药用地龙的研究进展

地龙为环节动物门钜蚓科动物参环毛蚓Pheretima aspergailum（E．Perrier）、通俗环毛蚓P．vulgaris Chen、威廉环毛蚓P.guillelmi（Michaelsen）或栉肓毛蚓Ppectinifera Michaels—en的干燥体。前一种习称“广地龙”,后三种习称“泸地龙”,主产于广西、广东、福建。     

蚓激酶治疗冠心病心绞痛的作用机制及其临床应用研究进展

冠心病心绞痛的发生发展主要与冠状动脉粥样硬化斑块破裂、出血、诱发局部血小板聚集、不完全堵塞性血栓形成而使管腔狭窄进一步加重有关[1],是当今严重威胁人类健康和生命安全的最常见的心血管疾病之一,因此寻找有效的冠心病心绞痛的治疗方法直接关系着人类的生命安全和身体健康。蚓激酶含有纤维蛋白溶酶和类似组织纤维蛋白溶酶原激活物,是从露天红赤子爱胜蚓、粉正蚓、双胸蚓、锯齿远蚓、参环毛蚓等中提取的一种具有纤溶活力的丝氨酸蛋白酶,近年来蚓激酶已广泛用于心、脑血管疾病、突发性耳聋、视网膜血管疾病、血管外科疾病等多种疾病的…     

两种蚯蚓及其不同部位的酶谱研究

采用聚丙烯酰胺凝胶电泳赤子爱胜蚓（Ｅｉｓｅｎｉａ ｆｏｅｔｉｄａ）和秉代环毛蚓（Ｐｈｅｒｅｔｉｍａ ｃａｒｎｏｓａ）不同部位的纤溶酶、酯酶及乳酸脱氢酶酶谱。纤溶酶主要存在于蝗蚓的体腔内。不同蝗蚓的纤溶酶酶谱不同。酯酶酶谱在两种蝗蚓及不同部位差异很大。蚯蚓及的乳酸脱氢酶较弱。     

参环毛蚓中纤溶活性蛋白酶研究

分离纯化参环毛蚓中纤溶活性蛋白酶，并对其活性及氨基酸序列进行分析。采用凝胶层析等方法进行蛋白质的分离纯化，SDSPAGE法进行分子质量的测定，纤维平板法测定纤溶活性，氨基酸自动分析仪分析氨基酸组成，并对一个具有纤溶活性的蛋白酶进行了序列分析。分离纯化得到4种酶，其中EQY-4活性较强。其N-端序列为：Gln-Ile-Gly-Gly-Thr-Asn-Ala-Ser-Pro-Gly-Glu-Phe-Pro-Pro-Gln-Leu-Ser-Gln-Thr。与文献报道中赤子爱胜蚓及粉正蚓中分离得到的蛋白酶对比，主要序列相近，但也有不同。     

地龙的化学及药理研究概况

中药地龙为巨蚓科动物参环毛蚓或正蚓科动物背暗唇蚓等的全体。性昧咸寒，有清热、平肝、正喘、通络之功效，主治高热狂躁、惊风抽搐、风热头痛、目赤、中风半身不遂、喘息、喉痛、关节疼痛、齿衄、小便不通、瘰疠、痄腮、疮疡等疾病。近半年来有文献报道，运用中药地龙对治疗癌症、消化性溃疡、烧伤、烫伤等20余种疾病均具有良好的疗效。本研究报道了有关地龙的化学及药理研究概况。     

黄芪与绿豆对砒石染毒大鼠的影响

目的 :为探讨中药黄芪与绿豆对砒石染毒大鼠毒副作用的拮抗情况及其保护作用与金属硫蛋白(MT)的关系。方法 :用黄芪、绿豆及阳性对照药物氯化镉 (CdCl2 )相比较 ,运用镉饱和法及RT PCR测定肝细胞MT的量及mRNA的表达 ,并通过检测丙氨酸氨基转移酶 (ALT)、血尿素氮 (BUN)、血清肌酐(SCR)观察其对砒石引起的肾脏、肝脏的保护作用。结果 :测的的MT的量与其mRNA表达的量具有一致性 ,砒石、黄芪、绿豆均能诱导MT的合成 ,单纯砒石诱导MT的量很少 ,增加黄芪、绿豆后表达量明显增加 ,黄芪与绿豆共同作用增加更明显 (P <0 .0 5 )。结论 :黄芪与绿豆对砒石染毒大鼠金属硫蛋白均有影响 ,对砒石毒性有一定的拮抗作用。     

