微囊化共培养诱导骨髓间充质干细胞体外定向成骨分化研究

为了模拟体内成骨微环境,为骨组织工程提供一种调控干细胞体外向成骨细胞定向分化的共培养新方法,SD大鼠骨髓间充质干细胞和包埋在海藻酸钠-聚赖氨酸-海藻酸钠(alginate-poly-lysine-alginate,APA)微胶囊中的SD大鼠成骨细胞进行体外共培养。共培养过程中,通过碱性磷酸酶(ALP)定量、定性分析以及钙化结节(von Kossa)染色等手段来评价骨髓间充质干细胞向成骨细胞定向分化。结果表明在体外微囊化共培养过程中,被诱导细胞的胞内ALP酶活性逐渐高于对照组的干细胞,接近于成骨细胞;ALP以及von Kossa定性染色证实被诱导细胞具有较高的ALP活性以及具有分泌钙基质的能力。微囊化成骨细胞和外部干细胞的共培养体系较好地模拟了体内干细胞向成骨细胞转化的成骨微环境,促进了干细胞向成骨细胞的体外定向分化;微胶囊膜将成骨细胞和干细胞进行了隔离,避免了两者的直接接触和可能的细胞交叉污染混合,同时利于分离目的细胞,这种微囊化共培养体系为骨组织工程提供了一种安全调控干细胞体外成骨定向分化的工程化新方法。     

去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞的分化

目的观察去上皮羊膜及其浸提液体外诱导骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)向上皮细胞的分化,并探讨其机制。方法从胎儿四肢长骨分离BMSCs,扩增后采用流式细胞术分析第3代(P3)细胞表面抗原(CD29、CD34、CD71和HLA-DR)的表达,并用4,6-乙酰基-2-苯基吲哚(DAPI)标记第4代BMSCs(P4-BMSCs)。机械法去除正常胎盘羊膜上皮,制成去上皮羊膜,并制备去上皮羊膜浸提液。将DAPI标记的BMSCs接种于羊膜上,设加或不加表皮细胞生长因子(Epidermal growth factor,EGF)、类胰岛素1号生长因子(Insulin-like growth factor 1,IGF-1)、羊膜浸提液诱导组及细胞爬片对照组,体外诱导培养后,采用免疫荧光组织(细胞)化学染色学法检测各组细胞角蛋白(Cytokeratin,CK)、EGF-R和IGF-1-R的表达,并于诱导后第10天计算CK阳性细胞率。结果原代BMSCs呈典型旋涡状生长,P3细胞表达CD29和CD71,不表达CD34和HLA-DR。羊膜组和细胞爬片组BMSCs在加入EGF或IGF-1诱导后,表达EGF-R和IGF-1-R的时间较未加生长因子的对照组提前2～4 d,表达CK的时间提前2～6 d,单用羊膜组或羊膜浸提液组的表达时间差异无统计学意义(P>0.05);诱导第10天,单用羊膜或羊膜浸提液诱导组的CK阳性细胞表达率明显高于细胞爬片对照组(P<0.05);羊膜与EGF、IGF-1联合诱导组高于单用羊膜组(P<0.05);EGF诱导组高于IGF-1诱导组(P<0.05)。结论羊膜及羊膜浸提液、外源性EGF和IGF-1在体外均可诱导BMSCs向上皮细胞分化,羊膜可能主要通过其所含的细胞因子诱导BMSCs向上皮分化。     

体外诱导微环境对骨髓间充质干细胞成神经分化的影响及分化后的自发逆转现象

目的体外定向诱导大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)成神经分化,并探讨诱导微环境对其分化的影响及分化后的自发逆转现象。方法体外分离培养大鼠MSCs,流式细胞仪检测细胞表面标志。采用改良神经元诱导液[Modified neuronal induction media(MNM)]定向诱导MSCs,免疫荧光检测神经细胞表面标志。观察胎牛血清(FBS)浓度、细胞密度、MNM剂量、新鲜与使用过的MNM等不同诱导微环境对MSCs成神经分化的影响。结果 MSCs经MNM诱导后,6h即可见尼氏体,表达神经元特异性表面标志神经元特异性烯醇化酶(NSE)、巢蛋白(Nestin)和微管相关蛋白-2(MAP-2)。随着诱导微环境的改变,MSCs成神经分化率及神经元表面标志表达亦发生改变,且分化后的神经元样细胞可自发逆转。结论 MSCs能够在MNM微环境中定向成神经分化,但诱导微环境的改变可以从量和质两个层面影响MSCs定向分化。     

