淫羊藿苷对人成骨细胞增殖及OPG蛋白表达的实验研究

目的：建立体外培养人成骨细胞系,观察淫羊藿苷对人成骨细胞增殖的影响,以及对人成骨细胞内骨保护素（Osteoprorotegerin,OPG）蛋白表达的影响,探讨淫羊藿苷促进人成骨细胞骨形成的可能作用机制。方法：将手术中取得的人股骨头松质骨骨片,使用酶消化法进行培养,取生长良好的第3代人成骨细胞进行实验。实验分为4组,对照组给予15%NCS-DMEM-F12（1：1）培养液培养;淫羊藿苷组分别于正常培养液内添加终浓度为10-6、10-8、10-10mol/L淫羊藿苷。噻唑蓝比色法（MTT）分别在培养1、3、5、7、9d后观察各组人成骨细胞的增殖情况,用蛋白免疫印迹方法（in-cell western blot）测定各组培养8、10、12d后人成骨细胞内OPG蛋白的表达情况。采用重复测量的方差分析方法,比较各组间在不同时间点的作用差异。结果：MTT结果,淫羊藿苷组具有促进人成骨细胞增殖的作用,并呈明显的量效关系,即随着淫羊藿苷浓度的增高,其促人成骨细胞增殖的能力增强,以10-6mol/L淫羊藿苷组（0.402±0.033）促成骨细胞增殖的作用最显著,与对照组（0.268±0.031）比较差异有统计学意义（P〈0.05）。在时效关系的研究中,随着培养天数的延长,人成骨细胞的量逐渐增加,人成骨细胞于第5天时进入快速生长期,第7天进入平台期;淫羊藿苷组从第5天开始促进人成骨细胞的增殖,第7、9天仍然具有明显的促成骨细胞增殖的作用,以第9天促增殖作用最强。其中,第9天10-6mol/L淫羊藿苷组（0.402±0.033）与对照组（0.268±0.031）比较有统计学意义（P〈0.01）。OPG表达结果,对照组人成骨细胞培养至第8天时检测到OPG蛋白的表达,表达量为1.01±0.08,第12天达到表达高峰为1.80±0.10,两个值比较有统计学差异（P〈0.05）;在观察的不同天数内,不同浓度的淫羊藿苷组人成骨细胞内OPG蛋白表达低于对照组,且随着淫羊藿苷浓度的降低,OPG蛋白的表达量逐渐降低,差异有统计学意义（P〈0.05）;干预12d,10-6mol/L淫羊藿苷组OPG量（1.67±0.13）和10-8mol/L淫羊藿苷组OPG量（1.64±0.05）比较,无统计学差异（P〉0.05）。结论：淫羊藿苷促人成骨细胞骨形成能力的作用途径之一,是通过提高人成骨细胞的增殖能力而实现。     

类风湿关节炎滑膜成纤维细胞通过高表达RANKL促进破骨细胞分化及活化

目的探讨类风湿关节炎滑膜成纤维细胞（RA-FLS）对破骨细胞（Oc）分化和活化的作用及机制。方法活动期RA滑膜体外分离培养FLS,以骨关节炎（OA）-FLS为对照,分别与健康人外周血单核细胞（MNC）共培养后,抗酒石酸酸性磷酸酶（TRAP）染色鉴定Oc并计数、甲苯胺蓝染色观察骨吸收陷窝情况。细胞免疫荧光染色检测FLS RANKL表达,Real-time PCR及W estern blot检测RANKL和OPG mRNA、蛋白表达。结果 RA-FLS与MNC共培养7 d时TRAP＋且细胞核≥3个的Oc很少,14 d时见较多的Oc,21 d甲苯胺蓝染色示清晰的骨吸收陷窝,而OA-FLS与MNC共培养后未见Oc,也未见骨吸收陷窝。细胞免疫荧光染色示RA-FLS较OA-FLS高表达RANKL（P〈0.05）。RA-FLS RANKL mRNA和蛋白表达较OA-FLS明显增高,而OPG mRNA和蛋白表达则明显降低,RANKL/OPG mRNA和蛋白比率较OA-FLS明显增高（均P〈0.05）。结论 RA-FLS可能通过高表达RANKL,促进外周血MNC向Oc分化,并促进Oc的骨吸收功能。     

