牡荆苷对骨髓间充质干细胞成脂分化的影响简

目的 探讨牡荆苷对大鼠骨髓间充质干细胞成脂分化的影响.方法 采用细胞的增殖检测（MTT）法检测骨髓间充质干细胞生存率,油红O染色检测细胞成脂分化,实时荧光定量PCR检测过氧化物酶体增殖物激活受体γ（PPARγ）和CCAAT增强子结合蛋白α（C/EBPα） mRNA表达.结果 牡荆苷浓度在1～50 μmol/L范围内对骨髓间充质干细胞生存率无显著影响;牡荆苷可抑制骨髓间充质干细胞成脂分化及PPARγ和C/EBPαmRNA表达.结论 牡荆苷可抑制骨髓间充质干细胞成脂分化,其作用机制可能与抑制PPARγ和C/EBPα基因表达有关.     

小檗碱对骨髓间充质干细胞成骨分化的影响

目的探讨小檗碱对体外培养SD大鼠骨髓间充质干细胞成骨分化的影响。方法应用MTT法检测不同浓度小檗碱对骨髓间充质干细胞增殖情况的影响,以对硝基苯磷酸盐法检测碱性磷酸酶活性,等离子体直读光谱仪检测钙沉积,放射免疫法检测骨钙素含量,茜素红法染色观察钙化结节形成。结果小檗碱在1×10-9～1×10-5mol.L-1浓度范围对骨髓间充质干细胞增殖无明显影响。小檗碱浓度为1×10-7和1×10-6mol.L-1培养3～7d能明显提高骨髓间充质干细胞内碱性磷酸酶活性。小檗碱在1×10-7～1×10-5mol.L-1浓度范围培养22d能明显促进骨髓间充质干细胞骨钙素合成和分泌。小檗碱在浓度为1×10-6和1×10-5mol.L-1培养28d时钙盐沉积量和钙化结节形成增加。结论小檗碱可促进骨髓间充质干细胞向成骨细胞方向分化,增强骨钙化。这可能为其防治骨质疏松的机制之一。     

过表达组蛋白去甲基化酶Jmjd3对成骨分化的调控作用

目的 研究过表达组蛋白去甲基化酶Jmjd3对间充质干细胞成骨分化的调控作用.方法 建立通过骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)诱导的间充质细胞系C3H10T1/2成骨分化模型;通过荧光定量PCR检测成骨分化转录因子,成骨细胞标记基因的表达;通过碱性磷酸酶活性测定其表达量;通过基因克隆构建Jmjd3的过表达载体,并转染到C3H10T1/2细胞实现过表达;通过免疫印迹方法鉴定蛋白质水平;通过荧光素酶报告基因测定Jmjd3对Runx2和Osx基因的转录调控.结果 BMP-2促进C3H10T1/2细胞内Jmjd3的表达;过表达Jmjd3促进碱性磷酸酶活性,以及成骨分化标记基因COLⅠ,Ocn,Bsp的表达;同时,过表达Jmjd3促进成骨转录因子基因Runx2和Osx的转录,进而促进其基因表达.结论 过表达组蛋白去甲基化酶Jmjd3对间充质干细胞的成骨分化有重要的正向促进作用.     

咯利普兰在骨髓间充质干细胞向成骨细胞分化中的作用

目的 研究咯利普兰在体外骨髓间充质干细胞向成骨细胞诱导分化过程中的作用.方法 体外分离培养小鼠股骨骨髓间充质干细胞,随机分为咯利普兰10 μmol/L组(A组)和对照组(B组).骨髓间充质干细胞向成骨诱导分化后,检测碱性磷酸酶(ALP)活性;Western blot检测Runx2和骨钙素表达;茜素红染色观察钙结节形成情况.结果 与B组相比,A组ALP活性及Runx2、骨钙素表达均增加(P＜0.01或P＜0.05),钙结节形成更加明显.结论 咯利普兰能提高体外骨髓间充质干细胞向成骨细胞诱导分化的能力.     

