酶解鹿茸肽的制备、纯化及抗氧化活性

利用一种工业用碱性蛋白酶对新鲜梅花鹿鹿茸进行水解制备生物活性肽。首先,考察了酶用量、底物浓度和反应时间对水解反应的影响,确定了最佳水解条件为酶浓度1：150,底物浓度1：13,水解时间60 min。其次,利用硫酸胺分级沉淀法制备了鹿茸多肽粗提液,同时采用缓冲液保护其活性,再经Sephadex G-25凝胶层析柱进行分离纯化,得到了具有最强抗氧化活性的鹿茸肽组分（VAP-B）。最后,利用制备型高效液相色谱柱对VAP-B进一步纯化,得到了分子量在800以内的小肽活性组分（VAP-B1）,其蛋白含量为70.45%。在此基础上,对小肽活性组分VAP-B1进行了理化性质分析,检测其抗氧化活性,包括清除超氧阴离子,羟自由基以及抗脂质过氧化的能力和还原能力。实验结果表明,鹿茸肽VAP-B1浓度为20mg/ml时,对邻苯三酚自氧化的抑制率达到91.9%,有明显的清除超氧阴离子的作用;浓度为10mg/ml时,对羟基自由基的清除作用达到100%,且在一定的浓度范围内呈剂量依赖关系。此外,鹿茸肽VAP-B1具有一定的防止脂质过氧化和还原力作用。     

高效液相色谱用于鹿茸中多肽的分离制备

研究证明,鹿茸中含有多肽。孙晓波等曾报道鹿茸总多肽具有明显的抗炎症作用。我们根据多肽的特性,利用高效液相色谱法(HPLC)从鹿茸中制备多肽。用HPLC进行分离纯化,得到两种纯多肽(P_1、P_2)。纯化后的P_1、P_2经PTH法鉴定只有一种末端氨基酸,药理实验证明,P_1肽具有抗炎活性,P_2肽无活性。P_1肽的抗炎活性表现在对右旋糖酐性足肿胀和角叉菜胶性足肿胀均有明显抑制作用。本法分离     

硝酸锶对原代培养的小鼠成骨细胞增殖、分化和矿化功能的影响

采用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色、Ⅰ型胶原测定以及矿化结节染色及定量分析等方法,研究了不同浓度的硝酸锶对原代培养的成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响。结果表明:硝酸锶对成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响与作用浓度和时间密切相关,但没有呈现出剂量依赖性。结果提示,硝酸锶对骨代谢的影响是复杂的,其具有保护还是损害作用取决于作用浓度和时间,而且它们是影响硝酸锶生物效应(从损伤到保护)转变的关键因素。     

肾上腺髓质素微球复合PLGA/纳米羟基磷灰石支架材料的制备及生物活性评价

利用离子乳化交联法制备了负载肾上腺髓质素的壳聚糖微球,应用热致相分离法制备了乳酸和乙醇酸共聚物/纳米羟基磷灰石(PLGA/nHA)支架材料并在其中包覆载药微球.通过扫描电子显微镜、体外释放行为、材料溶血行为、碱性磷酸酶(ALP)活性的测定、支架材料表面细胞荧光染色和MTT[3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐]比色法等手段综合评价载药支架材料的性能及生物活性.结果表明,微球直径均匀,载药支架孔径大小合适并相互穿通.支架材料的溶血率小于5%,符合医用材料的溶血实验要求.载药支架及支架材料本身对成骨细胞及血管内皮细胞的增殖以及成骨细胞的分化均有一定的促进作用.     

白藜芦醇白蛋白纳米粒的制备及其抗卵巢癌细胞增殖作用的研究

以牛血清白蛋白(BSA)为载体, 用去溶剂化-化学交联法制备白藜芦醇白蛋白纳米粒(RES-BSANP). 以原子力学显微镜(AFM)观察其形态, 用高效液相色谱法(HPLC)对制备的纳米微粒进行分析. 采用四甲基偶氮唑盐微量酶反应比色法(MTT)及流式细胞技术(FCM)比较RES-BSANP和RES对卵巢癌SKOV3细胞的抗增殖活性及对细胞周期和凋亡的影响. 结果表明, 获得的RES-BSANP纳米粒的平均粒径为400～500 nm, 表面光滑, 12 mg纳米粒中RES载药量为4.077 mg, 包封率33.97％, 24 h内的稳定性好, 水溶性较RES显著提高. 二者的抗肿瘤增殖作用呈剂量依赖性, 中高浓度组纳米粒组的抗增殖活性及凋亡细胞比率显著提高. 两种药物均使细胞周期阻滞于G0/G1+S期, 纳米组使进入S期细胞比率明显增加, 表明白藜芦醇白蛋白纳米粒在抗卵巢癌细胞增殖方面有广阔的应用前景.     

