雷奈酸锶通过TGF-β1/Smad通路促进骨髓间充质干细胞向成骨细胞分化

 [摘 要] 目的:研究转化生长因子β1（TGF-β1）/Smad通路在雷奈酸锶（strontium ranelate,Sr）促进大鼠骨髓间充质干细胞（BMSCs）向成骨细胞分化中的作用。方法: 在大鼠BMSCs向成骨细胞的诱导分化过程中,用Sr处理细胞,用Western blotting法检测磷酸化Smad2（phosphorylated Smad2,p-Smad2）和Runx2的表达。用TGF-β1特异性阻断剂SB431542或Smad2小干扰RNA（Smad2-siRNA）预处理BMSCs后加入Sr,再观察p-Smad2和Runx2表达的改变。应用试剂盒检测碱性磷酸酶（alkaline phosphatase,ALP）活性及钙结节水平。结果: 在大鼠BMSCs向成骨细胞的诱导分化过程中,Sr可增加p-Smad2和Runx2的表达,Sr 浓度为1 mmol/L、作用1 h时,p-Smad2表达最多;Sr 浓度为1 mmol/L、作用5 d时,Runx2表达最多;用SB431542或Smad2-siRNA预处理BMSCs后再加入Sr,不仅可以抑制p-Smad2和Runx2的表达,且ALP活性与钙结节数量也受到明显的抑制。结论: Sr可通过TGF-β1/Smad通路促进大鼠BMSCs向成骨细胞分化。     

骨髓脂肪细胞向成骨细胞的转分化

背景：骨髓脂肪细胞、成骨细胞共同来源于骨髓基质细胞,二者存在基因同源性,在一定的条件下可以相互转分化。 目的：观察骨髓细胞源性脂肪细胞在成骨诱导分化培养条件下转分化为成骨细胞的活性,探索股骨头坏死细胞水平治疗的新途径。 方法：将前脂肪细胞3T3-L1分别进行成骨诱导培养和成脂诱导培养。培养后不同时间点观察细胞形态的变化;并于诱导培养后5,21 d分别进行实时定量-聚合酶链反应检测成骨、成脂分化过程中,细胞中Runt相关基因转录因子2、氧化物增殖体激活物受体γ2、骨钙素和Ⅰ型胶原mRNA表达。并于成骨、成脂培养21 d后采用Wertern-blot法检测相关蛋白的表达。培养细胞爬片,分别进行碱性磷酸酶、钙结节茜素红、油红O染色,观察3T3-L1成骨转分化情况以及成骨细胞活性表达。 结果与结论：3T3-L1在成骨诱导培养5 d后,细胞由圆形逐渐演变成纺锤形和梭形;实时定量-聚合酶链反应检测结果与对照组相比,氧化物增殖体激活物受体γ2 mRNA表达减弱,而Runt相关基因转录因子2、骨钙素和Ⅰ型胶原mRNA表达微量增强。至21 d时,这种表达改变更加明显;Western-blot显示,Runt相关基因转录因子2、骨钙素和Ⅰ型胶原蛋白量增加,而氧化物增殖体激活物受体γ2微量甚至无表达;碱性磷酸酶染色阳性表达较多,茜素红钙结节染色可见多个散在分布的钙结节;油红O染色微量脂滴。提示小鼠骨髓基质细胞源性前脂肪细胞3T3-L1在成骨诱导培养下,可在一定程度上转分化为有生物活性的成骨细胞;小鼠骨髓基质细胞的脂肪细胞和成骨细胞二者之间存在着可塑性。     

MiR-183 regulates the differentiation of osteoblasts in the development of osteoporosis by targeting Smad4

