林蛙皮胶原蛋白的提取及其生物学特征分析

目的:建立林蛙皮胶原蛋白低温提取及纯化方法,保证胶原蛋白的活性,并对其生物学特性进行初步探讨。方法:采用低温酸酶提取结合的方法提取林蛙皮中的胶原蛋白,并对提取过程中过氧化氢的加入量进行考察;对纯化的胶原蛋白进行含量、相对分子质量分布等定性及定量检测,同时观察其对间充质干细胞增殖情况的影响。结果:在4℃经脱色脱脂处理(过氧化氢浓度为0.025%)后,用0.5 mol/L的醋酸提取48 h,沉淀再分别用0.67%、1.34%、2%的胃蛋白酶提取,在此工艺条件下,酸溶性胶原蛋白(ASC)的提取率为8.77%±0.44%,纯度为75.83%±3.78%;酶溶性胶原蛋白(PSC)的提取率为30.69%±0.83%,纯度为66.39%±1.79%。紫外扫描图谱表明提取物具有胶原蛋白的特征吸收。SDS-PAGE分析表明所得ASC与PSC均由2条α1链与1条α2链组成,为保持了三股螺旋结构特征的Ⅰ型胶原蛋白。对细胞生长影响的实验表明,ASC与PSC在0.05~20 mg/m L浓度范围对间充质干细胞的生长无抑制作用,并且在某些浓度下具有促进干细胞增殖的作用。结论:低温酸酶法可高效提取林蛙皮粉中的胶原蛋白,提取的胶原蛋白具有良好的生物学活性,本方法及提取的胶原蛋白在组织工程等领域具有良好的应用前景。     

黄连素对变异链球菌抑制作用的体外研究

目的分析黄连素对体外变异链球菌生长、产酸、粘附的影响,探讨其防龋作用。方法采用微量肉汤稀释法进行最小抑菌浓度(MIC)的测定;然后通过试管粘附法测定不同浓度药液对变异链球菌粘附作用的影响;最后计算不同浓度药液作用24h后pH值的变化。结果黄连素对变异链球菌的MIC为1.25mg/mL,MBC为5.00mg/mL。实验组对变异链球菌的粘附及产酸的抑制作用随着药物浓度的增加而增强,与阴性对照组相比差异有统计学意义(P0.05)。结论黄连素可抑制体外变异链球菌的生长、产酸及粘附。     

重组胶原蛋白在表皮修复中的作用机理研究

为探究重组胶原蛋白(RHC)用于表皮创伤修护的作用机理,按照国标方法对RHC促进细胞生长、细胞迁移、细胞黏附、促进组织吸收胶原蛋白的各项能力展开研究.结果显示,RHC促进细胞生长的起效量为0.0025％;以0.1％浓度为例,RHC在2 h内即有显著的细胞修复能力,24hRHC细胞修复比海洋胶原蛋白高21.1％;RHC浓...     

生物材料表面蛋白质微图形对人体软骨细胞形态及蛋白表达的影响

主要研究采用微接触印刷术在生物材料表面制备的细胞外基质蛋白微图形对人体软骨细胞粘附、铺展以及蛋白质表达等细胞行为的影响．研究结果表明,蛋白质微图形表面对细胞的粘附、铺展、排列以及细胞蛋白质表达具有明显的影响．细胞优先粘附在微图形蛋白区域,微图形形状以及尺度明显影响细胞的粘附形态以及铺展程度,同时也影响细胞生长过程中的Ⅱ型与Ⅵ型胶原蛋白的表达,细胞的铺展行为与细胞的蛋白质表达具有一定的正相关性,铺展较好的细胞表现出更好的Ⅱ型与Ⅵ型胶原蛋白表达．结果表明,通过在材料表面制备细胞外基质蛋白微图形可以有效调控人体软骨细胞的生长行为与功能．     

乌贼皮胶原蛋白的提取及结构表征

为了充分利用乌贼加工废弃物,分析了乌贼皮的基本组成成分,优化了从乌贼皮中提取胶原蛋白的工艺条件,并利用SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱对所提取的胶原蛋白进行了结构表征.结果表明,乌贼皮中含有大量胶原蛋白,可作为胶原蛋白来源的补充.采用酸酶复合提取胶原蛋白的最佳条件为:酒石酸浓度为0.1mol/L,胃蛋白酶添加量为1400U/g,料液比为1:20(m:V,原料),4℃提取18h,提取率为12.08％.SDS-PAGE垂直电泳、紫外扫描和傅里叶变换红外光谱的结果表明,采用酸酶复合法从乌贼皮中提取的胶原蛋白为I型胶原蛋白,保持了完整的三螺旋结构.     

