下调microRNA-34a增强对人脂肪干细胞成骨诱导分化作用的初步探索
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摘要
目的:颅颌面临界骨缺损是整复外科常见疾患,常常给患者的身心带来障碍和压力。为解决骨形成不足这一难题,我们拟通过表观遗传修饰的手段,调节microRNA-34a在人脂肪干细胞中的表达,来探讨microRNA-34a对人脂肪干细胞成骨诱导分化的影响。方法:分离培养原代人脂肪干细胞,以慢病毒为转染载体,分三组对其进行上调、下调microRNA-34a的表达及阴性对照。然后对其进行成骨诱导培养,于诱导的第7天行碱性磷酸酶(ALP)染色,第14天行茜素红(Alizarin Red)染色,定性比较转染后人脂肪干细胞成骨诱导分化的效果。结果:成功分离和培养原代人脂肪干细胞,并以慢病毒高效率转染调控microRNA-34a的表达。对比碱性磷酸酶和茜素红染色可见下调microRNA-34a组成骨效率最高,其次是阴性对照组,上调组效率最低。结论:1.人脂肪干细胞通过慢病毒转染获得高效率的表观遗传修饰;2.下调microRNA-34a的表达对人脂肪干细胞成骨的诱导分化有一定的促进作用。
Objective: Cranial critical-sized bone defect is commonly seen in plastic and reconstructive surgery causing dysfunctions and pressure on patients both physically and psychologically. In order to solve the difficulty, we regulated the expression of microRNA-34 a in human adipose-derived stem cells(hADSCs) by epigenetic modification to evaluate the effect of microRNA-34 a on hADSCs in osteoinduction differentiation. Methods: Extract and culture primary h ADSCs. Transfect h ADSCs with lentivirus respectively in three groups: microRNA-34 a up-regulation, knockdown and negative control. And then induce them to differentiate into osteoblasts.The osteogenic efficiency was qualitatively detected by ALP staining at day 7 and alizarin red staining at day 14 after osteoinduction.Results: hADSCs were successfully extracted and cultured; microRNA-34 a expression was regulated by means of high efficient transfection of lentivirus; staining results showed that microRNA-34 a knockdown group was most efficient in osteoinduction differentiation, control group came as the second, up-regulation group was the least efficient. Conclusion: 1.By transfecting with lentivirus, hADSCs acquired high efficiency of epigenetic modification; 2.microRNA-34 a knockdown to some extent can promote osteoinduction differentiation in hADSCs.
Objective: Cranial critical-sized bone defect is commonly seen in plastic and reconstructive surgery causing dysfunctions and pressure on patients both physically and psychologically. In order to solve the difficulty, we regulated the expression of microRNA-34 a in human adipose-derived stem cells(hADSCs) by epigenetic modification to evaluate the effect of microRNA-34 a on hADSCs in osteoinduction differentiation. Methods: Extract and culture primary h ADSCs. Transfect h ADSCs with lentivirus respectively in three groups: microRNA-34 a up-regulation, knockdown and negative control. And then induce them to differentiate into osteoblasts.The osteogenic efficiency was qualitatively detected by ALP staining at day 7 and alizarin red staining at day 14 after osteoinduction.Results: hADSCs were successfully extracted and cultured; microRNA-34 a expression was regulated by means of high efficient transfection of lentivirus; staining results showed that microRNA-34 a knockdown group was most efficient in osteoinduction differentiation, control group came as the second, up-regulation group was the least efficient. Conclusion: 1.By transfecting with lentivirus, hADSCs acquired high efficiency of epigenetic modification; 2.microRNA-34 a knockdown to some extent can promote osteoinduction differentiation in hADSCs.
