依普黄酮对骨质疏松症笼养蛋鸡OPG和RANKL表达的影响

选用200只24周龄健康海兰褐蛋鸡,随机分为4组:对照组(基础日粮中含钙3.55％)、低钙组(日粮合钙1.27％)、试验Ⅰ组(依普黄酮8 mg/kg,低钙日粮)和试验Ⅱ组(依普黄酮20 mg/kg,低钙日粮),观察依普黄酮对骨质疏松症笼养蛋鸡血清及骨骼骨保护素(Osteo protegerin,OPG)/核因子-相受体活化因子配体(Receptor activatorof NF-κB ligand,RANKL)表达的影响.结果显示:与低钙组相比,30 d时,试验Ⅰ组和Ⅱ组血清OPG分别显著升高33.12％和60.19％(P＜0.05)；60 d时,试验Ⅰ组和Ⅱ组血清OPG分别显著升高35.89％和61.59％ (P＜0.05)；30 d时,试验Ⅰ组和Ⅱ组血清RANKL分别极显著降低63.85％和70.87％(P＜0.01)；60 d时,试验Ⅰ组和Ⅱ组血清RANKL分别极显著降低69.98％和72.85％ (P＜0.01).与低钙组相比,试验Ⅰ组和Ⅱ组骨组织OPG平均光密度分别显著(P＜0.05)和极显著(P＜0.01)升高,试验Ⅰ组和Ⅱ组蛋鸡骨组织RANKL平均光密度极显著低于低钙组蛋鸡(P＜o.01).试验Ⅰ组和Ⅱ组OPG/RANKL值高于低钙组.结果表明,低钙日粮中添加依普黄酮对蛋鸡骨质疏松症可起到一定改善作用,这可能与OPG上调,RANKL下调,影响RANKL信号通路,使其骨量增加,骨结构改善密切相关.     

Echinacoside promotes bone regeneration by increasing OPG/RANKL ratio in MC3T3-E1 cells

Echinacoside (ECH), isolated from Cistanche tubulosa (Schrenk) R. Wight stems, was subjected to in vitro experiments to investigate its bioactivities on proliferation, differentiation and mineralization of the osteoblastic cell line MC3T3-E1. MTT assay, the alkaline phosphatase (ALP) activity and calcium deposition were determined, and the secretion of collagen I (COL I), osteocalcin (OCN), osteoprotegerin (OPG) and receptor activator of nuclear factor-κB ligand (RANKL) were also assayed by enzyme-linked immunosorbent assay (ELISA). The results showed that ECH caused a significant increase in cell proliferation, ALP activity, COL I contents, OCN levels and an enhancement of mineralization in osteoblasts at the concentration range from 0.01 to 10 nmol·L^− 1 (p < 0.05), suggesting that ECH has a stimulatory effect on osteoblastic bone formation or has potential activity against osteoporosis. In addition, the ratio of OPG/RANKL also could be enhanced by ECH. These findings provide the potent evidence that ECH can promote bone regeneration in cultured osteoblastic MC3T3-E1 cells, which might be done by elevating the OPG/RANKL ratio, and potential evidence for echinacoside to be a promising drug or a lead compound in the development of disease-modifying drug to prevent osteoporosis.     

OPG/RANKL/RANK系统的生理功能

骨是一个代谢活跃的器官,在整个生命周期中都在进行不断的更新与重建。在骨形态发生和重建的过程中主要受到细胞因子和激素的调节,同时骨代谢也受到OPG/RANKL/RANK系统的调控。论文主要对骨骼生理结构与骨组织细胞之间的关系,骨代谢和骨的形成过程,OPG/RANKL/RANK系统的生理功能,OPG/RANKL/RANK系统的影响因子以及骨代谢紊乱所引起的疾病几个方面作一综述。     

复方妥曲珠利缓释微囊的药效试验

为了探讨复方妥曲珠利缓释微囊对鸡球虫病的治疗效果及给药方案,确定首次给药剂量和给药频率。在鸡感染柔嫩艾美耳球虫后第3天(粪便变稀)开始给药,于饮水中添加复方妥曲珠利缓释微囊,期间分别以不同频率给予每组鸡药物,各组鸡给药总量相同。以抗球虫指数(ACI)、相对增重率、病变值等指标进行药效学考察研究。试验结果表明,与感染不给药组相比,各给药治疗组ACI均极显著(P<0.01);试验期间给药3次和2次组治疗效果均可达到高效抗球虫效果;发病后2次给药,且首次剂量加倍组抗球虫指数可达188.14,高于其它给药组的ACI(179.03、182.50、175.98),相对增重率达到90.80%,病变值为2.67,治疗效果显著,可作为最佳的推荐给药方案。     