广地龙两年来缘何价格持续上升

广地龙又叫地龙干，为距蚓科动物参环毛蚓除去内脏的干燥体，常用中药材，主产广西、广东两省区；广西颁于桂南和桂东南地区的横县、灵山、合浦、浦北、博白、陆川等到县；广东则产于粤西的湛江地区的部分县市。近两年来，广西市场上的广地龙的销势日趋看好，价格一直往上飚升，以大开广地龙这一等级来说，     

地龙要净制

地龙为蚯蚓科动物参环毛蚓或缟蚯蚓的干燥体。由于价格高，药农在捕捉时为增加其经济效益，有意未完全去除内脏及泥沙（非药用部分）。最近笔者对我院购进的地龙（经笔者鉴定为蚯蚓科动物参坏毛蚓的干燥体，习称广地龙）。接连3次（均为进货时），每次随意称取IOO0g，除去内脏及泥沙后得纯地龙分别是：7339、6329、6859，其中内脏、泥沙分别是2679、3689、3159，平均内脏、泥沙占原药材量的31．6％。如果用此配方，实际用药剂量就减少了约三分之一，这样必将影响临床效果。因此为提高地龙的质量，保证处方的用量准确，对地龙必须净制（除…     

膜型基质金属蛋白酶在胃癌中的表达意义

目的:探讨膜型基质金属蛋白酶-1(membrane-type matrix metalloproteinase-1,MT1-MMP)在胃癌和非癌性胃组织中的表达及与胃癌临床病理特征的关系。方法:用免疫组织化学方法(PV-6002法)检测140例胃癌手术病人及20例非癌性胃组织中MT1-MMP的表达。结果:非癌性胃黏膜和胃癌组织中的MT1-MMP蛋白表达的阳性率分别为10.0%(2/20)和54.3%(76/140),呈递增的趋势,两者差异有显著性(P=0.001)。MT1-MMP蛋白表达与Lauren分型、肿瘤浸润深度、TNM分期和淋巴结转移均呈显著相关(P<0.05)。结论:MT1-MMP与胃癌的发生发展有关,高表达提高了胃癌的侵袭转移能力。     

朱砂和朱砂安神丸对小鼠肝组织金属硫蛋白表达的影响

目的探讨朱砂及朱砂安神丸中汞对肝组织金属硫蛋白(MT)表达的影响。方法健康小鼠分别ig给予生理盐水、Hg S 0.2 g·kg-1、朱砂0.2和2 g·kg-1、朱砂安神丸1.8 g·kg-1、氯化汞(Hg Cl2)0.032 g·kg-1、甲基汞0.026 g·kg-1,每天1次,连续30 d,取肝组织。用原子荧光光谱法检测肝汞蓄积量,HE染色法观察肝组织病理变化,实时荧光定量PCR法检测肝组织Mt-1基因的表达,免疫组织化学和Western蛋白印迹法检测肝组织MT蛋白的表达,并分析肝汞蓄积量与肝组织Mt-1基因和MT蛋白表达的相关性。结果与正常对照组比较,Hg S、朱砂0.2 g·kg-1和朱砂安神丸组小鼠肝组织汞蓄积量未见明显增高,肝组织Mt-1基因和MT蛋白表达无明显变化,且未见明显病理改变。朱砂2 g·kg-1,Hg Cl2和甲基汞组小鼠肝汞蓄积量明显增高(P<0.05,P<0.01),分别为正常对照组的6.9,82.2和173.1倍;肝组织Mt-1mRNA表达明显增加(P<0.01),分别为正常对照组的3,11和45倍,同时MT蛋白表达明显增加(P<0.05,P<0.01);肝组织可见肝细胞水肿,淋巴细胞浸润。肝汞蓄积量与Mt-1基因和MT蛋白表达水平具有正相关性。结论对比不同形式的汞,ig给予小鼠Hg S、朱砂和朱砂安神丸后肝组织汞蓄积量远低于氯化汞和甲基汞;朱砂0.2 g·kg-1和朱砂安神丸未导致肝组织Mt-1基因和MT蛋白表达水平的变化,肝组织亦未出现病理变化。     

Intestinal epithelial cell injury induced by copper containing nanoparticles in piglets

