ELISA竞争法测定人尿金属硫蛋白的初步应用

探讨应用ELISA竞争法测定正常人尿金属硫蛋白含量的价值。 [方法 ]应用ELISA法测定了2 0～ 2 3岁健康在校大学生 93份 (男 49,女 44 )晨尿样品的MT。 [结果 ]本法的检测下限为 2 5ng/ml,操作内和操作间的变异系数分别为 2 2 5 %和 1 19% ,回收率为 97 6 %～ 10 0 0 % ;人尿MT含量为 (6 86± 6 0 2 )ng/ μmol肌酐。 [结论 ]本法具有较好的精密度和准确度 ;大学生尿MT含量无性别差异。     

Immunohistochemical localization of metallothionein and p53 protein in pancreatic serous cystadenomas

Introduction  The objective of this study was to determine the expression levels of metallothionein (MT) and p53 protein, recognized neoplastic transformation markers, in pancreatic serous cystadenomas (SCA) and adenomocarcinomas. Materials and Methods  Neoplastic pancreatic tissue was taken from 20 patients with diagnosed benign (SCA: 5 cases) or malignant tumors (adenomocarcinomas: 15 cases) and control pancreatic tissue from healthy persons who had died in car accidents. Sections were stained with hematoxylin-eosin. Immunohistochemical localization of MT and p53 protein was carried out by LSAB2-HRP using specific antibodies against MT and p53. Results  Metallothionein expression was observed only in the epithelial cells of the neoplastic tissue of SCAs. MT expression in the cystadenomas was weaker than in the healthy pancreatic tissue. No tissue was found with p53 protein expression. In the adenomocarcinomas, positive staining for MT was observed in 67% and p53 was positive in the carcinoma cells. Conclusion  The weak MT expression and lack of p53 protein expression in pancreatic SCAs confirms the lack of local invasive potential of the neoplastic lesion. Increased expressions of MT and p53 were observed in the less differentiated tumors. Thus the expression of MT may be a potential prognostic marker for tumors.     

In vitro bioavailability of heavy metals in pressure-treated wood dust.

Pressure treatment with chromium, copper, and arsenic (CCA) is the most prevalent method for protecting wood used in outdoor construction projects. Although these metals are tightly bound to the wood fibers and are not released under most conditions of use, we examined the bioavailability of metals in CCA pressure-treated wood dust in vitro. Cytotoxicity and metallothionein (MT) mRNA expression were examined in V79 Chinese hamster lung fibroblast cells incubated with respirable-size wood dust generated by sanding CCA-treated and untreated (control) Southern yellow pine. In colony survival studies, increased cytotoxicity (p < 0.05) occurred in V79 cells treated with CCA wood dust (351 +/- 77 microg/ml, mean +/- SE) compared with control wood dust (883 +/- 91 microg/ml). Increased cytotoxicity with CCA wood dust also occurred in an arsenic resistant subline of V79 cells, thus suggesting that arsenic was not responsible for the increased cytotoxicity. Metallothionein mRNA was significantly increased after 48 h of treatment with CCA wood dust compared with control wood dust. Incubation of CCA wood dust in cell culture media resulted in the transfer of copper, but not chromium or arsenic, into the media. Moreover, the treatment of cells with this filtered extract resulted in significantly increased metallothionein mRNA, suggesting that bioavailable copper is responsible for inducing metallothionein mRNA in V79 cells. Thus, these bioassays suggest that metals become bioavailable during in vitro culture of phagocytic V79 cells with CCA wood dust.     

