胰岛素样生长因子-1对脂肪间充质干细胞增殖的影响

目的：探讨胰岛素样生长因子-1（insulin-like growth factor-1,IGF-1）对脂肪间充质干细胞（adipose-derived stem cells,ADSCs）增殖的影响。方法：采用密度梯度离心法结合贴壁法分离脂肪间充质干细胞,接种于含体积分数为10%的胎牛血清的DMEM培养基中行贴壁培养。流式细胞仪检测ADSCs表面标志物（CD90、CD29、CD31、CD34、CD45）的表达情况,利用成骨、成脂诱导液诱导ADSCs向成骨细胞、成脂细胞分化,用碱性磷酸酶、油红O染色观察。采用终浓度为0、5、10、15、20、30 ng/mL IGF的培养基培养ADSCs,利用Edu染色标记ADSCs,分析不同浓度的IGF-1对ADSCs增殖的影响。结果：流式细胞术显示ADSCs的表型分子CD90、CD29呈阳性,CD31、CD45呈阴性,成骨诱导后碱性磷酸酶染色阳性,成脂诱导后油红O染色可见大量脂滴,表明培养的ADSCs具有成骨、成脂分化的能力。IGF-1促进ADSCs增殖的作用随IGF-1的作用浓度的增加而增加,并逐渐趋于饱和,在趋于15μg/mL的浓度时达到最大促增殖作用,且随着IGF-1作用时间的延长其促ADSCs增殖的作用逐渐增强。结论：本实验成功分离培养ADSCs,IGF-1对体外培养的ADSCs有促进增殖的作用。     

胰岛素样生长因子-1 对脂肪间充质干细胞增殖的影响

目的：探讨胰岛素样生长因子-1(insulin-like growth factor-1,IGF-1)对脂肪间充质干细胞(adipose-derived stem cells,ADSCs) 增殖的影响。方法：采用密度梯度离心法结合贴壁法分离脂肪间充质干细胞,接种于含体积分数为10%的胎牛血清的DMEM 培 养基中行贴壁培养。流式细胞仪检测ADSCs表面标志物(CD90、CD29、CD31、CD34、CD45)的表达情况,利用成骨、成脂诱导液诱 导ADSCs 向成骨细胞、成脂细胞分化,用碱性磷酸酶、油红O 染色观察。采用终浓度为0、5、10、15、20、30 ng/mL IGF的培养基培 养ADSCs,利用Edu 染色标记ADSCs,分析不同浓度的IGF-1 对ADSCs增殖的影响。结果：流式细胞术显示ADSCs的表型分子 CD90、CD29 呈阳性,CD31、CD45 呈阴性,成骨诱导后碱性磷酸酶染色阳性,成脂诱导后油红O染色可见大量脂滴,表明培养的 ADSCs具有成骨、成脂分化的能力。IGF-1 促进ADSCs 增殖的作用随IGF-1 的作用浓度的增加而增加,并逐渐趋于饱和,在趋于 15 滋g/mL的浓度时达到最大促增殖作用,且随着IGF-1 作用时间的延长其促ADSCs 增殖的作用逐渐增强。结论：本实验成功分 离培养ADSCs,IGF-1 对体外培养的ADSCs 有促进增殖的作用。     

低温冻存对兔脂肪间充质干细胞部分生物学特性的影响

目的：研究低温冻存对兔脂肪间充质干细胞部分生物学特性的影响。方法采用组织块法分离培养兔脂肪间充质干细胞。用倒置显微镜观察原代细胞的细胞形态,流式细胞仪检测兔脂肪间充质干细胞的免疫表型。取第3代兔脂肪间充质干细胞置于-196℃液氮保存半年,37℃复苏并传至第7代。实验分为两组,实验组为冻存复苏后传至第7代的兔脂肪间充质干细胞,对照组为未冻存的第7代兔脂肪间充质干细胞,用MTT绘制其生长曲线;添加成脂、成骨诱导液进行诱导,油红O、茜素红染色和碱性磷酸酶活性检测分别进行鉴定。结果体外培养的兔脂肪间充质干细胞呈梭形纤维样细胞形态,生长力旺盛。流式细胞仪检测显示,第3代兔脂肪间充质干细胞强表达CD44、CD90,阴性表达造血细胞相关的表面标志CD45。两组细胞生长曲线呈典型的“S”形,无统计学差异（P＞0.05）;成脂诱导14 d后,油红O染色呈阳性;成骨诱导2周时茜素红染色阳性,ALP表达活性随成骨诱导时间延长不断增加且无统计学差异（ P＞0.05）。结论冻存后的兔脂肪间充质干细胞体外生长及多向分化潜能未发生显著变化。     