氯化镨对原代培养的小鼠成骨细胞增殖、分化和矿化功能的影响

采用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色、I型胶原测定以及矿化结节染色及定量分析等方法 ,研究了不同浓度的氯化镨对原代培养的成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响。结果表明:氯化镨对成骨细胞增殖、分化、矿化功能以及横向分化为脂肪细胞的影响与作用浓度和时间密切相关,但没有呈现出时间和剂量依赖性。结果提示,氯化镨对骨代谢的影响是复杂的,其具有保护还是损害作用取决于作用浓度和时间。作用浓度和时间是影响氯化镨生物效应转变的关键因素。     

碱性磷酸酶的体外检测和体内成像研究进展

碱性磷酸酶(Alkaline phosphatase,ALP)是一种水解酶,可以催化蛋白质、核酸和碳水化合物中磷酸基团的去除.ALP广泛存在于原核生物和真核生物中,具有调节细胞分裂、增殖、凋亡和信号转导等生物功能.ALP的活性失调与心血管疾病、糖尿病和癌症等疾病密切相关,是一种重要的生物标志物.ALP的超灵敏检测对临床...     

Fe3+和Fe2+对原代培养的成骨细胞增殖、分化和矿化功能的影响

采用噻唑蓝(MTT)法、碱性磷酸酶(ALP)比活性测定、油红O染色和茜素红染色及定量分析,研究了不同浓度的Fe3+和Fe2+对原代培养的成骨细胞增殖、分化及矿化功能的影响.结果表明:浓度为1×10-9～1×10-4 mol·L-1的Fe3+和Fe2+促进成骨细胞增殖,但是在较高浓度1×10-3 mol·L-1时,它们则抑制成骨细胞增殖.与成骨细胞作用48 h,浓度为1×10-8～1×10-4 mol·L-1的Fe3+和Fe2+抑制其分化,但在较低的浓度1×10-9 mol·L-1时则对其分化没有影响:进一步延长作用时间为72 h,Fe3+对成骨细胞分化没有影响,除1×10-6mol·L-1浓度的Fe2+促进成骨细胞分化外,其他浓度的Fe2+则抑制其分化;测试浓度下的Fe3+对成骨细胞向脂肪细胞的横向分化表现为抑制或没有影响,而Fe2+的影响则依赖于浓度和作用时间.在1×10-8～1×10-5mol·L-1浓度范围内,Fe3+和Fe2+对矿化结节的影响表现出相反的效应.在较高浓度(1×10-4mol·L-1)下,它们促进矿化节结的形成,而在较低浓度(1×10-9mol·L-1)下,Fe3+抑制矿化节结的形成,Fe2+则没有影响.结果提示:浓度.作用时间和铁离子的价态都是影响Fe3+和Fe2+生物效应(从毒性到活性,从损伤到保护,从上调到下调)转变的关键因素.     

稀土化合物TbCl3对成骨细胞系MC3T3-E1增殖、分化和矿化功能的影响

在细胞和分子水平上,研究了稀土化合物氯化铽(TbCl_3)对成骨细胞MC3T3-E1增殖、分化及矿化功能的影响。结果表明,细胞水平上,浓度为0.000 1、0.001、0.01、0.1、1和10μmol·L-1的TbCl_3均促进MC3T3-E1细胞的增殖、分化及其矿化功能,然而,当浓度升至为100和1 000μmol·L-1时,TbCl_3表现出抑制作用。分子水平上,浓度为0.000 1和0.1μmol·L-1的TbCl_3明显上调成骨分化相关基因骨形成蛋白2(BMP-2),碱性磷酸酶(ALP),骨涎蛋白(BSP),Ⅰ型胶原蛋白(ColⅠ),骨钙素(OCN)和runt相关转录因子2(Runx2)的表达。浓度为1 000μmol·L-1的TbCl_3则抑制上述成骨分化相关基因的表达。浓度为0.000 1、0.1和1μmol·L-1的TbCl_3促进成骨分化相关蛋白Runx2,BMP-2和OCN的表达;结果显示,低浓度的TbCl_3促进MC3T3-E1细胞的成骨分化及矿化功能,而高浓度TbCl_3则呈现出抑制作用。TbCl_3通过调控Runx2的表达刺激早期成骨分化相关基因BMP-2、ColⅠ和晚期成骨分化相关基因ALP、OCN的表达,从而诱导MC3T3-E1成骨分化。     

纳米羟基磷灰石/丝素蛋白复合支架材料的降解特性及生物相容性研究

应用共混法制备了纳米羟基磷灰石/丝素蛋白复合支架材料, 通过体外降解和细胞培养实验研究了复合支架材料的降解特性和生物相容性. 体外降解实验结果显示, 复合支架材料具有稳定的降解能力; 在降解过程中, 羟基磷灰石由于与降解液发生钙、磷等离子的交换, 使其结晶得到了进一步生长和完善. 利用细胞计数法、四甲基偶氮唑盐(MTT)比色法和碱性磷酸酶(ALP)活性测定等分析了复合支架材料的生物相容性, 结果表明, MG63细胞在复合支架材料上具有良好的粘附、增殖能力, 并可引起早期的骨分化. 因此, 纳米羟基磷灰石/丝素蛋白复合支架作为骨组织工程的支架材料具有良好的应用前景.     

梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.     

Comparison of Structure and Biological Activity of Natural Polypeptide from Velvet Antlers of Cervus elaphus with Those of Synthesized Polypeptide

Biochemical techniques including ion-exchange chromatography, gel filtration chromatography and reversed-phase high-performance liquid chromatography(RP-HPLC) were used to isolate and purify the natural polypeptide from velvet antler(nVAP) of Cervus elaphus(C. elaphus), which has a molecular weight of 3215.8 and the primary structure of VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM. The homology of the protein sequence in nVAP with known protein sequence is less than 50%, suggesting that nVAP appears to be a new bioactive substance. At a level of 0.4―50 μg/mL, nVAP promotes mitosis in epidermal cells, chondrocytes and NIH3T3 fibroblasts primarily cultured in a significant way. Given that a yield of high-purity nVAP isolated from C. elaphus is 0.001%, nVAP is artificially synthesized to prepare synthetic velvet antler polypeptide(sVAP) according to its primary structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) of sVAP shows a single band, and its HPLC spectrum displays a single peak. The matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) was used to identify sVAP to be of a molecular weight of 3200 and the consistency between primary structures of sVAP and nVAP. Bioactivity test shows that at a dose of 5―40 μg/mL, sVAP promotes the proliferation of primarily cultured epidermal cells and NIH3T3 cell line. From the traditional Chinese medicine theory, velvet antler from Cervus nippon(C. nippon) and velvet antler from C. elaphus are considered as the same medicine, but differences between biochemical base and pharmacological effect of these two velvet antlers have been observed. We compared the total polypeptide mapping of the two velvet antlers, discovering that nVAP is active polypeptide and only exists in the velvet antler of C. elaphus. sVAP is similar to nVAP in physicochemical property and biological activity. These studies extend the possible utility of sVAP to be the promising compound to prepare velvet antler polypeptide of C. elaphus.     

麇鹿茸样品的制备和化学成分分析

建立了麋鹿茸样品制备的工艺路线和方法,获得了易保存的干燥粉末样品,对麋鹿茸中的水分、粗蛋白、粗脂肪、膳食纤维、氨基酸、必需无机元素以及维生素的含量进行了测定,并与马鹿和梅花鹿茸进行了比较。结果表明,麋鹿茸与马鹿和梅花鹿茸的化学成分及含量相近,麋鹿茸中膳食纤维和必需无机元素含量高于其它两种鹿茸。     

麋鹿茸、马鹿茸和梅花鹿茸营养成分的分析比较研究

对麋鹿茸,马鹿茸,梅花鹿茸中的水分,粗蛋白,粗脂肪,膳食纤维,水溶性和脂溶性维生素,氨基酸和无宏量及微量元素进行了测定,和比较,结果表明,麋鹿茸与马鹿茸和梅花鹿茸的化学分成及含量相近,鹿鹿茸中膳食纤维和必需无机元素含量高于其它两种鹿茸,填补了麋鹿研究的空白,为鹿资源的保护,发展,开发和利用提供了营养学依据。     

Effects of velvet antler with blood on bone in ovariectomized rats

In traditional Chinese medicine (TCM), both velvet antlers (VA) and VA blood can tonify qi, essence, and marrow, nourish the blood, and invigorate bones and tendons. In TCM, the combination of VA and VA blood is believed to have superior pharmacological effects. Scientific evidence supporting the traditional therapeutic preference for redder antler is needed. The effectiveness of the combination therapy of VA middle sections (VAMs) and VA blood (VAM-B) was first examined in promoting proliferation of mouse osteoblastic cells (MC3T3-E1). The anti-osteoporotic activity of VAM-B (ratio of VAM:VA blood = 1:0.2) was evaluated with ovariectomized (OVX) rats at a dose of 0.2 g/kg. In VAM-B-treated OVX rats, the body weight decreased 10.7%, and the strength of vertebrae and the femur respectively increased 18.1% and 15.4%, compared to the control. VAM-B treatment also recovered the estrogen-related loss of the right tibial trabecular bone microarchitecture. Alkaline phosphatase (ALP) significantly decreased, but estradiol did not significantly change in serum of VAM-B-treated OVX rats. We also provide an effective strategy to enhance the anti-osteoporotic activity of VAM. In conclusion, our results provide scientific evidence supporting the traditional therapeutic preference of redder antler and indicate that VAM-B is a potential therapeutic agent for managing osteoporosis.     

