珠子参总皂苷对大鼠成骨细胞的保护作用及其机制初探

离体培养大鼠原代成骨细胞,分别采用MTT法、EdU成像法、测定碱性磷酸酶法、茜素红染色法考察了不同浓度珠子参总皂苷对成骨细胞活力、增殖、分化、矿化等的影响;并采用Western bloting法研究了不同浓度珠子参总皂苷对成骨细胞OPG和RANKL蛋白表达的影响。结果表明,珠子参总皂苷浓度为100μg·mL~(-1)时可提高成骨细胞活力;浓度为50μg·mL~(-1)、100μg·mL~(-1)、200μg·mL~(-1)时可显著促进成骨细胞增殖、分化和矿化;珠子参总皂苷呈浓度依赖性地提高OPG/RANKL相对表达比值。     

维生素D_3对成骨细胞增殖和分化行为的影响

生理环境下的维生素D_3在骨质疏松的发病机制中发挥重要作用。目前,业界广泛研究的对象是维生素D_3对成骨细胞行为的影响作用。着重研究了维生素D_3对成骨细胞增殖和分化行为的影响。采用不同浓度的维生素D_3(0, 1, 10,100 nmmol/L)对成骨细胞进行刺激,观察维生素D_3对成骨细胞增殖和分化行为的影响。     

黄芪颗粒中总黄酮的含量测定

建立了黄芪颗粒中总黄酮的含量测定方法:以芦丁为参照,利用紫外分光光度法在371 nm波长测定黄芪颗粒中总黄酮的含量。芦丁在5.90～59.0μg/mL线性关系良好,线性方程为A=0.0103C+0.2707,γ=0.9963,平均回收率为98.65%,RSD%=0.85%。结果表明:紫外分光光度法测定黄芪颗粒中总黄酮的含量方法简便,快捷,准确,可用来做黄芪颗粒的质量评估的方法。     

淫羊藿苷调控BMP-2与OSX促进成骨细胞增殖的研究

目的:探讨淫羊藿苷(ICA)调控BMP-2与OSX对成骨细胞MC3T3-E1增殖的影响.方法:不同浓度淫羊藿苷处理成骨细胞后,细胞增殖实验(CCK-8)和碱性磷酸酶(ALP)检测评价成骨增殖分化能力,激光共聚焦显微镜观察成骨细胞的骨架形态,免疫印迹法(Western Blot)检测骨形态发生蛋白-2(BMP-2)和成骨...     

黄芪甲苷联用维生素D_3对二维回转培养大鼠原代成骨细胞增殖的影响

探讨了黄芪提取物黄芪甲苷与维生素D3联用对二维回转培养大鼠原代成骨细胞增殖的影响。离体培养大鼠原代成骨细胞后,用碱性磷酸酶染色、茜素红染色等方法对细胞进行鉴定;通过细胞回转器模拟微重力效应,采用MTT、细胞计数等方法研究不同浓度配比的黄芪甲苷与维生素D3混合液对大鼠原代成骨细胞增殖的影响。结果表明,单用黄芪甲苷或维生素D3均对正常培养成骨细胞增殖具有促进作用,最佳促增殖浓度分别为1×10-7 mol·L-1和1×10-8 mol·L-1,当1×10-7 mol·L-1黄芪甲苷分别配以1×10-9 mol·L-1或1×10-8 mol·L-1维生素D3时,其相对增殖率可达200%和196%;不同浓度配比的黄芪甲苷与维生素D3混合液对模拟微重力下二维回转培养成骨细胞的增殖有显著促进作用,其最佳促增殖浓度为1×10-7 mol·L-1黄芪甲苷配以1×10-9 mol·L-1维生素D3。     

黄芪多糖在变应性哮喘大鼠模型中对树突状细胞的免疫干预作用

目的探讨黄芪多糖(Astragalus polysacchaside,APS)对变应性哮喘(allergic asthma,AS)大鼠模型树突状细胞(dendritic cells,DCs)的免疫干预作用。方法选用卵清蛋白(ovalbumin,OVA)对30只大鼠进行连续雾化致敏,建立AS模型,其中10只为模型组,20只经腹腔注射APS,给药量分为100和200μg/kg(各10只),同时以10只未建模的大鼠为对照组。给药4 h后观察大鼠的症状,并按预设评估标准评价治疗效果;给药24 h后检测各组大鼠肺功能,并处死大鼠,取肺黏膜相关淋巴组织,用免疫组织化学技术检测CD80、CD86、IL-4、IL-10、IL-12的表达情况。结果根据预设的评估情况,100μg/kg APS干预组9只显效,1只缓解,200μg/kg APS干预组10只显效。与对照组比较,模型组的第1秒肺活量(forced expiratory volume in one second,FEV1)/用力肺活量(forced vital capacity,FVC)比值、最大呼气流速(peak expiratory flow rate,PEF)及IL-10、IL-12表达水平均明显下降(P<0.05),CD80、CD86、IL-4的表达水平均显著升高(P<0.05);与模型组比较,100及200μg/kg APS干预组FEV1/FVC比值、PEF及IL-10、IL-12表达水平显著升高(P<0.05),CD86、CD80及IL-4的表达水平明显降低(P<0.05);100与200μg/kg APS干预组间各指标差异均无统计学意义(P>0.05)。结论 APS在AS大鼠模型中可通过调整DCs的表型表达缓解AS病情。     

