小鼠肝脏金属硫蛋白Metallothioneins的分离纯化与鉴定

由健康小鼠通过皮下注射一定量的氯化镉,取肝脏匀浆,离心,经SephadexG-50和DEAE-Sephadex A-50柱层析分离,即可获得MT-Ⅰ和MT-Ⅱ两种金属硫蛋白。经鉴定:两个分子各含18个巯基,结合4个Cd原子和3个Zn原子,无金属蛋白质Thionein的分子量约为6kD,半胱氨酸含量约占氨基酸总量的28％,所提取到的MT-Ⅰ和MT-Ⅱ经氨基酸组成,HPLC和PAGE分析皆证明为高度均一的,且与有关文献报道基本相符。其中MT-Ⅱ已获得晶体。     

顺,反铂配合物诱导生物合成金属硫蛋白的研究

给家兔皮下注射不同结构的铂配合物溶液、对肝、肾组织的可溶性组分进行凝胶和离子交换层析分离,研究了它们诱导金属硫蛋白合成的性质,发现顺铂只选择性地诱导肝脏合成ＭＴ,反铂对肝,肾都不诱导,Ｋ２ＰｔＣｌ４同时可在肝和肾脏中诱导ＭＴ的合成。并通过用Ｚｎ（ＮＯ３）２预先诱导肝和肾中合成ＭＴ,然后每日以２、４、４和８ｍｇＰｔ／ｋｇ的剂量连续皮下注射４天顺铂或反铂溶液后,在肝和肾中分离出大量的铂化硫蛋白,且用反     

孕酮对家猫肝金属硫蛋白的诱导

胰高血糖素、血管紧张素、糖皮质激素均可诱导鼠、兔肝肾等器官金属硫蛋白（ｍｅｔａｌｏｔｈｉｏｎｅｉｎ,ＭＴ）的合成。兔在怀孕早期ＭＴ对从母体转移Ｚｎ至兔胎儿起调节作用。孕酮是家猫妊娠过程中的主要激素,它是否对肝ＭＴ有诱导作用,研究这一课题有助于阐明孕激...     

金属硫蛋白与医学

金属硫蛋白是一类富含巯基与金属的内源性蛋白,化学结构极为特殊,该蛋白的为具有很强的诱导性,金属,糖皮质激素,某些化的和应激状态均可诱导其生成,在炎症等应激过程中ＩＬ－６可能为诱导者。在不降低疗效的情况下该蛋白可解除抗肿瘤药物ＣＤＤＰ的抗药性密切有关,金属硫蛋白与炎性肠病,妊娠中毒症,肝脏疾患等多种疾病密切相关。     

镉诱导家兔肝脏含锌金属硫蛋白的纯化及鉴定

预先给家兔皮下注射CdCl_2四次,取肝脏经匀浆、离心、乙醇沉淀后,通过Sephadex G-50凝胶过滤层析,DEAE Sepharose Fast Flow离子交换层析和Sephadex G-25层析柱脱盐,得到MT的两个“亚型”—MT-1和MT-2,对其进行鉴定,其紫外扫描图谱示在280和250nm处无吸收峰,而最大吸收在220nm处。MT-1和MT-2均不含镉和铜,而每分子结合6个锌。用简化巯基试剂测定,MT-1含18.8个巯基,MT-2含19.1个巯基;HPLC分析MT主要以单体存在,也有少量二聚体。氨基酸组成与Cd-MT一致。整个纯化过程采用示波极谱法和火焰原子吸收光谱法测锌含量。结果表明前者可代替后者。     

金属硫蛋白融合蛋白的分离纯化

采用IPTG诱导新1#菌BL21（DE3）（pET21a-MT)高效表达Balb/C小鼠金属硫蛋白融合蛋白,并采用包涵体分离纯化方法及Sephadex G-75柱层析得到SDS－PAGE电泳为一条带的金属硫蛋白融合蛋白收集液。     

鲫鱼金属硫蛋白的提纯及性质研究

用氯化镉诱导鲫鱼(Carassius aurattus),应用层析介质 Sephacryl S 体系从鱼肝脏中提取金属硫蛋白(MT),并对其性质进行研究.实验表明鲫鱼 MT 的性质与哺乳动物 MT 的性质十分相似.分子量约10 000,含丰富的半胱氨酸,较多的赖氨酸,不含芳香族氨基酸.紫外250nm 处强吸收,280nm 处几乎没有吸收。鱼 MT 含7个金属每分子,并含有20个巯基.测得鲫鱼 MT 等电点 pI 在5.6左右.     

