牛磺酸通过ERK1/2信号途径促进成骨细胞分化

牛磺酸(taurine)增强成骨样MG63细胞碱性磷酸酶活性,促进骨钙素的分泌和矿化结节的形成.细胞信号转导研究发现牛磺酸诱导成骨细胞细胞外信号调节激酶1/2(ERKl/2)磷酸化,ERKl/2抑制剂PD98059可阻断牛磺酸对成骨细胞的促分化作用.提示牛磺酸通过ERKl/2信号转导途径促进成骨样MG63细胞的分化.     

2β-(3羟丙氧)-骨化三醇对OS-732人成骨样细胞增殖与分化的影响

目的观察2β(3羟丙氧)骨化三醇(ED71)对体外培养人成骨样细胞增殖与分化的影响,进一步探讨ED71治疗骨质疏松的机制。方法采用塞唑蓝(MTT)分析法检测ED71对体外培养OS732人成骨样细胞增殖作用与效价。采用培养细胞匀浆测单位蛋白碱性磷酸酶活性,培养基中骨钙素含量分析及原位杂交方法检测ED71对OS732人成骨样细胞分化的影响。结果培养第4天,10-7mol/LED71显著抑制成骨样细胞增殖(P<0.05),而10-8mol/L、10-9mol/LED71对成骨样细胞的作用不明显。培养4d后培养基中骨钙素水平在10-7mol/LED71组变化不大,在10-8mol/L、10-9mol/LED71组骨钙素水平显著增高(P<0.05或P<0.01)。原位杂交转化生长因子β(TGFβ1)表达明显增加,而碱性磷酸酶比活性无明显变化。结论ED71抑制成骨样细胞增殖高剂量作用显著,促进成骨样细胞分化较低剂量显著。     

骨髓基质细胞成脂分化及辛伐他汀对其影响的实验研究

目的　观察骨髓基质细胞的成脂分化潜能 ,研究辛伐他汀对骨髓基质细胞成脂分化的影响 ,探讨辛伐他汀刺激成骨的作用机制。　方法　体外培养成年小鼠骨髓基质细胞 ,脂肪细胞分化诱导剂 (HI)作用 6d ,检测细胞碱性磷酸酶 (ALP)比活性的变化。HI与不同浓度的辛伐他汀共同作用 72h ,RT PCR检测脂蛋白脂酶 (LPL)mRNA表达水平的变化 ;HI与不同浓度的辛伐他汀或 10 0μg/L重组人骨形态发生蛋白 2 (rhBMP 2 )共同作用 12d后 ,油红O染色、荧光活化的细胞分选(FACS)检测脂肪细胞分化比例。　结果　HI作用 6d后 ,细胞ALP比活性降低 ,约 4 0 3%。分别为( 383 6 1± 134 30 )U·g 1·L 1和 ( 891 5 1± 2 82 5 2 )U·g 1·L 1,P <0 0 1。在含HI的培养液中 ,辛伐他汀作用 72h后 ,LPLmRNA表达水平降低 ;辛伐他汀作用 12d后 ,脂肪细胞的分化比例显著减低 (P<0 0 1)。　结论　骨髓基质细胞可以分化为脂肪细胞 ,并伴随成骨活性减低 ;辛伐他汀抑制骨髓基质细胞的成脂分化 ,辛伐他汀治疗骨质疏松的作用机制可能与此有关。     

白细胞介素-23刺激的类风湿关节炎成纤维细胞对人破骨样细胞形成的研究

目的 探讨向细胞介素(IL)-23刺激的类风湿关节炎(RA)滑膜成纤维细胞(FLS)在人破骨样细胞形成中的作用.方法 用不同浓度(1、5和10 ng/ml)的IL-23刺激RA和骨关节炎(OA)患者滑膜FLS 72 h,实时荧光定量聚合酶链反应(real time-PCR)检测RA和OA滑膜FLS核因子κB受体激活剂配体(RANKL)mRNA表达.分离人外周血单核细胞(MN),与IL-23刺激的RA和OA滑膜FLS共培养,通过抗酒石酸酸性磷酸酶(TRAP)染色观察是否有破骨样细胞形成;同时,real time-PCR法检测破骨样细胞形成的分子标志.结果 不同浓度的IL-23均能上调RA滑膜FLS RANKL的表达;但几乎不诱导OA滑膜细胞RANKL的表达.在IL-23刺激的RA FLS和MN共培养体系中能观察到TRAP染色阳性的破骨样细胞形成;并且破骨样细胞形成的分子标志表达增高.而无IL-23刺激的RA FLS或IL-23刺激的OAFLS和MN共培养体系中未见TRAP染色阳性的破骨样细胞的形成.结论 IL-23通过诱导RA滑膜细胞RANKL的表达促进人MN分化衍生为破骨样细胞.     

