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。     

Low bone density and abnormal bone turnover in patients with atherosclerosis of peripheral vessels.

Patients with vascular calcifications often have low bone mineral density (BMD), but it is still uncertain if osteoporosis and peripheral vascular disease (VD) are interrelated and linked by a common pathomechanism. Moreover, data on bone turnover in patients with advanced atherosclerosis are lacking. We measured BMD by dual-energy X-ray absorptiometry (DXA) and quantitative bone ultrasound (QUS), as well as the serum levels of osteocalcin (OC), bone-specific alkaline phosphatase (BAP), osteoprotegerin (OPG) and its ligand RANKL, and the urinary concentration of the C-terminal telopeptides of type I collagen (CrossLaps), in 36 patient (20 male and 16 female) with serious atherosclerotic involvement of the carotid and/or femoral artery to investigate the underlying mechanism of vascular and osseous disorders. Thirty age-matched and gender matched healthy individuals served as controls. After adjustment for age, BMD was significantly reduced at the lumbar spine in 23/36 (63%) patients (mean T score –1.71±1.42) and at the proximal femur in 34/36 (93%) patients (neck mean T score –2.5±0.88). Ten patients (27%) had abnormal QUS parameters. Gender and diabetes had no effect on the relationship between vascular calcification and bone density at any site measured. VD subjects had OC and BAP serum levels lower than controls (13.3±3.1 vs 27.7±3.3 ng/ml, P<0.01, and 8.4±2.3 vs 12.5±1.4 g/l, P<0.01, respectively). Urinary CrossLaps excretion was not significantly different in patients with VD and in controls (257.9±138.9 vs 272.2±79.4 µg/mmol Cr, respectively). Serum OPG and RANKL levels were similar in patients and in controls (3.5±1.07 vs 3.4±1.05 pmol/l, and 0.37±0.07 vs 0.36±0.06 pmol/l, respectively). We proved high occurrence of osteoporosis in VD, with evidence of age and gender independence. Negative bone remodelling balance would be a consequence of reduced bone formation, with no apparent increased activation of the OPG–RANKL system.     

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。     

RANKL,a necessary chance for clinical application to osteoporosis and cancer-related bone diseases

Osteoporosis is a common bone disease characterized by reduced bone and increased risk of fracture. In postmenopausal women, osteoporosis results from bone loss attributable to estrogen deficiency. Osteoclast differentiation and activation is mediated by receptor activator of nuclear factor-κB ligand (RANKL), its receptor receptor activator of nuclear factor-κB (RANK), and a decoy receptor for RANKL, osteoprotegerin (OPG). The OPG/RANKL/RANK system plays a pivotal role in osteoclast biology. Currently, a fully human anti-RANKL monoclonal antibody named denosumab is being clinically used for the treatment of osteoporosis and cancer-related bone disorders. This review describes recent advances in RANKL-related research, a story from bench to bedside. First, the discovery of the key factors, OPG/RANKL/RANK, revealed the molecular mechanism of osteoclastogenesis. Second, we established three animal models: (1) a novel and rapid bone loss model by administration of glutathione-S transferase-RANKL fusion protein to mice; (2) a novel mouse model of hypercalcemia with anorexia by overexpression of soluble RANKL using an adenovirus vector; and (3) a novel mouse model of osteopetrosis by administration of a denosumab-like anti-mouse RANKL neutralizing monoclonal antibody. Lastly, anti-human RANKL monoclonal antibody has been successfully applied to the treatment of osteoporosis and cancer-related bone disorders in many countries. This is a real example of applying basic science to clinical practice.     

Gene therapy with human osteoprotegerin decreases callus remodeling with limited effects on biomechanical properties

Osteoprotegerin (OPG) is a naturally occurring protein, which prevents bone resorption by inhibition of osteoclastogenesis, function, and survival. Therefore, recombinant OPG may be an attractive drug in the treatment of chronic bone resorptive diseases such as osteoporosis. Gene therapy has the potential to achieve long-term treatment by delivering genes of anti-resorptive proteins to the recipient. The effects of OPG gene therapy on fracture healing have not been described previously.

