小鼠间充质干细胞诱导分化成脂肪细胞miRNA表达的变化

目的：探讨小鼠间充质干细胞（MSCs）定向诱导分化成脂肪细胞微小RNA（miRNA）表达的变化,为进一步研究miRNA调控MSCs向脂肪细胞分化的分子机制奠定基础。方法：采用全骨髓体外分离结合差速贴壁法纯化扩增C57BL／6小鼠MSCs,形态学观察细胞生长情况,并用免疫组化方法鉴定细胞表面抗原CD29、CIM4和CD34的表达。脂肪细胞分化诱导剂诱导MSCs分化为脂肪细胞,利用油红O染色,判断MSCs成脂分化情况。运用rrfiRNA芯片技术检测MSC8和脂肪细胞中差异表达的miRNA。结果：①倒置显微镜下观察,传5代后可获得均一性较高的MSCs;免疫组化显示90％以上的骨髓间质干细胞CD29、CD44阳性,CD34阴性。MSCs经脂肪诱导剂诱导后,胞内大量脂滴形成,油红O染色阳性;②基因微阵列分析表明,小鼠MSCs分化成脂肪细胞差异表达的miRNA共75个,其中20个表达上调、55个表达下调。结论：MSCs分化成脂肪细胞存在miRNA表达的变化,某些miRNA很可能具有重要的调控MSCs成脂分化的作用。     

脱细胞神经移植物对骨髓间充质干细胞的诱导分化

目的探讨脱细胞神经移植物诱导大鼠骨髓间充质干细胞分化为施旺细胞样细胞的可行性。方法将分离纯化的SD大鼠骨髓间充质干细胞进行体外培养扩增,行表型鉴定后,取第5代细胞,诱导组采用脱细胞神经移植物匀浆进行诱导,非诱导组加入等量无血清培养基,倒置相差显微镜观察诱导后细胞形态变化,免疫细胞化学染色检测诱导后细胞S-100,神经胶质纤维酸性蛋白(glial fibrillary acidic protein GFAP)的表达情况。结果BMSCs表型鉴定为CD44+、CD54+、CD34-,免疫细胞化学染色GFAP、S-100的阳性表达率分别为为(42±4)%和(64±5)%。结果 脱细胞神经移植物可诱导骨髓间充质干细胞分化为施旺细胞样细胞。     

贴壁法分离培养大鼠骨髓间充质干细胞的生物学特性

目的建立一种简便有效的体外分离纯化及培养扩增大鼠骨髓间充质干细胞(MSCs)的方法。研究MSCs的生物学特性,为血管组织工程提供理想的种子细胞。方法贴壁培养法分离纯化大鼠MSCs体外培养和连续传代,在倒置显微镜下连续观察细胞的形态变化;利用MTT法测定MSCs的生长曲线;行免疫组化方法鉴定MSCs膜抗原;分别加成骨、成脂肪诱导剂后MSCs体外培养1到3周,分别做碱性磷酸酶(ALP)、VonKossa染色及油红O染色,观察细胞形态变化、成骨及成脂肪分化结果。结果MSCs体外培养生长状况良好,呈均一的成纤维细胞样,表达波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA),不表达层粘连蛋白(Laminin)、CD34、VIII因子相关抗原(VIII)。经体外诱导后具有多向分化潜能。结论贴壁培养法能有效分离纯化大鼠MSCs,用此方法培养的细胞生长稳定,增殖能力活跃,具有MSCs的一般生物学特性,为其成为血管组织工程理想的种子细胞提供了进一步的支持。     