RNA干扰下调雌激素受体α亚基的人成骨样细胞模型的构建

目的 构建雌激素受体α亚基(ERα)下调的人成骨样细胞模型.方法 采用计算机辅助设计并合成ERα特异性小干扰RNA(siRNA)前体基因后,定向克隆入pSilencer 4.1-CMV质粒中,构建重组的ERα siRNA表达载体并测序鉴定.由脂质体介导重组质粒稳定转染人成骨样细胞株MG63,潮霉素筛选阳性抗性克隆细胞.逆转录聚合酶链反应(RT-PCR)法检测ERα基因在人成骨样细胞株MG63内的表达,抗生物素蛋白-生物素-过氧化物酶复合体法(ABC)分析ERα蛋白在人成骨样细胞株MG63内的表达与定位.MTT法测ERα基因被特异性下调后对MG63细胞生长的影响.流式细胞术分析ERα siRNA对人成骨样细胞株MG63细胞生长周期的影响.采用改良Gomori氏钙钻法分析ERα siRNA对人成骨样细胞株MG63表达碱性磷酸酶的影响.结果 构建表达ERαsiRNA的重组真核表达载体,并成功地下调了人成骨样细胞株MG63的ERα亚基基因,其中MG63细胞的G1期为68.6%,G2期为17.6%,S期为13.8%;ERα siRNA MG63细胞的G1期为68,6%,G2期为16.8%,S期为14.6%.而ERα siRNA下调人成骨样细胞株MG63的ERα亚基对细胞的生长特性无明显影响.结论 成功地构建了ERα下调的人成骨样细胞模型,该模型为进一步研究雌激素及其受体对骨代谢影响的分子机制提供了基础材料.     

破骨样细胞中骨保护素和NF-κB配体受体的表达及其意义

目的探讨破骨样细胞中骨保护素(OPG)和NF-κB配体受体(RANKL)表达情况。方法单独培养骨髓单核细胞前体,用巨噬细胞集落刺激因子(M-CSF)和维生素D诱导其转化为破骨样细胞,在0、5、10、15 d用光镜观察和TRAP染色(抗酒石酸染色)评估破骨样细胞的转化程度,用RT-PCR检测OPG和RANKL的表达情况;然后构建主动脉中膜平滑肌细胞(SMC)与骨髓单核细胞前体共培养的模型,用维生素C和β-磷酸甘油诱导SMC转化为成骨样细胞,并在0、5、10、15 d用同样方法再次检测共培养中破骨样细胞转化的情况,以及两种细胞中OPG和RANKL的表达情况。结果不管是单独培养、还是共培养,破骨样细胞中始终没有OPG和RANKL的表达。结论破骨样细胞的转化可能主要受成骨样细胞分泌的OPG和RANKL调控,本身并没有分泌OPG和RANKL进行自身调节的机制存在。     

生长激素与儿童骨发育

生长激素 (GH)通过双重作用机制增加成骨、破骨细胞的增殖 :GH促进前体细胞的分化 ,而胰岛素样生长因子 (IGF) 1促进已分化细胞的增殖。并且IGF 1、2和胰岛素样生长因子结合蛋白 (IGFBP)分别通过抑制和增加生长激素受体 (GHR)的活性来调节GH的作用。GH缺乏 (GHD)儿童的骨形成指标降低 ,但却不影响骨吸收指标。GH治疗促进儿童骨形成和骨吸收指标的上升 ,增加骨密度 ,减少GHD儿童成年后骨折的发生率。对GHD儿童用GH治疗的指标不仅要看其身高而且要看峰骨量是否形成。     