The nano copper has been widely used in modern clinical medicine practice. However, it has been noticed that nano copper particles induce cell injury and toxicity. The present study was designed to determine the effect of nano copper particles on cell injury of intestinal epithelial cells (IECs) in piglets. The IECs were treated with different doses of nano copper (5, 10, 20 and 40 μg/ml) for 24–48 h to observe cell injury and toxicity. Cell injury was measured based on morphological and other changes including oxidative stress and genes expression. The oxidative stress markers were assayed by differential expression levels of SOD, MDA and Metallothionein (MT) in addition to CTR1, SOD1, COX17, MT and ATOX1 genes expression. Cellular morphology showed an increasing growth of cells without nano copper treatment and nano copper showed significant damage to IECs with higher dose as compared to low dose. Higher doses of copper nanoparticles (10, 20 and 40 μg/ml) have membrane damaging effect on the intestinal epithelial cells, whereas MDA contents and MT value were significantly increased, and SOD activity was decreased with the increase in concentration of nanoparticles. Nano copper up-regulated the CTR1 and SOD1 genes and down-regulated the relative expression of COX17, MT and ATOX1 genes significantly in a dose-dependent manner. The findings of the current study provide important insights that nano copper plays an important role in intestinal epithelial cell injury and oxidative stress.     

褪黑素干预急性坏死性胰腺炎肺损伤及巨噬细胞移动抑制因子表达

目的：研究急性坏死性胰腺炎（acute necrotizing pancreatitis,ANP）大鼠肺组织巨噬细胞移动抑制因子（MIF）、TNF-α的变化及褪黑素（MT）的干预效果。方法：36只大鼠随机分为假手术组（SO组）、急性坏死性胰腺炎组（ANP组）和MT干预组（MT组）,每组12只。应用胰胆管内注射牛磺胆酸钠的方法诱导ANP模型,MT组于诱导模型前30min皮下注射MT。术后4h和24h分批处死大鼠（每个时间点n=6）,用RT—PCR检测肺组织MIF、TNF-αmRNA的表达情况,用SABC免疫组织化学法检测肺脏MIF蛋白表达情况,并行胰腺及肺脏的病理检查。结果：ANP组4、24h肺组织MIF、TNF-αmRNA表达较SO组明显升高（P〈0．05）,肺脏及胰腺病理损伤较严重;MT组较ANP组肺组织MIF、TNF-αmRNA表达下降（P〈0．05）,病理损伤减轻;MIF主要表达于肺脏支气管上皮细胞胞浆。结论：MIF可能参与急性胰腺炎肺损伤的发生,MT可减弱其肺内表达及胰腺炎相关肺损伤的严重程度。     

大黄素对重症急性胰腺炎肠黏膜屏障的保护作用及机制

目的从细胞凋亡方面,探讨大黄素对重症急性胰腺炎（SAP）肠黏膜功能障碍的保护作用及相关作用机制。方法SD 大鼠30 只,按随机数字表法分为假手术组、SAP 组和大黄素组。采用胰管逆行注射3%牛磺胆酸钠溶液制作SAP 模型,大黄素组制作SAP 模型1 h 后给予大黄素25 mg/kg 腹腔注射,2 h 后重复1 次。TUNEL 法检测回肠黏膜细胞凋亡;免疫组化法检测回肠黏膜细胞GRP78 蛋白表达。结果与假手术组相比,SAP 组大鼠肠黏膜细胞凋亡及GRP78 蛋白表达明显增加（P＜0.05）;与SAP 组相比较,大黄素组大鼠肠黏膜细胞凋亡减少（P＜0.05）, GRP78 蛋白表达差异无统计学意义（P＞0.05）。结论大黄素可减少SAP 时的肠黏膜细胞凋亡,保护肠黏膜屏障,但这种保护作用似乎并未涉及到内质网应激途径。     

Tissue- and cell-specific expression of metallothionein genes in cadmium- and copper-exposed mussels analyzed by in situ hybridization and RT-PCR

Metallothioneins (MTs) are metal-inducible proteins that can be used as biomarkers of metal exposure. In mussels two families of MT isoforms (MT10 and MT20) have been characterized. In this study, mussels (Mytilus galloprovincialis) were exposed to 200 ppb Cd and 40 ppb Cu for 2 and 9 days to characterize the tissue and isoform specificity of metal-induced MT expression. Non-radioactive in situ hybridization demonstrated that both MT isoforms were mainly transcribed in digestive tubule epithelial cells, especially in basophilic cells. Weaker MT expression was detected in non-ciliated duct cells, stomach and gill epithelial cells, haemocytes, adipogranular cells, spermatic follicles and oocytes. RT-PCR resulted in cloning of a novel M. galloprovincialis isoform homologous to recently cloned Mytilus edulis intron-less MT10B isoform. In gills, Cd only affected MT10 gene expression after 2 days of exposure while increases in MT protein levels occurred at day 9. In the digestive gland, a marked increase of both isoforms, but especially of MT20, was accompanied by increased levels of MT proteins and basophilic cell volume density (Vv(BAS)) after 2 and 9 days and of intralysosomal metal accumulation in digestive cells after 9 days. Conversely, although metal was accumulated in digestive cells lysosomes and the Vv(BAS) increased in Cu-exposed mussels, Cu exposure did not produce an increase of MT gene expression or MT protein levels. These data suggest that MTs are expressed in a tissue-, cell- and isoform-specific way in response to different metals.     