小鼠脐带间充质干细胞的体外诱导分化

目的分离扩增小鼠脐带间充质干细胞(mouse umbilical cord mesenchymal stem cells,m UCMSCs)探讨其是否可诱导成软骨、脂肪和成骨细胞。方法通过贴壁培养法将m UCMSCs体外分离、扩增、纯化,倒置显微镜下观察细胞的形态特征,运用流式细胞仪检测分析细胞的抗原标志表达进行鉴定。运用诱导培养液对分离的m UCMSCs分别定向诱导培养为软骨、脂肪和成骨细胞。结果运用组织贴块培养法可从新鲜脐带中分离到贴壁生长的成纤维样细胞,这些细胞高表达CD29、CD90和CD105,低表达CD34。成软骨诱导后阿新兰染色呈蓝色;成脂诱导后油红O染色,出现红色脂滴;茜红素染色成骨诱导的m UCMSC,可见红色结节。结论贴壁培养法分离培养所获得的m UCMSCs在体外可诱导分化为软骨、脂肪和成骨细胞。     

兔骨髓间充质干细胞的体外分离培养和生物学特性观察

目的:探讨兔骨髓间充质干细胞体外分离、培养和鉴定方法,观察其生物学特性.方法:采集兔股骨及胫骨骨髓组织,采用密度梯度离心法结合贴壁培养法体外分离、培养和扩增兔骨髓间充质干细胞,倒置相差显微镜观察细胞形态,绘制原代、第1、3、8代细胞生长曲线,流式细胞术检测细胞表面标志物,成骨和成脂肪诱导培养鉴定,观察细胞生物学特性.结果:培养的BMSCs呈纺锤形、长梭形,旋涡状排列、放射性生长,增值活跃.各代细胞生长曲线呈S型,细胞增值活跃.细胞表面标志物CD44分子阳性,CD34和CD45分子阴性.经成骨和成脂肪诱导后细胞碱性磷酸酶染色和油红O染色阳性.结论:成功建立了兔BMSCs体外分离、培养的有效方法,扩增的BMSCs仍保留多向分化潜能,是理想的组织工程种子细胞.     

大鼠骨髓基质干细胞的分离培养与向神经元样细胞诱导分化的实验研究

目的 探讨大鼠骨髓基质干细胞的提取、分离培养和体外扩增的最佳条件,研究其在体外培养中定向诱导分化为神经元样细胞的可能。方法 通过密度梯度离心和贴壁培养法从成年大鼠骨髓中分离骨髓基质干细胞,进行培养扩增,观察其生长特性;用2-巯基乙醇(β-mercaptoethanol,β-ME)对传代细胞诱导分化,并通过免疫细胞化学染色鉴定分化细胞的类型。结果 原代培养时形成由基质干细胞组成的细胞集落,细胞集落14d时接近融合,传代后,细胞体积变大,约5～7d传代一次。β-ME诱导后,70％以上的细胞在形态上呈神经元样,免疫细胞化学染色呈NSE阳性,GFAP阴性,说明诱导分化的细胞为神经元,而不是星形胶质细胞。结论 骨髓基质干细胞在体外培养条件下生长良好,并可连续传代;在β-ME作用下可被诱导分化为神经元样细胞。     

猪脂肪间充质干细胞的分离培养及其成脂分化

脂肪间充质干细胞(Adipose mesenchymal stemcell,AMSCs)是一类来源于脂肪组织并具有多向分化潜能的干细胞。近年来的研究证明,脂肪组织具有取材方便和干细胞含量高的优势,有望在研究与应用领域成为骨髓干细胞的替代物。猪是一种比啮齿类更接近人类的模式动物,具有较强的脂肪沉积能力。本研究探讨了猪脂肪间充质干细胞的体外分离纯化、培养扩增和向脂肪细胞诱导分化的条件。采用Ⅰ型胶原酶消化分离脂肪微管基质成分,传代培养扩增,流式细胞仪检测细胞表面标记。取第3-7代AMSCs,采用不同方法诱导AMSCs向脂肪细胞分化,光学显微镜下可观察到诱导后的细胞内有高折光性的小脂滴出现,油红O染色成阳性,不同诱导方法诱导率不同。被诱导细胞用RT-PCR可检测到脂肪细胞分化标志基因LPL和PPARγ的表达。结果表明可以从脂肪组织中分离培养出AMSCs,经传代后可提高其纯度。CD44、CD105表达呈阳性,CD14、CD34、S-100、HLA-DR呈阴性,在合适的诱导条件下,可向脂肪细胞分化。     