茶条槭悬浮培养体系的建立与没食子酸合成的优化条件

初步建立茶条槭（Acer ginnala）细胞悬浮培养体系：以茶条槭子叶为外植体,接种于WPM培养基中,对茶条槭愈伤组织进行诱导和继代培养。悬浮培养中,每代增长指数达到11．6,没食子酸含量达到1．518％。通过对比NT、IS、WPM、B5和MS培养基所含成分对茶条槭愈伤组织悬浮培养的影响,综合考虑悬浮细胞的生长速率和有效成分的含量,确定WPM为基本培养基。WPM培养基大量元素的浓度对细胞的生长和没食子酸的积累有显著影响,其浓度越高,促进作用越明显。3倍浓度的大量元素最有利于没食子酸的积累。蔗糖浓度为10g·L^-1最适于没食子酸的积累,浓度为30g·L^-1最适于茶条槭细胞生长和没食子酸合成。     

桦木酸对人胃癌MGC-803细胞增殖的影响

目的: 探究不同浓度桦木酸对人胃癌MGC-803细胞增殖的影响。方法: 将人胃癌 MGC-803 细胞分成 4 组,每组设置 3 个复孔,对照组细胞为加入浓度为0 μg /ml的桦木酸实验组细胞分别加入终浓度为10、20、30 μg /ml 的桦木酸,各组细胞在含5%的 CO₂ 培养箱中孵育 48 h 后,使用吉姆萨染色法和台盼蓝拒染法检测桦木酸对细胞克隆形成率和生长抑制率的影响;EdU法和流式细胞术分别检测细胞增殖和细胞周期变化;qRT-PCR和Western blot检测细胞周期调控因子CCNB1、CCND1的表达。结果: 与对照组相比,人胃癌MGC-803的克隆形成率显著降低(P＜0.01),生长抑制率明显升高,细胞增殖能力显著下降(P＜0.01);各实验组细胞随着桦木酸浓度的增加G1 期细胞所占比例逐渐降低, 而S 期细胞数量显著增多(P＜0.01);细胞周期调控因子CCNB1、CCND1 的mRNA和蛋白表达水平均显著降低(P＜0.01),30 μg /ml 的桦木酸处理组的表现最佳。结论: 在终浓度为 10～30 μg /ml 的范围内,桦木酸能够降低人胃癌MGC-803细胞增殖,抑制细胞生长,下调CCNB1和CCND1的表达将人胃癌 MGC-803细胞阻滞于G0/G1 期。     

理化因子对谷皮菱形藻细胞密度及中性脂含量的影响

以高脂微藻谷皮菱形藻(Nitzschia palea NY025)为实验材料,探讨了利用光密度法和尼罗红荧光染色法测定细胞生长和细胞中性脂含量的可行性,进而研究了温度、光强及培养基中N、P、Si含量对藻细胞生长和中性脂积累的影响。结果表明:(1)谷皮菱形藻在675nm处存在最大吸收峰,细胞密度与OD675之间存在良好的线性关系,利用光密度法和尼罗红荧光染色法表征谷皮菱形藻生物量和中性脂含量操作简单,适用于高通量样品的测定;(2)谷皮菱形藻在20℃,光强160μmol m-2s-1时生长最快,在20℃,光强200μmol m-2s-1时,有利于中性脂积累;(3)培养基中N、P、Si浓度分别为80、120、100 mg/L时,有利于谷皮菱形藻细胞生长,其中,N元素影响最大,其次是P、Si,且N、P、Si三因子以及交互作用N×P与P×Si对藻株生长作用均为显著。培养基中N、P、Si浓度分别为80、120、50 mg/L时,利于中性脂积累,其中,N元素影响最大,其次是Si、P,且因子N、Si及交互作用N×P、N×Si作用均为显著;(4)可采用两步培养法,先使谷皮菱形藻细胞大量增殖,而后适当改变培养条件,以增加脂质合成。     