大鼠软骨细胞系的培养研究

本课题对分离获取的大鼠关节软骨细胞进行体外培养研究。实验研究分四个方面:(1)软骨的获取。(2)软骨细胞的消化和接种。(3)软骨细胞的形态学观察。(4)软骨细胞分泌物的鉴定。实验结果表明,体外培养6~7天后软骨细胞即为单层覆盖,单层培养的软骨细胞经多次传代,会快速丧失其固有的软骨细胞特征性,细胞出现成纤维细胞样改变。这种去分化现象伴随着大量的化学物质的变化,Ⅱ型胶原和蛋白多糖的合成减少,Ⅰ型胶原合成增加。在进行软骨细胞体外实验研究时,选用第3、4代细胞较为适宜。     

不同炮制条件对槐米总黄酮含量影响的研究

探讨不同炮制条件对炒槐米中总黄酮含量的影响。取生槐米置锅中进行炒制,通过改变炒制温度、炒制时间和投样量,采用纤维素酶-超声波协同的方法提取其总黄酮,通过单因素实验和正交试验,选出最佳炮制条件。结果表明在炒制温度为140～160℃、炒制时间为10 min、投样量为20 g时为槐米中黄酮含量最高。本实验为槐米炒制条件工业化规范提供了一定的实验基础。     

逆境胁迫处理后山楂叶总黄酮含量变化及对心肌缺血性大鼠分子机制的研究

探讨不同品种山楂叶总黄酮含量变化及其作用于心肌缺血性大鼠的分子机制。选取长势良好,大小一致的不同品种山楂树幼苗,通过有机溶剂萃取法提取山楂叶总黄酮,使用HPLC及紫外分光光度计法测定其含量。通过紫外分光光度计测定其抗氧化能力;再将山楂叶总黄酮作用于心肌缺血性模型大鼠,通过心电图法、生化指标检测以及Real-Time PCR技术确定其作用分子机制。结果显示：“大金星”山楂叶总黄酮含量及其抗氧化能力显著高于其它品种;山楂叶总黄酮能够显著改善急性心肌缺血性大鼠的心电图ST段状态、生化指标以及血流动力学参数等;其能够显著影响心肌缺血性大鼠心肌CaSR、c-fos以及Cyt-C基因的表达。4种山楂叶品种中,“大金星”山楂叶总黄酮含量及抗氧化能力显著高于其它组,且能够显著改善心肌缺血性大鼠的状态,为将来遴选优质生药来源提供了理论基础和实践基础。     

提取条件对橘皮中总黄酮提取率的影响

实验采用不同浓度的乙醇提取橘皮中的黄酮,以芦丁为标准品,采用NaNO2-Al(NO3)3比色法,测定提取液中的总黄酮含量。结果表明,四种因素对提取率的影响分别是:乙醇浓度>料液比>提取时间>提取温度,乙醇提取法最佳工艺为:乙醇浓度95%,提取时间3 h,料液比1∶20,提取温度为60℃,黄酮提取率最高为0.68%。该工艺流程为橘皮中黄酮的工业化生产提供了参考。     

Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical,on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone’s ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called “BMP2 enhancers”. In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as “osteopromotive substance” in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.     

Effects of Triterpene Soyasapogenol B from Arachis hypogaea (Peanut) on Differentiation,Mineralization, Autophagy,and Necroptosis in Pre-Osteoblasts

Triterpenes are a diverse group of natural compounds found in plants. Soyasapogenol B (SoyB) from Arachis hypogaea (peanut) has various pharmacological properties. This study aimed to elucidate the pharmacological properties and mechanisms of SoyB in bone-forming cells. In the present study, 1–20 μM of SoyB showed no cell proliferation effects, whereas 30–100 μM of SoyB increased cell proliferation in MC3T3-E1 cells. Next, osteoblast differentiation was analyzed, and it was found that SoyB enhanced ALP staining and activity and bone mineralization. SoyB also induced RUNX2 expression in the nucleus with the increased phosphorylation of Smad1/5/8 and JNK2 during osteoblast differentiation. In addition, SoyB-mediated osteoblast differentiation was not associated with autophagy and necroptosis. Furthermore, SoyB increased the rate of cell migration and adhesion with the upregulation of MMP13 levels during osteoblast differentiation. The findings of this study provide new evidence that SoyB possesses biological effects in bone-forming cells and suggest a potentially beneficial role for peanut-based foods.     

