成年小鼠心脏干细胞的分离、培养鉴定和离子通道特征

目的：建立分离、培养和鉴定成年小鼠心脏干细胞的方法并初探其离子通道特性。方法和结果：（1）分离Langendorff法灌流小鼠心脏，用胶原酶酶解法制备单个心肌细胞，差速离心法将较大的细胞（如心室肌细胞）和较小的细胞分离卉。取用较小细胞，用c-kit抗体孵育后，然后用免疫磁珠分离法分离心脏干细胞。     

黄芪甲苷促进脐带血干细胞向心肌细胞分化作用的研究

目的探讨黄芪甲苷对脐带血间充质干细胞向心肌细胞分化的作用。方法采用贴壁法分离人脐带血间充质干细胞,用流式细胞术对细胞表型进行鉴定。通过免疫组化方法观察黄芪甲苷对间充质干细胞向心肌细胞分化的促进作用。结果流式细胞仪检测结果表明,分离的脐带血间充质干细胞表达CD29、CD44和CD105,不表达CD34和HLA-DR。黄芪甲苷（浓度200 mg/L）作用下,间充质干细胞表达特异性心肌细胞蛋白,包括结蛋白（desmin）、α横纹肌肌动蛋白（-αsarcomeric actin）和肌钙蛋白T（C-TnT）。结论黄芪甲苷对脐带血间充质干细胞分化的促进作用,分化的心肌细胞在心肌损伤性疾病的潜在的治疗价值值得进一步研究。     

光电成像系统在评价胚胎干细胞体外定向分化为心肌细胞中的应用

目的采用简易光电成像系统记录胚胎干细胞(ES细胞)体外定向分化心肌细胞的搏动频率,为药物诱导ES细胞体外定向分化心肌细胞提供量化评价指标。方法以简易光电成像系统记录心肌细胞搏动频率,并以维A酸和淫羊藿苷诱导ES细胞定向分化心肌细胞为例,收集分化过程中发育依赖性基因(α-肌球蛋白重链、心室肌球蛋白轻链及β-肾上腺素受体)和心肌特异性蛋白(α-辅肌动蛋白和肌钙蛋白T)表达情况,同步分析光电成像信号与分子生物学指标的一致性。结果光电成像系统可灵敏反映分化心肌细胞的搏动频率,在与维A酸和淫羊藿苷共培养体系中,搏动频率与心肌发育依赖性基因、蛋白表达和β-肾上腺素受体形成等一致,可体现心肌分化的不同时间段。结论简易光电成像系统能灵敏记录ES细胞定向分化心肌细胞的搏动频率,此搏动频率与细胞分化成熟程度一致,可望成为药物诱导分化心肌细胞初步评价的量化指标。     

舌鳞癌组织中肿瘤干细胞的分离培养及PIWIL4对其的影响

目的：从人舌鳞癌组织中分离培养肿瘤干细胞细胞（CSC）,鉴定其生物学特性,并研究PIWIL4在CSC中的表达和意义。方法收集9例不同临床分期舌鳞癌患者组织标本,通过酶消化和原代培养相结合等方法处理,采用无血清悬浮培养法分离培养获得含CSC的悬浮细胞球,流式细胞仪检测细胞表面分子标志CD133和CD44的表达,免疫磁珠分选系统分离CD133＋CD44＋细胞;采用半定量逆转录聚合酶链反应检测PIWIL4 mRNA在CSC中的表达;裸鼠皮下接种CSC,观察其成瘤能力。结果成功的从人舌鳞癌组织分离培养获得可悬浮生长、稳定传代的CSC,CSC高表达CD133和CD44,裸鼠皮下接种1×10^4、1×10^5个CSC均可全部成瘤,PIWIL4 mRNA在CSC的表达也显著高于正常舌组织细胞。结论采用无血清悬浮培养法成功从人舌组织中分离获得CSC,具有肿瘤干细胞特性,能高度表达PIWIL4,为靶向肿瘤干细胞的治疗提供了实验依据。     

