海藻糖的长期毒性试验研究

海藻糖是一种由两分子葡萄糖缩合而成的非还原性二糖,它作为生物制品、医药及食品的保护剂和添加剂,已在国内外许多文献中报道。本文通过对大白鼠的长期毒性试验研究,表明海藻糖对人体无潜在危害,是安全的。     

西藏凹乳芹根、叶提取物的急性及亚急性毒性研究

对西藏凹乳芹根、叶提取物进行急性毒性及亚急性毒性实验研究,先经预试验确定给药组数和剂量,然后灌胃给药,测试西藏凹乳芹根、叶提取物的半数致死量(LD₅₀)及最大耐受量(MTD);再分别以LD₅₀的1/20和1/40剂量连续灌胃给药做亚急性毒性实验。结果显示,西藏凹乳芹根和叶提取物的LD₅₀分别为154.81和58.27 g/kg,MTD分别为108.28和32.62 g/kg。西藏凹乳芹根、叶提取物的亚急性毒性实验各组的血液生化指标、脏器指数、体重及摄食量与对照组相比均没有明显变化。因此,西藏凹乳芹根、叶提取物属无毒级,长期服用未发现毒性反应,有较高的食用安全性。     

海藻糖分析方法的研究

对目前研究中应用的海藻糖的定性定量分析方法作了较为全面的介绍 ,并对各种方法的特点及适用性作了比较。     

海藻糖降解酶抑制因子的研究

研究了一系列因素对食尼古丁节杆菌D-97胞内海藻糖降解酵的抑制作用,发现有效霉素(井冈霉素)和乙酸能有效抑制海藻糖降解酶的活性,抑制率最大分别达到87．13％和85．16％．同时研究了它们对海藻糖转化率的影响,发现有效霉素能提高海藻糖转化率,最大增幅达17．44％,而乙酸却降低了转化率。     

海藻糖合酶的研究进展

海藻糖是一种天然存在的非还原性二糖, 对生物膜和蛋白质等大分子有独特的保护作用, 在食品、医药、化妆品等多个领域中都有广泛的发展空间。海藻糖合酶(TreS)是一类分子内转糖苷酶, 专一性地以麦芽糖为底物, 一步转化生成海藻糖, 操作工艺简单、底物价格低廉、应用前景良好。本文综述了海藻糖合酶的酶学性质、催化机理、基因工程以及目前存在的主要问题和拟解决方案。     

海藻糖微生物酶法合成机制的研究

来源于嗜酸热古菌芝田硫化叶菌(Sulfolobus shibatae)B12的麦芽寡糖基海藻糖合酶(MTSase)和麦芽寡糖基海藻糖海藻糖水解酶(MTHase)基因在大肠杆菌中获得表达。将获得纯化的两个酶,分别以麦芽寡糖和淀粉为转化底物,在pH5.5,60℃条件下合成海藻糖。从反应产物分析结果可知,两个酶合成海藻糖时能利用的最小底物是麦芽四糖,海藻糖产率与麦芽寡糖链长正相关。同时还发现两个酶都具有轻微的α-1,4-葡萄糖苷酶活性,能在麦芽寡糖还原末端水解α-1,4糖苷键,生成葡萄糖分子,其反应最小底物分别是麦芽三糖和四糖。推测海藻糖合成酶可能有两个不同的催化活性中心。     

海藻糖载入血小板的研究

将不可渗透型的保护剂海藻糖有效地载入血小板内部是用冷冻干燥法保存血小板重要的第一步。研究血小板对海藻糖的载入量随外部海藻糖浓度、孵化时间、孵化温度改变的变化规律,发现在细胞外海藻糖浓度为50mmol/L、孵化温度37℃、孵化时间4h的条件下,血小板能有效地吸收海藻糖,细胞内海藻糖浓度达到15mmol/L以上。对孵化后的血小板进行形态观察、血液学分析和膜联蛋白(annexin)V结合活化分析,结果表明孵化后的血小板保持了正常血小板的形态和功能。     

海藻糖——昆虫的血糖

海藻糖(trehalose)是由2个葡萄糖分子通过α,α-1,1糖苷键连接的一种非还原性双糖。海藻糖作为昆虫的血糖,对于生物的能量代谢和抗逆等方面具有重要的作用。文章从昆虫海藻糖的发现、海藻糖的化学性质、昆虫中海藻糖的生理作用、代谢途径等方面进行综述,并对昆虫中海藻糖的进一步研究作了展望。     

