【作者】 刘学娟；

【导师】 李玉林；

【作者基本信息】 吉林大学 ， 病理与病理生理学， 2013， 博士

【摘要】 干细胞能定向诱导分化为机体几乎所有组织中具有特定功能的终末细胞，进而修复或替代因外伤或疾病而丧失功能的组织或细胞是该领域研究的重大突破，但其准确的调控机制远未解决。本实验拟以人毛囊源性间充质干细胞（hHF-MSCs）为起始细胞，在将其诱导分化为平滑肌细胞(SMCs)的研究中，聚焦于miRNA在这一分化过程中所起的作用，并试图在miRNA的靶基因上寻找突破。材料取自引产胎儿的头皮，分离出hHF-MSCs，在对其干性及多向分化潜能做出鉴定之后，以5ng/ul TGF-β1诱导hHF-MSCs分化为SMCs，用免疫荧光、流式细胞术、Western blot、RT-PCR及qRT-PCR检测诱导后的SMCs特异性标记物表达；通过免疫荧光、qRT-PCR检测TGF-β1诱导miR-18b作用之后的hHF-MSCs向SMC分化特异性标记物的表达；通过双荧光素酶报告基因检测系统证实miR-18b在这一过程中的靶点。结果显示：1.多潜能性hHF-MSCs在TGF-β1诱导下分化为SMCs，mRNA和蛋白水平上表达α-SMA、calponin；2.在TGF-β1诱导hHF-MSCs向SMCs分化过程中，miR-18b表达逐渐降低；3.分别转染agomir-18b或antagomir-18b的hHF-MSCs，再用TGF-β1作用7天之后，α-SMA和calponin表达降低或升高；4.检测并验证SMAD2基因是miR-18b的靶点；5.将hHF-MSCs敲减SMAD2，再转染antagomir-18b之后，用TGF-β1诱导，发现抑制miR-18b不再促进SMCs分化，证明miR-18b通过SMAD2调节TGF-β1诱导hHF-MSCs向SMCs分化。综上所述，本实验建立了从毛囊中分离、培养及扩增hHF-MSCs的标准化技术；通过TGF-β1诱导hH-FMSCs分化为SMCs，并在此过程中研究了miR-18b对分化的作用；首次证实了miR-18b通过作用于靶基因SMAD2促进hHF-MSCs向SMCs分化。结果不仅为揭示干细胞定向诱导分化的分子机制提出了新的见解，对其在损伤修复与再生医学中的实际应用也具有重要指导价值。更多还原