人脂肪干细胞体外培养及细胞载体复合物诱导培养

目的探讨人脂肪干细胞体外培养方法以及对细胞载体复合物诱导成软骨分化的潜能。方法从人脂肪组织分离人脂肪间充质干细胞(hADSCs),噻唑盐(MTT)法检测细胞生长情况,流式细胞术hADSCs细胞表面分子标志,hADSCs成脂、成骨和成软骨的诱导及鉴定检测。hADSCs与不同浓度胶原纤维蛋白溶液混合制成细胞载体复合物凝胶,14 d后进行细胞鉴定。结果体外分离、培养出高活性hADSCs,增值曲线呈S形。细胞CD29、CD90、CD105呈阳性表达,CD34、CD45呈阴性表达。hADSCs成脂诱导后细胞呈类圆形,在细胞浆内有透亮的脂滴出现,油红O染色脂滴呈鲜红色。成骨诱导后细胞呈为不规则形,免疫荧光检测细胞表达骨唾液酸蛋白、骨桥蛋白、骨连接蛋白。hADSCs成软骨诱导后细胞形态变为铺路石样,甲苯胺蓝染色细胞基质呈蓝色,免疫组化检测Ⅱ型胶原蛋白染色呈阳性。阿利新蓝染色细胞载体复合物成蓝色,免疫组化检测Ⅱ型胶原蛋白结果显示,混合胶原蛋白组较单一胶原蛋白组更强阳性表达。结论本研究成功分离培养出高度同源性hADSCs,具有多分化潜能。复合胶原纤维蛋白载体诱导hADSCs成软骨细胞分化,可能作为修复关节软骨缺损的理想载体支架材料。     

Wnt/β-catenin抑制剂DKK1调控类风湿关节炎的研究进展

正类风湿关节炎(RA)是一种自身免疫炎症性疾病~([1])。Wnt通路通过调节细胞的各个功能,如生长、发育、增殖、分化和凋亡,同时参与调控成骨、软骨、滑膜和关节生理学等多方面的代谢调节。随着对Wnt信号通路的深入研究,发现此信号途径在RA患者成纤维样滑膜细胞(FLS)异常激活和增殖、成骨/破骨细胞平衡失调、软骨破坏和骨钙流失中发挥着及其重要的作用~([2])。近年来,内源性Wnt通路的负调控蛋白如     

炎症性肠病患者血清miR-181a-5p和miR-126表达水平及其与肠道菌群相关性的研究

目的 探讨miR-181a-5p、miR-126在炎症性肠病(IBD)患者血清中的表达及其与肠道菌群的相关性.方法 收集2016年10月至2018年12月保定市第二医院收治的100例活动期IBD患者作为研究组,根据IBD类型分为溃疡性肠炎(UC)组(60例)和克罗恩病(CD)组(40例),另选择同期在本院体检的50名健...     

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells

Background

Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs.

Methods and results

Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase.

Conclusion

We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.     