ObjectiveTo discuss the effect of miR-183 on osteoblast differentiation in the osteoporosis progression via targeting Smad4.MethodsOsteoporosis models were constructed on ovariectomized (OVX) mice to determine the expression of miR-183 and Smad4. Then, MC3T3-E1 cells and primary osteoblasts were divided into Mock, miR-control, miR-183 mimic, miR-183 inhibitor, siSmad4 and miR-183 inhibitor + siSmad4 groups. Alkaline phosphatase (ALP) staining were performed to determine ALP activity, alizarin red staining to evaluate the calcium deposit, while qRT-PCR and Western blotting were used to determine the expression of related molecules. Besides, MC3T3-E1 cells transfected with miR-control or miR-183 mimic were cultured with or without TGF-β1 to verify whether miR-183 regulates the TGF-β signaling pathway.ResultsMiR-183 was up-regulated with decreased Smad4 in the femur of OVX mice, and dual luciferase reporter gene assay showed that Smad4 was a target of miR-183. As compared to Mock group, MC3T3-E1 cells and primary osteoblasts in the miR-183 mimic group and siSmad4 group had significant reductions of OCN, OPN, Runx2 and Osx, as well as decreased ALP activity and calcium deposit. Contrarily, miR-183 and Smad4 were up-regulated and down-regulated respectively. However, cells in the miR-183 inhibitor group manifested the opposite changes. Besides, osteoblast differentiation in the miR-183 inhibitor + siSmad4 group was weakened evidently when compared to miR-183 inhibitor group. Pathway analysis indicated that miR-183 regulated osteogenic differentiation via TGF-β signaling pathway.ConclusionMiR-183 was up-regulated in osteoporosis, and miR-183 overexpression can inhibit osteoblast differentiation by targetedly down-regulating TGF-β pathway member Smad4 to trigger osteoporosis.     

低氧条件下载二甲基草酰甘氨酸电纺纤维促血管化及成骨性能研究

目的 通过聚L-丙交酯-己内酯电纺纤维（PLCL）负载二甲基草酰甘氨酸（DMOG）,研究其在低氧和常氧成骨诱导过程中对大鼠骨髓间充质干细胞（BMSC）的血管化和体外成骨分化的作用。方法 本研究经静电纺丝技术制备PLCL电纺纤维（P）和负载DMOG的PLCL电纺纤维（PD）,通过扫描电镜观察电纺纤维形貌;通过细胞骨架染色观察BMSC在不同电纺纤维上的黏附和生长状态;通过碱性磷酸酶和茜素红染色检测在不同电纺纤维上的BMSC经常氧和低氧成骨诱导7 d后的碱性磷酸酶表达和14 d时的钙沉积情况;通过RT-PCR检测在不同电纺纤维上的BMSC经常氧和低氧成骨诱导7 d和14 d时的成骨相关基因（ALP、Runx2、Col1和OCN）和促血管化相关基因（VEGF）的表达情况。结果 扫描电镜结果表明,P和PD具有纤维状形态并呈现为多孔结构。细胞实验表明,BMSC可在P和PD表面黏附生长,且低氧条件下在PD上表现出更好的形态。与常氧条件相比,在低氧条件下,P和PD在7 d时的碱性磷酸酶表达减少。但低氧条件成骨诱导14 d时PD仍能促进钙的沉积。在常氧条件下,电纺纤维P可上调ALP、Runx2、Col1、OCN和VEGF的表达,但低氧条件下其对上述基因的上调作用不明显。而电纺纤维PD在常氧和低氧条件下均可促进ALP、Runx2、Col1、OCN和VEGF的表达。结论 本研究制备的负载DMOG的PLCL电纺纤维在低氧条件下具有良好的体外促血管化和促成骨分化性能,预期可作为一种较好的成骨修复材料。     