牛脂肪间充质干细胞的分离、培养与鉴定

为了给组织工程提供种子细胞,对牛间充质干细胞(Adipose-derived stem cells,ADSCs)进行体外分离培养。首先应用胶原酶消化法分离牛ADSCs,进行体外培养、连续传代,并观察细胞的形态变化,通过细胞计数绘制生长曲线,细胞压片进行染色体分析,采用细胞免疫荧光化学方法检测细胞表面标记,利用成骨分化和成脂分化检测其分化能力。结果显示牛ADSCs体外培养时细胞形态呈成纤维细胞样,增殖稳定;Vimentin、CD49d、CD13表达呈阳性,CD34表达呈阴性;成骨诱导条件下的细胞碱性磷酸酶活性高,茜素红染色呈阳性;成脂诱导条件下细胞周围脂滴明显,油红-O染色呈阳性。结果证明牛ADSCs体外生长稳定、增殖速度快、定向分化能力强,简易的体外分离培养及诱导方法为其在组织工程中的应用奠定了基础。     

人羊水来源干细胞与骨髓间充质干细胞的对比研究

该研究对比人羊水来源干细胞(human amniotic fluid derived stem cells,hAFSCs)和人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)的细胞表型及成骨分化能力。分离培养hAFSCs和hBMSCs,通过光镜观察,CCK-8检测,流式细胞术,基因芯片等方法对比两组细胞表型,采用碱性磷酸酶(alkaline phosphatase,ALP)染色,茜素红(alizarin red S,ARS)染色,Real-time PCR,细胞免疫荧光等方法对比两组细胞体外成骨分化过程,再进一步通过裸鼠异位成骨来初步检测两种细胞体内成骨能力。结果显示,hAFSCs与hBMSCs在镜下均表现为梭形,且具有相似的增殖能力,都表达CD90和CD105,成骨诱导下两种细胞ALP活性及矿化结节均随着时间增加,同时成骨标志物RUNX2、OSX、COL I、ALP、OPN的mRNA水平也增高。基因芯片分析表明,两者在细胞黏附以及炎症反应方面的基因表达存在差异。类似地,体内裸鼠异位成骨结果也表明,hAFSCs与hBMSCs具有相近的成骨分化潜能。总之,人羊水来源干细胞与人骨髓间充质干细胞具有相似的细胞形态及增殖能力,均能在体内外成骨诱导分化环境下展现出良好的成骨功能。     

脐带间充质干细胞——组织工程的新视角

目的 从脐带中分离培养脐带间充质干细胞(mesenchymal stem cell, MSC) 并进行鉴定,阐明其多向分化的潜在作用.方法 收集健康胎儿脐带,分离培养脐带中的间充质干细胞,以流式细胞仪对培养的间充质干细胞进行细胞表面标志检测,多种成分联合诱导其向脂肪、成骨方向分化,细胞化学染色检测诱导后的细胞变化.结果 脐带中分离培养的间充质干细胞不表达造血细胞系的标志CD34、CD45、HLA-DR,强表达CD105、CD44、CD90,在适当的诱导条件下可向脂肪及成骨方向分化.结论 脐带中存在具有多向分化潜能的间充质干细胞.     

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.     

