人脂肪来源的间充质干细胞的生物学研究

目的探索从人脂肪中获取间充质干细胞的方法,通过观察人脂肪来源间充质干细胞(hADAS)的形态学、生长动力学、细胞表面标志,揭示其生物学特性。方法分离培养脂肪间充质干细胞并观察其形态学变化;四甲基偶氮唑盐(MTT)比色法测细胞活性;流式细胞仪测细胞周期和间充质干细胞表面抗原。结果脂肪干细胞呈成纤维细胞样;MTT比色法证实其有很强的增殖活性;流式细胞周期显示处于增生活跃期的细胞占很大比例;间充质干细胞表面抗原CD29、CD44随着传代表达逐渐增高,不表达内皮细胞标志CD31,造血干细胞标志CD34随细胞培养时间延长表达逐渐降低。结论通过酶消化法可从人脂肪中获取间充质干细胞,具有很强的增殖能力,表达干细胞表面标志。     

银屑病患者皮损间充质干细胞生长缓慢的原因探讨

目的通过比较银屑病患者与健康人皮肤间充质干细胞(DMSCs)原代培养和3代总培养时间,及培养上清液中碱性成纤维细胞生长因子(bFGF)的浓度,研究DMSCs增殖速度与bFGF含量的关系。方法Dispase酶消化结合贴壁法分离培养患者与健康人皮肤DMSCs,流式细胞术及多向分化法进行细胞鉴定;细胞生长达90%融合时胰酶消化传代,记录原代培养时间与3代总培养时间,酶联免疫吸附试验(ELISA)法检测细胞培养上清液bFGF的浓度,bFGF对正常DMSCs增殖的影响。结果银屑病组与对照组皮肤DMSCs的细胞形态相似,均具有多向分化能力,细胞表面抗原CD105、CD44、CD73、CD29及CD90表达阳性,CD34、CD45及人类白细胞抗原(HLA-DR)表达阴性。与对照组相比,银屑病患者DMSCs原代培养和3代总培养时间延长(P<0.01),分泌的bFGF降低(P<0.01),加入bFGF组DMSCs原代培养和3代总培养时间缩短(P<0.01)。结论银屑病皮损DMSCs分泌bFGF水平下降,直接影响其增殖速度,这可能是银屑病患者间充质干细胞生长缓慢的原因。     

大鼠骨髓间充质干细胞生物学特性的研究

目的研究体外大鼠骨髓间充质干细胞的生长特性。方法在无菌条件下,取大鼠骨髓,采用密度离心法和全骨髓贴壁法联合培养并结合传代获取、纯化细胞,倒置显微镜观察其形态,流式细胞术检测其表面抗原CD29、CD90及CD34的表达。CCK-8法测定第1、3代细胞的增殖情况,并绘制其生长曲线。结果经全骨髓贴壁法和密度梯度离心联合培养,所得到的原代细胞形态呈椭圆形、圆形、三角形,接近融合状态时可呈现均一的长梭形,排列规则。传至第8代时,细胞的增殖能力减弱,其形态宽大畸形,呈树枝状。经流式细胞仪检测,所分离的细胞CD29的表达率为94.97%,CD90的表达率为88.50%,CD34的表达率为2.23%。CCK-8法显示第3代细胞有较强的增殖能力。结论体外培养的大鼠BMSCs是骨组织工程的优良种子细胞。     

大鼠脂肪间充质干细胞的分化潜能鉴定及XIAP基因修饰*

目的分离培养扩增大鼠脂肪源间充质干细胞(ADSCs),以活体标记并鉴定其分化潜能,了解ADSCs的X连锁凋亡抑制蛋白（XIAP）基因修饰的可行性。方法无菌条件下取大鼠一侧腹股沟脂肪组织,Ⅰ型胶原酶消化法分离培养ADSCs,胰酶消化法传代扩增。检测细胞分化为脂肪细胞、软骨细胞及成骨细胞的潜能,转染XIAP表达质粒进入ADSCs,通过Western blotting等方法检测XIAP的表达能力。结果 ADSCs呈长梭形漩涡样生长,细胞流式鉴定显示CD29、CD44、CD90、CD105均呈高表达,并在特定诱导剂下分化为脂肪细胞、软骨细胞或成骨细胞。XIAP转染后显像经XIAP基因修饰的脂肪间充质干细胞在PVDF膜的相应分子质量区域出现相应的条带。结论脂肪源干细胞易于培养和传代扩增,并可活体标记,具有多向分化潜能,可作为组织工程的种子细胞。     