Metallothionein in cultured human epithelial cells and synovial rheumatoid fibroblasts after in vitro treatment with auranofin

Radioimmunoassay (RIA) and reversed-phase high-pressure liquid chromatography (HPLC) were used to investigate gold-binding proteins of possible metallothionein (MT) nature occurring upon auranofin exposure of cultured human cells. An epithelial cell line (HE) and two sub-strains were examined. The HEAF sub-strain had been made resistant to 2 mumole auranofin/l culture medium. The resistance was associated with the appearance of gold-binding substances with gel filtration characteristics like MT. The HE100 sub-strain had been made resistant to 100 mumole CdCl2/l and contained high amounts of cytosolic Cd-induced MT. In addition, cultured synovial fibroblasts, derived from normal (SN) and rheumatoid (SRA) synovial tissues, were investigated. Evidence was obtained by RIA that the low molecular weight (mol.wt. 6000-7000) gold-binding proteins occurring in the HEAF cells and SRA cells following auranofin exposure, were of MT nature. The relative amounts of MT in the epithelial cell lines were: HE:HEAF:HE100 = 1:18:100. The relative amounts in the synovial fibroblasts were: SN:SRA:SRA treated with auranofin = 1:3:10. The HPLC methods used were found suitable for isolation of Cd-MT in the HE100 cells, but not for the Au-MT in the HEAF cells. By HPLC, the Cd-MT in the HE100 cells was resolved into 3 MT-1 and 1 MT-2 iso-proteins exhibiting the amino acid composition typical of MT. Judged by HPLC, the MT in these cells constituted 0.4% of the cytosolic proteins.     

褪黑素对梗阻性黄疸肠组织ICM-1表达的影响

目的探讨褪黑素对大鼠梗阻性黄疸后肠组织细胞间粘附分子-1(ICAM-1)表达的影响。方法将36只大鼠随机分为3组(每组12只):假手术对照组(SO)、梗阻性黄疸组(OJ)、褪黑素治疗组(MT)。观察各组小肠组织中ICAM-1的表达及小肠组织形态学改变,并测量肠绒毛高度和黏膜厚度。结果梗阻性黄疸组小肠组织ICAM-1的表达明显增强,肠绒毛高度、黏膜厚度明显降低,与假手术对照组有显著性差异(P<0.01,P<0.01);褪黑素治疗组与梗阻性黄疸组比较小肠组织ICAM-1的表达明显减弱(P<0.01),肠绒毛高度、黏膜厚度增高,肠组织结构损害减轻(P<0.01)。结论褪黑素通过抑制梗阻性黄疸后肠组织ICAM-1的表达,减轻小肠黏膜的损伤。     

卵巢上皮性癌中p-AKT、PI3K和PTEN蛋白的表达及意义

目的：探讨磷酸化AKT（p-AKT）、3-羧基磷脂酰肌醇激酶（PI3K）与张力蛋白同源的第10染色体丢失的磷酸酶基因（ PTEN）在卵巢上皮性癌中的表达,分析它们之间的关系及其与卵巢上皮性癌患者预后的关系。方法应用免疫组织化学方法,检测10例正常卵巢组织,20例卵巢良性上皮性肿瘤,60例卵巢上皮性癌组织中p-AKT、PI3K和PTEN的表达。结果 P-AKT和PI3K在正常卵巢组织、卵巢良性上皮性肿瘤中的阳性表达显著低于卵巢上皮性癌组织,而PTEN蛋白在卵巢上皮性癌组织中的表达显著低于正常卵巢组织、良性上皮性肿瘤（ P均＜0．01）。P-AKT、PI3K和PTEN表达与临床分期、病理分级、是否存在淋巴结转移及远处转移有关（ P ＜0．01）,与年龄、病理类型及是否伴有腹水无关（ P ＞0．05）。 P-AKT和PI3K在卵巢上皮性癌中的表达呈正相关（ r ＝0．552, P ＜0．01）,P-AKT和PTEN卵巢上皮性癌中的表达呈负相关（ r ＝-0．497, P ＜0．01）,PI3K和PTEN卵巢上皮性癌中的表达呈负相关（ r ＝-0．535, P ＜0．01）。结论 P-AKT、PI3K的过表达伴随PTEN表达缺失参与卵巢上皮性癌的发生发展。     