人参皂苷对骨髓间充质干细胞通过旁分泌途径治疗心肌缺血的促进作用

目的探讨人参皂苷对骨髓间充质干细胞(bone marrow derived mesenchymal stem cell,BMSC)通过旁分泌途径治疗心肌缺血的促进作用。方法从SD大鼠胫骨和股骨中分离培养BMSC,取第3代BMSC,流式细胞术检测细胞表面抗原CD44、CD45、CD34和CD29的表达。建立大鼠心肌缺血模型,将模型鼠随机分为模型组(每天常规喂食水)和人参皂苷组[每天常规喂食水及人参皂苷液(100 mg/100 ml),每日3次,100 ml/次],给药的同时,均经大鼠尾静脉缓慢注射BMSC悬液(1×105个/ml),1 ml/只,另设空白对照组(未建模的SD大鼠)。分别采集模型组建模后24、48、72 h及人参皂苷组建模后72 h的大鼠心脏血,分离血清,ELISA法检测血清中干细胞因子(stem cell factor,SCF)、干细胞衍生因子(stromal cell derived factor-1,SDF-1)、血管内皮生长因子(vascular endothelial growth factor,VEGF)及趋化因子受体4(chemokine receptor 4,CXCR4)的分泌水平。体外迁移试验检测不同浓度SDF-1(1、10、100 ng/ml)对BMSC迁移的影响;取各组大鼠建模后24、48、72 h的心肌组织提取液,检测其对BMSC及经SDF-1抑制剂AMD3100作用的BMSC迁移的影响。结果大鼠BMSC表面表达CD44、CD29,不表达CD34、CD45。模型组大鼠血清中细胞因子SCF、SDF-1、VEGF、CXCR4的分泌水平随建模时间延长显著上升,人参皂苷组大鼠建模后72 h血清中各细胞因子的表达水平均明显高于模型组(P<0.05)。SDF-1可显著增加BMSC的迁移数,且呈浓度依赖性(P<0.05);模型组和人参皂苷组大鼠心肌组织提取液作用BMSC的迁移数随建模时间的延长显著上升(P<0.05),两组相同时间点间差异有统计学意义(P<0.05);人参皂苷组72 h的大鼠心肌组织提取液作用的AMD3100处理的BMSC的迁移数明显低于模型组72 h(P<0.05)。结论人参皂苷能促进BMSC旁分泌作用,提高BMSC对心肌缺血性疾病的疗效,可作为治疗该疾病的辅助药物。     

一种分离培养人脐带Wharton's jelly间充质干细胞的新方法

目的探讨一种分离培养人脐带Wharton′s jelly间充质干细胞(mesenchymal stem cell,MSC)的新方法。方法取人脐带组织,除去动静脉后剪碎成2～5 mm3,将组织碎片浸入4 g/LⅠ型胶原酶和1 g/L透明质酸酶的混合液中,于37℃处理1 h,再用0.25%胰蛋白酶在相同条件下消化30 min,得到的消化液经70μm细胞滤网过滤后,制备单个细胞悬液,培养并传代。取P1、P3、P7代脐带Wharton′s jelly MSC,绘制细胞生长曲线;取P3代细胞,采用流式细胞术检测细胞表面标记分子,并分别采用成骨和成脂诱导培养基进行成骨及成脂诱导分化,茜素红染色和油红O染色观察结果。结果 P1、P3代脐带Wharton′s jelly MSC的增殖能力强,且P1代细胞的增殖能力强于P3代,P7代细胞的增殖能力较P3代细胞有所减弱。P3代脐带Wharton′s jelly MSC高表达CD90(99.8%)、CD105(100%)和CD166(100%),低表达CD45(0.3%)、CD14(0.1%)、CD34(0.2%)和CD79a(0.3%),不表达HLA-DR。P3代Wharton′s jellyMSC经成骨诱导后,茜素红染色可见红色结节;经成脂诱导后,油红O染色可见脂质沉积。结论本方法获得的Wharton′s jelly MSC活性好,增殖能力强,为后续实验研究及临床应用提供了理想的种子细胞。     