黄芩苷通过Wnt/β-catenin信号通路对大鼠骨髓间充质干细胞成骨分化的促进作用

目的研究黄芩苷(baicalin)对大鼠骨髓间充质干细胞(rat bone marrow derived mesenchymal stem cells,r BMSC)成骨分化过程中Wnt/β-catenin信号通路的影响。方法采用贴壁筛选法体外培养r BMSC,给予黄芩苷3、5、7 d后,比较药物处理组与对照组之间碱性磷酸酶(ALP)的活性。同时检测给予黄芩苷对碱性磷酸酶阳性克隆和矿化结节形成的影响。提取总mRNA和总蛋白,用实时荧光定量PCR检测黄芩苷对Wnt10a、GSK-3β、β-catenin以及LEF1 mRNA水平的影响。免疫印迹检测药物处理对β-catenin以及Runx2蛋白表达量的影响。结果黄芩苷明显提高ALP的活性。10μmol·L-1浓度的黄芩苷还可增加碱性磷酸酶克隆数和钙化结节的形成。黄芩苷还可提高Wnt10a、β-catenin、GSK-3β、LEF1以及osteocalcin的mRNA水平,并提高β-catenin和Runx2的蛋白表达量。结论黄芩苷在0.1~50μmol·L-1的给药浓度下可促进rB MSC的成骨分化成熟,Wnt/β-catenin信号可能参与调控rB MSC的成骨分化。     

地塞米松诱导小鼠骨髓基质细胞成骨的分子机制

目的 研究骨髓间充质细胞成骨分化的分子机制，为将其作为基因治疗的载体细胞奠定理论基础。方法 取成年小鼠股骨骨髓，贴壁培养，分别用矿化诱导培养基（10^-7 M地塞米松、10mM β-甘油磷酸钠和50mg／ml抗坏血酸）与骨形成蛋白2诱导，用RT-PCR检测Runx2，Osx和骨钙蛋白基因表达，茜素红染色鉴定钙节结。结果 RT-PCR显示，矿化培养基诱导组仅有Osx和OCN表达，BMP2诱导组Runx2，Osx和OCN全部表达，而两组茜素红染色均呈阳性钙结节。结论 地塞米松诱导的成骨作用中可能通过不同于BMP2的信号通路起作用。     

转染bFGF基因对人骨髓间充质干细胞增殖特性的影响

目的 探讨作为组织工程种子细胞的人骨髓间充质干细胞,在体外培养条件下转染bFGF基因对其增殖特性的影响.方法 利用脂质体将含人pcDNA3.1-bFGF质粒转染到生长良好的P3代人BMSCs,G418筛选获得抗性克隆.采用荧光定量PCR和免疫荧光检测转染bFGF的骨髓间充质干细胞bFGF基因及其产物的表达,MTY法检测和流式细胞仪检测细胞的增殖情况和细胞增殖周期,并将转染和非转染BMSCs分别成骨诱导分化,对其碱性磷酸酶活性进行测定.结果 脂质体介导pcDNA3.1-bFGF重组表达质粒转染BMSCs,经荧光定量PCR和免疫荧光检测.证实转染细胞表达bFGF.转染细胞增殖活力加强,处于增殖周期的细胞比例更高（P〈0.05）.碱性磷酸酶活性检测结果表明转染细胞的碱性磷酸酶活性高于非转染细胞（P〈0.05）.结论 用脂质体转染法能将pcDNA3.1-bFGF成功导入体外培养的hBMSCs,bFGF基因改良的BMSCs可以改善其生存状态、促进其增殖,并可促进向成骨细胞分化.     

刺头复叶耳蕨总黄酮促进脐带间充质干细胞成骨分化

目的研究刺头复叶耳蕨总黄酮(total flavonoids from arachniodes exilis,TFAE)在人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UCMSCs)成骨分化中的作用。方法采用组织块贴壁法分离培养h UCMSCs,取P3代细胞进行实验,不同浓度的TFAE处理h UCMSCs,CCK-8法检测细胞活性;AMP法测定碱性磷酸酶(ALP)活力,茜素红染色检测钙结节形成;RT-PCR检测成骨相关基因I型胶原酶a1(collagen type I alpha1,Col1a1)、骨桥蛋白(osteopotin,OPN)、Runx2、Osterix(Osx)mRNA表达水平,Western blot检测Col1a1、OPN蛋白表达水平。结果在一定浓度范围内,TFAE(1、5 mg·L~(-1))促进细胞增殖;钙结节数量增多;ALP活力增强,成骨相关基因Col1a1、OPN、Runx2、OsxmRNA及Col1a1、OPN蛋白表达水平上调。结论一定浓度的TFAE促进h UCMSCs增殖及成骨分化。     