血浆miR-181a-5p可评估急性脑梗死的病情及预后

摘要 目的：探讨急性脑梗死（ACI）患者血浆中微小RNA(miR)-181a-5p的表达水平及临床价值。方法：收集148例ACI患者（ACI组）的临床资料，根据美国国立卫生研究院卒中量表（NIHSS）评分将其分为：轻度72例、中度35例、重度41例。ACI患者发病4.5h内用阿替普酶静脉溶栓治疗。根据出院后第90天的预后情况，将患者分为预后良好组（102例）和预后不良组（46例）。另选取同期健康体检者100例为对照组。用逆转录-聚合酶链反应（RT-PCR）检测血浆miR-181a-5p表达水平；酶联免疫吸附试验（ELISA）检测血浆中凋亡分子B淋巴细胞瘤-2基因（Bcl-2）和含半胱氨酸的天冬氨酸蛋白水解酶（caspase-1）表达水平。相关性检验用Pearson分析；用Logistic多因素回归分析影响预后的因素；绘制受试者工作特征（ROC）曲线，分析血浆miR-181a-5p预测预后的价值，计算曲线下面积（AUC）、灵敏度及特异性。结果：与对照组比较，ACI组血浆miR-181a-5p和caspase-1表达水平较高，而Bcl-2表达水平较低（P均＜0.05）。miR-181a-5p与caspase-1呈正相关（r=0.176，P=0.032），与Bcl-2呈负相关（r=-0.209，P=0.011）。轻度、中度和重度ACI患者血浆miR-181a-5p表达水平分别为（1.35±0.32）、（1.79±0.20）、（2.34±0.59），经单因素方差分析，组间比较差异有统计学意义（F=32.001，P＜0.001），病情越重，血浆miR-181a-5p表达水平越高。与预后良好组比较，预后不良组年龄更大，入院时NIHSS评分、血浆miR-181a-5p和caspase-1水平较高，而Bcl-2水平较低（P均＜0.05）。多因素分析显示，年龄、入院时NIHSS评分及血浆miR-181a-5p水平是ACI患者预后不良的独立影响因素（OR=1.532、1.832、3.111，P均＜0.05）。血浆miR-181a-5p预测不良预后的AUC为0.856（95%CI：0.815~0.886，P＜0.01），灵敏度为89.56%，特异性为70.12%。结论：ACI患者血浆中miR-181a-5p表达水平明显升高，对ACI病情和预后的早期评估有一定价值。     

LINC00370 modulates miR-222-3p-RGS4 axis to protect against osteoporosis progression

BackgroundWe aimed to determine the role of the LINC00370/miR-222-3p/RGS4 axis in modulating the process of adipose-derived stem cell (ADSC) osteogenic differentiation.MethodsWe first evaluated the differential expression of LINC00370, miR-222-3p and RGS4 between normal and osteogenically induced ADSCs. Moreover, we transfected ADSCs with LINC00370 siRNA and an miR-222-3p inhibitor to determine the role of LINC00370 in modulating the process of ADSC osteogenic differentiation. Finally, we analyzed the dual-luciferase reporter gene to identify the relationship between LINC00370 and miR-222-3p. We first created osteoporotic rat models by ovariectomy (OVX) and treated with pcDNA-LINC00370. HE and immunohistochemical staining of OCN were performed to assess the changes in bone microarchitecture.ResultsLINC00370 and RGS4 expression was remarkably upregulated in the osteogenic ADSC group compared with the normal medium group. On the other hand, miR-222-3p expression was remarkably decreased in the osteogenic group compared with the normal medium group. Knockdown of LINC00370 reduced the osteogenic differentiation of ADSCs. Moreover, the inhibitor of miR-222-3p partially reversed the reduction of osteogenic differentiation by LINC00370 knockdown. Knockdown of LINC00370 reduced the expression of p-Akt and p-PI3K. The inhibitor of miR-222-3p partially reversed the reduction of the expression of p-Akt and p-PI3K by LINC00370 knockdown. A dual luciferase reporter assay indicated that LINC00370 can directly bind miR-222-3p. LINC00370 suppressed OP progression in OVX and partially upregulated OCN protein expression.ConclusionCollectively, the above results confirm that LINC00370 promotes the process of ADSC osteogenic differentiation via the miR-222-3p/RGS4 axis. Moreover, LINC00370 could protect against OVX-induced OP.     

茶黄素对人骨髓基质干细胞的成骨诱导作用

目的探讨茶黄素对体外培养人骨髓基质干细胞（BMSCs）的成骨诱导作用及其能力。方法将第二代人BMSCs分为茶黄素组、对照组,茶黄素组加入浓度为10 mmol/L茶黄素,对照组不干预。对比观察两组干预后细胞形态,钙结节、碱性磷酸酶（ALP）、Ⅰ型胶原及骨钙素（OCN）免疫组化染色结果,并检测各组4、8、12、16 d各时点ALP、OCN活性。结果茶黄素组钙结节、ALP、Ⅰ型胶原及OCN免疫组化染色均呈阳性,对照组均为阴性;茶黄素组各时点ALP、OCN活性均高于对照组（P均〈0.05）。结论茶黄素可促进人BMSCs向成骨细胞分化。     

依托咪酯通过调控LncRNA CDKN2B-AS1/miR-199a-5p表达抑制胶质瘤细胞增殖、迁移和侵袭

目的 探讨依托咪酯对胶质瘤细胞的增殖、迁移和侵袭的影响及其作用机制.方法 用不同浓度的依托咪酯处理胶质瘤细胞U251,采用四甲基偶氮唑蓝(MTT)检测依托咪酯对U251细胞增殖能力的影响;Transwell实验检测依托咪酯对U251细胞迁移及侵袭的影响;实时荧光定量-聚合酶链反应(qRT-PCR)检测胶质瘤组织中长链非...     

