人脐带间充质干细胞体外分化为胰岛素分泌细胞

目的探讨人脐带间充质干细胞(UC-MSCs)向胰岛素分泌细胞分化的可能性。方法采用直接贴壁法从脐带中分离UC-MSCs,取第3代细胞作流式细胞术鉴定其表型。用β-巯基乙醇、尼克酰胺和碱性成纤维生长因子(bFGF)诱导UC-MSCs(诱导组),对照组在未添加任何诱导试剂的培养基中培养。倒置显微镜下观察UC-MSCs的形态学变化;取诱导后3周的细胞进行双硫腙染色,采用化学发光免疫法测定培养上清液中胰岛素水平,RT-PCR检测胰岛细胞相关基因的表达。结果细胞高表达UC-MSCs相关抗原CD90、CD105和CD13,而低表达造血细胞相关抗原CD34、CD45和HLA-DR。UC-MSCs经诱导后细胞形态由梭形变为圆形或椭圆形,双硫腙染色为阳性。诱导组胰岛素水平明显高于对照组[(0.305±0.065)μU/ml vs.(0.085±0.024)μU/ml](P<0.01)。RT-PCR显示诱导后细胞表达胰岛细胞相关基因。结论 UC-MSCs具有向胰岛素分泌细胞分化的潜能。     

体外诱导人脐带间充质干细胞向胰岛素分泌细胞分化

目的 通过体外培养将人脐带间充质干细胞诱导分化为胰岛素分泌细胞.方法 将体外培养的人脐带间充质干细胞分为诱导组和对照组.诱导组先后予以1 mmol·L-12-巯基乙醇培养2 d,10 μg·L-1表皮生长因子、10 μg·L-1碱性成纤维细胞生长因子及2% B27培养7 d,之后添加20 mmol·L-1尼克酰胺及艾塞那肽培养7 d.对照组不添加诱导剂,继续换液培养.通过观察细胞形态变化、RT-PCR体外扩增两组细胞的胰腺-十二指肠同源框-1（PDX-1）基因、放射免疫法测定两组细胞胰岛素分泌量三种方法来鉴定胰岛素分泌细胞.结果 （1）通过形态学观察,诱导组细胞类似胰岛样细胞,对照组细胞则无明显改变;（2）应用RT-PCR方法,诱导组细胞可扩增出PDX-1基因,而对照组则没有.（3）诱导组细胞培养上清液可检测出胰岛素分泌,7 d时胰岛素浓度为（6.32±0.18）mIU·L-1,21 d时增至（34.57±4.54）mIU·L-1,而对照组则未测出.结论成功在体外微环境下将人脐带间充质干细胞诱导分化为胰岛素分泌细胞.     

神经节苷脂-1与β-巯基乙醇体外联合诱导脂肪间充质干细胞向神经元样细胞分化的实验研究

目的探讨脂肪间充质干细胞（ADSCs）向神经元样细胞分化的规律,以及神经节苷脂-1（GM- 1）在诱导分化过程中的作用。方法取成年兔颈后脂肪组织采用胶原酶消化法原代培养脂肪间充质干细胞,取第5代脂肪间充质干细胞分别采用β-巯基乙醇及GM-1与β-巯基乙醇联合作用的方式向神经元样细胞诱导。免疫细胞化学法检测神经元特异性烯醇化酶（NSE）的表达,细胞化学染色检测尼氏小体的生成。结果成功分离出脂肪间充质干细胞,通过β-巯基乙醇及GM-1与β-巯基乙醇联合作用均可使ADSCs定向诱导为神经元样细胞,联合诱导组NSE及尼氏小体表达的阳性率均高于单纯β-巯基乙醇诱导组,差异有统计学意义（P＜0．05）。结论从兔脂肪组织中能够分离出脂肪间充质干细胞,通过诱导可向神经元样细胞分化,GM-1具有促进脂肪间充质干细胞向神经元样细胞分化的能力。     