几种抗球虫药物对獭兔球虫感染和幼兔生长性能的影响

在福建省福安市某獭兔场共检测出16种艾美耳球虫,均为混合感染。选取210只断乳1周、球虫卵囊阳性的獭兔幼兔,随机分为7组,进行抗球虫药物试验。在抗球虫药物试验组中,2.5%妥曲珠利组的獭兔幼兔按有效剂量30 mg/kg体重、5%肠球双效(盐酸氨丙啉+胺喹噁啉钠)组和5%优球乐(癸氧喹酯)组按有效剂量50 mg/kg体重灌服,分为1次用药组和2次用药组;对照组灌服等量生理盐水。结果显示,妥曲珠利组獭兔幼兔用药后第1周,粪便中的球虫卵囊数(OPG值)比用药前减少78.39%,也显著低于其他药物组和对照组(P<0.01);用药后第2周,球虫OPG值比用药前减少88.35%,感染率也降至最低;用药后第8周,粪便中球虫OPG值比用药前减少92.99%。至用药后第8周,各试验组与对照组獭兔幼兔球虫OPG值均转归为相对较低水平,感染率则仍保持较高水平;妥曲珠利组獭兔幼兔死亡率显著低于对照组(P<0.05),其他组间死亡率差异不显著;其平均增重显著高于对照组(P<0.05),料重比则显著低于对照组(P<0.05)、极显著低于5%肠球双效1次用药组(P<0.01)。     

Dietary phosphorus restriction affects bone metabolism,vitamin D metabolism and rumen fermentation traits in sheep

Homeostasis of calcium (Ca) and phosphate (P_i) is maintained by a concerted interplay of absorption and reabsorption via the gastrointestinal tract and the kidney and by storage and mobilization from the bone regulated mainly by parathyroid hormone (PTH), 1,25-dihydroxycholecalciferol and calcitonin. The present study aimed at characterizing the effects of dietary P restriction on bone, vitamin D metabolism and rumen fermentation traits reflecting the endogenous P cycle maintaining the ruminal P supply for microbial metabolism. The experiments were done in eleven female, non-pregnant, non-lactating four- to nine-year-old Black Headed Mutton sheep allotted to two feeding groups: "P-restricted" (0.11% P/kg DM and 0.88% Ca/kg DM) and "Control" (0.38% P/kg DM and 0.88% Ca/kg DM). Dietary P restriction did not lead to hypophosphataemia, probably due to a compensation by bone mobilization, demonstrated by increased serum concentrations of a resorption marker and altered gene expression in bone tissue. In addition, the RNA expression of fibroblast growth factor 23, a bone-derived factor involved in the regulation of vitamin D metabolism, was significantly reduced with dietary P restriction. Furthermore, several genes related to vitamin D metabolism and plasma concentrations of 1,25-(OH)₂D were associated with serum concentrations of phosphate (P_i). In the parotid gland, the expression of the P_i transporter NaPi2b was negatively associated with serum P_i and positively with parathyroid PTH expression. Although P_i concentrations in saliva and the gastrointestinal tract were significantly reduced, we found no adverse effects of the P-restricted ration on the production of short chain fatty acids, but slight differences in the production of butyrate as well as its relationship to rumen P_i and ammonia concentrations that might indicate an impact on ruminal fermentation.     

