Exendin-4通过JNK和ERK信号通路增强大鼠脂肪来源干细胞的增殖

目的:探讨Exendin-4对大鼠脂肪来源干细胞的(Adipose-derived stem cells,ADSCs)增殖作用及其机制。方法:体外分离培养SD大鼠腹股沟处脂肪组织,流式细胞学方法鉴定分离的ADSCs,使用Exendin-4(Ex-4,0-50 nm/L)对P4代(第四代)ADSCs进行干预,采用MTT检测ADSCs的增殖情况,Western blot检测MAPK通路中JNK和ERK的磷酸化水平,使用相应的阻断剂来观测JNK和ERK通路对ADSCs增殖作用的影响。结果:分离培养的ADSCs高表达CD29、CD90和CD105,低表达CD31、CD34和CD45,符合间充质干细胞的表型。Ex-4以浓度依赖方式促进ADSCs体外增殖,10 nm/L为最适促增殖处理浓度。同时,Ex-4可以提高细胞中c-Jun和ERK的磷酸化水平,而给予相应阻断剂后,磷酸化蛋白表达减弱,细胞增殖能力也明显减弱。结论:Ex-4可以通过JNK和ERK通路增强大鼠脂肪来源干细胞的增殖。     

TGF-β1基因转染大鼠脂肪干细胞向软骨细胞分化的实验研究

目的TGF-β1基因转染大鼠的ADSCs(adipose stromal cells,ADSCs),诱导其向软骨细胞分化,为软骨组织工程学种子细胞提供新方法。方法分离、培养大鼠的ADSCs,免疫荧光法进行鉴定;TGF-β1基因转染ADSCs,对转染后细胞进行筛选;MTT法测定筛选后细胞的增殖活性;RT-PCR、Western blot对筛选后细胞的表达进行检测。结果成功分离、培养大鼠的ADSCs;细胞表面标志物CD29和CD44表达阳性,CD106和CD34表达阴性;基因转染后细胞的增殖力变强;转染TGF-β1的ADSCs在目的基因TGF-β1、SOX9、Aggrecan、Collagen II mRNA的表达和软骨特异性基质-Collagen II的分泌增强,明显高于对照组和转染空载体组;结果TGF-β1基因转染后ADSCs具有了软骨细胞的表型特征,可以用作软骨组织工程的种子细胞。     

重组腺病毒介导 HGF 修饰 ADSCs 对大鼠肝损伤模型的治疗作用

摘要 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。     

microRNA-21靶向PDCD4/PTEN对大鼠ADSCs凋亡的抑制作用

该研究探究miR-21对大鼠ADSCs凋亡的影响,为提高ADSCs移植存活率提供依据。该研究建立大鼠ADSCs体外凋亡模型,采用qRT-PCR检测miR-21的表达。在ADSCs中采用Lipofectamine 2000转染miR-21 mimics,qRT-PCR检测miR-21 mimics转染效率。CCK-8、Hoechst 33258检测大鼠ADSCs过表达miR-21对细胞存活率的影响;Western blot检测大鼠ADSCs过表达miR-21后凋亡相关蛋白表达量。结果显示H_2O_2诱导细胞凋亡后,miR-21在ADSCs中的表达明显下调。大鼠ADSCs转染miR-21 mimics后,ADSCs中miR-21的表达量较对照组显著提高了7.4倍。与miR-21 scramble组相比,ADSCs过表达miR-21后,ADSCs存活率显著升高,促凋亡蛋白Caspase-3和Bax的表达量显著降低,而抗凋亡蛋白Bcl-2的表达水平明显升高。与对照组相比,miR-21过表达后,PDCD4和PTEN mRNA无显著变化,但PDCD4和PTEN蛋白表达明显下调。利用PDCD4 siRNA和Phen阻断PDCD4和PTEN的表达后,Caspase-3、Bax的表达水平显著降低,Bcl-2的表达水平显著升高。该研究得出miR-21通过靶向PDCD4/PTEN增强大鼠ADSCs对凋亡的抑制作用。     

