无血清悬浮分离培养法分离、培养、鉴定脊髓源性神经干细胞

背景：目前临床上尚未发现完全治愈脊髓损伤,使其达到结构及功能恢复的治疗方法。已有的神经营养因子治疗方法促进神经细胞再生的效果有限,而干细胞移植治疗可能是现阶段修复脊髓损伤的有效方法之一。 目的：无血清悬浮分离培养法培养胎鼠脊髓源性神经干细胞,采用形态学、免疫荧光技术和多向分化能力进行神经干细胞鉴定。 方法：应用悬浮分离培养法,培养纯化孕13.5 d C57BL/6胎鼠的脊髓源性神经干细胞。倒置显微镜观察细胞形态学变化;CCK-8法检测细胞的增殖能力。免疫荧光技术检测Nestin和Sox2表达;使用自然分化法验证第4代脊髓源性神经干细胞多向分化特性,明确其分化能力。 结果与结论：采用无血清悬浮分离培养法可以顺利获取脊髓源性神经干细胞。培养的脊髓源性神经干细胞增殖活性良好,高表达Nestin和Sox2,且两种标记物与DAPI核染高度均匀吻合,说明细胞纯度高。进一步诱导分化显示,细胞可以向GFAP和Tuj1阳性细胞分化,证明脊髓源性神经干细胞分化潜能较好。该实验可建立一套体外培养脊髓源性神经干细胞分离、培养、纯化及鉴定体系,为后续神经干细胞的应用研究奠定基础。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程      

无血清培养体系原代培养脐带间充质干细胞

背景：以含血清培养基培养的脐带间充质干细胞,存在着进一步应用的障碍。 目的：建立无血清培养体系原代培养脐带间充质干细胞。 方法：取脐带华通氏胶(Wharton’s Jelly),采用机械法将组织胶切碎,1-3 mm3大小,分别培养到含胎牛血清完全培养液中以及无血清培养液中。培养第11,14,17天收获细胞并进行相关检测。在符合2006年制定的干细胞最低评估标准的基础上,以集落形成单元指标评估何种培养体系能获得较多高质量的原代细胞。 结果与结论：无血清培养体系相对于经典的含血清的培养体系而言,细胞生长速度更快。另外,培养第11天,集落形成实验表明无血清培养体系下能获得更多的集落。因此,无血清培养体系可以保持间充质干细胞的特性,为替代含血清培养体系进行脐带间充质干细胞原代培养提供了可能。     

无血清培养新生Wistar大鼠神经干细胞及免疫荧光鉴定

目的在无血清条件下,分离培养新生1d的Wistar大鼠神经干细胞,观察其生长及分化情况,并对其生物学特性进行鉴定。方法取新生1d的大鼠海马组织,机械分离法分离神经干细胞,加入含有表皮生长因子、碱性成纤维生长因子、B27的DMEM/F12无血清培养基中培养、增殖。倒置显微镜下观察神经干细胞的增殖分化情况,采用免疫荧光染色鉴定其生物特性。结果从新生大鼠海马组织分离培养的神经干细胞在无血清培养基中不断增殖,免疫荧光染色显示巢蛋白呈阳性表达。诱导分化后,免疫荧光染色可见高分子量神经丝蛋白、胶质纤维酸性蛋白和髓鞘碱性蛋白阳性表达细胞。结论无血清条件分离培养的神经干细胞具有自我更新和增殖能力,能分化为神经元、星形胶质细胞和少突胶质细胞。     

人胚胎干细胞无血清无饲养层培养体系的建立

目的 拟建立人胚胎干细胞无血清无饲养层的培养体系.方法 将干细胞分别置于Knockout培养基(第1组)、自制无血清培养基(第2组)和添加bFGF及肝素的改良无血清培养基(第3组)中培养25代后观察比较3组干细胞形态学变化、克隆数目及大小.并借助细胞免疫组化、流式细胞术和体外拟胚体形成等方法检测干细胞的未分化状态和全能性.结果 传代25次后,第2组克隆逐渐分化.第1组和第3组的干细胞均呈典型的人胚胎干细胞形态特征;细胞表达Nanog,Oct-4,Tra-1-60,SSEA-4,但不表达SSEA-1;流式检测也显示SSEA-4高表达,SSEA-1低表达;体外分化能形成拟胚体.通过单位视野下克隆个数及大小的比较,结果显示第2组与第1组和第3组间的差异显著.结论 本研究通过改良本实验室已获得国家专利的无血清培养基,初步建立了人胚胎干细胞无血清无饲养层培养体系,培养效果与公认培养体系无明显差异.     