大鼠骨髓间充质干细胞的体外分离培养与鉴定

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。     

血小板裂解液大规模扩增人脐带间充质干细胞

目的用血小板裂解液(platelet lysate,PL)大规模扩增人脐带间充质干细胞(umbilical cord mesenchymal stem cell,UCMSC),并检测其生物学特性,为临床应用提供实验依据。方法分别采用PL和胎牛血清(FBS)低密度扩增人UCMSC,比较两组UCMSC的细胞形态、大小、克隆形成率、增殖能力、细胞表型和分化能力。结果 PL扩增的UCMSC形态细长;直径明显小于FBS扩增的UCMSC(P<0.05);克隆形成率与FBS扩增的UCMSC差异无统计学意义(P>0.05);有更高的细胞累积群倍数;与FBS扩增的UCMSC有相似的细胞表型;与FBS扩增的UCMSC均具有成骨、成脂诱导分化能力,但PL扩增的UCMSC成骨分化能力更强。结论 PL可取代FBS用于大规模扩增UCMSC。     

大鼠骨髓间充质干细胞向肝样细胞的定向诱导分化

目的 探讨肝细胞生长因子(Hepatocyte growth factor,HGF)和碱性成纤维细胞生长因子(Basic fibroblast growthfactor,bFGF)诱导大鼠骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BM-MSCs)分化为肝样细胞的可行性。方法取SD大鼠股骨骨髓,直接贴壁法分离纯化BM-MSCs,并体外传代,流式细胞术和成骨诱导对其进行鉴定。取第3代BM-MSCs,分为2组:实验组用HGF(20 ng/ml)和bFGF(10 ng/ml)进行诱导,阴性对照组不加诱导剂,倒置显微镜下观察细胞形态变化;RT-PCR法检测诱导后细胞甲胎蛋白(Alpha fetoprotein,AFP)和白蛋白(Albumin,ALB)基因mRNA的转录水平;免疫细胞化学染色法检测诱导后细胞的AFP和ALB蛋白的表达。结果第3代BM-MSCs表型标志和功能特性均符合MSCs的特点。BM-MSCs经HGF和bFGF诱导后呈肝样细胞形态。实验组细胞可检测出AFP和ALB基因mRNA的表达。实验组细胞诱导后第7天,AFP蛋白开始表达,第14天时表达降低,第21天时不表达;ALB于诱导后第14天出现表达,并随诱导时间的延长表达逐渐增加。结论 HGF和bFGF具有体外诱导BM-MSCs向肝样细胞分化的作用。     

骨髓间充质干细胞向视网膜神经节样细胞的分化

目的探讨乳鼠视网膜细胞条件分化液诱导骨髓间充质干细胞(BMSCs)的神经分化情况,以期为视网膜退行性疾病提供治疗方案。方法体外分离培养Wistar大鼠乳鼠BMSCs,观察BMSCs的增殖情况并进行鉴定;制备乳鼠视网膜细胞条件分化液,以其诱导BMSCs,观察BMSCs的神经分化情况,并行免疫组化鉴定。结果体外培养获得了较纯的BMSCs;在乳鼠视网膜细胞条件分化液的环境中,诱导后72h,BMSCs胞体收缩成锥形或球形,细胞突起变细、变长,呈神经细胞的典型形态;免疫组化结果显示,部分细胞呈神经元特异性烯醇化酶(NSE)、巢蛋白(nestin)和Thy1.1阳性反应。结论乳鼠视网膜细胞条件分化液可诱导BMSCs分化成视网膜神经节样细胞。     

cAMP-PKA-CREB信号通路在骨形态发生蛋白9诱导小鼠间充质干细胞成骨分化中的作用

目的探讨cAMP-PKA-CREB信号通路在骨形态发生蛋白9(bone morphogenetic protein 9,BMP9)诱导小鼠间充质干细胞(mesenchymal stem cells,MSCs)C3H10T1/2成骨分化过程中的作用及其机制。方法将C3H10T1/2细胞分别加入不同浓度的cAMP-PKA-CREB信号通路抑制剂H89(1、2.5、5和10μmol/L),检测其对碱性磷酸酶(alkaline phosphatase,ALP)活性的影响;通过ALP定量和钙盐沉积试验分别检测H89对BMP9诱导C3H10T1/2细胞早期和晚期成骨分化的影响;经Western blot法检测H89对C3H10T1/2细胞中磷酸化CREB、骨钙素(Osteocalcin,OCN)和成骨关键转录因子Runx2表达水平的影响;通过Wentern blot及荧光素酶活性的检测,观察H89对经典信号通路BMPs-smad1/5/8的影响。结果随着H89浓度的增加,对BMP9诱导的C3H10T1/2细胞ALP的抑制作用明显增强(P0.05),且呈剂量依赖性;ALP定量和钙盐沉积试验结果表明,H89可明显抑制BMP9诱导的C3H10T1/2细胞早期及晚期成骨分化;H89可显著抑制BMP9诱导的C3H10T1/2细胞中磷酸化CREB、OCN及Runx2蛋白的表达(P0.05),与AdBMP9组比较,H89对经典BMPs-smad1/5/8信号通路无明显影响(P0.05)。结论阻断cAMP-PKA-CREB信号通路可抑制BMP9诱导的MSCs C3H10T1/2的成骨分化,为BMP9的临床应用奠定了理论基础。     