低氧对大鼠骨髓间充质干细胞HIF-1α表达影响的研究

目的通过体外不同氧浓度下培养大鼠骨髓间充质干细胞,RT-PCR方法检测,观察正常氧浓度和低氧浓度对HIF-1α(缺氧诱导因子-1α)表达的影响。方法分离雄性Wistar大鼠骨髓间充质干细胞体外培养,鉴定其CD34、CD44的表达,分为正常氧浓度组和低氧浓度组培养24h,RT-PCR方法检测,不同氧浓度对细胞HIF-1α表达的影响。结果贴壁法培养的细胞表达CD44,不表达CD34;正常氧浓度培养的细胞较少有HIF-1α的表达,低氧浓度培养的细胞HIF-1α的表达明显增加。结论细胞低氧可激活HIF-1α的表达,低氧浓度培养的细胞的HIF-1α表达较正常氧浓度培养的细胞的表达明显增加。     

SD大鼠脂肪干细胞的体外分离培养及实验研究

目的探索从脂肪组织中分离、培养SD大鼠脂肪干细胞(Adipose tissue-derivedstromal cells,ADSCs)的方法,同时观察其生物学特性。方法取SD大鼠腹股沟区皮下脂肪组织,0.1%Ⅰ-型胶原酶消化分离、培养ADSCs,流式细胞术测定CD44、CD45和CD49d抗原的表达,MTT比色法测定细胞生长活力,诱导培养基向脂肪细胞定向分化,Oil Red O染色鉴定。结果分离出的细胞CD44表达阳性,CD49d表达弱阳性和CD45表达阴性,其生长曲线呈倒"S"形,成脂诱导培养基定向诱导分化,经Oil Red O染色呈红色。结论本次实验所分离出来的SD大鼠ADSCs在体外具有生长稳定,增殖较快并能诱导分化的特点。     

成体大鼠骨髓间充质干细胞的表型研究

目的文献报道大鼠骨髓间充质干细胞（MSCs）不表达CD45,而本实验中却发发现CD45高度表达,针对这一现象做初步分析：方法成体大鼠骨髓MSCs的分离培养,对原代、第3代和第6代细胞做流式细胞仪检测MSCs的表面标记物以及染色体分析。结果体外培养的大鼠骨髓MSCs呈长梭型、有突起,排列成旋涡状。流式细胞仪检测示原代细胞：CD29（99．9％）,CD90（48．5％）,CD45（996％）,CD34（202％）;第3代细胞：CD29（100％）,CD90（97．1％）,CD45（100％）,CD34（089％）;第6代细胞：CD29（99．7％）,CD90（99．6％）,CD45（99．7％）,CD34（0．49％）。上述3代细胞的染色体分析床细胞核型为二倍体。结论体外培养的成体大鼠骨髓MSCs同时表达CD29,CD90和CD45,不表达CD34。     

体外诱导成人脂肪间充质干细胞分化为平滑肌细胞的实验研究

目的体外培养成人脂肪间充质干细胞(ADMSCs),并应用血小板衍生生长因子-BB(PDGF-BB)诱导ADMSCs分化为平滑肌细胞。方法采用酶消化法和贴壁培养法分离培养ADMSCs,流式细胞仪对第5代细胞进行表面抗原和细胞周期的检测,然后对第5代细胞进行PDGF-BB诱导,于诱导后2周进行免疫组织化学鉴定。结果体外培养的ADMSCs呈梭形,细胞形态均一,传代稳定。干细胞相关标志CD29,CD44表达阳性,内皮细胞相关标志CD31和造血干细胞相关标志CD34表达阴性。ADMSCs中G0/G1,S,G2/M期的细胞分别占90.14%,3.77%,6.09%。定向诱导后倒置显微镜下观察细胞呈长梭状,胞膜清晰,无空泡,可重叠生长,融合后细胞形成"峰"和"谷"状,免疫荧光化学显示诱导组细胞α平滑肌肌动蛋白表达阳性。结论成人脂肪组织中含有间充质干细胞,且可经PDGF-BB诱导分化为平滑肌细胞。     