金属硫蛋白与肿瘤

近年来的研究发现,一些肿瘤组织的金属硫蛋白含量增加。致癌过程中ＭＴ的写表达与致癌素作用,癌基因激活和肿瘤迅速生长有关。ＭＴ的加旺表达也是细胞产生耐药的机制之一天肿瘤预防和治疗中,ＭＴ可保护细胞低抗重金属和烷化剂的致癌,致突变作用,ＭＴ还可用于肿瘤临床辅助治疗和用作肿瘤标志物。     

锌诱导金属硫蛋白在文蛤不同组织中的表达

采用体外暴露法对文蛤Meretrixmeretrix进行锌(0、1.5mg/L、3mg/L、6mg/L)染毒,研究不同染毒浓度的锌离子在不同染毒时间下,诱导金属硫蛋白(metallothionein,MT)在文蛤足、肝胰腺、鳃、外套膜组织中的表达差异。取经不同浓度的锌溶液在染毒2d及4d后的不同组织匀浆、离心后经Bio-GelP-10凝胶柱层析纯化,用TU-1810紫外可见分光光度计测定每个样品中金属硫蛋白的吸光值。实验结果显示:同一浓度锌离子染毒后,MT在文蛤4个不同组织中的表达量差异较显著,一般为足>肝胰腺>外套膜>鳃;不同染毒浓度的锌离子在不同染毒时间下诱导MT在不同组织中的表达也不同,具有一定的组织差异性和规律性。     

Liver, Brain, and Heart Metallothionein Induction by Stress

To date, stress has been reported to induce metallothionein (MT) synthesis in the liver only. In the present experiment, the effects of food and water deprivation alone or of immobilization stress plus food and water deprivation on liver, brain, and heart MT have been studied in adult male rats. Liver and brain MT levels were increased by immobilization stress as soon as 6 h after the onset of stress. Eighteen hours of immobilization, which is accompanied by food and water deprivation, further increased liver and brain MT levels and significantly increased heart MT content. A specific effect of immobilization was evident in all three tissues, because the effect of food and water deprivation alone was significantly lower than that of immobilization plus starvation. Changes in MT apparently were not related to changes in cytosolic Zn.     

鼠肝中金属硫蛋白的纯化

旨在探索鼠肝中金属硫蛋白(MT)提取工艺并加以改进。按照经典方法工艺提取MT,用中空纤维柱超滤方法加以改进,依据MT自身特点进行分离和鉴定。结果显示,粗品符合MT特点:分子量在6.5kD左右;无280nm特征吸收峰,加酸后紫外吸收(200-300nm)肩峰消失;通过离子交换可以实现MT1、MT2的分离;通过原子吸收测定,含有锌、铜、镉3种金属,锌含量最高,具有重要的病理生理意义。因此,与其它方法比较,改进后方法更适宜实验室制备。     

Involvement of Oxidative Stress in Paraquat-Induced Metallothionein Synthesis Under Glutathione Depletion

The inhibition of glutathione (GSH) synthesis by -buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

水压转染法在小鼠不同肝叶的表达效率

为了解水压转染法(hydrodynamics-based transfection,HDT)在小鼠肝脏不同肝叶的转染效率,将容量为小鼠体重的10%,绿色荧光蛋白基因质粒pEGFP-C1含量为35μg/只的生理盐水溶液以0.4 mL/s的速度从小鼠尾静脉注射,于注射后不同时间取小鼠各肝叶制备冰冻切片,在荧光显微镜下观察,计数各肝叶的绿色荧光蛋白表达情况。结果显示注射后12 h,绿色荧光蛋白阳性细胞比例最高,从24 h起表达量逐渐减少,至48 h时各肝叶均基本难以检测出绿色荧光蛋白阳性细胞。在12 h观察各肝叶的转染效率如下:右叶、蒂状叶的绿色荧光蛋白阳性细胞约为22%,左叶、中叶、尾状叶约为15%取材部位不同会造成数据分析的极显著差异。     

神经生长因子在不同周龄小鼠睾丸组织中的表达

目的研究神经生长因子在小鼠不同周龄睾丸组织中的定量和定位表达。方法分别剖取不同周龄雄性小鼠的睾丸组织,部分提取总RNA,real-time PCR相对定量分析神经生长因子mRNA的表达量;另外部分组织固定、包埋,进行SABC法免疫组化分析,以观察神经生长因子蛋白在各周睾丸组织中的定位。结果Real-timePCR定量分析表明:小鼠生后1周龄睾丸组织有神经生长因子mRNA的表达,生后3周龄表达量达峰值,5周之后随鼠龄的增加呈下降趋势,成年小鼠睾丸组织的神经生长因子mRNA表达维持在一定水平。免疫组化定位分析显示:睾丸组织的神经生长因子蛋白表达于小鼠出生后的各个时期内,1周龄睾丸组织免疫阳性反应主要位于支持细胞,精原细胞也有着色;3周龄睾丸组织的间质细胞、各级生精细胞、支持细胞、管周肌样细胞表达均呈现阳性;5周后的睾丸组织内神经生长因子呈低水平表达,主要表达于间质细胞和生精细胞内。结论神经生长因子mRNA的表达量随着小鼠睾丸的生长发育期存在着一定的规律性变化;神经生长因子蛋白的表达在小鼠睾丸生长发育的不同时期其主要表达部位不同。     