尿源性干细胞移植修复慢性肝损伤裸鼠肝脏功能

该研究探讨尿源性干细胞(urine-derived stem cells, USCs)的生物学性状及移植治疗慢性肝损失模型的可能。分离培养USCs,观察细胞形态、流式细胞术检测干细胞表面标记,碱性磷酸酶(alkaline phosphatase, ALP)染色、茜素红染色、油红O染色、ICG(indocyanine green)摄取实验、PAS(periodic acid-Schiff)染色等评估其成骨、成脂和成肝分化。建立四氯化碳(carbon tetrachloride,CCL4)诱导的慢性肝损伤模型,尾静脉4次移植USCs,计算肝脏指数,检测血清ALT、AST, HE及Masson染色,评估治疗效果。结果表明, USCs为米粒状贴壁生长细胞,表达多种间充质干细胞标志物:CD24、CD29、CD73、CD90和CD105,表达细胞周期表面标志物CD146,不表达造血细胞表面标志物CD31、CD34、CD45。成骨成肝诱导的USCs后ALP染色、茜素红染色、油红O染色阳性,单纯成肝诱导后几乎无ICG摄取及PAS染色阳性的细胞,而与肝干细胞共培养的USCs诱导组中,约10%细胞有ICG摄取及PAS染色阳性。与模型组相比, USCs移植组肝脏指数显著降低, ALT、AST降低但无统计学意义,肝细胞退行性变及纤维增生明显改善。该研究成功分离培养出增殖能力强并具有多向分化潜能的USCs,移植入慢性肝损伤裸鼠,可在一定程度上修复肝脏损伤。     

大鼠脂肪间充质干细胞的分离鉴定与生物学特性研究

脂肪间充质干细胞(adipose-derived mesenchymal stem cells,ADSCs)是一类具有多向分化潜能的成体干细胞,在组织器官修复方面具有潜在应用前景。体外培养条件下脂肪间充质干细胞增殖活性和分化潜能的维持对其应用研究具有重要意义。该研究通过大鼠腹股沟部脂肪组织分离获得了脂肪间充质干细胞,并对其细胞生长曲线、细胞群体倍增时间、细胞克隆形成等进行了检测,从而筛选出细胞生长最适的血清及浓度;再进一步对筛选条件下培养的大鼠脂肪间充质干细胞进行了免疫荧光检测、流式细胞术分析和诱导分化能力鉴定。结果显示,大鼠脂肪间充质干细胞在20%血清浓度下生长状态最好,增殖较快,细胞群体倍增时间较短,细胞克隆形成率可达(27.33±0.58)%。分离的大鼠脂肪间充质干细胞中CD44、CD90、CD106的阳性率分别为99.12%、99.59%、65.81%,CD34分子呈阴性。诱导后,细胞经油红O、碱性磷酸酶、阿新兰染色,结果表明,细胞具有向脂肪、骨、软骨方向分化的潜能。以上结果表明,成功建立了大鼠脂肪间充质干细胞系。     

AGEs和TNF-α对人牙周膜干细胞骨向分化能力的协同抑制作用

本实验旨在研究糖基化终末产物(AGE-BSA)和TNF-α对人牙周膜干细胞增殖及骨向分化能力的影响。本实验通过体外组织块酶消化法和有限稀释法克隆化培养牙周膜干细胞,使用流式细胞仪检测细胞表型分子stro-1、CD146、CD44、CD90的表达而对其进行干细胞鉴定后,取第3代人牙周膜干细胞在100μg/mL AGE-BSA及10 ng/mL TNF-α刺激下进行增殖能力检测;同时矿化诱导,设A组(AGE-BSA刺激组),T组(TNF-α刺激组),AT组(AGE-BSA/TNF-α共同刺激组),不含AGE-BSA/TNF-α的常规矿化诱导组作为对照;于诱导的21d茜素红染色观察钙结节形成情况,诱导7 d,碱性磷酸酶染色观察ALP活性、实时定量聚合酶链反应(real time PCR)和Western blotting检测成骨相关基因及蛋白表达情况。流式细胞仪显示细胞阳性表达STRO-1、CD146、CD44、CD90;成骨诱导21 d后茜素红染色和定量分析显示,AT组骨结节形成量最低,A组及T组相对于对照组骨结节形成量存在下降;差异均有统计学意义(p0.05)。成骨诱导7 d后ALP染色,ALP活性变化趋势与茜素红定量分析相同。成骨诱导7 d后RT-PCR检测成骨相关基因BSP、OCN、ALP mRNA表达,AT组表达水平最低,A组及T组有下降趋势,差异均有统计学意义(p0.05)。Western blotting检测显示,各组总蛋白BSP蛋白表达趋势与RT-PCR趋势相同。AGEs与TNF-α均具有对HPDLSC的骨向分化能力的抑制作用,两者共同刺激对HPDLSC骨向分化能力存在协同抑制作用。     

Isolation, identification and multipotential differentiation of mouse adipose tissue-derived stem cells