转化生长因子β1联合生长分化因子-5体外诱导骨髓间质干细胞向类髓核细胞分化的实验研究

目的探讨转化生长因子β1联合生长分化因子-5体外诱导骨髓间质干细胞向类髓核细胞分化的可能性。方法取SD大鼠骨髓间质干细胞,流式细胞仪检测两种干细胞CD105、CD90、CD44、CD29、CD45、CD34、CD24的表达。将增殖至第三代的BMSCs分为对照、TGF-β1、GDF-5、TGF-β1+GDF-5四组,分别以含不同细胞因子诱导液培养14d后,采用RT-PCR检测各组细胞Ⅱ型胶原、蛋白多糖、SOX-9基因的表达。结果两种干细胞CD105、CD90、CD44、CD29表达阳性;CD45、CD34、CD24表达阴性。向类髓核细胞诱导培养14d后,TGF-β1、GDF-5、TGF-β1与GDF-5三组的Collagen typeⅡ、Aggrecan、SOX-9基因表达水平较对照组均有明显升高,差异有统计学意义(P0.05)。联合诱导组经过诱导后的Collagen typeⅡ、Aggrecan、SOX-9基因表达水平明显高于TGF-β1组及GDF-5组,差异有统计学意义(P0.05)。结论 GDF-5与TGF-β1都具有诱导BMSCs向类髓核细胞分化的能力,且二者之间具有协同作用,联合应用可以更好的诱导BMSCs向类髓核细胞分化。     

大鼠骨髓间充质干细胞的体外分离与培养

目的 建立体外分离纯化及培养扩增大鼠骨髓间充质干细胞的方法.方法 应用全骨髓贴壁培养法分离培养大鼠骨髓间充质干细胞,并进行细胞传代培养.倒置显微镜下观察细胞形态及生长特征,测定细胞生长曲线,通过流式细胞仪检测细胞表面标志物,取第3代细胞分别加入成骨、成脂诱导剂并采用碱性磷酸酶染色及Von Kossa染色鉴定成骨能力,以油红O染色鉴定成脂能力.结果 获取的大鼠骨髓间充质干细胞形态呈均一成纤维细胞样,并呈集落样生长.传至第3代细胞纯度可达97%以上,其细胞表型CD29、CD44、CD105、CD166呈阳性表达,CD34、CD80、CD86呈阴性表达.经成骨诱导后细胞碱性磷酸酶染色及Von Kossa染色呈阳性,成脂诱导后细胞油红O染色呈现阳性.结论 应用全骨髓贴壁法可以分离培养出高纯度的大鼠bMSCs,培养的细胞扩增迅速、生物学特性稳定,是一种较为理想的体外分离扩增bMSCs的培养体系.     

Histamine produced by macrophage and T lymphocyte: a new type of signal transducer

Macrophages (M phi) produce histamine (Hm) when activated by bacterial endotoxin (LPS) through induced histidine decarboxylase (HDC). Among the cytokines tested, GM-CSF or IL-3 specifically augmented the LPS-dependent HDC induction by M phi. Hm formed by M phi regulates synthesis of cytokines such as IL-1, IL-6, G-CSF and M-CSF by the cells per se and may modulate immune reactions and division and differentiation of various hematopoietic cells. Kupffer cells, M phi-like cells in the liver, also synthesize Hm in mice injected with hepatotoxins such as tetradecanoylphorbol acetate or LPS. Hm thus produced by Kupffer cells may participate in the regeneration of the injured liver through induction of hepatocyte growth factor. Concanavalin A (Con A) enhanced Hm formation by T lymphocytes. GM-CSF or IL-3 also enhanced the Hm synthesis by CD4+ and CD8+ T cells. Hm formed by T cells regulates immune reactions such as lymphocyte blastogenesis. In animals infected with gram(-) bacteria Hm is produced by the M phi-T cell system and may regulate immune competence to the bacteria. In addition, Hm may act as a signal transducer between the peripheral immune system and hypothalamus-pituitary-adrenal system, leading to GC secretion, in order to prevent occurrence of tissue injury caused by excess immune reactions.     