引文
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[2]Totir M,Ciuluvica R,Dinu I,et al.Biomaterials for orbital fractures repair[J].J Med Life,2015,7(Spec Iss 4):41-43
[3]Schmitz J P,Hollinger J O.The critical size defect as an experimental model for craniomandibulofacial nonunions[J].Clinical Orthopaedics&Related Research,1986,205(205):299-308
[4]Mishra R,Goel S K,Gupta K C,et al.Biocomposite cryo gels as tissue-engineered biomaterials for regeneration of critical-sized cranial bone defects[J].Tissue Engineering Part A,2014,20(3-4):751-762
[5]Boeloni J N,Ocarino N M,Goes A M,et al.Comparative study of osteogenic differentiation potential of mesenchymal stem cells derived from bone marrow and adipose tissue of osteoporotic female rats[J].Connective Tissue Research,2014,55(2):103-114
[6]Banyard D A,Salibian A A,Widgerow A D,et al.Implications for human adipose-derived stem cells in plastic surgery[J].Journal of Cellular&Molecular Medicine,2015,19(1):21-30
[7]Feisst V,Meidinger S,Locke M B.From bench to bedside:use of human adipose-derived stem cells[J].Stem Cells&Cloning Advances&Applications,2015,8(default):149-162
[8]Behr B,Tang C,Germann G,et al.Locally Applied Vascular Endothelial Growth Factor A Increases the Osteogenic Healing Capacity of Human Adipose Derived Stem Cells by Promoting Osteogenic and Endothelial Differentiation[J].Stem Cells,2015,29(2):286-296
[9]Mohr A M,Mott J L.Overview of microRNA biology[J].Seminars in liver disease,2015,35(1):3-11
[10]Simonson B,Das S.MicroRNA Therapeutics:the Next Magic Bullet?[J].Mini Reviews in Medicinal Chemistry,2015,15(6)467-474
[11]Hammond S M.An overview of microRNAs[J].Adv Drug Deliv Rev,2015,87:3-14
[12]Hu R,Li H,Liu W,et al.Targeting miRNAs in osteoblas differentiation and bone formation[J].Expert Opin Ther Targets2010,14(10):1109-1120
[13]Farooqi A A,Tabassum S,Ahmad A.MicroRNA-34a:A Versatile Regulator of Myriads of Targets in Different Cancers[J].Internationa journal of molecular sciences,2017,18(10)
[14]Li C,Holmstr M K,Qiu W,et al.MicroRNA 34a Inhibits Osteoblas Differentiation and In Vivo Bone Formation of Human Stromal Stem Cells[J].Stem Cells,2014,32(4):902-912
[15]Al-nawas B,Schiegnitz E.Augmentation procedures using bone substitute materials or autogenous bone-a systematic review and meta-analysis[J].European Journal of Oral Implantology,2014,7Suppl 2(2):S219-S234
[16]Myeroff C,Archdeacon M.Autogenous bone graft:donor sites and techniques[J].Journal of Bone&Joint Surgery American Volume2011,93(23):2227-2236
[17]Smith B,Goldstein T,Ekstein C.Biologic adjuvants and bone:current use in orthopedic surgery[J].Current Reviews in Musculoskeletal Medicine,2015,8(2):193-199
[18]Jr D L W,Einhorn T A,Koval K,et al.Bone grafts and bone graf substitutes in orthopaedic trauma surgery.A critical analysis[J]Journal of Bone&Joint Surgery-american Volume,2007,89(3):649-658
[19]Dimitriou R,Mataliotakis G I,Angoules A G,et al.Complications following autologous bone graft harvesting from the iliac crest and using the RIA:A systematic review[J].Injury-international Journal of the Care of the Injured,2011,42(5):S3-S15
[20]Crane G M,Ishaug S L,Mikos A G.Bone tissue engineering[J].Nature medicine,1995,1(12):1322-1324
[21]Hu N,Feng C,Jiang Y,et al.Regulative Effect of Mir-205 on Osteogenic Differentiation of Bone Mesenchymal Stem Cells(BMSCs):Possible Role of SATB2/Runx2 and ERK/MAPK Pathway[J].International Journal of Molecular Sciences,2015,16(5):10491-10506
[22]Sun J,Jieyun L,Chichi L,et al.Role of bone morphogenetic protein-2 in osteogenic differentiation of mesenchymal stem cells[J].Molecular Medicine Reports,2015,12(3):4230-4237
[23]Zhuang W,Ge X,Yang S,et al.Upregulation of lncRNA MEG3Promotes Osteogenic Differentiation of Mesenchymal Stem Cells From Multiple Myeloma Patients By Targeting BMP4 Transcription[J].Stem Cells,2015,33(6):1985-1997
[24]丁勇,许超,吴季辉,等.表观遗传学研究进展[J].中国科学:生命科学,2017,47:3-15
[25]Mohr A M,Mott J L.Overview of microRNA biology[J].Seminars in liver disease,2015,35(1):3-11
[26]Yang Z,Qu X,Li H,et al.MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2[J].Febs Letters,2012,586(16):2375-2381
[27]Li J,Hu C,Han L,et al.MiR-154-5p regulates osteogenic differentiation of adipose-derived mesenchymal stem cells under tensile stress through the Wnt/PCP pathway by targeting Wnt11[J].Bone,2015,78:130-141
[28]Gao J,Li N,Dong Y,et al.miR-34a-5p suppresses colorectal cancer metastasis and predicts recurrence in patients with stage II/IIIcolorectal cancer[J].Oncogene,2015,34(31):4142-4152
[29]Xu X,Chen W,Miao R,et al.miR-34a induces cellular senescence via modulation of telomerase activity in human hepatocellular carcinoma by targeting FoxM1/c-Myc pathway[J].Oncotarget,2015,6(6):3988-4004
[30]Wang Y,Jia L S,Yuan W,et al.Low miR-34a and miR-192 are associated with unfavorable prognosis in patients suffering from osteosarcoma[J].American Journal of Translational Research,2015,7(1):111-119