Bone marrow and adipose tissue have provided two suitable sources of mesenchymal stem cells. Although previous studies have confirmed close similarities between bone marrow-derived stem cells (BM-MSCs) and adipose tissue-derived stem cells (ADSCs), the molecular phenotype of ADSCs is still poorly identified. In the present study, mouse ADSCs were isolated from the inguinal fat pad of 12-14 weeks old mice. Freshly isolated and three passaged ADSCs were analyzed for the expression of OCT4, Sca-1, c-kit and CD34 by RT-PCR. Three passaged ADSCs were analyzed by flow cytometry for the presence of CD11b, CD45, CD31, CD29 and CD44. Moreover, cardiogenic, adipogenic and neurogenic differentiation of ADSCs were induced in vitro. Freshly isolated ADSCs showed the expression of OCT4, Sca-1, c-kit and CD34, and two days cultured ADSCs were positively immunostained with anti-OCT4 monoclonal antibody. After three passages, the expression of OCT4, c-kit and CD34 eliminated, while the expression of Sca-1 showed a striking enhancement. These cells were identified positive for CD29 and CD44 markers, and they showed the lack of CD45 and CD31 expression. Three passaged ADSCs were differentiated to adipocyte-, cardiomyocyte- and neuron-like cells that were identified based on the positive staining with Sudan black, anti-cardiac troponin I antibody and anti-map-2 antibody, respectively. In conclusion, adipose tissue contains a stem cell population that seems to be a good multipotential cell candidate for the future cell replacement therapy.     

Differentiation potential of rabbit CD90-positive cells sorted from adipose-derived stem cells in vitro

To investigate the differentiation potential of purified CD90⁺ cells sorted from adipose-derived stem cells (ADSCs), CD90⁺ cells were sorted from rabbit ADSCs using flow cytometry. Then, cell expansion of CD90⁺ cells and unsorted ADSCs was observed using an inverted microscope. Furthermore, cell surface markers including CD40, CD105, and CD90 on CD90⁺ cells and unsorted ADSCs were quantified using flow cytometry. Additionally, multi-lineage differentiation ability between CD90⁺ cells and unsorted ADSCs was compared, and expression of adipocyte-related genes PPAR-r and CEBPA as well as stem cell-related gene SOX2 in CD90⁺ cells and unsorted ADSCs was determined using real-time quantitative PCR. We found that CD90⁺ cells had a stronger cell proliferation ability than unsorted ADSCs. CD90⁺ cells showed a stronger ability of osteoblast and chondrocyte differentiation than unsorted ADSCs and CD90^? cells, whereas the adipose differentiation ability of CD90⁺ cells was similar to that of ADSCs and CD90^? cells. CD14, CD105, and CD90 on CD90⁺ cells were expressed more highly than those on ADSCs. Additionally, the mRNA expression level of SOX2 in CD90⁺ cells was significantly higher than that in ADSCs, whereas the expression of PPAR-r and CEBPA was markedly lower than that in ADSCs. These results suggested that the purified CD90⁺ cells sorted from ADSCs exhibit a stronger differentiation potential than the unsorted ADSCs.     

重组腺病毒介导HGF修饰ADSCs对大鼠肝损伤模型的治疗作用

目的：建立重组腺病毒介导肝细胞生长因子HGF促ADSCs 定向分化肝细胞的方法,并对其参与肝损伤修复能力进行验证,为作为治疗肝损伤细胞来源提供参考。方法：采用消化培养的方法,分离SD 大鼠腹股沟脂肪组织ADSCs 细胞,连续传代3 次对其进行纯化培养,利用形态学鉴定、流式细胞术检测ADSCs 表面标志物方法对其间充质干细胞样特征进行鉴定,加入成脂肪细胞诱导液观察其分化成脂肪细胞的能力;构建腺病毒表达HGF载体Adeno-HGF-EGFP,并转染ADSCs 细胞,利用免疫细胞化学染色方法检测肝细胞标志分子表达水平;最后建立大鼠肝损伤动物模型,观察Adeno-HGF-EGFP 转染的ADSCs 细胞参与肝损伤修复能力情况。结果：分离的ADSCs 细胞形态较为一致,绝大多数呈梭形,排列不规则。流式细胞术结果显示,该细胞表达CD29、CD90、CD106 等间充质干细胞细胞表面标记物,低表达造血干细胞细胞表面标记物CD34、CD45,同时,分离的ADSCs 细胞具有诱导分化成脂肪细胞能力;Adeno-HGF-EGFP 转染ADSCs后,AFP、ALB、CK18 等肝细胞特异性分子表达水平升高;经尾静脉注射ADSCs 细胞后,肝损伤大鼠的AST、ALT、TBIL 等分子表达水平恢复正常。结论：建立了重组腺病毒介导肝细胞生长因子HGF促ADSCs定向分化肝细胞的方法,并且表达HGF的ADSCs 细胞具有修复大鼠肝损伤模型能力,这为通过细胞治疗肝损伤提供了新的细胞来源。     