淫羊藿次苷Ⅱ促进大鼠骨髓间充质干细胞成骨性分化过程中iNOS活性和NO生成量的变化

研究淫羊藿次苷II(icariside II,ICS II)对大鼠体外培养骨髓间充质干细胞(rat bone marrow stromal cells,rBMSCs)成骨性分化过程中诱导性一氧化氮合酶(induced nitric oxide synthase,iNOS)表达及NO生成的影响。贴壁筛选法体外培养rBMSCs,待铺满80%皿底时,进行成骨性诱导培养,同时采用1×10-5 mol.L-1 ICS II进行药物干预,比较ICS II组、L-NAME组、ICS II+L-NAME组和不加药的对照组之间的iNOS的活性、NO生成量,对比各组之间的成骨性指标,包括碱性磷酸酶活性、碱性磷酸酶阳性克隆数(CFU-FALP)及钙化结节数量。提取总RNA,实时荧光定量RCR(real-time PCR)检测Osterix(OSX)、Runx-2及iNOS mRNA的表达情况;同时提取总蛋白,Western blotting法检测Ⅰ型胶原蛋白和iNOS的分泌量。ICS II可显著增强碱性磷酸酶(ALP)活性,增加钙化结节和CFU-FALP数量,与成骨性分化相关的因子OSX和Runx-2的基因表达量也显著升高,同时I...     

HSV-1体外感染骨髓间充质干细胞的实验研究

目的:在体外原代培养大鼠骨髓间充质干细胞,观察单纯疱疹病毒1型(HSV-1)感染骨髓间充质干细胞情况。方法:分离骨髓间充质干细胞,并对其作鉴定。用HSV-1感染骨髓间充质干细胞,提取总DNA,PCR法扩增骨髓间充质干细胞内的HSV-1特异性片段。结果:骨髓间充质干细胞经14d诱导后,碱性磷酸酶含量增高,形成钙结节,表现出成骨细胞特性。HSV-1感染骨髓间充质干细胞,细胞出现典型的病变,PCR法成功扩增出骨髓间充质干细胞内的HSV-1特异性片段。结论:大鼠骨髓间充质干细胞在体外可以向成骨细胞方向分化,可作为组织工程学的种子细胞。HSV-1可以在体外感染骨髓间充质干细胞。     

Geniposide promotes the proliferation and differentiation of MC3T3-E1 and ATDC5 cells by regulation of microRNA-214

The research plans to make sure how Geniposide (GEN) functions in osteoblast proliferation and differentiation. The MC3T3-E1 and ATDC5 cells were treated with the GEN, XAV-939 and/or transfected with microRNA (miR)-214 mimic or corresponding control. Cell viability was detected with the CCK-8. The CyclinD1, Runx2, Osx, Ocn, Wnt3a and β-catenin were individually quantified via western blot. The cell cycle was tested by cell cycle analysis assay. The ALP activity was tested by ALP assay. qRT-PCR was used to examine the miR-214 expression level. The cell viability and the expressions of the CyclinD1, Runx2, Osx, Ocn Wnt3a and β-catenin, as well as the ALP activity were individually and significantly promoted by the GEN. Besides, miR-214 was down-regulated by the GEN. The XAV-939 or the miR-214 mimic destroyed the promotional effect of GEN on these elements above. In conclusion, GEN induced the proliferation and differentiation of the MC3T3-E1 and ATDC5 cells by targeting the miR-214 through Wnt/β-catenin activation.     