己酮可可碱在大鼠肠缺血再灌注损伤中的保护作用及机制

目的:探讨己酮可可碱(pentoxifylline,PTX)在大鼠肠缺血再灌注损伤中的保护作用及机制。方法:Wistar大鼠48只随机分成6组,每组8只。(1)假手术组(S); (2)缺血组(I); (3 )缺血再灌注2h组(IR2h); (4)缺血再灌注4h组(IR4h);(5)缺血再灌注2h+PTX组(P2 ); (6)缺血再灌注4h+PTX组(P4 ),测定各组动物血清肿瘤坏死因子α(TNF α)水平、肠组织细胞粘附分子(ICAM 1 )表达、肠组织过氧化物酶(MPO)活性及观察病理形态。结果:IR2h及IR4h组大鼠血清TNF α水平、肠组织ICAM 1表达及肠MPO活性均明显高于S组(P<0. 01)。给予PTX后, 血清TNF α水平有所下降,P4 组与IR4h组比较差异有非常显著意义(P<0. 01);肠组织MPO活性明显降低,IR2h组与P2 组比较或IR4h组与P4 组比较差异均有非常显著意义(P<0. 01);肠组织ICAM 1积分光密度值比较,P2,P4 组明显低于IR2h及IR4h组(P<0. 01)。结论:已酮可可碱可降低血TNF α水平及肠组织ICAM 1表达,减轻中性粒细胞在肠内聚集、活化,从而防治肠缺血再灌注损伤。     

TRAP1对结直肠癌细胞增殖和凋亡的影响

摘要：目的　探讨肿瘤坏死因子受体相关蛋白1（TRAP1）在结直肠癌中的表达情况及对结直肠癌细胞增殖和凋亡的影响。方法　通过Oncomine数据库分析TRAP1在结直肠癌组织和正常肠黏膜组织中的表达,Western blot检测TRAP1在结直肠癌组织及其配对癌旁黏膜组织、LOVO、HT29、HCT116、RKO、SW480、NCM460细胞中的表达及干扰TRAP1对细胞周期相关蛋白、凋亡相关蛋白及PI3K/AKT通路的影响,CCK-8检测TRAP1对结直肠癌细胞增殖的影响,平板克隆实验检测TRAP1对结直肠癌细胞克隆形成能力的影响,流式细胞术检测TRAP1对细胞周期及凋亡的影响。结果　生物信息学分析显示TRAP1在结直肠癌中的表达高于正常肠黏膜组织,Western blot结果显示TRAP1在结直肠癌组织和细胞中蛋白表达水平均高于正常组织和细胞（P＜0.05）,干扰TRAP1的表达可以显著抑制SW480细胞的增殖及克隆形成能力（P＜0.05）,并且将细胞阻滞在G0/G1期,增加细胞凋亡（P＜0.01）,细胞周期相关蛋白CyclinD1表达降低,凋亡相关蛋白Cleaved-Casp3表达增加,p-PI3K和p-AKT表达降低（P＜0.05）。结论　TRAP1在结直肠癌中高表达,干扰TRAP1的表达可以通过抑制PI3K/AKT通路的激活,从而降低结直肠癌细胞的增殖能力,促进细胞凋亡。     

Cigarette smoking and metallothionein expression in invasive breast carcinomas

Studies indicate that smoking is significantly related to breast cancer mortality. One hypothesis is that this association is due to the elevated expression of proteins associated with resistance to cancer therapies. The metallothioneins (MTs) are a family of proteins that appear to play a role in conferring resistance to certain cancer therapies. This study was carried out to assess whether smoking was associated with MT expression in breast carcinomas. Archival breast tissues with accompanying clinical and epidemiological information were collected from three different cancer centers for 123 women diagnosed with invasive breast cancer. MT expression was assessed using immunohistochemical procedures and semi-quantified using an immunoreactivity score that was obtained by multiplying the percentage of MT-positive tumor cells by MT staining intensity. The results showed that 27.6% of the women were current smokers. Among women whose tissues were collected at the cancer center where enrolled women had primarily early stage, low grade breast cancer, smokers had increased odds, although not significantly, of having a MT-positive tumor compared to non-smokers, independent of cancer stage. This association was not observed among the women whose tissues were collected from the other cancer centers. These findings suggest that among specific groups of women, smoking at the time of breast cancer diagnosis may be associated with an increase in breast tissue MT.