MSCs from amniotic fluid and compared their differentiation ability and immunosuppression effect on PHA-activated PBMC with those of MSCs isolated from umbilical cords. Amniotic fluid MSCs were isolated and cultured on commercial AFC medium and classic MSC medium, and the number and size of colonies were used to evaluate differences in primary and passaged culture. Rate of proliferation, population doubling time, cell morphology, cell surface markers and mRNA expression were measured in subcultured cells. Furthermore, a comparative study was performed with umbilical cord MSCs to assess the ability of differentiation and immunosuppressive effect of PHA-stimulated PBMCs. Amniotic fluid MSCs were isolated and expanded by three methods, and exhibited nearly all the characteristics of umbilical cord MSCs. Compared with umbilical cord MSCs, amniotic fluid MSCs had an enhanced osteogenic and chrondrogenic differentiation capability, and stronger immunosuppression effect of inhibition of PHA-activated PBMC division. Culture with commercial AFCs medium yielded the highest percentage of CD105 expression and showed some advantages in primary cell isolation, cell source-specific marker retention and cell proliferation. We demonstrated that amniotic fluid MSCs exhibited some advantages over umbilical cord MSCs, and different culture media caused cell proliferation, cell surface marker and cell morphology change, but were not associated with varying differentiation capability and immune effects.