无血清培养促进脂肪干细胞向血管内皮细胞分化

背景：无血清培养对大鼠脂肪干细胞向血管内皮细胞诱导分化影响的报道甚少。 目的：观察无血清培养大鼠脂肪干细胞后向血管内皮细胞诱导分化的情况。 方法：采用酶消贴壁培养法获得雄性SD大鼠脂肪干细胞,传代培养至第3代。实验组细胞无血清培养24 h,对照组用含体积分数10%胎牛血清的L-DMEM完全培养基培养。然后应用血管内皮细胞诱导培养基培养3周。 结果与结论：大鼠脂肪干细胞经体外培养可呈多角形或梭形贴壁生长,并能稳定传代。传代后大鼠脂肪干细胞极低表达细胞表面标志CD31。大鼠脂肪干细胞定向血管内皮细胞诱导分化后细胞呈鹅卵石样,CD31阳性表达明显上升且实验组明显高于对照组。实验组诱导后大鼠脂肪干细胞能够吞噬Dil标记的乙酰化低密度脂蛋白及在基质胶上形成2D小管样结构,其能力明显强于对照组。结果证实无血清培养可促进体外大鼠脂肪干细胞向血管内皮细胞诱导分化。     

悬浮组织块法分离培养SD 大鼠脂肪干细胞的实验研究

目的:建立SD 大鼠脂肪干细胞悬浮组织块分离培养法。方法:取正常SD 大鼠股沟处脂肪组织,用悬浮组织块法和组织块贴壁法分离培养SD 大鼠脂肪干细胞,观察细胞的生长状况及形态特征。取第3 代对数期细胞,CCK-8 法绘制其生长曲线;免疫荧光法检测其表面抗原CD29、CD44、CD45 的表达情况;分别用成脂、成骨诱导培养液进行诱导分化,油红O、茜素红染色定性鉴定。结果:两种方法体外培养得到的SD 大鼠脂肪干细胞形态为梭形纤维样,生长力旺盛,生长曲线为典型的“S冶型;免疫荧光法检测显示第3 代细胞强表达CD29、CD44,CD45 表达阴性;细胞成脂诱导培养14 d 后,油红O 染色为阳性;成骨诱导培养14 d 后,茜素红染色为阳性。结论:悬浮组织块法可以分离培养出SD 大鼠脂肪干细胞,方法简便,为体外分离培养SD 大鼠脂肪干细胞提供了一种新的方法。     

杂交瘤细胞的无血清无蛋白培养

随着单克隆抗体在体内治疗中的广泛应用，杂交瘤细胞的无血清无蛋白培养日益受到重视。本文讨论了杂交瘤细胞无血清无蛋白培养基的常用添加成分和它们的功能，综述了用无血清无蛋白培养基培养杂交瘤细胞技术的进展     

无血清培养基SYL-SF对间充质干细胞的培养和扩增作用

目的探讨新型无血清培养基SYL-SF对脐带间充质干细胞的体外培养和扩增作用。方法机械分离方法获得脐带间充质干细胞,从P1代开始将含小牛血清的原代培养基SYL-NBCS更换为实验组SYL-SF和对照组MSCM-SF两种无血清培养基对细胞进行培养、传代、扩增和维持。利用Alexa Fluor 488 annexin V检测细胞凋亡率,CCK-8法比较传代细胞的增殖效率,并对细胞进行形态、表型、染色体核型和分化能力等方面的检测和评价。结果分离得到的单个脐带间充质干细胞在原代培养基SYL-NBCS中培养8~15d可获得原代(P0)细胞,更换为无血清培养基后,SYL-SF组细胞凋亡率明显低于MSCM-SF组;在细胞传代过程中SYL-SF组细胞增殖能力较强且维持MSCs特异性表面标志和向脂肪、骨、软骨等多向分化的潜能,并且在传代过程中维持正常染色体核型。结论 SYL-SF无血清培养基可以对UC-MSCs进行良好地体外扩增和维持。     