The influence of OPG gene therapy on callus formation, callus tissue structural strength, apparent material properties, and histology of tibia fractures in rats was investigated after 3 weeks and 8 weeks of healing. Intramuscular administration of adeno-associated virus (AAV) vector-mediated OPG resulted in increased levels of OPG in serum of approximately 100 ng/ml throughout the study period. Control animals with fractures received transduction with an AAV reporter gene construct (AAV-enhanced green fluorescent protein (eGFP)), and in this group serum OPG levels remained at baseline (<10 ng/ml). After 3 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (33%, P < 0.001). However, AAV-OPG treatment did not influence callus dimensions, callus bone mineral content (BMC), fracture structural strength, or apparent callus tissue material properties. After 8 weeks of healing, AAV-OPG treatment reduced the number of osteoclasts in the callus tissue (31%, P < 0.001) compared with AAV-eGFP fractures. Furthermore, deposition of new woven bone at the fracture line of the original cortical bone was hampered (new woven bone present: in all AAV-eGFP animals, in 41% of AAV-OPG-treated animals, P < 0.001). AAV-OPG treatment also increased callus BMC (18%, P = 0.023) compared with AAV-eGFP fractures. AAV-OPG did not influence callus dimensions, structural strength of the fractures, or ultimate stress, whereas elastic modulus was reduced in the AAV-OPG groups (37%, P = 0.039). The experiment demonstrates that AAV-OPG gene therapy decreases the fracture remodeling, but this does not influence the structural strength of healing fractures.     

Decreased osteoprotegerin and increased bone turnover in young female patients with major depressive disorder and a lifetime history of anorexia nervosa

Low bone mineral density (BMD) is a frequent, often persistent complication in patients with major depressive disorder (MDD) and anorexia nervosa (AN) that increases the risk of pathologic fractures. The pathogenetic process underlying osteopenia in MDD and AN is still unclear, although several factors, including a dysbalance of cytokines, are associated with loss of bone mass. Alterations in the serum levels of cytokines have been observed in patients with MDD, AN, and other psychiatric disorders. Therefore, we examined serum levels of cytokines, markers of bone turnover, and BMD in 13 patients with MDD and a lifetime history of AN. Bone turnover markers (osteocalcin and C-terminal degradation products of type I collagen) and tumor necrosis factor (TNF-) in patients were significantly increased compared with those of the control group. Osteoprotegerin (OPG) in patients was significantly decreased. Eight of 13 patients (62%) displayed osteopenia at the lumbar spine. TNF- correlated significantly with C-terminal degradation products of type I collagen, an osteoclastic marker, but significantly negatively with OPG. Our data suggest that TNF- and OPG may play a role in the pathogenetic process underlying osteopenia in these patients.     

OPG/RANKL system imbalance in a case of hepatitis C-associated osteosclerosis: the pathogenetic key?

Hepatitis C-associated osteosclerosis (HCAO) is an impressive example of acquired diffuse osteosclerosis in adults, recently described in ten patients infected with hepatitis C virus (HCV). Its hallmark is a painful and generalized increase of bone mass. Bone biopsies show enhanced accretion rate, usually without histological abnormalities. The HCAO pathogenesis is hitherto unknown. HCV might induce a slow bone cell infection and the production of bone growth factors, such as insulin-like growth factors. Recently, receptor activator of nuclear factor-B (RANK), its ligand (RANKL), and soluble decoy receptor osteoprotegerin (OPG) have been identified as a pivotal cytokine system in the bone remodeling control. We describe the 11th case of HCAO. Notably, the patients bone biopsy showed the presence of a high number of OPG-positive osteoblasts, a slight increase of RANKL-positive stromal cells, and a dramatic reduction of the osteoclasts. Moreover, OPG serum levels were increased. These findings reported here for the first time are consistent with a pathogenetic role of the OPG/RANKL system imbalance in HCAO.     

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。     

补肾壮骨胶囊含药血清对大鼠成骨细胞增殖及骨保护素mRNA表达的影响

目的:观察补肾壮骨胶囊含药血清对SD大鼠成骨细胞(OB)增殖、骨保护素(OPG)mRNA表达的影响,探讨其防治骨质疏松的机理。方法:将40只3月龄SD雌性大鼠分为补肾壮骨胶囊灌胃组(高、中、低剂量)和空白对照组制备含药血清和空白对照血清;取1天龄SD大鼠颅盖骨,分离、培养成骨细胞(OB),分别加入各实验血清组培养液培养OB,倒置相差显微镜下观察OB生长情况,MTT法测定OB的OD值,RT-PCR检测各组成骨细胞OPGmRNA表达。结果:3-8天时,补肾壮骨胶囊高、中、低含药血清组成骨细胞OD值均高于空白对照组(P<0.05)。各含药血清组成骨细胞OPGmRNA表达均高于空白对照组(P<0.01);补肾壮骨胶囊高、中、低含药血清组间比较,差异无显著意义(P<0.05)。结论:补肾壮骨胶囊含药血清组能促进OB增殖、上调OPGmRNA表达,可能是其防治骨质疏松的机制之一。     

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。