OPG RANKL在2型糖尿病大鼠血清龈沟液中的表达

目的:通过检测2型糖尿病、牙周炎大鼠龈沟液和血清中骨保护素(OPG)和核因子-κB受体活化因子配体(RANKL)的表达水平,探讨OPG和RANKL在2型糖尿病牙周炎发生、发展中所起的作用。方法:通过结扎、喂食高脂高糖、注射链脲佐菌素的方法,分别建立单纯牙周炎和2型糖尿病大鼠实验模型,并通过血糖、胰岛素敏感性指数、牙周情况的检查,确认建模成功。第8周末检测龈沟液和血清中OPG和RANKL的表达水平并进行统计学分析。结果:与对照组相比,牙周炎大鼠龈沟液OPG表达减少,RANKL表达增加(P<0.05);2型糖尿病大鼠龈沟液OPG表达减少,RANKL表达增加(P<0.05);血清中OPG、RANKL表达均增加(P<0.05)。结论:OPG与RANKL的失衡可能是导致牙周炎发生发展的主要原因之一,而作为全身因素的2型糖尿病可能造成宿主本身对牙周炎的高度易感性,加快OPG与RANKL的失衡。     

罗格列酮通过p-ERK通路抑制类风湿关节炎滑膜成纤维细胞表达核因子κB受体激活配体

目的 探讨罗格列酮对类风湿关节炎患者滑膜成纤维细胞(RA-FLS)核因子κB受体激活配体(RANKL)和骨保护素表达的影响及信号传导通路。方法 原代培养RA-FLS,不同浓度罗格列酮（0、5、10、15、20 μmol/L）干预3 d,检测罗格列酮对RA-FLS增殖及RANKL、骨保护素mRNA和蛋白表达的影响,并检测0、15、20 μmol/L罗格列酮对RA-FLS p-ERK、p-p38、p-JNK蛋白表达的影响。多组间比.较采用单因素方差分析,两两比较采用Bonferroni法。结果 5、10、15、20 μmol/L罗格列酮干预可抑制RANKL mRNA (0.503±0.005、0.438±0.031、0.161±0.042、0.050±0.018）和蛋白(1.72 ±0.09、1.58±0.05、1.46±0.11、1.22±0.14)表达（F=200.820,F=13.602,均P＜0.01）,上调骨保护素mRNA (2.8±0.5、7.0±3.2、8.4±2.3、25.7±5.1)和蛋白(1.21±0.11、1.52±0.16、1.63±0.11、1.91 ±0.03)表达（F=26.531,F=24.872,均P＜0.01）,RANKI/骨保护素mRNA和蛋白比率降低（F=453.425,P＜0.01;F=4.173,P＜0.05）,且呈剂量依赖性;15、20μmol/L罗格列酮明显抑制p-ERK蛋白表达（0.55±0.06、0.45±0.06,F=6.991,P＜0.05）。结论 罗格列酮可能通过p-ERK信号途径抑制RA-FLS表达RANKL并上调骨保护素的表达。     

黄芩苷对人牙周膜细胞功能活性调节的实验研究

目的：通过观察中药黄芩的有效成份黄芩苷对人牙周膜细胞（human periodontal ligament cells,hPDLCs）增殖、矿化成骨等功能活性的影响,研究黄芩对人牙周膜细胞的调节作用。方法：采用组织贴块联合胶原酶消化的方法,体外分离、培养、纯化和鉴定hPDLCs后,通过四甲基偶氮唑蓝（MTT）比色法、酶联免疫吸附、半定量逆转录聚合酶链反应（RT-PCR）等方法,检测不同浓度的黄芩苷（10、1.0、0.1、0.01mg/L）对hPDLCs增殖、矿化成骨、骨保护素（osteoprotegerin,OPG）、细胞核因子-κB受体活化因子配基（receptor activator of nuclear factor-κB ligand,RANKL）mRNA表达等方面的影响。结果：0.1mg/L的黄芩苷对于增加人牙周膜细胞的增殖活性、碱性磷酸酶活性和Ⅰ型胶原蛋白的表达作用最强;适宜浓度的黄芩苷可以降低RANKL/OPGmRNA比值。结论：黄芩苷能促进hPDLCs的增殖和成骨作用,该作用可能与RANKL/OPG信号通路相关。     