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。     

电磁脉冲诱导大鼠心肌闰盘Cx43连接蛋白的上调

目的:探讨电磁脉冲(EMP)对SD大鼠心肌闰盘及主要连接蛋白Cx43的影响.方法:将雄性SD大鼠35只,随机分为对照组和EMP辐照组,EMP辐照组又分为照后即刻、1h、3h、6h、12h、24h组,每组5只大鼠.应用EMP模拟发生器对EMP辐照组大鼠进行辐照,场强为200 kV/m、前沿15 ns、脉宽7-8 ns、脉冲次数200次,脉冲间隔为7s,麻醉后取大鼠左心室,采用硝酸镧示踪法和透射电子显微镜观察大鼠心肌闰盘(ID)结构的变化,用Real-Time PCR方法和Western blot方法检测EMP辐照后,大鼠连接蛋白Cx43在转录水平和蛋白水平表达量的变化情况.结果:电镜下正常对照组大鼠心肌ID处未见硝酸镧颗粒沉积;EMP组大鼠ID处可见硝酸镧颗粒,且随时间的延长,硝酸镧颗粒沉积量逐渐增多,以照后6h组硝酸镧颗粒沉积量达最大,然后硝酸镧颗粒沉积量随时间延长逐渐减少,24h组恢复正常.心肌细胞Cx43的mRNA表达水平在EMP照后lh和6h明显增高,分别是对照组的1.95、3.10倍(P＜0.05);照后即刻、3h、12h、24h表达水平与对照组相比差异无统计学意义(P＞0.05).Western blot结果为照后即刻、1h、3h和6h组大鼠心肌细胞Cx43蛋白表达量与对照组比较显著增加,12h、24h与对照组相比差异无统计学意义(P＞0.05).结论:EMP可使心肌闰盘结构发生改变,上调Cx43蛋白的mRNA水平和蛋白表达量.     

三七总皂苷通过抗炎和调血脂作用抑制大鼠动脉粥样硬化形成

目的:利用高脂饮食叠加炎症刺激诱发大鼠动脉粥样硬化(AS)模型,在转录水平及信号转导方面探讨三七皂苷(PNS)防治炎性因素诱发AS的分子机制。方法:实验分为对照组、模型组和治疗组3组,分别腹腔注射给予无菌医用液体石蜡、酵母多糖(Zym,20mg/kg,1次/3天)、Zym(20mg/kg,1次/3天) PNS(100mg/kg,1次/天)。所有大鼠均喂食含3%胆固醇的高脂饲料。9周后,取血测定血脂水平和血液粘度;应用定量PCR法测定腹主动脉组织中抑制性核转录因子(IκBα)mRNA、心房肽(ANF)mR- NA、基质金属蛋白酶7(MMP7)mRNA以及炎性因子和脂肪酸合成酶(FAS)mRNA的表达;用Western Blotting法检测IκBα的表达。结果:Zym刺激引起大鼠血清总胆固醇、甘油三酯、全血粘度与血浆比粘度均显著升高,IκBαmRNA及其蛋白表达均明显降低,ANF mRNA、MMP7 mRNA和FAS mRNA表达均明显升高。与模型组相比,PNS能明显升高血脂水平、全血及血浆粘度,促进IκBαmRNA与蛋白以及ANF mRNA表达,抑制MMP7 mRNA和FAS mRNA表达。结论:PNS对炎症免疫诱发的AS有显著防治作用,其作用机制与促进IκBα的表达从而抑制相关炎症因子的生成有关,PNS对FAS的表达调节可能是其降脂的主要机制之一。     

结核分枝杆菌H37Rv刺激巨噬细胞后差异表达lncRNA分析及鉴定

分析毒性结核分枝杆菌(Mycobacterium tuberculosis,M.tb)H37Rv刺激RAW264.7巨噬细胞的表达谱芯片以及筛选和鉴定M.tb H37Rv感染巨噬细胞RAW264.7后差异表达的lncRNA。首先,分析热灭活的H37Rv刺激RAW264.7细胞24h后lncRNA表达谱芯片,并对差异表达的lncRNA和mRNA进行生物信息分析,然后采用实时定量聚合酶链反应对16条在芯片中差异表达的lncRNA进行细胞水平验证;进一步在H37Rv感染小鼠的脾脏和肺脏中检测差异表达的lncRNA。结果显示,表达谱芯片中4 730条lncRNA的表达水平上调,9 558条lncRNA的表达水平下调。生物信息分析筛选的16条lncRNA,其染色体定位于附近蛋白质编码基因的基因间区或者与外显子区域有重叠,mRNA功能注释显示差异表达的mRNA主要集中于转录调节、磷酸化、凋亡等生化过程以及丝裂原活化蛋白激酶(MAPK)等抗结核的信号通路中。在H37Rv作用下的细胞水平和动物感染模型的组织中RT-q PCR验证出与芯片结果相同的4条lncRNA,其中上调的有3条,下调的有1条。研究中异常表达的lncRNA可为巨噬细胞在结核分枝杆菌感染中的功能紊乱提供线索,为后续的研究奠定基础,进一步的研究将集中于发掘lncRNA在宿主调控中发挥的功能。     

RNA修饰对长链非编码RNA的调控作用

长链非编码RNA (lncRNA)能在表观遗传、转录以及转录后水平上调控基因表达,与疾病的发生、发展和防治有着密切的联系。RNA修饰介导的表观转录组学调控是表观遗传的新领域,可以在转录后水平调控基因表达,并且可以作为一种重要的修饰手段对lncRNA进行调控。RNA修饰可以通过对lncRNA表达水平、剪切方式及二级结构的调控,影响各种生物学进程。现回顾和展望RNA修饰对lncRNA的调控作用和其潜在的生物学功能。     