Ultrastructural Changes in the Kidney of Rats with Acute Exposure to Cadmium and Effects of Exogenous Metallothionein

Ultrastructural changes in the kidneys of rats after acute cadmium exposure and the effects of exogenous metallothionein (MT) were studied by transmission electron microscopy. Thirty-six adult Wistar rats were divided into three groups. Cadmium chloride (CdCl₂) (3.5 mg/kg/day) was injected subcutaneously in the first group. In the second group, 30 μmol/kg MT was administered in addition to CdCl₂. Control rats received 0.5 ml subcutaneous saline solution. Four rats from each group were killed on days 1, 3, 5, and 7 after administration of the compounds. Kidney tissues were taken and fixed in 2.5% glutaraldehyde solution for electron microscopic observations. Tissue damage in kidney increased as time passed since the administration of CdCl₂ in the first group. Degeneration in the proximal and distal tubules was observed. Increased apoptosis was seen in the proximal tubules epithelium, especially on day 7. Peritubular capillaries became dilated, there was degeneration of the endothelial cells, and the amount of intertubular collagen fibers was increased. On day 1, irregular microvilli in the proximal tubules, deepening of the basal striations, and myelin figures; on day 3, multiple vesicular mitochondria and regions of edema around tubules; on days 5 and 7, increased apoptotic cell in the proximal tubules and widened rough endoplasmic reticulum of the endothelial cells of glomerular capillaries were observed. We observed that the structural alterations that increased depending on the day of Cd administration decreased after exogenous MT administration, the dilation of the peritubular capillaries persisted, and there were degenerated proximal tubules. It was established that cadmium chloride was toxic for kidney cortex and caused structural damage. Exogenous MT partly prevents CdCl₂-induced damage.     

Different responses of metallothionein and leptin induced in the mouse by fasting stress

Changes in metallothionein (MT) and leptin under fasting stress were studied. MT content in the liver of mice increased markedly during continuous fasting periods up to 66 h. Hepatic content of MT increased significantly in mice during the first three cycles of alternate daily fasting-feeding, and then the rate of increase gradually decreased with repetition of this cycle. At the end of 10 cycles, the hepatic MT content was still greater in stressed mice than in the control, although the rate of increase decreased. On the other hand, the plasma concentration of leptin decreased dramatically during continuous fasting. The plasma leptin level recovered to the basal level at the end of 10 cycles of fasting-feeding. These data indicate that MT induction was strongly increased, but leptin was scarcely induced under the continuous fasting stress, and that rates of the changes in MT and leptin levels were gradually reduced under the repeated fasting stress, which may result from an inborn tolerance. Fasting presumably causes translocation of zinc from intracellular to extracellular space, from which it is taken into target organs. The increased zinc-bound MT under the continuous fasting may partly result from the need to maintain zinc and protect tissues against oxidative damage.     

Antibody Responses in Sera of Different Mouse Strains Experimentally Infected with Neodiplostomum seoulense

To examine humoral immune responses in the host, we measured serum antibody levels in different strains of mice (ICR, BALB/c, and C3H) experimentally infected with Neodiplostomum seoulense. Specific IgG antibody levels were increased remarkably with little difference among 3 strains of mice infected with N. seoulense from day 7 to 35 post-infection. More target proteins of adult parasites reacted with IgG at the time when the worm recovery decreased compared with other times. More than 20 protein bands, from 14 kDa to 94 kDa in size, were separated from the crude antigen of N. seoulense adults by SDS-PAGE, and among them 26, 30, 35, 43, 54, 67, and 94 kDa proteins were the major antigenic proteins. The results suggest that significant IgG antibody responses occur against N. seoulense in mice and this may be related with expulsion of worms.     

高温应激下补充L-精氨酸对小鼠免疫功能的影响

探讨补充L 精氨酸对高温应激小鼠免疫功能的影响 ,结果显示 :1.高温应激后各项免疫指标均有显著下降 ,其中 2～ 8h免疫功能受到最大损害 ;2 .补充适量L 精氨酸可减轻小鼠胸膜和脾脏急性萎缩 ;3.补充L 精氨酸后淋巴细胞增殖活性、IL - 2浓度和IL - 2R的表达与对照组比较有显著上升。 4.补充L 精氨酸组的活化胸腺细胞 [Ca2 + ]显著高于给水对照组 ,说明L 精氨酸对热应激小鼠胸腺具有保护作用。实验观察到当补充L 精氨酸浓度达到 1.5mg/g .bw时 ,四项指标效果最好 ,揭示该浓度可最大程度缓解热应激对小鼠免疫功能的抑制