肝癌相关基因HTA的重组表达及多克隆抗体的制备

目的将肝癌相关基因HTA进行克隆、表达并制备多克隆抗体,为进一步研究其作为肝癌导向治疗靶点的可行性及临床意义奠定基础。方法应用RT-PCR技术,从人肝癌细胞系HepG2细胞中扩增得到HTA3＋cDNA,再将其克隆到原核表达载体pET21a（＋）-MBP内,用IPTG诱导其在大肠杆菌BL21（DE3）中表达。His-tag磁珠纯化试剂盒纯化重组蛋白MBP-HTA,通过Westernblot和ELISA方法检测重组蛋白的抗原性。将纯化的重组蛋白免疫BALB／c小鼠制备多克隆抗体,并采用ELISA、Westernblot和免疫组化的方法检测抗体的灵敏度和特异性。结果成功地构建了表达MBP-HTA融合蛋白的原核表达质粒pET21a（＋）-MBP-HTA。重组MBP-HTA融合蛋白在大肠杆菌BL21（DE3）内得以高效表达,且以包涵体的形式存在。His-tag磁珠纯化试剂盒纯化和Westernblot分析,得到了分子量约为52kDa的目的蛋白。获得了高效价的特异性多克隆抗体,经ELISA检测抗体的效价为1：3200。用Westernblot检测的效价为1：400。免疫组织化学检测表明：肝癌组织中HTA蛋白的阳性表达率明显高于正常肝组织俨〈0．01）。结论成功地制备出抗MBP-HTA多克隆抗体,该抗体有较高的效价和特异性;能用于免疫组织化学的检测,且HTA在肝癌组织中的阳性表达率明显高于正常肝组织。HTA蛋白有望成为肝癌导向治疗的潜在靶点。     

心肌干细胞研究进展

传统观点认为,成年哺乳动物的心肌细胞高度分化,没有再生能力,只能通过肥大等重构方式代偿.1994年,Soonpaa等[1]发现将小鼠胚胎干细胞移植到成年小鼠发生梗死的心肌部位,能够限制疤痕发展以及预防梗死后心衰的发生;继而有人发现心肌梗死后,在梗死灶边缘及正常心肌组织中有少量心肌细胞发生有丝分裂,说明病理状态下心肌细胞有自我更新的能力[2];提示成年心脏中大部分心肌细胞不可再分裂,但是仍然包含了部分可以再生的细胞,并不是终分化器官,而有内在的再生潜能.近年来,将不同来源包括心肌细胞来源的干/祖细胞体外培养诱导分化为心肌细胞,应用于心肌梗死或者心肌肥大症等,已经成为研究的热点.本文将就心肌干细胞(Cardiacstem cells,CSC)的调控、分化及其在心脏疾病治疗方面的进展进行简要综述.     

新生小鼠心肌细胞的纯化培养与鉴定

目的建立新生小鼠心肌细胞(CM)的分离纯化、培养及鉴定的方法。方法采用酶消化法分离消化心室组织,差速贴壁法和化学方法纯化CM;培养7d后,利用免疫荧光技术和RT-PCR技术检测部分CM特异基因在蛋白和mRNA水平的表达;改进透射电镜的常规操作方法,观察CM超微结构。结果采用0.08%胰蛋白酶和0.04%胶原酶Ⅱ消化心室组织后,细胞成活率为98%,贴壁培养第3d出现同簇细胞的同步跳动;抗心肌肌钙蛋白(cTnT)免疫荧光染色阳性率为99.07%;以骨髓间充干细胞为阴性对照,RT-PCR结果显示CMα和β两型心肌肌球蛋白重链、cTnT基因表达呈阳性;经3%~4%琼脂预包埋法处理可以对少量细胞进行超薄切片的制作,CM具有正常的超微结构。结论建立了新生小鼠CM分离纯化培养和鉴定方法。     