胰高血糖素样肽1和Extentin-4体外诱生人骨髓间充质干细胞分化为分泌胰岛素细胞

目的 探讨并优化人骨髓间充质干细胞（hBMSCs）向分泌胰岛素细胞（IPCs）定向分化的条件,为糖尿病替代疗法寻找新出路.方法 hBMSCs复苏培养后,联合应用胰高血糖素样肽1（GLP-1）、Ex-tentin-4等细胞因子通过三步诱导方案进行体外向IPCs诱导分化.应用反转录-聚合酶链反应检测与β细胞发育和功能相关的基因表达;免疫细胞化学、双硫腙染色证实IPCs的生成;电化学发光法检测细胞胰岛素的分泌量.结果 诱导分化18d后,镜下观察到胰岛样细胞团的生成,双硫腙及免疫细胞化学染色胰岛素均呈现阳性反应;诱导分化的细胞在第18天则可以观察到胰十二指肠同源盒基因1、胰岛素的高表达;诱导分化的细胞接受葡萄糖刺激后能够分泌胰岛素,且胰岛素的分泌量随葡萄糖浓度的提高而增加,高糖胰岛素分泌量[（188.7±1.5）mU/L]与低糖胰岛素分泌量[（60.1±0.9）mU/L]相比,差异有统计学意义（P＜0.05）.结论 体外联合应用GLP-l、Extentin-4、尼克酰胺等细胞因子采用三阶段诱导方案可以大大提高胰岛素细胞诱导分化率,并能促进IPCs的成熟和胰岛素的释放.     

体外培养人脐带血间充质干细胞向胰岛细胞分化及对糖尿病治疗的实验研究

目的研究人脐带血间充质干细胞向胰岛素分泌细胞分化的潜能及其移植后对糖尿病大鼠的治疗效果。方法体外分离培养HUCB-MSCs,在胰岛细胞培养条件下经药物定向诱导其分化;免疫组化对诱导细胞进行胰岛β细胞标记鉴定;双硫腙染色鉴定锌离子表达及检测胰岛样细胞的移植效果。结果 HUCB-MSCs经诱导后,免疫细胞化学染色显示表达人胰岛素;双硫腙染色呈棕红色;移植后2周,胰岛样细胞组血糖浓度明显降低。结论 HUCB-MSCs在体外诱导培养条件下,具有向胰岛素分泌细胞分化的潜能,这种细胞可能为Ⅰ型糖尿病提供一条新的治疗途径。     

全反式维甲酸联合细胞因子诱导骨髓间充质干细胞分化为神经元样细胞的作用

目的 探索全反式维甲酸(ATRA)、碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)在诱导骨髓间充质干细胞(BMSCs)分化为神经元样细胞过程中的作用.方法 应用Ficoll分离骨髓中单核细胞,贴壁培养,观测形态并用流式细胞仪检测其细胞表面标志;用含ATRA、bFGF、EGF的条件培养基对培养的细胞向神经细胞诱导分化;采用免疫荧光染色法对分化的细胞进行鉴定.结果 分离培养的贴壁细胞具有典型的BMSCs形态和细胞表面标志,诱导后细胞表达神经元和星形胶质细胞标志神经元特异性烯醇化酶(NSE)和胶质纤维酸性蛋白(GFAP) .结论 ATRA联合细胞因子具有诱导BMSCs在体外分化为神经元样细胞的能力.     

HGF＋EGF联合诱导大鼠BMSCs分化肝样细胞的研究

目的在成功分离并培养大鼠骨髓间充质干细胞的基础上,用HGF、EGF以及HGF＋EGF诱导其向肝细胞方向分化,并采用不同方法鉴定细胞分化能力,试图为肝细胞移植等寻找新的种子细胞来源。方法应用HGF、EGF、HGF＋EGF诱导大鼠骨髓间充质干细胞分化为肝样细胞。实验分组：A组：未加任何诱导因素的BMSCs;B组：HGF（20ng/ml）,诱导分化BMSCs;C组：EGF（10ng/ml）,诱导分化BMSCs;D组：HGF（20ng/ml）＋EGF（10ng/ml）,诱导分化BMSCs。相差显微镜下观察细胞形态的变化;在诱导第7、21天行免疫细胞化学染色检测肝细胞标志AFP和CK18。结果在加入各种诱导成分后,梭形或成纤维细胞样细胞随着诱导时间的延长,变成纺锤形、不规则圆形或多角形细胞,数量逐渐变多,形似肝细胞。在诱导分化第7天和21天免疫细胞化学染色各诱导组均可检测出AFP和CK18表达,图像分析表明HGF＋EGF联合诱导分化的AFP、CK18阳性率最高,HGF次之,EGF则较弱。结论 HGF、EGF、HGF＋EGF均能诱导大鼠骨髓间充质千细胞分化为肝样细胞,分化后的肝样细胞能分泌肝细胞特异性产物AFP、CK18。     