角质细胞无血清培养基促进大鼠毛囊干细胞的增殖

背景：以往研究培养毛囊干细胞多使用DMEM/F12+体积分数10%胎牛血清培养基,而进年来开发出的角质细胞无血清培养基也可应用于毛囊干细胞的培养。 目的：观察3种不同培养基对大鼠毛囊干细胞增殖情况及干细胞纯度的影响。 方法：取大鼠触须部皮肤组织,体式显微镜下分离出毛囊组织,中性蛋白酶Ⅱ与胰蛋白酶和乙二胺四乙酸混合液“两步酶法”消化。所得细胞悬液按细胞数平均分为3份,分别使用角质细胞无血清培养基、DMEM/F12+体积分数10%胎牛血清及角质细胞无血清培养基+体积分数10%胎牛血清共3种培养基培养,Ⅳ型胶原差速贴壁法筛选毛囊干细胞,进行传代培养。 结果与结论：培养毛囊干细胞传至第3代,锥虫蓝染色计数法检测结果显示,此3组间细胞活率差异无显著性意义(P > 0.05)。CCK8比色法检测细胞生长曲线显示,培养前2 d,此3组细胞生长均较缓慢;培养4 d,细胞生长进入对数生长期,3种培养基培养的细胞增殖活性：角质细胞无血清培养基+10%胎牛血清组>DMEM/F12+10%胎牛血清>角质细胞无血清培养基(P < 0.05)。流式细胞仪检测显示,角质细胞无血清培养基组CD34的表达高于角质细胞无血清培养基+10%胎牛血清组(P < 0.05)。DMEM/F12+10%胎牛血清组中CD34、β1-整合素(CD29)及CK15标记物的表达低于其他2组(P < 0.05)。结果表明,角质细胞无血清培养基较DMEM/F12+体积分数10%胎牛血清能培养出纯度更高的毛囊干细胞,并且在此培养基培养的基础上加入血清,能够促进毛囊干细胞的增殖。     

Enrichment of Cancer Stem Cells Based on Heterogeneity of Invasiveness

Cancer stem cells (CSCs) have multiple potentials in carcinogenesis and tumor progression. However, it is rather difficult to enrich and amplify CSCs either from tumor cell lines or even primary tumor tissues. Therefore, establishing new methodologies for isolation and enrichment based on the functional properties of CSCs is of great importance for studies on CSCs. According to the findings that CSCs possess more infiltrative capability as compared with their differentiated descendants, we propose a novel strategy based on heterogeneity of cancer cell invasiveness for isolation and enrichment of CSCs from committed cancer cell population. In addition, we hypothesize that existence of CSCs might be the real root of tumor invasion and metastasis.     

Targeting Cancer Stem Cells in Cancer Prevention and Therapy

The cancer stem cell hypothesis is an attractive framework within which one may think about cancer initiation, recurrence, and metastasis, and methods to devise treatment strategies for cancers. Although all cancers do not appear to sustain themselves with cancer stem cells, but also through a dominant cell population, creating strategies for cancer treatment which include cancer stem cells as targets seems reasonable. In this perspective we discuss possible strategies for controlling the viability and tumorigenecity of cancer stem cells, and extend our discussion to strategies approaching the prevention of cancer.     

乳腺癌干细胞BCRP的表达及意义

目的探讨乳腺癌干细胞耐药蛋白BCRP(breast cancer resistance protein)的表达情况及意义。方法应用流式细胞分选技术从人乳腺癌组织中分离出乳腺癌干细胞和非干细胞,实时定量PCR(realtime-PCR)技术测定不同细胞亚群的BCRP表达情况。结果与乳腺癌非干细胞相比,乳腺癌干细胞的BCRP表达水平明显增强(P<0.01),并且化疗后干细胞比例增加(P<0.01)。结论乳腺癌干细胞可以通过高表达BCRP具有更强的化疗耐药性,是乳腺癌化疗失败和复发的关键因素之一。     

人角膜缘干细胞的体外原代培养和生物学鉴定

目的　建立人角膜缘干细胞体外培养方法,观察其生物学特性。方法　用含20%胎牛血清1640培 养液对人角膜缘干细胞进行体外培养,进行形态学观察和免疫细胞化学鉴定。结果　植块3～4d后细胞开始贴壁 生长,10～15d形成良好的单层,细胞呈圆形、卵圆形,类角膜上皮细胞;免疫细胞化学染色显示,培养第11d,少量 细胞表达AE5,大量细胞表达p63。结论　应用20%胎牛血清1640培养液组织培养的方法获得的原代培养细胞具 有与正常角膜缘干细胞相一致的生物学特性。     