羟基磷灰石诱导脂肪间充质干细胞成骨分化的实验研究

目的 探究羟基磷灰石（HA）对脂肪来源的间充质干细胞（ADSCs）向成骨分化的影响。方法 分离、纯化 并鉴定C57BL/6小鼠的ADSCs，将HA与ADSCs共培养，CCK-8法检测不同浓度（0、5、10、20、50、100、500 mg/L）的HA 对ADSCs增殖的影响；碱性磷酸酶（ALP）染色检测不同浓度HA对ADSCs成骨分化的影响；实时荧光定量逆转录-聚 合酶链反应（qRT-PCR）检测ADSCs成骨相关基因骨钙素（BGLAP）、碱性磷酸酶（ALP）、Ⅰ型胶原（COL1A1）、骨桥蛋 白（OPN）、Runt相关转录因子2（Runx2）的mRNA表达情况。结果 低浓度HA（≤20 mg/L）对ADSCs增殖的影响较 小，随着HA浓度增加，细胞的增殖活性下降。HA与ADSCs共培养可显著增加其ALP活性，并促进成骨相关基因的 表达（P＜0.01），且HA为20 mg/L时诱导效果较好。结论 HA具有诱导ADSCs向成骨细胞分化的能力，为两者混合 制作成新的骨支架修复材料提供了理论基础。     

miR-21对绝经后骨质疏松骨髓间充质干细胞成骨能力影响的研究

目的 探讨miR-21对绝经后骨质疏松骨髓间充质干细胞（PMOP-hBMMSC）成骨能力影响.方法 分离培养正常人（H-hBMMSCs）和绝经后骨质疏松患者（PMOP-hBMMSCs）的骨髓间充质干细胞,并对两组细胞的成骨能力进行比较.转染上调PMOP-hBMMSCs中miR-21表达,观察分析其对成骨能力的影响.Real time RT-PCR和Western Blot比较miR-21在三组细胞间的表达差异,同时检测三组成骨标志性基因Runx2和Osterix的表达.结果 PMOP-hBMMSCs组中的miR-21表达水平、ALP的活性以及钙结节染色面积都高于PMOP组（P＜0.05）,与H-hBMMSCs正常对照组差异无统计学意义（P＞0.05）;RT-PCR和Western Blot结果显示成骨诱导7d后,转染后高表达Runx2和Osterix mRNA和蛋白水平均高于PMOP组（P＜0.05,P＜0.01）,与H-hBMMSCs正常对照组差异无统计学意义（P＞0.05）.结论 转染miR-21后的PMOP-hBMMSCs成骨能力增强.     

Icariin regulates miR-23a-3p-mediated osteogenic differentiation of BMSCs via BMP-2/Smad5/Runx2 and WNT/β-catenin pathways in osteonecrosis of the femoral head

Icariin is commonly used for the clinical treatment of osteonecrosis of the femoral head (ONFH). miR-23a-3p plays a vital role in regulating the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The present study aimed to investigate the roles of icariin and miR-23a-3p in the osteogenic differentiation of BMSCs and an ONFH model. BMSCs were isolated and cultured in vitro using icariin-containing serum at various concentrations, and BMSCs were also transfected with a miR-23a inhibitor. The alkaline phosphatase (ALP) activity and cell viability as well as BMP-2/Smad5/Runx2 and WNT/β-catenin pathway-related mRNA and protein expression were measured in BMSCs. Additionally, a dual-luciferase reporter assay and pathway inhibitors were used to verify the relationship of icariin treatment/miR-23a and the above pathways. An ONFH rat model was established in vivo, and a 28-day gavage treatment and lentivirus transfection of miR-23a-3p inhibitor were performed. Then, bone biochemical markers (ELISA kits) in serum, femoral head (HE staining and Digital Radiography, DR) and the above pathway-related proteins were detected. Our results revealed that icariin treatment/miR-23a knockdown promoted BMSC viability and osteogenic differentiation as well as increased the mRNA and protein expression of BMP-2, BMP-4, Runx2, p-Smad5, Wnt1 and β-catenin in BMSCs and ONFH model rats. In addition, icariin treatment/miR-23a knockdown increased bone biochemical markers (ACP-5, BAP, NTXI, CTXI and OC) and improved ONFH in ONFH model rats. In addition, a dual-luciferase reporter assay verified that Runx2 was a direct target of miR-23a-3p. These data indicated that icariin promotes BMSC viability and osteogenic differentiation as well as improves ONFH by decreasing miR-23a-3p levels and regulating the BMP-2/Smad5/Runx2 and WNT/β-catenin pathways.     