EtMIC-2表达产物对鸡三种艾美耳球虫的保护试验

鸡艾美耳球虫病由隶属顶复门7种艾美耳球虫（E.tenella、Enecatrix、E.acervulina、E.naxima、E.brunetti、E.praecox、E.mitis）引起，常混合感染，有很高的发病率和死亡率，轻者则影响鸡的增重和饲料转化率。微线是顶器的结构之一，位于子孢子和裂殖子前端，EtMIC-2是其50kDa左右的酸性蛋白，在虫体入侵时表达，该蛋白可能在虫体入侵时起重要作用。用Pet载体经原核表达该蛋白，7、14日龄经口服、肌注不同剂量工程活菌和菌体裂解蛋白二次免疫鸡，免疫后1周用3种艾美耳球虫（E.tenella、E.acervulina、E.maxima）攻虫，发现相对增重率随菌体蛋白含量增加呈现增加趋势。E.tenella攻虫表现最有效的是口服PBS200ug活菌组为96%，E.ac-ervulina、E.maxima分别为96%、87%。从攻虫后7～14d卵囊排出曲线看，每天排卵囊数和相对卵囊产量均低于红对照；相对卵囊产量,E.tenella攻虫后，口服PBS 200ug活菌组最低，为17%，E.acervulina、E.maxima攻虫后，口服PBS 200ug裂解菌组最高，分别为90%、93%。从增重与卵囊产量来看，EtMIC-2对E.tenella、E.ac-ervulina和E.maxima这3种球虫有一定的免疫保护作用，尤其口服200ugPBS活菌时，对E.tenella保护作用最明显。说明来源于E.tenella子孢子的Etmic-2基因的表达产物对E.tenella、E.acervulina和E.maxima3种球虫有一定的免疫保护作用。     

Impact of Pueraria candollei var. mirifica and its potent phytoestrogen miroestrol on expression of bone-specific genes in ovariectomized mice

Miroestrol (MR) is a highly active phytoestrogen isolated from tuberous root of Pueraria candollei var. mirifica (PM). Modulatory effects of PM and MR on osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) mRNAs which are bone-specific genes were investigated in ovariectomized female ICR mice. After ovariectomy, expression of OPG mRNA was suppressed but that of RANKL was induced. Estradiol benzoate (E2) recovered OPG expression to the level comparable to the sham while that of RANKL was suppressed in ovariectomized mice. PM crude extract (PME) significantly down-regulated the expression of RANKL mRNA with no change in the OPG level whereas MR elevated the expression of OPG mRNA with lowering level of RANKL mRNA, resulting in the increased OPG/RANKL ratio, and consequently lead to lowering progression of osteoporosis at molecular level. These findings revealed potential of PME and MR on bone loss prevention via increasing the ratio of OPG to RANKL (osteoformation/osteoresorption) in liver of ovariectomized mice. Therefore, using PME and MR as alternative hormone replacement therapy of E2 might be beneficial recommended due to advantageous on regulation of osteoporosis related genes.     

OPG/RANKL/RANK系统的研究进展

OPG/RANKL/RANK系统是近年来骨科研究领域中的重大突破,研究发现许多激素、细胞因子等均通过直接或间接的调节骨保护素(osteoprotegerin,OPG),核因子-κB受体活化因子配体(receptor activator ofNF-κB ligand,RANKL)的表达,调控OPG、RANKL和核因子-κB受体活化因子(recep-tor activator of NF-κB,RANK)之间的比例,从而介导破骨细胞的分化和功能而达到抗骨质疏松或致骨质疏松的作用。现就OPG/RANKL/RANK系统在骨质疏松中的作用做一综述。     

鸡骨保护素（chOPG）的原核表达、纯化及抗体制备

【目的】骨保护素（OPG）是一种新发现能抑制前体破骨细胞分化及成熟破骨细胞活性的蛋白,本文旨在从体外扩增、表达鸡骨保护素（chOPG）蛋白,并制备其多克隆抗体,为深入研究它的生物学功能奠定基础。【方法】以体外培养的鸡胚成骨细胞中提取的总RNA为模板,利用RT-PCR方法,扩增出chOPG基因,测序后克隆至原核表达载体pET-32a（+）,成功构建了原核表达质粒pET-32a（+）-OPG,重组质粒转化Rosetta-gami（DE3）pLysS,经IPTG诱导表达。【结果】表达产物经SDS-PAGE电泳,发现目的蛋白大小约为63 kD,占菌体总蛋白的11.9%,Western blotting分析显示,表达的chOPG蛋白具有良好的免疫反应性。用镍离子亲和层析的方法可获得纯化的蛋白,以纯化的蛋白免疫家兔制备多克隆抗体,ELISA结果显示效价达到1﹕10240。Western印迹结果表明,该抗体可以和chOPG酵母表达产物特异性结合。【结论】成功地实现了鸡骨保护素在大肠杆菌中的表达,并获得其多克隆抗体。