体外培养成人脂肪间充质干细胞生物学特性及诱导分化为心肌细胞的研究

目的通过分离、培养脂肪间充质干细胞观察其生物学特性及诱导分化为心肌细胞,为心肌再生提供良好的干细胞来源。方法胶原酶消化分离成人脂肪来源的间充质干细胞并进行传代培养,倒置相差显微镜观察细胞形态,流式细胞仪测定CD29、CD31、CD34、CD44及细胞周期,MTT绘制细胞生长曲线。用第3代细胞进行诱导分化,观察不同浓度5-氮杂胞苷(5-Aza,1,3,5,10,15,20μmol/L)及不同作用时间(12,24,48,72h)诱导其向心肌细胞分化的差别,采用最佳浓度10μmol/L,最佳作用时间24h进行实验,分别在第7,14,21,28天用免疫细胞荧光染色鉴定心肌细胞α-横纹肌、肌球蛋白重链(MHC)、心肌肌钙蛋白I(cTnI)表达,第14天反转录-聚合酶链反应(RT-PCR)法检测心肌发育相关基因NKX2.5的表达。结果倒置相差显微镜下观察原代细胞,可见细胞呈梭型、核圆形或椭圆形,偶见双核。传代细胞核原代细胞形态相似,排列有了一定的方向性。流式细胞仪检测结果显示,第1、3、5代细胞均高表达CD29和CD44;而CD31始终表达很弱,可认为呈阴性表达;CD34在第1、3代细胞弱表达,在第5代细胞表达逐渐减弱为阴性。细胞生长曲线显示前3d处于细胞潜伏状态,第4天进入对数生长期,第10天达到顶峰。细胞周期检测结果显示G1期细胞为85.93%,S期为7.24%,G2期为6.83%。10μmol/L5-Aza诱导后7d进行免疫细胞荧光染色,未见有α-横纹肌、MHC、cTnI表达。14d少量细胞α-横纹肌和MHC阳性表达,cTnI阴性表达。21d表达α-横纹肌和MHC的细胞数量增多,并可见少量cTnI阳性表达。28dα-横纹肌、MHC、cTnT阳性表达数目均增多,RT-PCR结果显示NKX2.5呈阳性表达。结论成人脂肪中可以分离出脂肪间充质干细胞并且可以在体外培养传代,经过5-Aza的诱导可以向心肌细胞分化,为干细胞移植治疗和组织工程学种子细胞提供了更多的选择。     

小鼠诱导多能干细胞向雄性生殖细胞样细胞诱导分化的研究

目的：对小鼠诱导多能干细胞向原始生殖细胞样细胞进行诱导培养,并对获得的原始生殖细胞样细胞进行鉴定。方法：在诱导干细胞培养基中对小鼠诱导多能干细胞进行培养,用蛋白质印迹法(Western blot)对Oct-4及C-kit蛋白表达水平进行鉴定,用定时定量PCR对Mvh, Fragilis, Stella的mRNA水平进行鉴定。结果：由小鼠诱导多能干细胞体外诱导分化获得的原始生殖细胞样细胞特异性表达Oct-4蛋白、C-kit蛋白以及Mv hmRNA,Fragilis RNA,Stella RNA。结论：由小鼠诱导多能干细胞成功诱导培养出原始生殖细胞样细胞,为进一步研究其向精子细胞的转化奠定了较好的基础。     

SFRP1通过抑制Wnt/β-catenin通路促进心肌细胞增殖的分子机制

目的 探讨SFRP1是否通过抑制Wnt/β-catenin通路促进心肌细胞增殖。方法 选取c57BL/6雄性小鼠18只,出生后1 d、7 d及28 d的c57BL/6小鼠各6只分为3组,分离和提取心肌组织,检测心肌组织中SFRP1的mRNA及蛋白表达水平。利用出生后1 d的乳鼠心脏培养小鼠原代心肌细胞,予SFRP1-shRNA慢病毒转染细胞,免疫荧光检测心肌细胞增殖标志物Ki67和PH3表达变化,Western blot检测胞浆β-catenin蛋白表达变化。结果 SFRP1 mRNA和蛋白水平在小鼠出生后逐渐下降(P<0.05)。转染SFRP1-shRNA慢病毒后,SFRP1蛋白表达水平均下降(P<0.05),其中shRNA-2干扰效果最好。与空载组相比,shRNA组细胞Ki67和PH3表达下降,同时胞浆β-catenin蛋白表达水平升高(P<0.05)。结论 SFRP1通过抑制Wnt/β-catenin信号通路促进心肌细胞增殖。     