Targeting Cancer Stem Cells to Modulate Alternative Vascularization Mechanisms

Recently, many papers have shown that tumor vascularization can be explained by angiogenesis, recruitment, cooption, vasculogenic mimicry and by mosaic vessels. In particular, vasculogenic mimicry seems to be different from mosaic blood vessels, where tumor cells form a part of the surface of the vessel while the remaining part is covered by endothelium. In this case, tumor cells in apparent contact with the lumen do not show an endothelial phenotype. More recently, vasculogenic mimicry was proposed to occur in patients with multiple myeloma due to bone marrow macrophages. Herein, all these data are, for the first time, discussed critically in comparison to cancer stem cells-which show high trans-differentiative capacity-and bone-marrow derived stem cells. In fact, the presence of alternative vasculogenic patterns might be due to the presence of stem cell population (cancer stem cells or bone-marrow stem cells). In this connection, the literature is discussed extensively and possible models are proposed. Pharmacological perspectives will also discuss.     

Mesenchymal Stem Cells in Cancer

It is becoming increasingly evident that stromal cells such as macrophages, mast cells, adipocytes and mesenchymal cells associated with tumors significantly contribute to tumorigenesis. Some types of cancer indeed profoundly rely on extrinsic signals afforded by infiltrating or neighbouring cells for survival, proliferation and dissemination. Tissue disruption that results from tumor growth further activates tissue repair and inflammatory reactions that significantly shape the nature of the developing tumors. Over the past recent years, several studies have revealed that mesenchymal stem cells (MSCs) are recruited to tumors and play a particularly important role in the regulation of both solid and haematological malignancies. The tumor-homing properties of MSCs have further led to studies investigating their therapeutic use as targeted delivery vehicles of gene products. I hereafter discuss the role of MSCs in cancer.     

胎盘来源的间充质干细胞体外分离培养及向软骨细胞分化的研究

目的对人胎盘间充质干细胞体外分离培养及向软骨细胞分化进行研究,为软骨组织工程提供种子细胞。方法取人正常分娩后的胎盘,用组织块培养法分离获取胎盘间充质干细胞,对所获细胞进行体外培养和表面标志的流式细胞仪分析。对经过证实的骨髓间充质干细胞用含TGF-β_3、维生素C、胰岛素、地塞米松、转铁蛋白的诱导培养基处理7-28d,对诱导细胞进行甲苯胺蓝染色及Ⅱ型胶原染色,鉴定诱导后的软骨细胞表型。结果培养的人胎盘间充质干细胞均一表达CD_(29)和CD_(44),而CD_(31)、CD_(34)、CD_(45)和HLA-DR呈阴性。细胞经诱导液作用14d后,甲苯胺蓝染色及Ⅱ型胶原染色阳性。结论采用组织块培养法能分离获取高纯度的人胎盘间充质干细胞,该细胞经诱导液作用可定向分化为软骨细胞。     

Stemming Cancer: Functional Genomics of Cancer Stem Cells in Solid Tumors

Cancer stem cells (CSCs) were discovered about 15 years ago in hematopoietic cancers. Subsequently, cancer stem cells were discovered in various solid tumors. Based on parallels with normal stem cells, a developmental process of cancer stem cells follows paths of organized, hierarchical structure of cells with different degrees of maturity. While some investigators have reported particular markers as identification of cancer stem cells, these markers require further research. In this review, we focus on the functional genomics of cancer stem cells. Functional genomics provides useful information on the signaling pathways which are consecutively activated or inactivated amongst those cells. This information is of particular importance for cancer research and clinical treatment in many respects. (1) Understanding of self-renewal mechanisms crucial to tumor growth. (2) Allow the identification of new, more specific marker for CSCs, and (3) pathways that are suitable as future targets for anti-cancer drugs. This is of particular importance, because today’s chemotherapy targets the proliferating cancer cells sparing the relatively slow dividing cancer stem cells. The first step on this long road therefore is to analyze genome-wide expression-profiles within the same type of cancer and then between different types of cancer, encircling those target genes and pathways, which are specific to these cells.