鼻黏膜间充质干细胞的培养、鉴定及诱导分化

目的建立Sprague Dawley(SD)大鼠鼻黏膜间充质干细胞(NM-MSCs)的获取、分离、培养方法,初步了解其生物学特性。方法通过SD大鼠鼻黏膜贴壁法体外分离、培养NM-MSCs,光镜下细胞形态学观察,用免疫荧光技术检测间充质干细胞和神经干细胞标记物,用流式细胞术检测细胞表面标记物,再诱导其向骨组织及脂肪组织方向分化,最后对其进行细胞周期检测及分析。结果分离培养的NM-MSCs光镜下以梭形与多角形细胞为主,呈放射状排列,且生长旺盛;免疫荧光染色显示NM-MSCs同时表达STRO-1和Nestin;第4代NM-MSCs不表达CD19、CD31、CD34、CD45及HLADR细胞表面标记物,但表达CD90、CD105基质细胞标记物;NM-MSCs经成骨、成脂诱导后,茜素红染色和油红O染色均呈阳性;细胞周期分析显示NM-MSCs符合干细胞生长的特性。结论 SD大鼠NM-MSCs组织块贴壁法获取容易,操作简单,且可大量扩增用于细胞实验研究,经体外诱导后具有多向分化潜能,具有间充质干细胞的一般生物学特性。SD大鼠鼻黏膜组织块贴壁法为组织细胞工程研究提供了充足的种子细胞来源。     

胰腺导管单克隆上皮样干细胞的多分化潜能

体外诱导源于成年大鼠的胰腺导管单克隆上皮样干细胞分化形成胰岛、神经、脂肪及成骨细胞,探讨干细胞的多分化潜能。扩增培养源于成年大鼠的胰腺导管单克隆上皮样干细胞,采用不同的诱导液体外诱导其向胰岛、神经、脂肪及成骨细胞分化,并通过DTZ染色、糖刺激试验、免疫荧光反应、油红O染色、茜素红染色或Vonkossa染色的方法对分化细胞进行检测。结果显示,体外诱导培养干细胞分化形成类胰岛,DTZ染色阳性,糖刺激分泌胰岛素、C-肽;分化形成类神经细胞,表达神经元特异性烯醇化酶;分化形成类脂肪细胞,油红O染色阳性;分化形成类成骨细胞,其分泌物呈岛状矿化结节,茜素红和Vonkossa染色阳性。这表明,该源于成年大鼠的胰腺导管上皮样干细胞系具有多分化潜能。     

人脐带和骨髓间充质干细胞体外分离培养及生物学特性比较

目的体外分离培养、扩增人脐带间充质干细胞(h UC-MSC)和人骨髓间充质干细胞(h BM-MSC),并对其生物学特性进行比较。方法采用组织块贴壁法从足月胎儿脐带分离、纯化和培养h UC-MSC,健康成人骨髓肝素抗凝后,采用密度梯度离心法分离、纯化和培养h BM-MSC;用倒置显微镜观察两种细胞的形态及细胞生长增殖情况,流式细胞仪分析检测第3代细胞表面标志的表达,Von?Kossa染色及油红O染色检测分化潜能。结果镜下两种细胞均为贴壁生长,形态为均一的成纤维细胞样,取相同数量的细胞传代接种后,h UC-MSC的增殖速率快于h BM-MSC,两种细胞具有均一的细胞表型,均表达CD29、CD44、CD105,不表达CD45、CD34、HLA-DR、HLA-G、CD80、CD86,两种细胞都有成骨、成脂分化潜能,但h UC-MSC的分化潜能更强。结论 h UC-MSC与h BM-MSC具有相似的生物学特性,且前者具有更强的增殖能力和分化潜能,h UC-MSC有望成为h BMMSC理想的替代来源。     