Developmental toxicity of dietary zinc deficiency in New Zealand white rabbits

Chemically induced maternal Zn deficiency has been shown previously to cause terata and increase embryonic loss in rodents. To examine the potential effects of Zn deficiency in the rabbit, a major developmental toxicity test species, rabbit dams were fed an ethylenediamine-tetraacetic acid-washed alfalfa-based Zn-deficient diet (−Zn) or the same diet replete with 80 ppm Zn (control) from Gestation Day (GD) 0 through 20. A third group of animals was pair fed to match the mean daily feed consumption levels of the <2 ppm Zn group. By GD 7, maternal serum Zn levels of the − Zn dams were decreased 56% and reached a nadir with a 75% decrease of serum Zn by GD 14. Zinc concentrations in the visceral yolk sac and visceral yolk sac-exoceolomic fluid were decreased 30% and 50%, respectively, by GD 11. Although GD 11 embryonic Zn levels were not affected, the embryos from Zn-deficient dams exhibited decreased head length, somite number, and total protein. On GD 28, a significant increase in resorptions/litter was noted in the − Zn group, and the incidence of totally resorbed litters of the −Zn group was greater than laboratory historical control values. No terata were observed in GD 28 fetuses. This study indicates that Zn deficiency occurring during the standard dosing period of guideline rabbit developmental toxicity studies may be associated with a modest increase in resorption rate and a transient inhibition of embryonic growth, but in contrast to rodent species, does not appear to be teratogenic.     

镉饱和法测定小白鼠肝中金属硫蛋白

金属硫蛋白（ＭＴ）是一种低分子的蛋白质，含大量的半胱氨酸，并对金属有较高的亲合力，镉饱和法测定金属硫蛋白正是利用这些性质。在本研究中，小白鼠经口给醋酸锌，１６小时后取出肝脏并用镉饱和法测其ＭＴ的含量。结果显示方法有较好的重复性，而且肝ＭＴ的含量随Ｚｎ剂量的增加而增加。     

同型半胱氨酸和金属硫蛋白对血管内皮细胞的影响

目的 :观察同型半胱氨酸 (Hcy)血症时小鼠肝、心、肾组织金属硫蛋白 (MT)含量的变化 ,以及MT对Hcy损伤内皮细胞的影响。 方法 :①昆明种小鼠 12只 ,分为对照组和高Hcy组 ,高Hcy组小鼠腹腔注射Hcy(16μmol/kg) ,复制高Hcy血症模型 ,对照组同法注入等量生理盐水。以Cd 血红素饱和法测肝、心和肾组织MT水平。②培养人血管内皮细胞 ,分为空白对照组 ,单用Hcy(1mmol/L)组及MT(2× 10 -5mol/L)和Hcy(1mmol/L)同时应用组 ,孵育人血管内皮细胞 6h测定细胞存活率、培养液乳酸脱氢酶 (LDH)活性和蛋白漏出量。结果 :Hcy组小鼠肝、心和肾组织MT水平较对照组分别增加 2 10 %(P <0 .0 1)、133%(P <0 .0 5 )和 6 0 %(P <0 .0 1)。MT +Hcy组培养的内皮细胞存活率较Hcy组培养的内皮细胞升高 13.9%(P <0 .0 1) ,培养液LDH和蛋白漏出量较Hcy组分别降低 2 7.1%(P <0 .0 5 )和 31.4%(P <0 .0 1)。结论 :Hcy诱导小鼠肝、心、肾组织MT生成 ,MT拮抗Hcy的细胞损伤作用。     

Effect of oral zinc supplementation on metallothionein and superoxide dismutase concentrations in patients with inflammatory bowel disease