Effect of myelopoietic and pleiotropic cytokines on colony formation by blast cells of children with acute lymphoblastic leukemia

The aim of this study was to see whether pleiotropic or myeloid hematopoietic growth factors, which do not stimulate normal lymphoid cells, can induce proliferation of blast cells of the acute lymphoid leukemia (ALL) of childhood. Bone marrow cells of 13 children with untreated ALL (nine common ALL, two myeloid antigen positive ALL and two early T-cell ALL) formed colonies of leukemic blast cells in primary methylcellulose cultures. Spontaneous growth was observed in three of 13 cases, whereas phytohemagglutinin-stimulated leukocyte conditioned medium (PHA-LCM), a conventional source of various natural human cytokines, induced colony formation in ten of 13 cases. A similar rate of responsiveness was seen with recombinant human granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and stem cell factor (SCF); a combination of these three cytokines induced colony formation in all cases studied. The effect of these growth factors on colony formation seemed to be dose-dependent in some cases. Of the stimuli studied, GM-CSF induced the smallest number of colonies, whereas the effects of G-CSF, SCF and PHA-LCM were similar in this respect. Combination of cytokines proved to be even more efficient in inducing clonal proliferation of leukemic lymphoblasts. In double combinations, G-CSF and GM-CSF as well as G-CSF and SCF were able to potentiate each other's effects. Triple combination of these cytokines mediated the most potent growth stimulus. Our results demonstrate that myeloid and pleiotropic cytokines are able to stimulate clonal proliferation of pediatric leukemic lymphoblasts. This may present a potential hazard to children with ALL while on adjuvant therapy with hematopoietic growth factors. In vitro colony assays performed prior to or in parallel with the administration of hematopoietic growth factors to ALL patients may help to forecast their possible effects on leukemic cells in vivo.     

Relationship between endogenous colony stimulating factors and apoptosis in human colon cancer cells: role of cyclo-oxygenase inhibitors.

1. Nonsteroidal anti-inflammatory drug (NSAID) usage is associated with gastrointestinal inflammatory damage and aggravation of gut inflammatory conditions. NSAIDs also exert a preventive effect against colon cancer that seems to be due to increased colon cell apoptosis. NSAIDs have been shown to modulate the release of colony stimulating factors (CSFs) in some cells. In the present study we analysed the effect of these drugs on secretion of CSFs and apoptosis in human colon epithelial cells (HT-29). 2. HT-29 cells secreted bioactive levels of GM-CSF, G-CSF and M-CSF when stimulated with IL-1ss and TNF-alpha, and diclofenac (10(-7)-10(-4) M), indomethacin (10(-7)-10(-4) M) and sodium salicylate (10(-5)-10(-2) M) induced concentration-dependent increases in GM-CSF secretion. 3. Reduced secretion of G-CSF and M-CSF and increased cell apoptosis were observed with the highest concentrations of these non-selective NSAIDs. 4. No changes in any CSF release or HT-29 cell apoptosis were detected in the presence of the COX-2 selective inhibitor DFP (10(-7)-10(-4) M). 5. Neither the exogenous addition of CSFs nor the blockade of secreted CSFs modified apoptosis in HT-29 cells stimulated with cytokines and/or NSAIDs. 6. These results suggest that colon epithelial cells can contribute to local inflammatory responses by releasing CSFs and thus extend the life span of local leukocytes. Modulation of CSF levels by non-selective NSAIDs may be involved in the pro-inflammatory effects of these agents in the gut.     