再生障碍性贫血患者骨髓间充质干细胞生物学特性的初步研究

目的:研究再生障碍性贫血(aplasticanemia,从)患者骨髓间充质干细胞(mesenchymalstemcells,MSCs)的生物学特性和初步探讨其异常和AA发生的可能关系。方法:取AA患者骨髓间充质干细胞,测定其生长曲线和倍增时间;流式细胞仪检测其细胞周期和免疫表型;体外定向诱导其向脂肪、成骨、内皮和神经细胞分化;用real-timePCR及油红O染色法比较AA和正常对照组MSCs的成脂分化的不同。结果:AA患者和正常成人的MSCs均呈梭形贴壁生长;AA组细胞倍增时间长于对照组;CD105、CD44、CD29、CD106、FlK-1均阳性;96．51％的细胞处在G0／G1期;AA患者的MSCs保持了多向分化潜能,体外诱导形成脂滴较对照组早,诱导早期的脂蛋白脂酶表达增高。结论:再生障碍性贫血患者的骨髓间充质干细胞增殖能力较正常成人弱,骨髓间充质干细胞的易成脂性可能参与了再障的发病环节。     

The differentiation of human placenta-derived mesenchymal stem cells into dopaminergic cells in vitro

Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.     

脐血间充质干细胞诱导向神经元样细胞分化的实验研究

目的：研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法：采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果：纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论：分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。     

人脐带间充质干细胞的成脂诱导分化及其相关基因鉴定

目的:探讨人脐带间充质干细胞(hUC-MSCs)的分离提取、体外诱导分化为脂肪细胞的能力及其相关基因的表达情况。方法:取新生儿脐带经组织培养法提取后分离培养于αMEM完全培养基中,经大量纯化与扩增后,采用倒置显微镜观察其形态与细微结构;流式技术检测其细胞周期及表面标志;以含有成脂诱导剂的αMEM培养基对P3的hUC-MSCs进行培养,诱导其向脂肪细胞方向分化,对其对诱导后的细胞进行检测。应用油红"O"染色对其进行定性鉴定;应用实时定量RT-PCR对LPL、Leptin的基因含量进行测定。结果:经过组织培养法后,细胞呈贴壁生长,细胞呈梭性或旋涡状,形态不规则,多数有凸起,细胞核较大,核仁明显;第7代以前的细胞具有较强的生长活性;流式技术检测发现此类细胞高表达CD44、CD73和CD105等细胞表面标记,而几乎不表达CD34、CD45、CD31和HLA-DR。培养至第3代的细胞约72.724%的细胞处G1期、S期的细胞仅占18.069%,第7代时G1期细胞约为83.875%、S期为9.606%左右;经成脂诱导剂诱导分化后,细胞经油红"O"染色,分化为脂肪细胞的细胞着色并呈红色,实时定量RT-PCR结果显示该部分细胞表达脂肪细胞的标志性基因LPL和Leptin。结论:P7以前的hUC-MSCs具有较强的生长分化能力,可以向脂肪细胞进行分化并表达一定量的特定标志性基因。     

Colon cancer mesenchymal stem cells modulate the tumorigenicity of colon cancer through interleukin 6

Multipotent mesenchymal stem cells (MSCs) have been isolated from several tumors and are implicated to play critical roles to increase malignant cell growth, invasion and metastasis. Here, we show that the MSC-like cells were isolated from human colon cancer tissues. These isolated hCC-MSCs (human colon cancer-derived mesenchymal stem cells) shared similar characteristic features with bone marrow-derived MSCs, which include cell morphology, surface antigens and specific gene expression. Additionally, the hCC-MSCs could differentiate into osteocytes or adipocytes under appropriate culture conditions. The conditioned medium collected from the cultured hCC-MSCs was shown to enhance the migration and invasive activity of HCT-116 colon cancer cells in vitro. Besides, transplantation of HCT-116 cells along with hCC-MSCs in nude mice increased the tumor growth and metastasis. Further study revealed that IL-6 present in the hCC-MSC-conditioned medium sufficiently induced the levels of Notch-1 and CD44 in HCT-116 and HT-29 cells, which contribute to enhance tumorigenic activity of HCT-116 and HT-29 cells. By using immunohistochemical staining, the intense co-expression of IL-6, Notch-1 and CD44 was predominantly detected in human colon cancer tissues. Taken together, our findings suggest the importance of the IL-6/Notch-1/CD44 signaling axis in the interaction between hCC-MSCs and colon cancer cells.     