不同浓度降钙素基因相关肽对大鼠成骨细胞OPG mRNA表达的影响

目的 观察不同浓度降钙素基因相关肽(calcitonin gene-related peptide,CGRP)对大鼠成骨细胞OPG mRNA的表达影响.方法 采用新生大鼠颅盖骨组织块培养成骨细胞,加入不同浓度CGRP干预体外培养成骨细胞,观察成骨细胞增殖和ALP活性,检测OPG mRNA及RANKL mRNA的表达水平.结果 CGRP在浓度为1×10-8mol/L时对成骨细胞增殖的促进作用最强,在1×10-9mol/L时对成骨细胞ALP活性的促进作用最强;CGRP不仅能明显增强OPG mRNA的表达水平(P＜0.05),还能明显抑制RANKL mRNA的表达水平(P＜0.05),以10-8和l0-7浓度时表达最为明显,均呈剂量依赖性;且CGRP可明显上调OPG/RANKL的比值(P＜0.05).结论 CGRP对成骨细胞增殖和活性均有促进作用,可能通过上调成骨细胞OPG/RANKL,最终发挥抑制骨吸收的作用.     

Screening of osteoprotegerin-related feature genes in osteoporosis and functional analysis with DNA microarray

Background

Osteoporosis affects 200 million people worldwide and places an enormous economic burden on society. We aim to identify the feature genes that are related to osteoprotegerin in osteoporosis and to perform function analysis with DNA microarray from human bone marrow.

Methods

We downloaded the gene expression profile {"type":"entrez-geo","attrs":{"text":"GSE35957","term_id":"35957"}}GSE35957 from Gene Expression Omnibus database including nine gene chips from bone marrow mesenchymal stem cells of five osteoporotic and four non-osteoporotic subjects. The differentially expressed genes between normal and disease samples were identified by LIMMA package in R language. The interactions among the osteoprotegerin gene (OPG) and differentially expressed genes were searched and visualized by Cytoscape. MCODE and Bingo were used to perform module analysis. Finally, GENECODIS was used to obtain enriched pathways of genes in an interaction network.

Results

A total of 656 genes were identified as differentially expressed genes between osteoporotic and non-osteoporotic samples. IL17RC, COL1A1, and ESR1 were identified to interact with OPG directly from the protein-protein interaction network. A module containing ERS1 was screened out, and this module was most significantly enriched in organ development. Pathway enrichment analysis suggested genes in the interaction network were related to focal adhesion.

Conclusions

The expression pattern of IL17RC, COL1A1, and ESR1 can be useful in osteoporosis detection, which may help in identifying those populations at high risk for osteoporosis, and in directing treatment of osteoporosis.     