Cancer stem cell-targeted chimeric antigen receptor (CAR)-T cell therapy: Challenges and prospects

Cancer stem cells (CSCs) with their self-renewal ability are accepted as cells which initiate tumors. CSCs are regarded as interesting targets for novel anticancer therapeutic agents because of their association with tumor recurrence and resistance to conventional therapies, including radiotherapy and chemotherapy. Chimeric antigen receptor (CAR)-T cells are engineered T cells which express an artificial receptor specific for tumor associated antigens (TAAs) by which they accurately target and kill cancer cells. In recent years, CAR-T cell therapy has shown more efficiency in cancer treatment, particularly regarding blood cancers. The expression of specific markers such as TAAs on CSCs in varied cancer types makes them as potent tools for CAR-T cell therapy. Here we review the CSC markers that have been previously targeted with CAR-T cells, as well as the CSC markers that may be used as possible targets for CAR-T cell therapy in the future. Furthermore, we will detail the most important obstacles against CAR-T cell therapy and suggest solutions.KEY WORDS: Chimeric antigen receptor T cell, Cancer stem cell, Immunotherapy, Tumor associated antigens, Combination therapy, Off-tumor toxicity, Clinical trial, Tumor immunosuppressive microenvironment     

Cancer stem cells and colorectal cancer: an overview

Normal tissue homeostasis involves a careful balance between the normal cell loss and renewal. Stem and progenitor cells help maintain this precise and fine balance through their ability of self-renewal in a tightly regulated manner. In this regard, the gastrointestinal epithelium is unique in that cell proliferation, differentiation, and apoptosis occur in an orderly fashion along the crypt-villus axis. The colonic crypt is primarily a proliferative compartment, is monoclonal and is maintained by stem cells. The concept of tissue stem cells capable of giving rise to all differentiated cells within a given tissue has led to the concept of a cellular hierarchy in tissues and in tumors including colorectal cancer (CRC). Thus, only a few cells may be necessary and sufficient for tissue repair or tumor regeneration. However, such a proposition also raises questions regarding the precise methods and markers to identify such population and to define the circumstantial evidences and place for the origin and establishment of the early mutant stem-cell population. Thus, it is imperative that we understand what cancer stem cells (CSC) are and their potential association with cancer in a tissue specific manner. In this review, we have summarized the current knowledge of stem cell organization and CSC within the colonic epithelium and discussed the potential role of CSC in the development and/or progression of CRC and as targets for therapeutic interventions.     

微载体—基质细胞造血模型中小鼠骨髓细胞c—kit基因的表达

目的：检测小鼠原始造血细胞c-kit基因表达以评价体外模拟骨髓造血微环境。方法：建立微载体－基质细胞体外造血模型。根据已发表的小鼠c-kit基因序列设计引物，采用RT－PCR方法检测c-kit mRNA水平，并测定祖细胞集落产率。结果：小鼠骨髓造血细胞2周培养实验显示，粒系－巨噬系造血祖细胞集落产率（CFU－GM／10^5）：模型组比液体悬浮培养和单纯微载体基质细胞培养对照组集落产率之总和高2．1倍（t=2.869,P＜0．05）；原始造血细胞的c-kit基因表达水平（OD比率）：模型组比两个对照组之总和高3．1倍（t=2.858,P＜0．05）。结论：在没有外加细胞因子的条件下，微载体－基质细胞造血模型可抑造血干、祖细胞过度分化与耗竭，维持其c-kit mRNA较高的表达水平。     