年龄相关性白内障晶状体上皮细胞中miR-181a和SIRT1的表达关系及意义

目的探讨白内障晶状体上皮细胞微小RNA-181a(miR-181a)与沉默信息调节因子(SIRT)1的表达关系,研究其在年龄相关性白内障患者的诊治及病情评估方面的临床意义。方法选取诊断为年龄相关性白内障患者62例为白内障组,另选取同期近视矫正手术患者42例为对照组,利用RT q-PCR法,检测两组晶状体上皮细胞miR-181a与SIRT1表达水平;Pearson法测定miR-181a与SIRT1相关性;利用Lipofectamine 2000转染miR-181a模拟物和抑制剂,调节人晶状体上皮细胞miR-181a表达水平,RT q-PCR法检测SIRT1表达水平;酶联免疫吸附试验(ELISA)检测晶状体上皮细胞中超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱氨酸(GSH)、谷胱氨酸过氧化物酶(GSH-Px)水平。结果与对照组相比,白内障组miR-181a表达水平显著升高,SIRT1表达水平显著降低(P<0.05);Pearson相关分析显示,miR-181a和SIRT1呈负相关关系(r=-0.719,P<0.05);miR-181a模拟物组SIRT1表达水平显著低于模拟物阳性对照组(P<0.05),miR-181a抑制剂组,SIRT1表达水平显著高于抑制剂阴性对照组(P<0.05);白内障组SOD、GSH、GSH-Px水平明显低于正常组(P<0.05),MDA水平明显高于正常组(P<0.05);miR-181a表达水平与SOD、GSH、GSH-Px因子呈负相关关系(r=-0.702,-0.739,-0.776),与MDA水平呈正相关关系(r=0.679);SIRT1表达水平与SOD、GSH、GSH-Px呈正相关关系(r=0.699,0.743,0.763),与MDA水平呈负相关关系(r=-0.684)。结论白内障晶状体上皮细胞中miR-181a高表达,SIRT1低表达,miR-181a可调控人晶状体上皮细胞中SIRT1表达;SIRT1低表达可促使晶状体上皮细胞凋亡,从而影响或导致了年龄相关性白内障的发病。     

p53 negatively regulates the osteogenic differentiation of vascular smooth muscle cells in mice with chronic kidney disease

Aim

To investigate the osteogenic differentiation of vascular smooth muscle cells (VSMCs) in mice with chronic kidney disease (CKD) and to evaluate the effects of p53 on the osteogenic differentiation of the VSMCs.

Methods

Experimental models of CKD-associated vascular calcification generated by five-sixth (5/6) nephrectomy (Nx) and a high-phosphate (HP) diet were used in p53+/+ and p53–/– mice. Following 5/6 Nx, aortic calcification, markers of osteogenic differentiation, VSMCs and p53 protein in aortic tissues were studied.

Results

Aortic calcification was observed after eight weeks following 5/6 Nx in mice of both genotypes, and expression of the markers of osteogenic differentiation in the VSMCs was increased. These changes were continuously observed up to 12 weeks after 5/6 Nx, and particularly after 5/6 Nx + HP. Compared with p53+/+ mice, aortic calcification in p53–/– mice was more severe (p < 0.001). Expression of the markers of osteogenic differentiation was noticeably increased (p < 0.001), while expression of the marker of VSMCs had decreased (p < 0.001). Statistical analysis demonstrated that the markers of osteogenic differentiation were negatively correlated with p53, and the marker of VSMCs was positively correlated with p53 (p < 0.001).

Conclusion

p53 has the potential to negatively regulate the osteogenic differentiation of VSMCs in CKD mice.