马齿苋中总黄酮提取工艺优化及其抗氧化活性研究

采用直接回流法提取马齿苋中总黄酮,通过单因素实验与正交实验对提取工艺进行了优化;并以芦丁为对照品,采用分光光度法对马齿苋中的总黄酮含量进行了检测;同时研究了马齿苋总黄酮提取液对羟基自由基(·OH)和超氧阴离子自由基(·O-2)的清除效果,并与Vc的抗氧化活性进行了比较。确定马齿苋中总黄酮最优提取工艺为:提取时间50min、水浴温度90℃、提取溶剂为30%乙酸乙酯、固液比1∶50(g∶mL),在此条件下,马齿苋总黄酮提取率可达7.15mg·g-1。当马齿苋总黄酮浓度为0.18 mg·mL-1时,对·OH的清除率高达73.12%,对·O-2的清除率可达49.02%,相同浓度时,其抗氧化活性低于Vc。     

Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation

The Low-Affinity Nerve Growth Factor Receptor (LNGFR), also known as CD271, is a member of the tumor necrosis factor receptor superfamily. The CD271 cell surface marker defines a subset of multipotential mesenchymal stromal cells and may be used to isolate and enrich cells derived from bone marrow aspirate. In this study, we compare the proliferative and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells. Mesenchymal stromal cells were isolated from bone marrow aspirate and adipose tissue by plastic adherence and positive selection. The proliferation and differentiation potentials of CD271⁺ and CD271⁻ mesenchymal stromal cells were assessed by inducing osteogenic, adipogenic and chondrogenic in vitro differentiation. Compared to CD271⁺, CD271⁻ mesenchymal stromal cells showed a lower proliferation rate and a decreased ability to give rise to osteocytes, adipocytes and chondrocytes. Furthermore, we observed that CD271⁺ mesenchymal stromal cells isolated from adipose tissue displayed a higher efficiency of proliferation and trilineage differentiation compared to CD271⁺ mesenchymal stromal cells isolated from bone marrow samples, although the CD271 expression levels were comparable. In conclusion, these data show that both the presence of CD271 antigen and the source of mesenchymal stromal cells represent important factors in determining the ability of the cells to proliferate and differentiate.     

黄芪多糖对胃癌细胞上清液中培养的树突状细胞分化成熟及功能的影响

目的探讨黄芪多糖对胃癌细胞SCG-7901上清液中培养的树突状细胞(Dendritic cells,DC)分化成熟及功能的影响,分析黄芪多糖抗癌的作用机制。方法将人外周血分离的单核细胞加入含重组人粒细胞-巨噬细胞集落刺激因子(rhGMCSF)和重组人白细胞介素的培养液中,随机分为空白组(以RPMI1640培养液培养)、干预组(以黄芪多糖干预后的胃癌细胞上清液培养)、对照组(以胃癌细胞上清液培养)。混合培养48h后,显微镜下观察DC成熟过程中的形态学变化,流式细胞术检测DC的表型(CD40、CD80),混合同种淋巴细胞增殖反应(Mixed lymphocyte reaction,MLR)检测DC的增殖效应。结果与对照组比较,空白组和干预组DC的CD40和CD80的表达均明显增加(P<0.05),刺激同种淋巴细胞增殖效应明显增强(P<0.05);干预组DC CD40和CD80的表达阳性率及刺激同种淋巴细胞增殖效应均明显低于空白组(P<0.05)。结论在体外,黄芪多糖可有效对抗胃癌细胞上清液引起的对DC分化成熟和功能的抑制。     

Ablation of KDM2A Inhibits Preadipocyte Proliferation and Promotes Adipogenic Differentiation

Epigenetic signals and chromatin-modifying proteins play critical roles in adipogenesis, which determines the risk of obesity and which has recently attracted increasing interest. Histone demethylase 2A (KDM2A) is an important component of histone demethylase; however, its direct effect on fat deposition remains unclear. Here, a KDM2A loss of function was performed using two unbiased methods, small interfering RNA (siRNA) and Cre-Loxp recombinase systems, to reveal its function in adipogenesis. The results show that the knockdown of KDM2A by siRNAs inhibited the proliferation capacity of 3T3-L1 preadipocytes. Furthermore, the promotion of preadipocyte differentiation was observed in siRNA-treated cells, manifested by the increasing content of lipid droplets and the expression level of adipogenic-related genes. Consistently, the genetic deletion of KDM2A by Adipoq-Cre in primary adipocytes exhibited similar phenotypes to those of 3T3-L1 preadipocytes. Interestingly, the knockdown of KDM2A upregulates the expression level of Transportin 1(TNPO1), which in turn may induce the nuclear translocation of PPARγ and the accumulation of lipid droplets. In conclusion, the ablation of KDM2A inhibits preadipocyte proliferation and promotes its adipogenic differentiation. This work provides direct evidence of the exact role of KDM2A in fat deposition and provides theoretical support for obesity therapy that targets KDM2A.