Rapid qualitative and quantitative evaluation of deer antler (Cervus elaphus) using near-infrared reflectance spectroscopy

Near-infrared (NIR) spectroscopy has been applied for both the qualitative and quantitative evaluation of the velvet deer antler. The most important parameters of determining the quality of velvet antler are the habitat (the country of origin) and ash content. Conventionally, the habitat is determined by examining the appearance of samples (by human eye), which lacks objectivity. Ash content is measured by an ignition method (measurement ash residue), however, it is too slow (4–5 h) to be used for rapid at-site measurement. Velvet antlers from three different habitats (China, New Zealand, and Russia), albeit the same species of Cervus elaphus, were evaluated in this paper. Soft independence modeling of class analogies (SIMCA) and partial least squares (PLS) were used for classification of habitat and determination of ash content. The habitat was successfully identified with over 80% accuracy, and the ash content prediction result using PLS regression showed good correlation with the reference ignition method with a standard error of prediction (SEP) of 1.264%.     

Electrochemical Monitoring of Saos‐2 Cell Differentiation on Pyrolytic Carbon Electrodes

In this study we establish an electrochemical platform based on two dimensional (2D) pyrolytic carbon electrodes for in vitro analysis of osteoblast differentiation. Electrochemical impedance spectroscopy (EIS) was used to monitor cell adhesion and proliferation, while an electrochemical assay based on square wave voltammetry (SWV) was applied to measure the activity of the differentiation marker alkaline phosphatase (ALP). 2D pyrolytic carbon electrodes were fabricated and used to monitor Saos‐2 cell differentiation for a period of up to 21 days. With this method it was possible to detect a faster increase of ALP activity for cells cultured in medium supplemented with differentiation factors compared to cells cultured in growth medium. This was confirmed by the results obtained with Alizarin Red staining, showing that cells subjected to osteogenic medium went through the entire differentiation process, from proliferation to mineralization. Finally, for the first time, real‐time monitoring of ALP activity combined with continuous EIS monitoring of the same cell culture was achieved using the pyrolytic carbon electrodes.     

A Novel Polypeptide from Cervus elaphus Linnaeus

A novel polypeptide having stimulant effect on some cell proliferation was isolated from the velvet antler (Cervus elaphus Linnaeus). The velvet antler polypeptide consists of a single chain of 32 amino acid residues. Amino acid sequence of the polypeptide was identified as:VLSAADKSNVKAAWGKVGGNAPAFGAEALLRM.     

In-Vitro Biocompatibility Evaluation of Collagen-Hyaluronic Acid/Bioactive Glass Nanocomposite Scaffold

A nano-structured scaffold was designed for bone repair using collagen, hyaluronic acid (HYA) and nano-bioactive glass (NBaG) as its main components. The collagen-HYA/NBaG scaffold was prepared by using a freeze-drying technique and characterized by scanning electron microscopy (SEM). Osteoblastls were seeded on these scaffolds and their proliferation rate, alkaline phosphatase (ALP) activity and ability to form mineralized bone nodules were compared with those osteoblasts grown on cell culture plastic surfaces. The cross-section morphology shows that the collagen-HYA/NBaG scaffold possessed a three-dimensional (3D) interconnected homogenous porous structure. The results obtained from biological assessment show that this scaffold did not negatively affect osteoblasts proliferation rate and improves osteoblasts function as shown by increasing the ALP activity and calcium deposition and formation of mineralized bone nodules. Therefore, the composite scaffolds could provide a favorable environment for initial cell adhesion, maintained cell viability and cell proliferation, and had good in-vitro biocompatibility.     

Cyclooxygenase-2 promotes cell proliferation, migration and invasion in U2OS human osteosarcoma cells

Osteosarcoma is the most common primary bone tumor, but the pathogenesis is not well understood. While cyclooxygeanse-2 (COX-2) is known to be closely associated with tumor growth and metastasis in several kinds of human tumors, the function of COX-2 in osteosarcoma is unclear. Therefore, to investigate the function of COX-2 in osteosarcoma, we established stable cell lines overexpressing COX-2 in U2OS human osteosarcoma cells. COX-2 overexpression as well as prostaglandin E2 treatment promoted proliferation of U2OS cells. In addition, COX-2 overexpression enhanced mobility and invasiveness of U2OS cells, which was accompanied by increases of matrix metalloproteinase-2 and -9 (MMP-2 and -9) activities. Selective COX-2 inhibitors, NS-398 and celecoxib, inhibited cell proliferation and abrogated the enhanced mobility, invasiveness and MMP activities induced by COX-2 overexpression. These results suggest that COX-2 is directly associated with cell proliferation, migration and invasion in human osteosarcoma cells, and the therapeutic value of COX-2 inhibitors should be evaluated continuously.