银灵通胶囊调节实验性高脂饮食大鼠血脂的研究

目的:探究银灵通胶囊对脂质代谢的影响及其机制。方法:建立大鼠高脂模型,用药后检测其血脂、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性,检测肝脏组织的高密度脂蛋白受体SR-BI、低密度脂蛋白受体(LDLR)、氧化低密度脂蛋白(ox-LDH)受体CD36蛋白表达的mRNA表达水平,检测血管组织学变化。结果:高脂饮食明显升高大鼠血清中总胆固醇(CH)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)和动脉硬化指数(AI值),银灵通胶囊组可降低上述指标,且呈一定的浓度依赖;高脂饮食可增加肝脏中SR-BI及CD36表达,降低LDLR表达,银灵通胶囊引起SR-BI的过度表达,使LDLR表达增加,CD36表达下降。高脂饮食使血清中MDA的含量增加,给予银灵通胶囊后,明显降低血清MDA的含量。结论:银灵通胶囊具有调节脂质代谢,抗动脉粥样硬化(AS)及抗脂质过氧化作用。其机制与银灵通胶囊能引起肝脏中SR-BI的过度表达及LDLR表达增加,降低肝脏中CD36表达和血清MDA含量有关。     

Effect of a subset of adipose-derived stem cells isolated with liposome magnetic beads to promote cartilage repair

This study aimed to investigate the ability of CD146⁺ subset of ADSCs to repair cartilage defects. In this study, we prepared CD146⁺ liposome magnetic beads (CD146⁺LMB) to isolate CD146⁺ADSCs. The cells were induced for chondrogenic differentiation and verified by cartilage-specific mRNA and protein expression. Then a mouse model of cartilage defect was constructed and treated by filling the induced cartilage cells into the damaged joint, to evaluate the function of such cells in the cartilage microenvironment. Our results demonstrated that the CD146⁺LMBs we prepared were uniform, small and highly stable, and cell experiments showed that the CD146⁺LMB has low cytotoxicity to the ADSCs. ADSCs isolated with CD146⁺LMB were all CD146⁺, CD105⁺, CD166⁺ and CD73⁺. After chondrogenic induction, the cells showed significantly increased expression of cartilage markers Sox9, collagen Ⅱ and aggrecan at protein level and significantly increased Sox9, collagen Ⅱ and aggrecan at mRNA level, and the protein expression and mRNA expression of CD146⁺ADSCs group were higher than those of ADSCs group. The CD146⁺ADSCs group showed superior tissue repair ability than the ADSCs group and blank control group in the animal experiment, as judged by gross observation, histological observation and histological scoring. The above results proved that CD146⁺LMB can successfully isolate the CD146⁺ADSCs, and after chondrogenic induction, these cells successfully promoted repair of articular cartilage defects, which may be a new direction of tissue engineering.     

一种体外快速分离脂肪来源干细胞的方法

目的:以小鼠为模型,建立一种基于流式细胞仪为检测手段的快速分离脂肪来源干细胞的方法,解决间充质干细胞进入实际应用过程中遇到的难题。方法:取BALB/c小鼠腹股沟内侧的皮下脂肪组织,采用Ⅰ型胶原酶消化等系列措施,获取脂肪干细胞(adipose-derived stem cells,ADSCs)。所得细胞分离培养4代后,流式细胞仪分选出CD73+CD45-ADSCs后成骨诱导分化。碱性磷酸酶染色和实时荧光定量PCR检测其分化情况。结果:刚分离培养的ADSCs细胞普遍呈圆形或椭圆形,传至第三代的细胞,非MSCs细胞逐渐被淘汰,剩余的细胞形态逐渐变得一致,细胞形态呈梭形。流式细胞仪检测发现ADSCs细胞表面抗原标记CD73+CD45-为20.7%。所得的ADSCs成骨诱导分化后碱性磷酸酶(alkaline phosphatase,ALP)染色呈阳性,实时荧光定量PCR检测成骨标志基因发现它们表达上调,其中ALP的表达高达22倍。结论:本方法可以获得纯度较高的ADSCs,且耗时少成本低;且提示可采用该方法来获得大量的人源ADSCs用于组织工程修复。     

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.     