Abstract Oxygen-derived free radicals may contribute to intestinal tissue damage in inflammatory bowel disease. The concentrations of metallothionein and superoxide dismutase, two copper and zinc containing proteins involved in the scavenging of free radicals, were previously found to be decreased in the intestinal mucosa of patients with this disorder. The plasma zinc concentration is often decreased also in these patients. Since zinc is reported to be an efficient inducer of metallothionein synthesis, and probably of superoxide dismutase, we evaluated the effect of oral zinc supplementation on metallothionein and superoxide dismutase levels in patients with inflammatory bowel disease. Fourteen patients with inactive to moderately active inflammatory bowel disease received oral zinc supplementation (300 mg zinc aspartate, equal to 60 mg elemental zinc per day) for 4 weeks in a placebo-controlled double-blind cross-over trial. The plasma zinc concentration of these patients was low at the start of the study (12.2 ± 1.7 μmol/L, P <0.05), when compared to that of 22 healthy controls (13.6 ± 2.3 μmol/L), but increased (P <0.05) towards the levels of controls during the supplementation period (13.3 ± 2.5 μmol/L). The concentrations of metallothionein and superoxide dismutase in plasma and in erythrocytes did not change in relation to the supplementation. The metallothionein concentration in both inflamed and non-inflamed intestinal mucosa was slightly higher after zinc supplementation but the superoxide dismutase concentration in the tissue was not altered. The histological inflammation score of intestinal biopsies, plasma albumin levels, and the disease activity index of the patients did not change during the study. Thus, although zinc supplementation therapy increased plasma zinc concentrations, there was no effect on the plasma, erythrocyte and mucosal metalloprotein levels in inactive to moderately active patients with inflammatory bowel disease.     

锌对小鼠肝金属硫蛋白合成的影响

本文研究了小鼠在宽的剂量范用内（１０，２０，４０，８０，１００，１２０，１４０，１６０，１８０，２００ｍｇ／ｋｇｂｗ）口服锌（Ｚｎ）盐时，对肝金属硫蛋白（ＭＴ）合成的影响。结果显示：因服用过量Ｚｎ（２＋），按剂量──效应关系发生肝ＭＴ的诱导合成，而且在肝Ｚｎ浓度和肝ＭＴ浓度之间存在正向关系。表明Ｚｎ是一个ＭＴ的诱导剂。在口服Ｚｎ８０～１００ｍｇ／ｋｇ的剂量范围时，无论是肝ＭＴ水平或肝Ｚｎ水平均有明显的改变。提示：在Ｚｎ诱导ＭＴ合成中，存在一个敏感的剂量范围。     

PP2A B56α亚基介导镉诱导的细胞毒性

目的： 探讨蛋白磷酸酶2A(PP2A)B56α亚基在调控CdCl2诱导的细胞毒性中的作用及其机制。方法：利用病毒感染法在人胚肾上皮细胞HEK上构建PP2A B56α表达降低的细胞株模型(HEK-SHB56α-1和HEK-SHB56α-2),采用改良四甲基偶氮唑盐(MTT)法检测CdCl2诱导的细胞毒性。用不同浓度(0、10、20、40和80 μmol/L)CdCl2联合或不联合c-Jun氨基末端激酶(JNK)抑制剂SP600125染毒细胞不同时间(0、2、6、12和24 h),采用免疫印迹检测B56α亚基、金属硫蛋白(MT)的表达和JNK的磷酸化水平。结果：免疫印迹结果证实细胞株HEK-SHB56α-1和HEK-SHB56α-2构建成功。与对照组比较,PP2A B56α表达抑制导致镉诱导的细胞毒性减小,JNK的磷酸化水平和MT的表达水平均显著增加。用不同剂量CdCl2处理细胞株12 h,发现B56α表达抑制导致JNK磷酸化(p-JNK)分别增加了2.78和1.26倍(P<0.05),MT的表达也分别增加了1.36和1.19倍(P<0.05)。用p-JNK抑制剂SP600125作用时,p-JNK的表达降低了35%~38%(P<0.05),MT的表达下降了13%~35%(P<0.05), CdCl2诱导的细胞毒性显著增加(P<0.05)。此外,用CdCl2处理细胞不同时间点,B56α的表达逐渐降低(P<0.05),而p-JNK和MT的表达水平呈逐渐增加趋势(P<0.05)。结论：PP2A B56α在调控CdCl2诱导的细胞毒性中起着重要作用,其作用机制可能通过介导JNK的去磷酸化调控MT的表达而发挥作用。本研究揭示了PP2A参与重金属应激的关键靶点和信号通路的调控。