Runx2通过抑制细胞巨自噬以诱导C2C12细胞向成骨细胞分化

 目的：探讨细胞巨自噬与Runx2诱导C2C12细胞成骨分化的关系。方法： 在强力霉素(doxycycline, Dox)诱导Runx2表达的细胞系C2C12/Runx2Dox中进行研究。Dox (10 mg/L) 处理0 d、1 d、3 d及6 d后,real-time qPCR检测LC3b、Beclin-1、p62和LAMP-2表达情况,Western blotting分析LC3-I/LC3-II比值。设置不同的3-甲基腺嘌呤(3-methyladenine, 3-MA)或雷帕霉素(rapamycin, Rap)浓度,Dox处理14 d后分析碱性磷酸酶(alkaline phosphatase,ALP)活性。用3-MA (5 mmol/L)或Rap (10 μmol/L)与Dox共同处理1 d、3 d及6 d后检测ALP及骨钙素 (osteocalcin,OC)表达情况。结果： (1) C2C12细胞向成骨分化时,LC3b 与Beclin-1显著下调,p62与LAMP-2无明显变化;(2) LC3-I向LC3-II转换的过程被抑制;(3) 3-MA (5 mmol/L)可增强ALP 活性,而Rap(10 μmol/L)则抑制其活性;(4) 3-MA可上调ALP及OC表达,Rap则下调二者表达。结论： Runx2通过下调LC3和Beclin-1、抑制LC3-I向LC3-II转换的方式阻碍自噬体形成,以诱导C2C12细胞分化为成骨细胞。     

Involvement of p38MAPK/NF-κB Signaling Pathways in Osteoblasts Differentiation in Response to Mechanical Stretch

Bone morphogenetic proteins (BMPs) are known to be important in osteoblasts' response to mechanical stimuli. BMPs/Smad signaling pathway has been demonstrated to play a regulatory role in the mechanical signal transduction in osteoblasts. However, little is currently known about the Smad independent pathway in osteoblasts differentiation in mechanical loading. In this study, MC3T3-E1 cells were subjected to mechanical stretch of 2000?micro-stain (με) at 0.5?Hz, in order to investigate the involvement of p38MAPK and NF-κB signaling pathways in mechanical response in osteoblasts. We found BMP-2/BMP-4 were up-regulated by mechanical stretch via the earlier activation of p38MAPK and NF-κB signaling pathways, which enhanced osteogenic gene expressions including alkaline phosphatase (ALP), collagen type I (Col I) and osteocalcin (OCN), and the expressions of these osteogenic genes were remarkably decreased with Noggin (an inhibitor for BMPs signals) pretreatment. Furthermore, BMP-2/BMP-4 expressions were suppressed by PDTC, an inhibitor of NF-κB pathway and SB203580, an inhibitor of p38MAPK pathway, respectively, leading to the declined levels of ALP, Col I and OCN. Interestingly, blocking in p38MAPK pathway can also cause the inactivation of NF-κB pathway in mechanical stretch. Collectively, the results indicate during mechanical stretch p38MAPK and NF-κB signaling pathways are activated first, and then up-regulate BMP-2/BMP-4 to enhance osteogenic gene expressions. Moreover, p38MAPK and NF-κB signals have cross-talk in regulation of BMP-2/BMP-4 in mechanical response.     

LPS激活TLR4抑制BMP9诱导iMEFs成骨分化

目的研究脂多糖(LPS)激活的Toll样受体4(TLR4)信号对骨形态发生蛋白9(BMP9)诱导永生化小鼠胚胎成纤维细胞(i MEFs)成骨分化的影响。方法细胞免疫荧光检测TLR4/NF-κB信号通路的激活;LPS,BAY11-7082和BMP9处理iMEFs,ALP染色和活性检测i MEFs早期成骨分化能力;茜素红S染色检测晚期成骨分化能力;半定量PCR和Western blot检测晚期成骨基因OCN和OPN表达;Western blot检测Smad1/5/8磷酸化水平;半定量PCR和Western blot检测成骨关键转录因子Runx2和Dlx5的表达。结果 LPS成功激活TLR4/NF-κB信号通路;LPS抑制BMP9诱导的ALP染色和活性(P0.01)、钙盐沉积、OCN的mRNA和蛋白质表达(P0.05)、OPN的mRNA(P0.01)和蛋白质(P0.05)表达、Smad1/5/8信号通路激活(P0.01)、Runx2的mRNA和蛋白质表达(P0.05)、Dlx5的mRNA(P0.01)和蛋白质(P0.05)表达,BAY11-7082可以部分逆转LPS的抑制作用(P0.05)。结论 LPS激活TLR4可以通过NF-κB信号通路抑制BMP9诱导的iMEFs成骨分化。     