多药耐药基因在肝细胞癌中的表达及与p53、PCNA表达的相关性研究

目的　研究肝细胞癌 (HCC)组织中多药耐药基因MDR 1与p5 3蛋白及增殖细胞核抗原 (PCNA)表达的关系 ,旨在从基因水平进一步探讨预测化学治疗效果的可行性。方法　利用免疫组织化学方法 (S P法 )研究 30例肝穿活检的肝癌组织中MDR 1、p5 3和PCNA的表达。结果　 30例肝细胞癌中MDR 1的阳性表达率为 5 6 6 7% ,MDR 1阳性表达与组织学分级无关 (P >0 0 5 )。MDR 1表达与 p5 3、PCNA表达间无相关性 (P >0 0 5 )。结论　通过检测MDR 1基因可以对肝细胞癌病人进行化学治疗敏感性预测 ;肝细胞癌中MDR 1基因表达不依赖于 p5 3和细胞增殖这些因素。     

大鼠肝癌发生过程中增殖细胞核抗原（PCNA／Cyclin）的表达

应用免疫组织化学的ＡＢＣ法,对二乙基亚硝胺（ＤＥＮ）诱发大鼠发生过程中增殖细胞核抗原在肝组织中的表达进行了系统观察。结果显示：正常大鼠肝组织中仅见极少数ＰＣＮＡ阳性肝细胞,阳性率为０．０８％,随着诱癌进程发展,大鼠肝组织中ＰＣＮＡ阳性肝细胞逐渐增多,诱癌第４、８、１２周,大鼠肝组织中ＰＣＮＡ阳性肝细胞百分率分别为１．６％、３．８％、１６．２％,诱癌晚期癌结节内大部分肝癌细胞里ＰＣＮＡ阳性表达,阳性率为８０．６％。本研究结果表明原位检测ＰＣＮＡ表达比传统依据形态学分化程度来判断肿瘤发生可能性更为客观、可靠。     

Physical and functional interaction of human nuclear uracil-DNA glycosylase with proliferating cell nuclear antigen

Uracil residues arise in DNA by the misincorporation of dUMP in place of dTMP during DNA replication or by the deamination of cytosine in DNA. Uracil-DNA glycosylase initiates DNA base excision repair of uracil residues by catalyzing the hydrolysis of the N-glycosylic bond linking the uracil base to deoxyribose. In human cells, the nuclear form of uracil-DNA glycosylase (UNG2) contains a conserved PCNA-binding motif located at the N-terminus that has been implicated experimentally in binding PCNA. Here we use purified preparations of UNG2 and PCNA to demonstrate that UNG2 physically associates with PCNA. UNG2 co-eluted with PCNA during size exclusion chromatography and bound to a PCNA affinity column. Association of UNG2 with PCNA was abolished by the addition of 100 mM NaCl, and significantly decreased in the presence of 10 mM MgCl(2). The functional significance of the UNG2.PCNA association was demonstrated by UNG2 activity assays. Addition of PCNA (30-810 pmol) to standard uracil-DNA glycosylase reactions containing linear [uracil-(3)H]DNA stimulated UNG2 catalytic activity up to 2.6-fold. UNG2 activity was also stimulated by 7.5 mM MgCl(2). The stimulatory effect of PCNA was increased by the addition of MgCl(2); however, the dependence on PCNA concentration was the same, indicating that the effects of MgCl(2) and PCNA on UNG2 activity occurred by independent mechanisms. Loading of PCNA onto the DNA substrate was required for stimulation, as the activity of UNG2 on circular DNA substrates was not affected by the addition of PCNA. Addition of replication factor C and ATP to reactions containing 90 pmol of PCNA resulted in two-fold stimulation of UNG2 activity on circular DNA.     

Proliferating cell nuclear antigen in normal urothelium and urothelial lesions of the urinary bladder: a quantitative assessment using a true color image analysis system