外源色氨酸对海水胁迫长春花幼苗根CCO活性和叶绿体超微结构的影响

以长春花[Catharanthus roseus (L.) G.Don]幼苗为材料,在温室条件下研究了不同浓度色氨酸对20%海水处理14 d后长春花幼苗生长、线粒体细胞色素c氧化酶(CCO)活性、细胞超微结构及长春碱含量的影响.结果显示:(1)与对照相比,20%海水中加入不同浓度的色氨酸,长春花幼苗生长均受到进一步显著抑制,线粒体细胞色素c氧化酶活性下降,但幼苗长春碱含量显著增加.(2) 在20%海水中增加250和500 mg/L色氨酸时,叶绿体片层松散、变稀,色氨酸浓度增大到750 mg/L时,叶绿体变形,片层扭曲、变形,叶绿体模糊、濒临解体.研究表明,在20%海水中增加500 mg/L的色氨酸时,长春花生长受到抑制较小,叶绿体超微结构受损伤程度较轻,而长春碱含量提高幅度最大,从而较有利于长春花幼苗的生长和长春碱的积累.     

云南红豆杉细胞的悬浮培养

在云南红豆杉细胞悬浮培养中,适宜的培养基为B5,接种量为0．5～0．8g干重细胞／100ml培养基,2,4－D浓度为1．0mg／L;培养细胞的生长周期约30d;培养基中较高浓度的蔗糖（40g／L）可提高紫杉醇含量;添加的椰子汁（CM）、酪蛋白氨基酸（C）和水解乳蛋白（LH）3种有机添加剂均能提高培养细胞中紫杉醇的含量,但只有CM和CA能促进细胞的生长。于B5培养基中添加不同浓度的NH4NO3对培养细胞无明显影响。     

牛蛙致病变形菌的鉴定及其敏感药物筛选

【目的】分离鉴定引起牛蛙(Rana catesbeiana)皮肤溃疡病症的致病菌,筛选抗菌药物。【方法】采用无菌解剖组织划线分离法分离致病菌,通过人工感染试验、菌体和菌落形态观察、API20E系统鉴定、16S r RNA基因序列分析,确定分离菌的致病性及分类地位,并对该菌进行药物敏感性试验。【结果】从患病濒死牛蛙体内分离细菌NWG20141026,通过形态特征观察、生理生化试验、API 20E系统鉴定和16S r RNA基因序列相似性分析,认为NWG20141026菌株为普通变形菌(Proteus vulgaris)。人工感染试验显示,NWG20141026菌株不仅可以通过皮肤伤口感染引起蛙体表溃疡溃烂病症,也可通过消化道感染引起蛙肠炎病。药敏实验结果表明,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑、恩诺沙星、红霉素等7种药物对普通变形菌具有较好的抑杀菌作用。【结论】引起牛蛙(R.catesbeiana)皮肤溃疡病症的致病菌NWG20141026为普通变形菌(P.vulgaris),所患疾病命名为牛蛙变形菌病。普通变形菌对健康牛蛙具有较强致病性,氟苯尼考、四环素、多西环素、萘啶酸、磺胺异噁唑等5种药可作为防治牛蛙变形菌病的候选药物。     

颌下腺导管细胞与胶原海绵细胞相容性的实验研究

为探讨胶原海绵对颌下腺 (submandibulargland ,SMG)导管细胞的细胞相容性 ,采用HE染色光镜观察及免疫组化观察SMG导管细胞接种于胶原海绵后 ,细胞的生长情况。光镜下可见接种后第 1d细胞数量较少 ,分散于胶原海绵支架中间 ,第 7d细胞数量明显增加 ,免疫组织化学染色抗IV型胶原抗体染色呈阳性 ,说明细胞与支架材料之间已经有细胞外基质产生。胶原海绵具有良好的细胞相容性 ,是一种理想的支架材料。与胶原海绵复合培养 ,颌下腺导管细胞仍可保持良好的增殖能力。     