Tbx18 能使兔脂肪干细胞向心肌样细胞分化

目的:研究Tbx18是否能成功转染脂肪干细胞并使脂肪干细胞向心肌细胞分化。方法:分离培养来源于日本大耳兔腹股沟部脂肪的兔脂肪干细胞,用搭载有Tbx18的腺病毒载体转染脂肪干细胞,诱导分化后检测向心肌细胞的分化情况,同时将转染了含GFP的腺病毒组与未转染组作为对照。采用用流式细胞仪检测转染效率,采用免疫荧光法检测平滑肌肌动蛋白α-SMA,采用实时定量PCR法检测兔肌钙蛋白TNNT2的表达。结果:转染后荧光显微镜下可观察到荧光表达,且持续时间较长。流式细胞仪检测转染效率为70%左右;诱导分化后,脂肪干细胞内出现了α-SMA和TNNT2的表达。结论:Tbx18可成功转染入脂肪干细胞,且能在细胞内稳定表达;Tbx18可诱导脂肪干细胞向心肌样细胞分化。     

Neural Differentiation of Rat Adipose-Derived Stem Cells in Vitro

It is reported that adipose-derived stem cells (ADSCs) had multilineage differentiation potential, and could differentiate into neuron-like cells induced by special induction media, which may provide a new idea for restoration of erectile dysfunction (ED) after cavernous nerve injury. The aim of this research was to explore the neuronal differentiation potential of ADSCs in vitro. ADSCs isolated from inguinal adipose tissue of rat were characterized by flow cytometry, and results showed that ADSCs were positive for mesenchymal stem cell markers CD90 and CD44, but negative for hematopoietic stem cell markers. ADSCs maintained self-renewing capacity and could differentiate into adipocytes and neurocytes under special culture condition. In this research, two methods were used to induce ADSCs. In method 1, ADSCs were treated with the preinduction medium including epithelium growth factor, basic fibroblast growth factor, and brain derived neurotrophic factor (BDNF) for 3?days, then with the neurogenic induction medium containing isobutylmethylxanthine, indomethacin, and insulin. While in method 2, BDNF was not used to treat ADSCs. After induction, neuronal differentiation of ADSCs was evaluated. Neuronal markers, glial fibrillary acidic protein (GFAP), and ??-tubulin III (Tuj-1) were detected by immunofluorescence and Western Blot analyses. The expressions of GFAP and Tuj-1 in method 1 were obviously higher then those in method 2. In addition, the positive rate of the neuron-like cells was higher in method 1. It suggested that ADSCs are able to differentiate into neural-like cells in vitro, and the administration of BDNF in the preinduction medium may provide a new way to modify the culture method for getting more neuron-like cells in vitro.     

Nurr-1基因转染促进脂肪干细胞的神经分化

目的:研究孤儿核受体相关基因1(Nurr-1)对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的潜在作用。方法:流式细胞术与成骨、成脂诱导技术鉴定脂肪干细胞;Nurrr-1基因转染脂肪干细胞后,应用神经特异性标志物MAP-2,β-tubulin的免疫荧光染色评估其向神经方向分化的能力。结果:流式细胞术结果表明培养的细胞CD29,CD44表达90%以上,CD45,CD90表达均低于1.5%,经过诱导后,油红O、茜素红S染色均呈阳性,表明所培养的细胞为脂肪干细胞;慢病毒转染Nurr-1基因后,免疫荧光染色检测MAP-2,β-tubulin的免疫荧光强度显著增加;RT-PCR结果显示Nurr-1转染的脂肪干细胞的MAP-2、β-tubulin、NF200的表达量显著提高。结论:Nurr-1基因转染能促进脂肪干细胞向神经方向分化,为神经损伤和神经退行性病变的治疗提供了新途径。