Mesenchymal stromal cells from unconventional model organisms

Mesenchymal stromal cells (MSCs) are multipotent, plastic, adherent cells able to differentiate into osteoblasts, chondroblasts and adipocytes. MSCs can be isolated from many different body compartments of adult and fetal individuals. The most commonly studied MSCs are isolated from humans, mice and rats. However, studies are also being conducted with the use of MSCs that originate from different model organisms, such as cats, dogs, guinea pigs, ducks, chickens, buffalo, cattle, sheep, goats, horses, rabbits and pigs. MSCs derived from unconventional model organisms all present classic fibroblast-like morphology, the expression of MSC-associated cell surface markers such as CD44, CD73, CD90 and CD105 and the absence of CD34 and CD45. Moreover, these MSCs have the ability to differentiate into osteoblasts, chondroblasts and adipocytes. The MSCs isolated from unconventional model organisms are being studied for their potential to heal different tissue defects and injuries and for the development of scaffold compositions that improve the proliferation and differentiation of MSCs for tissue engineering.     

Differentiation potential of bone marrow mesenchymal stem cells in duck

The bone marrow mesenchymal stem cells （MSCs） are multipotent stem cells which can differentiate into mesenchymal cells in vitro. In this study, MSCs in duck were isolated from bone marrow by density gradient centrifuge separation, purified and expanded in the me- dium. The primary MSCs were expanded for 11 passages. The different-passage MSCs were induced to differentiate into osteoblasts and neuron-like cells. Karyotype analysis indicated that MSCs kept diploid condition and the hereditary feature was stable. The different- passage MSCs expressed CD44, ICAM-1 and SSEA-4, but not CD34, CD45 and SSEA-1 when detected by immunofluorescence staining There was no significant difference among the positive rates of passages 2, 6 and 8 （P 〉 0.05）, but a significant difference existed among those of passages 2, 6, 8 and 11 （P 〈 0.05）. After the osteogenic inducement was added, the induced different-passage MSCs expressed high-level alkaline phosphatase （ALP）, and are positive for tetracycline staining, Alizarin Red staining and Von Kossa staining. After the neural inducement was added, about 70% cells exhibited typical neuron-like phenotype, the induced different-passage MSCs expressed Nestin, neuron-specific enolase （NSE） and glial fibrillary acidic protein （GFAP） when detected by immunofluorescence staining. There was no significant difference among the positive rates of passages 3, 4 and 6 （P〉0.05）, but a significant difference existed among those of passages 3, 4, 6 and 8 （P〈0.05）. These results suggest that MSCs in duck were capable of differentiating into osteoblasts and neuron-like cells in vitro.     

具有间充质干细胞特征的CD105+细胞在胎儿多种组织器官的分布

CD105是骨髓间充质干细胞的特征性表型之一。为了研究机体各组织器官也存留有间充质干细胞,首先检测胎儿各组织CD105^ 细胞的分布,进而分离胎儿各组织CD105^ 细胞。将CD105^ 细胞向脂肪和成骨细胞诱导分化。结果表明胎儿心、肝、肺、血管、肌肉、皮肤等组织含有CD105^ 间充质干细胞。在间充质干细胞分化为脂肪细胞时,CD105表达明显下降。地塞米松可以促进脂肪细胞形成并提高了培养液中甘油三酯的含量。而向成骨细胞分化时,诱导的成骨细胞胞浆内外有电子密度高的钙盐沉积。以上结果提示,分布于多种组织的间充质干细胞异常分化可能与疾病的发生有关。