Characterization of the cDNA encoding bullfrog, Rana catesbeiana, osteocalcin and two forms of the protein isolated from bone

A full-length cDNA clone encoding osteocalcin from the bullfrog, Rana catesbeiana (bone Gla-protein, BGP) has been isolated, and the complete coding sequence for the 100-amino-acid pre-pro-osteocalcin protein was determined. The amino acid sequence of Rana catesbeiana osteocalcin, especially the mature 49-amino acid sequence, is closer to the mammalian than to the fish, Sparus osteocalcin. Rana mature osteocalcin has a similarity of 67% with human or 59% with rat osteocalcin, and only 42% with fish mature osteocalcin. The 51-amino-acid pre-pro-peptide contains the expected hydrophobic leader sequence and the dibasic Arg-Arg sequence preceding the NH2-terminal Ser of the mature 49-amino-acid Rana osteocalcin. The pro-peptide sequence also contains the expected motif of polar and hydrophobic residues, which targets vitamin K-dependent gamma-carboxylation of three specific Glu residues at positions 17, 21, and 24 in the mature protein. At the native protein expression levels, extraction from Rana cortical bone in the presence of protease inhibitor cocktail resulted in the isolation of two distinct forms of osteocalcin, P-1 and P-2, with a 3:2 distribution. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and amino acid sequence analysis of the N-terminal domain, we confirmed that P-1 is the intact 49-residue osteocalcin with N-terminal SNLRNAVFG., and that P-2 lacks four amino acids from the N-terminus, (NAVFG.). These results demonstrate the existence of a form of osteocalcin lacking four N-terminal amino acids in Rana bone, and that mature Rana osteocalcins remained highly conserved in their molecular evolution, especially with respect to the conservation of the C-terminal domain (residues 14-49).     

Localization of a Cell Adhesion Site on Collagen XIV (Undulin)

Cell adhesion to collagen XIV is implied to be mediated by proteoglycans as cellular receptors (T. Ehniset al.,1996,Exp. Cell Res.229, 388–397). In order to define the cell binding region(s), fusion proteins expressed inEscherichia coliand covering the large noncollagenous domain NC3 of collagen XIV were used as substrates for the adhesion of skin fibroblasts. A prominent cell binding site could be localized in the N-terminal fibronectin type III repeat of collagen XIV and its immediate C-terminal extension. Since this region also mediates the binding of the small chondroitin/dermatan sulfate proteoglycan decorin (T. Ehniset al.,1997,J. Biol. Chem.272, 20414–20419), our finding could provide the molecular basis for the observation that decorin serves as inhibitor and potential modulator of cellular interactions with collagen XIV.     

Cell adhesive activity of two animal lectins through different recognition mechanisms

Cell adhesive activity of two animal lectins, frog (Rana catesbeiana) S-type 14K lectin and echinoidin (a C-type lectin from sea urchin plasma), was studied with human rhabdomyosarcoma (RD) cells. RD cells attached to and spread on plastic plates coated with each lectin. Cell adhesion by the frog lectin was completely inhibited by the addition of lactose or asialofetuin glycopeptide. Echinoidin-induced cell adhesion was only inhibited by peptide GRGDS. Since echinoidin is known to contain an RGD-sequence, our results clearly indicate that this sequence is active as the cell adhesive signal. These results suggest that some of the animal lectins may function as a cell adhesive molecule rather than using the carbohydrate-recognition mechanism.     