人脐血间充质干/祖细胞诱导为神经样细胞的实验研究

目的探讨人脐血间充质干细胞用于神经细胞再生的可行性与人脐血间充质干/祖细胞最佳的诱导培养条件。寻找一种合适的细胞来源进行移植以代替受损的神经元、星形胶质细胞和少突胶质细胞来修复神经损伤。方法将脐血单个核细胞置于低血清(2%)达氏修正依氏培养基中培养,生成贴壁细胞层,依传代方法,用相同培养条件进行扩增,扩增后的贴壁细胞置入细胞培养液(加入新生大鼠神经细胞分离培养上清液)中,并添加表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)进行诱导分化。采用流式细胞术、免疫组织化学法分别检测被诱导的神经样细胞的表面标志及细胞特异性标志、结构,并进行分析。结果脐血间充质干/祖细胞克隆在脐血单个核细胞中出现频率为0.5×10-6,在传20代时,可有效扩增1.5×106倍,诱导后,70%的脐血间充质干/祖细胞分化为神经样细胞。结论采用上述扩增与诱导条件,脐血间充质干/祖细胞可得到有效扩增,并可高效向神经样细胞分化,从而证实脐血是一种细胞替代治疗前景光明的细胞来源。     

鹿茸精诱导大鼠骨髓间充质干细胞分化为神经元样细胞

目的:探讨鹿茸精体外定向诱导SD大鼠骨髓间充质干细胞分化为神经元样细胞。方法:通过贴壁法分离大鼠骨髓间充质干细胞,体外扩增培养。鹿茸精定向诱导分化为类神经元样细胞。光镜下观察细胞形态,免疫细胞化学检测神经细胞特异性抗原标志物神经元烯醇酶(NSE)、巢蛋白(Nestin)、胶质纤维酸性蛋白(GFAP)的表达。结果:大鼠骨髓间充质干细胞可通过贴壁法成功分离并可在体外大量扩增。鹿茸精诱导3小时后大部分骨髓间充质干细胞转变为神经元样细胞,出现胞体和突起,免疫细胞化学染色NSE及Nestin呈阳性,GFAP阴性。结论:鹿茸精可在体外诱导大鼠骨髓间充质干细胞分化为神经元样细胞。     

大鼠胰腺导管上皮细胞体外诱导分化

目的 探讨上皮细胞生长因子(EGF)、尼克酰胺(NIC)、肝细胞生长因子(HGF)、葡萄糖(Glu)、角脘蛋白生长因子(KGF)和β细胞素在大鼠胰腺导管上皮细胞体外诱导分化的作用.方法 采用健康SD大鼠胰腺导管上皮细胞,分于70个培养孔中,加入含有多种营养因子(不含血清)的PRMI 1640培养液中培养.每个孔中6种诱导剂均进行随机组合.在胰岛样细胞团(ICCs)形成前后的不同天数进行细胞计数、电镜观察、胰岛素测定以及免疫细胞化学和荧光分析.结果 胰腺导管上皮细胞培养7d后,单个贴壁上皮细胞分裂增殖,呈鹅卵石样细胞片,单层覆盖;加入诱导和促生长因子后,单层覆盖上皮细胞中逐渐产生一定数量类似胰岛样球状细胞团,直径为80～130μm.结论 6种促生长和诱导因子中,Glu、NIC、KGF、EGF作用较强;在所设定的浓度范围内,其作用均随着浓度的增加而增强.     

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。     

Expression of cytochrome P450 and transcription factors during in vitro differentiation of mouse embryonic stem cells into hepatocytes

Hepatocyte differentiation markers were expressed in the cells differentiated from mouse embryonic stem (ES) cells. In the differentiating ES cells, Cyp1a1 mRNA was highly expressed during the early to middle stage; Cyp2c29, Cyp2e1, Cyp3a11 and Cyp7a1 mRNAs were expressed only at the late stage; Cyp7b1 mRNA was expressed throughout all stages. Alpha-fetoprotein and albumin were co-expressed with Cyp3a and Cyp1a, respectively. Aryl hydrocarbon receptor, aryl hydrocarbon receptor nuclear translocator and glucocorticoid receptor mRNAs were detected in differentiating ES cells throughout the culture period. Pregnane X receptor mRNA was detected only in cells cultured for more than 24 days. The expression levels of Cyp2c29, Cyp3a11 and Cyp7a1 and G6p mRNAs were increased in embryoid bodies that were cultured with culture medium containing acid fibroblast growth factor, hepatocyte growth factor (HGF) and oncostatin M for 12 or 18 days, then the medium was replaced by that without HGF. These findings suggested that the expression levels of Cyp genes in hepatocytes differentiated from ES cells were markedly changed in individual enzymes during the course of differentiation, and that the duration of incubation with the addition of HGF affected the expression of Cyps and hepatocytes marker proteins.     