兔骨髓基质干细胞的分离培养及鉴定

目的探讨兔骨髓基质干细胞(MSC)体外分离培养及鉴定。方法自兔髂骨抽取骨髓,采用密度梯度离心法分离纯化出MSC,并增殖。观察MSC的生长情况及形态学特点,流式细胞仪检测第3代MSC表面抗原的表达情况。结果体外培养的兔MSC贴壁生长,呈长梭形,可增殖形成克隆;MSC阳性表达CD29,CD90,但CD34,CD45呈阴性。结论利用密度梯度离心法获取的MSC具有大量增殖的能力,表达CD29,CD90,不表达CD34,CD45。     

Comparing the Osteogenic Potential and Bone Regeneration Capacities of Dedifferentiated Fat Cells and Adipose-Derived Stem Cells In Vitro and In Vivo: Application of DFAT Cells Isolated by a Mesh Method

Background: We investigated and compared the osteogenic potential and bone regeneration capacities of dedifferentiated fat cells (DFAT cells) and adipose-derived stem cells (ASCs). Method: We isolated DFAT cells and ASCs from GFP mice. DFAT cells were established by a new culture method using a mesh culture instead of a ceiling culture. The isolated DFAT cells and ASCs were incubated in osteogenic medium, then alizarin red staining, alkaline phosphatase (ALP) assays, and RT-PCR (for RUNX2, osteopontin, DLX5, osterix, and osteocalcin) were performed to evaluate the osteoblastic differentiation ability of both cell types in vitro. In vivo, the DFAT cells and ASCs were incubated in osteogenic medium for four weeks and seeded on collagen composite scaffolds, then implanted subcutaneously into the backs of mice. We then performed hematoxylin and eosin staining and immunostaining for GFP and osteocalcin. Results: The alizarin red-stained areas in DFAT cells showed weak calcification ability at two weeks, but high calcification ability at three weeks, similar to ASCs. The ALP levels of ASCs increased earlier than in DFAT cells and showed a significant difference (p < 0.05) at 6 and 9 days. The ALP levels of DFATs were higher than those of ASCs after 12 days. The expression levels of osteoblast marker genes (osterix and osteocalcin) of DFAT cells and ASCs were higher after osteogenic differentiation culture. Conclusion: DFAT cells are easily isolated from a small amount of adipose tissue and are readily expanded with high purity; thus, DFAT cells are applicable to many tissue-engineering strategies and cell-based therapies.     

干细胞分化为胰岛细胞的研究进展

糖尿病是以高血糖为代谢特征的严重影响人类健康的疾病。目前,糖尿病较理想的治疗方法是胰腺移植,但受供体缺乏和免疫排斥两方面的限制而不能普遍应用。干细胞由于具有自我更新和多分化潜能,其作为糖尿病细胞替代治疗的种子资源,已成为目前研究的热点。本文对近年来干细胞分化为胰岛细胞的研究进展作一综述。     

Characterization of Extracellular Vesicles from Preconditioned Human Adipose-Derived Stromal/Stem Cells

Cell-free therapy using extracellular vesicles (EVs) from adipose-derived mesenchymal stromal/stem cells (ASCs) seems to be a safe and effective therapeutic option to support tissue and organ regeneration. The application of EVs requires particles with a maximum regenerative capability and hypoxic culture conditions as an in vitro preconditioning regimen has been shown to alter the molecular composition of released EVs. Nevertheless, the EV cargo after hypoxic preconditioning has not yet been comprehensively examined. The aim of the present study was the characterization of EVs from hypoxic preconditioned ASCs. We investigated the EV proteome and their effects on renal tubular epithelial cells in vitro. While no effect of hypoxia was observed on the number of released EVs and their protein content, the cargo of the proteins was altered. Proteomic analysis showed 41 increased or decreased proteins, 11 in a statistically significant manner. Furthermore, the uptake of EVs in epithelial cells and a positive effect on oxidative stress in vitro were observed. In conclusion, culture of ASCs under hypoxic conditions was demonstrated to be a promising in vitro preconditioning regimen, which alters the protein cargo and increases the anti-oxidative potential of EVs. These properties may provide new potential therapeutic options for regenerative medicine.     