To evaluate proliferating cell nuclear antigen (PCNA) staining for assessing proliferative activity in routine pathology specimens of urinary bladder, the bladder carcinoma cell line J82 and a total of 122 specimens of normal bladder and urothelial lesions were stained with the antibody clone PC10 against proliferating cell nuclear antigen. In in vitro plateau cultures the proportion of PCNA-positive cells exceeded that of Ki-67-positive cells, and only very few cells were negative. In formalin-fixed tissues, the PCNA staining pattern, which should be confined to replicon units in the nucleus, was optimized by 1 h postfixation in an organic solvent (methacarn). Sections showed positive nuclear staining confined to basal and some suprabasal cells in normal urothelium and grade 1 dysplasias, but more generalized nuclear staining in all other neoplastic lesions. In addition, stromal cells adjacent to invasive tumors showed nuclear positivity in some instances. Using quantitative true color image analysis of sections counterstained with hemalum, the degree of brown staining of the PCNA reaction product is contrasted with the blue staining of the nuclear area. With this method low contrast specific staining not appreciated optically can be reliably detected. Image analysis data confirmed observations made on noncounterstained sections and showed significant differences between grade 1 and 2 dysplasias as well as between grade 1 dysplasia and all grades of papillary tumor. Furthermore, a significant difference in PCNA staining indices was found between grade 1 and 3 bladder carcinomas. The results indicate that PCNA staining using the PC10 antibody is not confined to the proliferative fraction of neoplastic urothelium. In contrast with data from normal tissue and malignant hematological neoplasms, the amount of PCNA is regulated differently in urothelial neoplasms, emphasizing the biological differences between the following two sets: mild dysplasia and moderate dysplasia; mild dysplasia and papillary carcinomas. The use of image analysis to standardize the detection process after controlled staining conditions is advisable in order to provide reliable data. Supported by the DFG project: Knuechel/Urothelcarcinom 263     

CD47基因对食道癌细胞增殖的影响

本研究将CD47-si RNA转染至食道癌细胞,采用蛋白免疫印迹检测食道癌细胞中CD47蛋白的表达,MTT法检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞增殖状态,蛋白免疫印迹检测CD47-siRNA转染组和空质粒转染组(对照组)食道癌细胞中PCNA蛋白表达,DCFDA染色流式细胞仪检测食道癌细胞CD47-siRNA转染组和空质粒转染组(对照组)中ROS水平,以探究CD47基因对食道癌发生发展的影响。研究结果表明,CD47-si RNA转染组食道癌细胞中CD47蛋白明显低于对照组;CD47-siRNA转染组食道癌细胞增殖率显著低于对照组(p<0.05);CD47-siRNA转染组细胞增殖相关蛋白PCNA低于对照组(p<0.01);CD47-siRNA转染组食道癌细胞中ROS水平明显高于对照组(p<0.05)。本研究初步认为:CD47-siRNA可降低食道癌细胞中CD47蛋白表达,抑制食道癌细胞的增殖并增加食道癌细胞中ROS水平。     

Growth of rat pancreatic acinar cells quantitated with a monoclonal antibody against the proliferating cell nuclear antigen

The monoclonal antibody PC10 raised against the proliferating cell nuclear antigen (PCNA) was used to study acinar cell replication in the pancreas of rats under different functional conditions. In Western blots, the antibody recognized a single band of 37 kDa in pancreatic homogenates indicating its specificity in this particular species and organ. Three conditions of growth were chosen for immunohistochemical analysis: pancreatic preand postnatal development, pancreatic regeneration after injury, and cholecystokinin-stimulated acinar cell proliferation. The time course of acinar cell replication under each condition was the same as that obtained after tritiated thymidine incorporation with subsequent autoradiography, indicating that the percentage of PCNA-positive cells reflects the pool of cycling cells in the models investigated. However, the absolute number of PCNA-positive cells was two to ten times higher than comparable labeling indices from ³H-thymidine autoradiography. This finding might reflect the half life of PCNA, which exceeds the duration of the S-phase. Thus, PCNA-positive cells not only represent S-phase cells, but also cells that have recently completed the cell cycle.     

PCNA、P27蛋白在老年胃癌中的表达及其临床意义

目的：探讨增殖细胞核抗原（PCNA）和P27蛋白（P27）在老年胃癌中表达及其临床意义。方法：采用免疫组织化学SP法检测58例老年胃癌组织中PCNA、P27蛋白表达情况,分析它们与老年胃癌临床生物学行为的关系。结果：PCNA在老年胃癌组中的阳性表达率明显高于对照组的阳性表达率,P27在老年胃癌组中的阳性表达率明显低于对照组的阳性表达率。PCNA蛋白阳性表达与老年胃癌是否有浆膜浸润、组织分化程度、淋巴结转移以及TNM分期密切相关（P〈0.01）,而P27蛋白阳性表达与老年胃癌类型、是否有浆膜浸润、组织分化程度、淋巴结转移以及TNM分期密切相关（P〈0.01）。老年胃癌组织中PCNA、P27的表达呈负相关（r=-0.536,P〈0.05）。结论：P27表达下调、PCNA表达增强在老年胃癌的发生、发展过程中具有重要的作用,且两者具有相互作用。     

Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.