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。     

Adipose‐derived mesenchymal stem cells therapy for acute kidney injury induced by ischemia‐reperfusion in a rat model

Acute kidney injury (AKI) represents a group of complicated syndromes with a high mortality rate. The administration of adipose‐derived mesenchymal stem cells (ADMSCs) has been tested as a possible treatment method for AKI. The long‐term evaluation of AKI induced by ischemia/reperfusion (IR) and the probable renal protection of ADMSCs are limited. In this study we have established a rat AKI model induced by IR and investigated the possible protective effects of ADMSCs. Adult Sprague‐Dawley (SD) rats were divided into three groups (n = 6/each group). The MOCK group was as the normal control. Rats in the IR‐AKI and IR‐AKI+ADMSCs groups were subjected to IR injury by clamping both renal pedicles for 40 minutes. Rats in the MOCK and IR‐AKI groups were injected with PBS via the tail vein as negative treatment controls. Rats in the IR‐AKI+ADMSCs group received ADMSCs therapy (2 × 10⁶ cells were injected into the rats via the tail vein). We found that ADMSC transplantation restored the pathologic morphology induced by IR‐AKI to normal compared with the MOCK group, suggesting the reparative function of ADMSCs in kidney tissues. Compared with IR‐induced AKI alone, ADMSC treatment significantly decreased the number of apoptotic cells, the level of total urinary protein and serum creatinine, the expression of pro‐inflammatory cytokines (IL‐6, TNF‐α, IL‐1β, IFN‐γ, TNF‐α, IFN‐γ, and TGF‐β), and the inflammation‐associated proteins (HGF and SDF1), but increased the expression of the anti‐inflammatory cytokine, IL‐10, and the anti‐apoptotic regulator, Bcl‐2. Our data have indicated that ADMSC transplantation may protect against IR‐induced AKI by anti‐apoptotic and anti‐inflammatory effects.     

An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. I: Establishment of the differentiation protocol

The aim of the present study was to establish an experimental protocol to differentiate murine embryonic stem (ES) cells into endothelial cells in vitro. The spinner flask technique as well as the hanging drop method were used to generate so-called embryoid bodies (EBs). In order to find out the optimal differentiation environment, EBs were cultured under various experimental conditions for up to 14 days. The influence of basic fibroblast growth factor (bFGF) alone, vascular endothelial growth factor (VEGF) alone, bFGF and VEGF together and a cocktail consisting of bFGF, VEGF, interleukin-6 (IL-6) and erythropoietin (Epo) on the induction of differentiation of ES cells into endothelial cells was studied. Different concentrations of growth factors and times of treatment were applied. Endothelial cells were characterized by analyzing the expression of platelet-endothelial cell adhesion molecule (PECAM-1), the endothelial-specific vascular endothelial cadherin (VE-Cadherin), the angiopoietin receptor Tie-2, VEGF receptors 1 and 2 (Flt-1 and Flk-1, respectively) and the soluble form of Flt-1 (sFlt) at the mRNA level. PECAM-1 and VE-Cadherin were also studied at the protein level. The data clearly showed that EBs generated by the hanging drop method, followed by their transfer into suspension culture on day 3 of differentiation and their subsequent plating on day 5 is the best of the studied methods to differentiate ES cells into endothelial cells. Addition of VEGF alone or a cocktail consisting of VEGF, bFGF, IL-6 and Epo resulted in the strongest gene expression levels of the above mentioned endothelial cell markers in the differentiated ES cells.     

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX

Aim:

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX.

Methods:

Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell–derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium.

Results:

The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell–derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium.

Conclusion:

We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.     

血管内皮祖细胞用于构建血管新生的体外三维模型

目的 采用血管内皮祖细胞(EPCs)体外构建血管新生的三维模型,证实体外获得的EPCs参与血管新生.方法 (1)密度梯度离心法获取兔外周静脉血的单个核细胞,贴壁筛选法分离EPCs,于添加了Singlequotes的EBM-2培养液中扩增,对培养14 d的细胞进行免疫荧光及免疫组织化学鉴定;(2)用上述同样的方法 获取EPCs,并接种于Matrigel三维基底膜胶,Singlequotes诱导,倒置相差显微镜观察血管形成.结果 (1)培养7～9 d,光电显微镜可见细胞集落形成,呈现干细胞特件;贴壁细胞表达血管性假血友病因子(vWF)、血管内皮生长凶子受体2(VEGFR-2)和血管内皮钙黏蛋白(VE-cadherin),呈现内皮细胞系特征;贴壁细胞用碳化青染料(DIL)标记的乙酰化低密度脂蛋白(DIL-ac-LDL)及FITC标记的Ⅰ型荆凝集素(F1TC-UEA-1)双荧光阳性,显现EPCs的生物学特性;(2)动态观察;培养2周左右,在三维胶体中形成血管结构.结论 外周血米源的EPCs,经过体外诱导后.具有成血管能力,可应用于制备体外血管新生的三维模型.