Comprehensive Comparison of Amnion Stromal Cells and Chorion Stromal Cells by RNA-Seq

Mesenchymal stromal cells derived from the fetal placenta, composed of an amnion membrane, chorion membrane, and umbilical cord, have emerged as promising sources for regenerative medicine. Here, we used next-generation sequencing technology to comprehensively compare amniotic stromal cells (ASCs) with chorionic stromal cells (CSCs) at the molecular and signaling levels. Principal component analysis showed a clear dichotomy of gene expression profiles between ASCs and CSCs. Unsupervised hierarchical clustering confirmed that the biological repeats of ASCs and CSCs were able to respectively group together. Supervised analysis identified differentially expressed genes, such as LMO3, HOXA11, and HOXA13, and differentially expressed isoforms, such as CXCL6 and HGF. Gene Ontology (GO) analysis showed that the GO terms of the extracellular matrix, angiogenesis, and cell adhesion were significantly enriched in CSCs. We further explored the factors associated with inflammation and angiogenesis using a multiplex assay. In comparison with ASCs, CSCs secreted higher levels of angiogenic factors, including angiogenin, VEGFA, HGF, and bFGF. The results of a tube formation assay proved that CSCs exhibited a strong angiogenic function. However, ASCs secreted two-fold more of an anti-inflammatory factor, TSG-6, than CSCs. In conclusion, our study demonstrated the differential gene expression patterns between ASCs and CSCs. CSCs have superior angiogenic potential, whereas ASCs exhibit increased anti-inflammatory properties.     

Injectable Gel Form of a Decellularized Bladder Induces Adipose-Derived Stem Cell Differentiation into Smooth Muscle Cells In Vitro

Biologic scaffolds composed of extracellular matrix components have been proposed to repair and reconstruct a variety of tissues in clinical and pre-clinical studies. Injectable gels can fill and conform any three-dimensional shape and can be delivered to sites of interest by minimally invasive techniques. In this study, a biological gel was produced from a decellularized porcine urinary bladder by enzymatic digestion with pepsin. The enzymatic digestion was confirmed by visual inspection after dissolution in phosphate-buffered saline solution and Fourier-transform infrared spectroscopy. The rheological and biological properties of the gel were characterized and compared to those of the Matrigel^TM chosen as a reference material. The storage modulus G’ reached 19.4 ± 3.7 Pa for the 30 mg/mL digested decellularized bladder gels after ca. 3 h at 37 °C. The results show that the gel formed of the porcine urinary bladder favored the spontaneous differentiation of human and rabbit adipose-derived stem cells in vitro into smooth muscle cells to the detriment of cell proliferation. The results support the potential of the developed injectable gel for tissue engineering applications to reconstruct for instance the detrusor muscle part of the human urinary bladder.     

bFGF促增殖后骨髓间充质干细胞向多巴胺能神经元样细胞的分化

目的探讨碱性成纤维细胞生长因子(Basic fibroblast growth factor,bFGF)体外作用于骨髓间充质干细胞(Mesen-chymal stem cells,MSCs)后,诱导其向神经元样细胞和多巴胺能神经元样细胞定向分化的情况。方法从鼠骨髓中获得MSCs,培养传代,用MTT法检测bFGF对骨髓MSCs生长的影响。10 ng/ml bFGF作用2 d后,通过IBMX、细胞因子bFGF、GDNFI、L-1β、中脑神经胶质细胞条件培养基和中脑神经细胞膜碎片等分组联合诱导骨髓MSCs向神经元样细胞、多巴胺能神经元样细胞分化,免疫细胞化学方法鉴定特异标志NSE、MAP-2a,b和TH的表达。结果在一定范围内,bFGF对骨髓MSCs具有明显的促增殖作用。分化的神经元样细胞表达NSE、MAP-2a,b和TH,联合应用GDNFI、L-1β、中脑条件培养基和中脑神经细胞膜碎片诱导7 d后,NSE阳性率为(27.774±2.747)%,MAP-2a,b为(28.006±3.080)%,TH为(3.098±0.352)%。结论体外骨髓MSCs被诱导分化成神经元样细胞和多巴胺能神经元样细胞,为帕金森等中枢神经系统疾病的细胞移植治疗奠定了基础。