体外诱导人肾间质成纤维细胞成骨分化

 目的: 观察在体外培养条件下,使用成骨诱导培养液或不同浓度钙离子诱导人肾间质成纤维细胞发生成骨分化的效果,初步探讨肾脏Randall斑形成可能的细胞机制。方法: 体外培养人肾间质成纤维细胞,实验分为5组:成骨诱导组(加成骨诱导液)、CaⅠ组(加0.5 mmol/L Ca²⁺液)、CaⅡ组(加1.5 mmol/L Ca²⁺液)、Ca Ⅲ 组(加2.5 mmol/L Ca²⁺液)和对照组(加PBS)。各组细胞分别培养至第1、3、6、9天时,采用MTT法检测细胞活力;诱导至第9天时,用细胞钙茜素红染色液和钙钴法磷酸酶染色液对各组细胞进行染色,观察钙结节形成和碱性磷酸酶的表达情况。另外,分别采用real-time PCR和Western blot检测各组细胞不同时间点Runt相关转录因子2(Runx2)的 mRNA和蛋白表达水平。 结果: 在1.5 mmol/L 和2.5 mmol/L Ca²⁺浓度条件下细胞的活力明显受抑制。细胞染色结果证实成骨诱导液组和钙离子组均可以见到典型的红色钙结节和黑色块状的硫化钴沉淀物。成骨诱导组细胞Runx2的mRNA和蛋白相对表达量(0~9 d)逐渐升高(P<0.05);诱导至第9天时,3个钙离子组细胞Runx2的mRNA和蛋白表达呈浓度依赖性升高(P<0.05)。结论: 在体外培养环境下,人肾成纤维细胞可以在成骨诱导培养液的刺激作用下发生成骨分化;另外,在高钙离子环境,人肾成纤维细胞也发生了类似的成骨分化。肾乳头组织中的成纤维细胞在高钙离子等因素的作用下发生成骨分化,这可能是肾脏Randall斑形成的细胞学基础。     

犬骨髓间充质干细胞的生长特点和在诱骨条件下的成骨特性

为了研究犬骨髓间充质干细胞（canine mesenchymal stem cells，cMSCs）的生长特点和在诱导条件下的成骨特性。使用密度梯度法分离成年犬骨髓间充质干细胞进行培养，保留贴壁细胞传代，观察，以地塞米松、β甘油磷酸钠、抗坏血酸为成骨诱导剂。利用倒置光学显微镜和透射电镜观察细胞形态特征，四甲基偶氮盐（MTT）比色测定增殖，用碱性磷酸酶（Alkaline phosphatase，ALP）活性及骨钙素（Osteocalcin，OCN）含量来研究细胞分化情况。形态学观察表明，cMSCs贴壁细胞呈集落生长，有成纤维细胞样外观，透射电镜可见成骨诱导后cMSCs具有分泌型细胞的特征；推测成骨诱导剂可促进骨髓间充质干细胞成骨，表现为ALP活性、OCN含量明显升高。本实验表明所培养的cMSCs保持了未分化状态，并在成骨诱导剂的作用下可向成骨细胞分化。     

The roles of extracellular signal-regulated kinase 1/2 pathway in regulating osteogenic differentiation of murine preosteoblasts MC3T3-E1 cells on roughened titanium surfaces