Collagen VI Regulates Normal and Transformed Mesenchymal Cell Proliferationin Vitro

Suggestions exist that, in addition to traditional growth factors, the extracellular matrix (ECM) of a cell can regulate its proliferation. This hypothesis was investigated with normal and transformed fibroblasts because they exhibit specific intracellular responses after adherence to ECM and produce large quantities of ECM proteins. Although cells cultured on different ECM proteins grew more rapidly than those on plastic, adherence and cell growth on an individual ECM protein were not correlated. To test if ECM can stimulate cell growth, soluble ECM proteins were given to cells after plating. In this culture system only collagen VI (CVI), at a concentration of 20 μg/ml in medium, increased 3T3 cell number to 402% of control by 72 h. Similar increases of human fibroblasts and HT 1080 cell numbers were noted. DNA synthesis of all three cell types increased 24 h after addition of soluble CVI. A mixture of CVI single chains, yielded by reduction and alkylation, was not stimulatory. However, this mixture efficiently inhibited the DNA synthesis induced by native CVI. Antibody inhibition studies showed that the region of CVI stimulating proliferation differs from the site bound by the integrin receptor α2β1, which mediates cell adhesion to immobilized CVI. Heparin inhibited a portion of CVI-induced proliferation. These data demonstrate that CVI can stimulate mesenchymal cell growth via a pathway that is independent of the integrin α2β1 and that the stimulatory region appears to be within the native helical portion of the collagen.     

奥尼罗非鱼胶原蛋白及其酸解液美拉德反应产物的研究

以奥尼罗非鱼鱼皮中胶原蛋白为研究对象,采用氨基酸自动分析仪和GC-MS分析了胶原蛋白的游离氨基酸组成、主要风味成分及其酸解液美拉德反应产物的风味成分.结果表明：鱼皮中胶原蛋白的提取率为6.61%,酸解后的游离氨基酸组成与一般胶原蛋白的氨基酸组成一致,其中风味氨基酸占52.57%;美拉德反应产物中含3,3-二甲基正辛烷、2,6,10-三甲基十二烷等20种风味成分.     

Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasing peptides. Cholecystokinins (CCKs) are neuroendocrine peptides known for their production in the gastrointestinal tract and central nervous system of mammalians. Several CCKs have been identified from two amphibians, Rana catesbeiana and Xenopus laevis. These amphibian CCKs are found to be express in brain and in the gastrointestinal tract, but not in skin. In the current report, a cholecystokinin (CCK) isoform was identified from skin secretions of the frog, Rana nigrovittata. Its amino acid sequence is RVDGNSDQKAVIGAMLAKDLQTRKAGSSTGRYAVLPNR PVIDPTHRINDRDYMGWMDF, which is the same with that of CCK from R. catesbeiana. Four different cDNAs (GenBank accession nos. EF608063-6) encoding CCK precursors were cloned from the cDNA library of the skin of R. nigrovittata. The present data demonstrated that amphibian CCK could also be expressed in gastrointestinal tract, central nervous system and skin as other amphibian skin-gut-brain triangle peptides.     

Recombinant Human Collagen XV Regulates Cell Adhesion and Migration

The C-terminal end of collagen XV, restin, has been the focus of several studies, but the functions of full-length collagen XV have remained unknown. We describe here studies on the production, purification, and function of collagen XV and the production of a monoclonal N-terminal antibody to it. Full-length human collagen XV was produced in insect cells using baculoviruses and purified from the cell culture medium. The yield was 15 mg/liter of cell culture medium. The collagen XV was shown to be trimeric, with disulfide bonds in the collagenous region. Rotary shadowing electron microscopy revealed rod-like molecules with a mean length of 241.8 nm and with a globular domain at one end. The globular domain was verified to be the N-terminal end by N-terminal antibody binding. The molecules show flexibility in their conformation, presumably due to the many interruptions in their collagenous domains. The ability of collagen XV to serve as a substrate for cells was tested in cell adhesion assays, and it was shown that cells did not bind to collagen XV-coated surfaces. When added to the culture medium of fibroblasts and fibrosarcoma cells, however, collagen XV rapidly bound to their fibronectin network. Solid phase assays showed that collagen XV binds to fibronectin, laminin, and vitronectin and that it binds to the collagen/gelatin-binding domain of fibronectin. No binding was detected to fibrillar collagens, fibril-associated collagens, or decorin. Interestingly, collagen XV was found to inhibit the adhesion and migration of fibrosarcoma cells when present in fibronectin-containing matrices.