佐剂性关节炎大鼠骨密度及滑膜OPG/RANKL蛋白的改变

目的观察佐剂性关节炎大鼠的骨密度及踝关节OPG和RANKL蛋白的表达。方法取SD大鼠,制成佐剂性关节炎模型,设正常组、模型1组、模型2组。观察各组大鼠造模后第7、14、21、28天不同时间点的关节炎指数。第21天处死模型1组全部大鼠;第28天处死正常组和模型2组的全部大鼠,分别检测取大鼠整体右侧胫骨和距胫骨远端约1cm为兴趣区的骨密度,同时取双侧踝关节滑膜Western-blotting法检测OPG/RANKL蛋白的表达。结果模型1组,模型2组和正常组比较,各时间点关节炎指数积分差异均有统计学意义(P<0.01)。模型1组大鼠兴趣区的骨密度较正常组低(P<0.01);模型2组大鼠兴趣区的骨密度较模型1组和正常组均显著降低(P<0.01);3组大鼠整体右侧胫骨骨密度值无明显差异。模型1组,模型2组的OPG蛋白表达与正常组差异均有统计学意义(P<0.01)。模型1组的RANKL蛋白表达与正常组无差异,而模型2组的与正常组相比差异有统计学意义(P<0.01)。结论佐剂性关节炎大鼠在造模后第21天存在胫骨远端骨丢失,第28天该病变更为加重,而同时整体胫骨的骨密度均未发生明显变化。造模后第21天和28天大鼠关节滑膜的OPG蛋白表达降低。造模后第28天大鼠滑膜的RANKL蛋白高于正常。     

不稳定性心绞痛患者血清骨保护素和超敏C反应蛋白水平的变化

目的探讨不稳定性心绞痛（UAP）患者血清骨保护素（OPG）、超敏C反应蛋白（hs-CRP）水平变化及其临床意义。方法随机选取行冠状动脉造影（CAG）检查UAP患者102例作为UAP组,另设稳定性心绞痛（SAP）组34例和正常冠脉组31例。UAP组根据CAG结果分为单支、双支、三支病变组。检测血清OPG、hs-CRP浓度,分析其在各组中水平变化。结果 UAP组、SAP组血清OPG、hs-CRP水平均高于正常冠脉组,UAP组高于SAP组,差异均有统计学意义（P〈0.05）。UAP组中三支、双支病变组血清OPG水平均高于单支病变组,三支病变组高于双支病变组,差异均有统计学意义（P〈0.05）,而hs-CRP水平3组比较差异无统计学意义（P〉0.05）。结论血清OPG、hs-CRP均与UAP密切相关,但OPG对预测UAP冠脉严重程度有更好的临床价值。     

Elevated osteoprotegerin is associated with inflammation, malnutrition and new onset cardiovascular events in peritoneal dialysis patients

Backgrounds

Osteoprotegerin (OPG) is known to regulate bone mineral metabolism and to be also associated with inflammation, cardiovascular disease (CVD) and mortality. Malnutrition-inflammation-atherosclerosis (MIA) syndrome is commonly found and closely linked to mortality in dialysis patients. The aim of this study was to investigate the associations between OPG and MIA syndrome in prevalent peritoneal dialysis (PD) patients.

Methods

Prevalent PD patients for more than 6 months were prospectively followed up from March 2005 to May 2010. At baseline, OPG, hs-CRP, albumin, and %lean body mass (LBM) by creatinine kinetics were checked, and subjective global assessment (SGA) was performed. New-onset cardiovascular events were evaluated during the study period. Based on the median level of OPG, patients were classified as lower OPG (LO) group (n = 88) and higher OPG (HO) group (n = 88).

Results

A total of 176 patients (age 52.0 ± 11.8 years, male 50.6%, duration of PD 105.3 ± 67.2 months) were recruited and followed. In HO group, age, hs-CRP level and Charlson's comorbidity indices were higher, whereas serum albumin level, %LBM and SGA score were significantly lower than LO group. OPG levels were positively correlated with inflammatory markers, whereas negatively correlated with nutritional status. Cardiovascular events occurred in 51 patients during the study period. Newly developed cardiovascular events were significantly common in HO group (n = 36, 40.9%) than LO group (n = 15, 17%, p = 0.002). Cox regression analysis revealed that higher OPG level (per 1-SD increase in OPG, HR: 1.44; 95% CI: 1.03–2.00; p = 0.034) was a significant risk factor for cardiovascular events even after adjustments for demographic and biochemical parameters.

Conclusion

OPG was significantly correlated with markers of systemic inflammation and malnutrition and was a significant predictor of CVD in PD patients. These findings suggest OPG might be a prognostic indicator of MIA syndrome in prevalent PD patients.