CD133 as a target for colon cancer

INTRODUCTION: Recent evidence based on cancer stem cell (CSC) models, is boosting the progress of translational research and providing relevant clinical implications in many tumour types, including colorectal cancer. The current failure of standard therapies is attributed to a small fraction of the primary cell population with stem-like characteristics, such as self-renewal and differentiation. Identification of CSCs is based on two different criteria of selection: stemness-selective conditions and direct isolation based on putative stem cell markers expression. CD133, a transmembrane glycoprotein, was associated with tumor-initiating cells derived from several histological variants of tumors, including colon. AREAS COVERED: In this review the current understandings about CD133 as putative marker of tumour-initiating cells in colorectal cancer (CRC) is described. The focus of the discussion is on the need for additional markers to better identify the cell population able to recapitulate the parental tumor in immunocompromised mice. EXPERT OPINION: Identification and characterization of CSCs represents a relevant issue to define innovative therapeutic approaches, overcoming the emergence of cancer cell clones capable of evading standard therapy.     

Targeting embryonic signaling pathways in cancer therapy

INTRODUCTION: The embryonic signaling pathways (ESP), Hedgehog, Notch and Wnt, are critical for the regulation of normal stem cells and cellular development processes. They are also activated in the majority of cancers. ESP are operational in putative cancer stem cells (CSC), which drive initial tumorigenesis and sustain cancer progression and recurrence in non-CSC bulk subpopulations. ESP represent novel therapeutic targets. A variety of inhibitors and targeting strategies are being developed. AREAS COVERED: This review discusses the rationale for targeting ESP for cancer treatment, as well as specific inhibitors under development; mainly focusing on those approaching clinical use and the challenges that lie ahead. The data sources utilized are several database search engines (PubMed, Google, Clinicaltrials.gov), and the authors' involvement in the field. EXPERT OPINION: CSC research is rapidly evolving. Expectations regarding their therapeutic targeting are rising quickly. Further definition of what constitutes a true CSC, proper validation of CSC markers, a better understanding of cross-talk among ESP and other pathways, and interactions with tumor non-CSC and the tumor microenvironment are needed. The appropriate patient population, the right clinical setting and combination strategies to test these therapies, as well as the proper pharmacodynamic markers to measure, need to be further established.     

Metastatic cancer stem cells: new molecular targets for cancer therapy

The cancer stem cell (CSC) hypothesis, predicts that a small subpopulation of cancer cells that possess "stem-like" characteristics, are responsible for initiating and maintaining cancer growth. According to the CSC model the many cell populations found in a tumour might represent diverse stages of differentiation. From the cellular point of view metastasis is considered a highly inefficient process and only a subset of tumour cells is capable of successfully traversing the entire metastatic cascade and eventually re-initiates tumour growth at distant sites. Some similar features of both normal and malignant stem cells suggest that CSCs are not only responsible for tumorigenesis, but also for metastases. The CSC theory proposes that the ability of a tumour to metastasize is an inherent property of a subset of CSCs. The similar biological characteristics shared by normal stem cells (NSCs) and CSCs mainly implicate self-renewal and differentiation potential, survival ability, niche-specific microenvironment requirements and specific homing to metastatic sites and may have important implications in terms of new approaches to cancer therapy in the metastatic setting. There are several agents targeting many of these CSC features that have shown to be effective both in vitro and in vivo. Although clinical trials results are still preliminary and continue under investigation, these new therapies are very promising. The identification of new therapeutic targets and drugs based on CSC model constitutes a great challenge.     

The mechanisms for lung cancer risk of PM2.5: Induction of epithelial‐mesenchymal transition and cancer stem cell properties in human non‐small cell lung cancer cells

Fine particulate matter (PM_2.5) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM_2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM_2.5 by targeting the induction of epithelial‐mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non‐small cell lung cancer cell line A549. We found that both acute and chronic PM_2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM_2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM_2.5 exposure. CSC properties induced by chronic PM_2.5 exposure characterized with increased cell‐surface markers (CD44, ABCG2), self‐renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness‐associated microRNAs, Let‐7a, miR‐16 and miR‐34a, were found to be significantly downregulated by chronic PM_2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM_2.5, and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM_2.5.