Icariin regulates miR-23a-3p-mediated osteogenic differentiation of BMSCs via BMP-2/Smad5/Runx2 and WNT/β-catenin pathways in osteonecrosis of the femoral head

Icariin is commonly used for the clinical treatment of osteonecrosis of the femoral head (ONFH). miR-23a-3p plays a vital role in regulating the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). The present study aimed to investigate the roles of icariin and miR-23a-3p in the osteogenic differentiation of BMSCs and an ONFH model. BMSCs were isolated and cultured in vitro using icariin-containing serum at various concentrations, and BMSCs were also transfected with a miR-23a inhibitor. The alkaline phosphatase (ALP) activity and cell viability as well as BMP-2/Smad5/Runx2 and WNT/β-catenin pathway-related mRNA and protein expression were measured in BMSCs. Additionally, a dual-luciferase reporter assay and pathway inhibitors were used to verify the relationship of icariin treatment/miR-23a and the above pathways. An ONFH rat model was established in vivo, and a 28-day gavage treatment and lentivirus transfection of miR-23a-3p inhibitor were performed. Then, bone biochemical markers (ELISA kits) in serum, femoral head (HE staining and Digital Radiography, DR) and the above pathway-related proteins were detected. Our results revealed that icariin treatment/miR-23a knockdown promoted BMSC viability and osteogenic differentiation as well as increased the mRNA and protein expression of BMP-2, BMP-4, Runx2, p-Smad5, Wnt1 and β-catenin in BMSCs and ONFH model rats. In addition, icariin treatment/miR-23a knockdown increased bone biochemical markers (ACP-5, BAP, NTXI, CTXI and OC) and improved ONFH in ONFH model rats. In addition, a dual-luciferase reporter assay verified that Runx2 was a direct target of miR-23a-3p. These data indicated that icariin promotes BMSC viability and osteogenic differentiation as well as improves ONFH by decreasing miR-23a-3p levels and regulating the BMP-2/Smad5/Runx2 and WNT/β-catenin pathways.     

辛伐他汀对成骨细胞分化和成骨基因的影响

目的 探讨辛伐他汀对成骨细胞分化及成骨基因表达的影响.方法 选取成骨肉瘤细胞株 MG-63,采用不同浓度辛伐他汀（0.0625、0.125、0.25、0.5和 1.0 μmol/L）处理成骨细胞,ALP活性检测辛伐他汀对成骨细胞分化作用的影响;实时定量 RT-PCR和免疫印迹检测细胞成骨基因 mRNA的表达.结果 通过不同浓度辛伐他汀处理成骨细胞后,不同浓度辛伐他汀组与对照组比较,成骨细胞ALP 活性差异均有统计学意义 （ P〈0.05）,其中 0.25 μmol/L 浓度组作用对成骨细胞碱性磷酸酶活性的影响最为显著.采用 0.25 μmol/L辛伐他汀处理成骨细胞后,辛伐他汀组与对照组比较,成骨细胞骨钙蛋白、ALP、I型胶原 mRNA表达差异均有统计学意义 （ P〈0.05）.结论 辛伐他汀可能通过上调成骨基因mRNA的表达水平来促进其分化,这为他汀类药物治疗骨质疏松症提供新的干预靶点.     

3种牙髓生物活性材料对小鼠间充质干细胞骨向分化的影响

目的 评估3种牙髓生物活性材料——三氧化聚合物（MTA）、Bioaggregate（BA）和Biodentine（BD）的生物相容性并观察其各自对小鼠间充质干细胞（MSCs）向成骨分化的影响。方法 采用XTT实验和ALP染色检测小鼠MSCs生存能力、分化矿化能力,观察MTA、BA及BD对小鼠MSCs向成骨分化的影响。结果 BD的细胞生存能力在浓度1、1/2、1/4时明显低于MTA和BA（P<0.001）,3种材料的细胞生存能力在材料浓度降至1/10和1/50时,差异无统计学意义（P>0.05）。MTA、BA和低浓度BD在显示分化矿化能力的ALP染色检测方面,与对照组相比染色值均升高,差异有统计学意义（P<0.05）。结论 MTA、BA以及低浓度BD与小鼠MSCs有良好的生物相容性;MTA、BA和低浓度BD在小鼠MSCs向成骨方向分化过程中有促进分化矿化作用,可以作为根管的根尖封闭材料。