人上睑眶隔脂肪来源的脂肪干细胞的分离培养及鉴定

目的探讨人上睑眶隔来源的脂肪提取脂肪干细胞(ADSCs)的可行性,并进行分离、培养及鉴定。方法取健康成年人上睑眶隔脂肪组织,用胰酶进行消化后收集细胞接种于培养瓶内,采用差速贴壁法纯化细胞。用HE染色、免疫荧光进行细胞鉴定,并进行成脂、成软骨诱导。结果 HE染色显示细胞形态为长梭形,呈漩涡状生长。免疫荧光鉴定结果显示细胞表面抗原CD44、CD29阳性表达,CD106、CD34阴性表达,说明分离培养的细胞是脂肪干细胞。成脂、成骨诱导实验证明所得细胞有多向分化的能力。结论可以从人上睑眶隔脂肪组织提取出ADSCs,并有多向分化能力,为今后ADSCs的取材提供了新的路径。     

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。     

Effects of naturin 2 on colony formation by hematopoietic progenitor cells in human umbilical cord blood mediated by induction of release of growth factors

Naturin 2, a health drink, contains a mixture of Chinese herb plants and is a potent immunomodulator. It has anti-tumor effects mediated by immune system. In the present study, low density (LD) and purified CD34+ cells (enriched for hematopoietic stem [HSC] and progenitor [HPC]) from human umbilical cord blood (CB) were assayed for colony formation in response to Naturin 2. First, we examined the in vitro activity of Naturin 2 on HPC. Naturin 2 by itself stimulated colony formation derived from either LD or CD34+ CB cells. The stimulatory effect by Naturin 2 was mediated by both direct and indirect action on HSCs/HPCs. The indirect action is via releasing of cytokines in the 5 day-conditioned media by LD CB cells with Naturin 2. The stimulatory activities in the 5 day-conditioned media (CM) could be blocked by the neutralization antibodies against interleukin (IL)-3, granulocyte macrophage (GM)-colony stimulating factor (CSF), IL-1 alpha, IL-1 beta and M-CSF. Therefore, the stimulatory activities detected in the media conditioned are due to the cytokines released in the cultures. In addition, we have also determined that addition of Naturin 2 to the cultures with LD or CD34+ cells stimulated by IL-3 and/or GM-CSF resulted in a decrease of colony formation. The inhibitory effect of Naturin 2 was mediated, at least in part, by releasing suppressive cytokines, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, in the 3 day--CM, since antibodies against these two suppressive cytokines partially blocked the inhibitory activity. These results demonstrate that Naturin 2 has differential effects on hematopoiesis.     

人脂肪间充质干细胞分离培养及其生物学特性的实验研究

目的探索人脂肪间充质干细胞（adipose tissue—derived mesenchymal stem cells，ADMSCs）分离培养的方法及体外扩增的条件，观察ADMSCs的生物学特性。方法以腹部手术患者皮下脂肪组织为材料，采用I型胶原酶消化法及贴壁法分离培养ADMSCs，在含10％胎牛血清的低糖DMEM培养基中贴壁培养，倒置显微镜观察，流式细胞仪检测细胞表面标记CD29、CD44、CD105、CD31、CD34、CD106的表达，透射电镜及扫描电镜下观察ADMSCs超微结构，流式细胞仪测定细胞周期。结果原代和传代细胞呈梭形外观，生长增殖能力良好。CD29、CD44、CD105均呈阳性表达，阳性率分别为95.3％、98．6％和86．5％；而CD31、CD34、CD106阳性率分别为3．5％、2．6％、1．3％。透射电镜观察显示ADMSCs表现出早期幼稚细胞形态的特点，流式细胞仪检测显示84．8％的细胞处于G0/G1期。结论酶消化法能有效地从人脂肪组织分离培养人ADSCs，细胞生长稳定，增殖能力活跃，为今后ADMSCs的分离培养提供了更简单有效的方法。