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。     

Evaluation of CD49f as a novel surface marker to identify functional adipose‐derived mesenchymal stem cell subset

ObjectivesCD49f is expressed on a variety of stem cells and has certain effects on their cytological functions, such as proliferation and differentiation potential. However, whether CD49f is expressed on the surface of adipose tissue‐derived mesenchymal stem cells (ADSCs) and its effect on ADSCs has not been clarified.Materials and methodsThe effects of in vitro culture passage and inflammatory factor treatment on CD49f expression and the adhesion ability of ADSCs from mice and rats were investigated. CD49f⁺ cells were selected from rat ADSCs (rADSCs) by magnetic‐activated cell sorting (MACS), and the cellular functions of CD49f⁺ ADSCs and unsorted ADSCs, including their clonogenic, proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities, were compared.ResultsCD49f expression and the adhesion ability of ADSCs decreased with increasing in vitro culture passage number. TNF‐α and IFN‐γ treatment decreased CD49f expression but increased the adhesion ability of ADSCs. After CD49f was blocked with an anti‐CD49f antibody, the adhesion ability of ADSCs was decreased. No significant difference in clonogenic activity was observed between unsorted ADSCs and CD49f⁺ ADSCs. CD49f⁺ ADSCs had greater proliferation, adipogenic and osteogenic differentiation, migration and anti‐apoptotic capacities than unsorted ADSCs.ConclusionIn the current study, the expression of CD49f on ADSCs was identified for the first time. The expression of CD49f on ADSCs was influenced by in vitro culture passage number and inflammatory factor treatment. Compared with unsorted ADSCs, CD49f ⁺ ADSCs exhibited superior cellular functions, thus may have great application value in mesenchymal stem cell (MSC)‐based therapies.     

Adipose Stem Cells Promote Smooth Muscle Cells to Secrete Elastin in Rat Abdominal Aortic Aneurysm

Background

Abdominal aortic aneurysm (AAA) is a life-threatening disease and its prevalence rate increases with social aging. The degradation of elastic is an important factor in the formation of AAA.

Methods

Adipose derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs) were isolated from rats, and identified by Oil red O and alizarin red staining after adipogenesis and osteogenesis induction. In addition, ADSCs were also identified by flow cytometry with CD markers. AAA model in rats was established, and smooth muscle cells (SMCs) were isolated from AAA aortic wall and identified by immunohistochemistry. ADSCs or BMSCs were co-cultured with AAA aortic wall for in vitro experiment, and ADSCs were injected into AAA model for in vivo test. Then orcein staining was used for observing the morphology of elastic fiber, Western blot and real-time PCR were used respectively to detect the protein and gene expression of elastin, gelatinases spectrum analysis was used to detect the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9.

Results

Lots of red lipid droplets were visible by Oil red O staining after adipogenesis induction, and black calcium nodules appeared by alizarin red staining after osteogenesis induction. The results of flow cytometry showed that ADSCs expressed CD44 and CD105, but exhibited negligible expression of CD31 and CD45. SMCs exhibited spindle-like morphology and α-actin protein was positive in cytoplasm. After co-cultured with ADSCs or BMSCs, the elastic fiber recovered normal winding shape, both the gene and protein expression of elastin increased, and the activity of MMP-2 decreased. The in vivo result was similar to that of in vitro.

Conclusions

ADSCs promote the expression of elastin in SMCs and contribute to the reconstruction of elastic fiber, which may provide new ideas for treating AAA.     

GDNF-ADSCs-APG embedding enhances sciatic nerve regeneration after electrical injury in a rat model

The pluripotency of adipose-derived stem cells (ADSCs) makes them appropriate for tissue repair and wound healing. Owing to the repair properties of autologous platelet–rich gel (APG), which is based on easily accessible blood platelets, its clinical use has been increasingly recognized by physicians. The aim of this study was to investigate the effect of combined treatment with ADSCs and APG on sciatic nerve regeneration after electrical injury. To facilitate the differentiation of ADSCs, glial cell line–derived neurotrophic factor (GDNF) was overexpressed in ADSCs by lentivirus transfection. GDNF-ADSCs were mingled with APG gradient concentrations, and in vitro, cell proliferation and differentiation were examined with 5-ethynyl-2′-deoxyuridine staining and immunofluorescence. A rat model was established by exposing the sciatic nerve to an electrical current of 220 V for 3 seconds. Rat hind-limb motor function and sciatic nerve regeneration were subsequently evaluated. Rat ADSCs were characterized by high expression of CD90 and CD105, with scant expression of CD34 and CD45. We found that GDNF protein expression in ADSCs was elevated after Lenti-GDNF transfection. In GDNF-ADSCs-APG cultures, GDNF was increasingly produced while tissue growth factor-β was reduced as incubation time was increased. ADSC proliferation was augmented and neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) expression were upregulated in GDNF-ADSCs-APG. In addition, limb motor function and nerve axon growth were improved after GDNF-ADSCs-APG treatment. In conclusion, our study demonstrates the combined effect of ADSCs and APG in peripheral nerve regeneration and may lead to treatments that benefit patients with electrical injuries.