Surface roughness of titanium-based implants may enhance osteogenic differentiation of cells in vitro and bone-to-implant contact in vivo. Nevertheless, how surface roughness regulates the signaling pathway of osteoblasts is little understood. The study intended to investigate specifically the roles of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating osteogenic differentiation of MC3T3-E1 murine preosteoblast cells on Ti surfaces. Substrates applied were two groups of titanium disks: (1) sand-blasted and acid-etched rough surfaces (SLA) and (2) smooth pretreated Ti surfaces (PT). Surface morphology of the two groups was examined by scanning electron microscope, and cell morphology cultured on Ti disks was observed by confocal microscope. The levels of alkaline phosphatase (ALP) activity and calcium deposition were measured and compared between the two groups. Real-time polymerase chain reaction was applied to detect the expression levels of osteogenic genes including runt related protein 2 (Runx2), osterix (OSX), osteocalcin (OCN) and osteoprotegerin (OPN) of the cells cultured on the two groups of substrates and on SLA surfaces treated with ERK1/2 inhibitor, PD98095. ERK1/2 activities in MC3T3-T1 cells were measured by Western-blotting on the two surfaces with or without PD98095. Cells cultured on rougher SLA surfaces displayed a more differentiated morphology. ALP activities at 7 days and 14 days and the calcium deposition at 28 days were significantly higher on SLA surfaces. The expression levels of Runx2, OSX, OPN and OCN were upregulated by the effect of surface roughness and PD98095 further upregulated the expression levels of these osteogenic genes on SLA surfaces. ERK1/2 phosphorylation was continuously inhibited by surface roughness at 2 days, 4 days and 6 days. In contrast, no marked alterations in ERK1/2 phosphorylation on PT surfaces were observed. PT surfaces treated with PD98095 (50 μM) and SLA surfaces without PD98095 both demonstrated reduced ERK1/2 phosphorylation of the cells, and the inhibitive effect of SLA surfaces was milder than that of PD98095. In conclusion, ERK1/2 pathway may be a negative regulator of cell differentiation in a dosage-dependent manner, and the enhancing effect of surface roughness on osteoblastic differentiation may be mediated through inhibiting ERK1/2 pathway.     

Cyclic tension promotes osteogenic differentiation in human periodontal ligament stem cells

Orthodontic forces result in alveolar bone resorption and formation predominantly on the pressure and tension sides of the tooth roots, respectively. Human periodontal ligament stem cells (PDLSCs) have demonstrated the capacity to differentiate into osteoblasts, and they play important roles in maintaining homeostasis and regenerating periodontal tissues. However, little is known about how PDLSCs contribute to osteoblastogenesis during orthodontic tooth movement on the tension side. In this study, we applied a 12% cyclic tension force to PDLSCs cultured in osteoinductive medium. The osteogenic markers Runx2, ALP, and OCN were detected at the mRNA and protein levels at different time points using real-time PCR and western blot analyses. We discovered that the mRNA and protein levels of Runx2, ALP and OCN were significantly up-regulated after 6, 12 and 24 hours of mechanical loading on PDLSCs compared to levels in unstimulated PDLSCs (P < 0.05). This study demonstrates, for the first time, the effects of mechanical tensile strain on the osteogenic differentiation of PDLSCs, as examined with a Flexcell FX-4000T Tension Plus System. Our findings suggested that cyclic tension could promote the osteogenic differentiation of PDLSCs. Furthermore, the effects of orthodontic force on alveolar bone remodeling might be achieved by PDLSCs.     

ERK信号通路在檞皮素促大鼠MSCs成骨分化中的作用

目的:观察细胞外信号调节激酶(ERK)信号通路在檞皮素(QUE)促进SD大鼠骨髓间充质干细胞(MSCs)成骨分化过程中的作用。方法:(1)用0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L和100μmol/LQUE干预MSCs,MTT法检测各浓度QUE对MSCs增殖的影响,碱性磷酸酶(ALP)测定试剂盒检测各浓度QUE对MSCsALP表达的影响;(2)用ERK1/2抑制剂干预后,加入QUE,用ALP测定试剂盒检测ALP的表达,ELISA法检测Ⅰ型胶原(ColⅠ)和骨钙素(BGP)的表达,Westernblotting检测ERK1/2的表达,荧光定量PCR检测转化生长因子β₁(TGF-β₁)mRNA、骨形成蛋白2(BMP-2)mRNA和核心结合因子α1(Cbfα1)mRNA表达。结果:(1)0.1μmol/L、1μmol/L和10μmol/LQUE剂量依赖性地促进MSCsALP的表达,同时能促进MSCs的增殖;(2)与空白组相比,QUE组ALP、BGP和ColⅠ表达均增加(P<0.01),加入ERK1/2抑制剂后,磷酸化的ERK1/2表达减少(P<0.05),同时ALP、BGP和ColⅠ表达降低(P<0.01);(3)与空白组比较,QUE组TGF-β₁mRNA、BMP-2mRNA和Cbfα1mRNA的表达均增加(P<0.05),加入ERK1/2抑制剂后这3个基因的表达都下降(P<0.05)。结论:一定浓度的QUE能促进MSCs的增殖和成骨分化,ERK通路的激活在此过程中起到了重要的作用。     

柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对MAPK信号通路的影响

目的: 研究中药单体柚皮苷(NG)对体外培养的大鼠骨髓间充质干细胞(MSCs)向成骨细胞分化过程中MAPK信号通路的影响。方法: 观察在正常、加入p38、ERK和JNK通路抑制剂SB203580、PD98059、SP600125及3种抑制剂全部加入的情况下,各组碱性磷酸酶(ALP)、骨钙素(BGP)、I型胶原(Col I)等骨向分化指标的差异。用Western blotting技术检测各组p38、ERK1/2和JNK蛋白的磷酸化水平,用荧光定量PCR技术检测细胞因子转化生长因子β₁(TGF-β₁)、骨形成蛋白2(BMP-2)和核心结合因子α1(Cbfα1) mRNA的表达。结果: (1)10^-7mol/L为本实验中NG的最佳促骨向分化浓度。(2) NG最佳浓度组的ALP和BGP含量比其它各组都高(P<0.05),Col I含量无明显差异(P>0.05);与NG组相比,加入不同抑制剂组的ALP、BGP和ColⅠ表达量出现不同程度的降低。(3)与空白组相比,NG组JNK蛋白的磷酸化水平升高(P<0.05),p38蛋白的磷酸化水平降低(P<0.01),ERK1/2蛋白的磷酸化水平无明显差异(P>0.05)。与NG组相比,加入不同抑制剂组的p38、ERK1/2和JNK蛋白的磷酸化水平有升高也有降低。(4) NG组上调BMP-2的表达(P<0.05),下调Cbfα1的表达(P<0.05),而对TGF-β₁的表达无明显影响(P>0.05)。与NG组相比,加入不同抑制剂组的TGF-β₁、BMP-2和Cbfα1 mRNA表达量出现不同程度的降低。结论: NG主要通过激活MAPK信号通路中ERK通路、JNK通路以及上调BMP-2的表达,促进MSCs的骨向分化。NG上调BMP-2的表达受MAPK通路中p38通路的影响较大。     

环状RNA-42398通过调控TGF-β1/Smads信号通路抑制肝星状细胞活化

目的:探讨小鼠环状RNA-42398(mmucirc42398)对肝星状细胞活化的影响及机制是否与调节TGF-β1/Smads信号通路有关。方法:小鼠肝星状细胞株JS1,分成正常对照组、空载体阴性对照组(vector组)和mmucirc42398过表达组(mmucirc42398组),体外构建mmucirc42398过表达载体,通过脂质体瞬时转染法转入JS1细胞,48 h后RT-qPCR检测mmucirc42398的表达变化,PCR产物经一代测序验证其环化位点,Western blot检测α-平滑肌肌动蛋白(α-SMA)、I型胶原(Col I)、转化生长因子β1(TGF-β1)、Smad2、Smad3、p-Smad2和p-Smad3蛋白表达。结果:与vector组相比,mmucirc42398组的mmucirc42398表达增加(P<0.01),PCR产物测序验证了其环化位点,提示mmucirc42398过表达质粒成功转入JS1细胞并高效表达;在JS1细胞中过表达mmucirc42398后,α-SMA和Col I蛋白表达显著降低(P<0.01),TGF-β1、Smad2和Smad3蛋白表达无显著变化(P>0.05),但p-Smad2和p-Smad3蛋白水平显著降低(P<0.01)。结论:mmucirc42398能抑制肝星状细胞的活化,其机制与调控TGF-β1/smads信号通路有关。