【Abstract】 In regenerative medicine applications,the origin problem of stem cells withself-renewal and differentiation potential must be solved.Embryonic stem cellsapplication have been limited because of the ethical controversies and problems ofimmunological rejection.Adult stem cells have gradually become a research hotspot.Adult stem cells exist not only in the peripheral blood, gastrointestinal tract, liver,glands, pulp, derm, skeletal muscle, blood and fat tissue and hair follicles and so on,and in almost all developmental stages.The researchers need to look for an abundantand convenient stem cell origin that tiny injury or no trauma to the body should to bethought about, Our research group has isolated, cultured and expanded multipotentmesenchymal stem cells from hair follicles successfully. MiRNAs in the regulation ofstem cell self-renewal, multipotent differentiation and phenotypic transformation areof great significance, and interact with signaling pathways, epigenetic modificationsto regulate almost all biological processes. Vascular tissue engineering, cardiovasculardiseases and disorders of the urinary tract and so forth required smooth muscle cellsources. In order to find a highly efficiently standardized method to improve smoothmuscle cells (SMCs) differentiation induced from human hair follicule mesenchymalstem cells (hHF-MSCs).Successfully miRNAs important for smooth muscle celldifferentiatio have been identified and validated through the gain and loss of functionexperiment.The pluripotent hHF-MSCs provides ample experimental materials for the nextwork. HHF-MSCs differentiated into SMCs by the induction by TGFβ1.Theexpression of SMCs markers α-SMA and calponin was detected.The differentiationof SMCs was confirmed.And the effect of miR-18b on differentiation was examinedin this differentiation process.The target gene of miR-18b was searched for. We willstudy how miR-18b play role in differentiation mechanism of SMCs from MSCs. Methods:1.Isolation,culture and identification of hHF-MSCsFlow cytometry was used to detect the expression of stem cells surface markers.Oil red O staining,Alizarin red staining,Alcian blue staining were used to detect theadipogenic,osteogenic and chondrogenic differentiation.2. Induction of differentiation of hHF-MSCs into SMCsα-SMAand calponin mRNAexpression were detected by RT-PCR and qRT-PCR. α-SMA and calponin protein expression were detected by immunofluorescence,flow cytometry and Western blot.3.The miR-18b important for hHF-MSCs differentiation to SMCs in vitro byTGF-β1inductionThe expression of miR-18b was detected during differentiation process byqRT-PCR. α-SM and calponin expression were detected in hHF-MSCs transfectedwith agomir-18b or antagomir-18b after the induction of differentiation byfluorescence and qRT-PCR. MiR-18b target gene was predicted with the onlineTargetscan and miRBase software and confirmed by the dual luciferase reporter assaysystem.HHF-MSCs Smad2protein was knocked down by means of the transfectionof SMAD2shRNA(h) lentivirus particles and comfirmed by Westernblot.Fluorescence and qRT-PCR detected the expression of SMCs specific markers inth hHF-MSCs knocked down SMAD2by the induction of TGF-β1.Results:1. Isolation,culture and identification of hHF-MSCsPluripotent adherent hHF-MSCs expressed CD90and CD105. Lipid dropletswere detected by Oil red O after adipogenic induction. Alizarin red staining showedosteogenic cells and intercellular calcium salt deposition. Green blue acidic mucussubstance in cellular gaps after chondrogenic induction.2. Induction of differentiation of hHF-MSCs into SMCsCompared with the control group, the group induced by TGF-β1showing greenfluorescence expressed the SMCs specific markers,for example, α-SMA andcalponin.70%-80%cells induced expressed α-SMA and calponin. At mRNA andprotein level, compared with the control group,the expression of α-SMA and calponinwere increased significantly with statistical significance.3.The miR-18b important for hHF-MSCs differentiation to SMCs in vitro by TGF-β1inductionScreening miRNAs having effect on the SMAD2target. Compared with thecontrol group,miR-18b reduce SMAD2expression obviously.The possibility ofmiR-18b target genes was predicted and validated.miR-18b have been detected indifferentiation of hHF-MSCs to SMCs by TGF-β1(5ng/ul) induction for7days,MiR-18b decreases gradually during this process.Agomir-18b or antagomir-18bwas transfected into hHF-MSCs, and then induced by the TGF-β1(5ng/ul) for7days. Overexpression of miR-18b significantly inhibited TGFβ1induceddifferentiation of hHF-MSCs to SMCs and reduced α-SMA and calponin expression.However, miR-18b antagonists inhibiting its effect increased significantly α-SMAand calponin expression. But in hHF-MSCs SMAD2knocked down, inhibition ofmiR-18b do not improve expressions of SMCs markers.In sum, The stem cell origin of multipotential hHF-MSCs solated from hair isabundant and convenient.TGF β1induced hHF-MSCs differentiation to SMCs.Ourdata firstly demonstrates that miR-18b is downregulated during the SMCdifferentiation of hHF-MSCs and represses differentiation by binding to targetsequences in the3’UTR of SMAD2. This will greatly enhance our understanding ofdifferentiation mechanisms of SMCs. The results not only reveal the molecularmechanism of directed differentiation of stem cells but also provide new insights intothe mechanism.There is an important guidance value for its practical application inwound repair and regeneration medicine.更多还原