斯钙素1在镉诱导牛肾细胞中的抗凋亡作用研究
发布时间：2018-02-26 23:09
本文关键词： 斯钙素1 镉 牛肾细胞 细胞凋亡 活性氧 出处：《华中农业大学》2015年硕士论文 论文类型：学位论文
【摘要】：斯钙素1(STC-1)是一种糖蛋白激素,在哺乳动物体内的多种器官组织都有分布与表达,属于多效因子。STC-1可以通过肠道与肾脏调节钙磷代谢。在肾脏中,主要是通过抑制钙的吸收,同时增加磷的重吸收来调节钙磷稳态。在多种细胞组织中的研究表明,STC-1还具有抗凋亡的作用,分子机制尚存在争议。镉是一种有蓄积性毒性的重金属元素。人的镉中毒已经受到普遍关注,然而对哺乳动物的镉中毒研究还很缺乏。与人相比,哺乳动物的镉中毒具有高耐受性和隐蔽性的特点。镉的主要蓄积部位是动物肾脏并引起肾脏损伤。因此,研究镉作用细胞后,STC-1在其中的相关作用,为STC-1功能研究提供参考,另外,研究STC-1对镉诱导牛肾细胞(MDBK细胞)的作用还可以有助于保障哺乳动物健康和规范动物食品安全检测。本实验对牛肾细胞(MDBK细胞)进行培养,在细胞对数生长期添加氯化镉处理,通过荧光定量PCR与Western-Blot方法对STC-1的基因与蛋白的表达量进行测定。通过添加定量氯化镉和不同浓度的人斯钙素(h STC-1)重组蛋白处理细胞,检测凋亡基因Caspase3、Bcl-2和解偶联蛋白2(UCP-2)的m RNA表达量变化,流式细胞仪检测细胞凋亡率和活性氧(ROS)产生量,结果如下:1.镉对牛肾细胞STC-1 m RNA和蛋白表达的影响通过不同浓度的镉处理细胞:STC-1基因有不同程度的上调并具有明显的剂量与时间依赖性。2h后,STC-1表现为上调显著,其中,20μg/m L镉诱导细胞8h后,与对照组相比,STC-1基因表达量是对照组的53倍,差异极显著,即(P0.01),30μg/m L和50μg/m L组上调26和16倍,差异显著(P0.05)。通过定量镉处理细胞:STC-1蛋白表达量上调明显并随时间延长上调越明显,2h组、4h组、8h组和12h组与对照组相比STC-1蛋白上调1.6、17、26.1和26.2倍。2.h STC-1重组蛋白对镉诱导的细胞凋亡过程的影响通过定量镉与不同浓度h STC-1重组蛋白共同处理细胞:有h STC-1重组蛋白共同处理的实验组与只有定量镉处理的对照组相比,Caspase-3、Bcl-2和UCP-2基因有明显的表达量变化。实验组的Caspase-3表现为下调,其中,4h后,0.5μM和2μM组基因下调为对照组的0.11和0.18倍,差异显著,即(P0.05),8h后,0.5μM和2μM组基因下调为对照组的0.13和0.24倍,差异显著,即(P0.05)。实验组的Bcl-2表现为上调,其中,2h后,0.5μM组基因上调为对照组的3.2倍,差异显著,即(P0.05),4h后,0.5μM组基因上调为对照组的2.4倍,差异显著,即(P0.05)。实验组的UCP-2表现为上调,2μM组基因上调为对照组的1.9倍,差异显著,即(P0.05),8h后,0.5μM组基因上调为对照组的3倍,差异显著,即(P0.05)。因此,h STC-1重组蛋白在镉诱导细胞凋亡过程中,早期上调Bcl-2和UCP-2明显,晚期下调Caspase-3明显,作用浓度为0.5μM-2μM。通过定量镉与不同浓度h STC-1重组蛋白共同处理细胞:0.5、2、10μM h STC-1重组蛋白处理的实验组的早期凋亡率分别为8.39%、4.23%和4.64%,细胞ROS产生量的平均值分别为9.88、3.53和8.58,与对照组早期凋亡率13.95%和ROS产生量的平均值19.8相比,说明h STC-1重组蛋白可以明显抑制凋亡与ROS产生,作用浓度2μM-10μM。结论:镉诱导细胞凋亡中STC-1基因蛋白表达上调显著,提示可以参考STC-1表达量来判断镉的蓄积情况。h STC-1重组蛋白可以有效抑制镉诱导的细胞凋亡,分子机制包括凋亡早期Bcl-2表达和UCP-2基因的表达,凋亡晚期时抑制促凋亡基因Caspase-3表达并抑制ROS的产生。本实验为进一步研究STC-1生物功能提供参照。
[Abstract]:Stanniocalcin 1 (STC-1) is a glycoprotein hormone in various organs and tissues of mammals have distribution and expression, belong to multiple effect factor.STC-1 can regulate calcium and phosphorus metabolism through the intestine and kidney. In the kidney, primarily by inhibiting the absorption of calcium, phosphorus absorption and increase to regulate calcium and phosphorus homeostasis research in a variety of tissues showed that STC-1 also has anti apoptosis, the molecular mechanism is still controversial. Cadmium is a heavy metal accumulation toxicity. The cadmium poisoning has been widespread concern, but research on cadmium poisoning in mammals is still lacking. Compared with others, it has the characteristics of high tolerance and concealment in mammals. The main site of cadmium accumulation of cadmium poisoning is animal kidney and cause kidney injury. Therefore, study on the effect of cadmium in STC-1 cells, the function of the related, provide a reference for the further study of STC-1 function, In addition, the research of STC-1 on cadmium induced bovine kidney cells (MDBK cells) function can also help to ensure the healthy and standard mammalian animal food safety detection. The experiment of bovine kidney cells (MDBK cells) were cultured with cadmium chloride treatment in the logarithmic growth period, were determined by fluorescence quantitative PCR and Western-Blot expression the gene and protein of STC-1. By adding different concentrations of cadmium chloride and quantitative human stanniocalcin-1 (H STC-1) recombinant protein treated cells, apoptosis gene Caspase3, Bcl-2 and uncoupling protein 2 (UCP-2) expression of M RNA and flow cytometry to detect cell apoptosis and reactive oxygen species (ROS) production, the results are as follows: 1. effects of cadmium on renal expression of STC-1 cells m RNA and protein by different concentrations of cadmium treated cells: the STC-1 gene was increased in different degrees and has obvious dose and time dependent.2h After STC-1 was up-regulated, among them, 20 g/m L induced by cadmium in 8h cells, compared with control group, STC-1 gene expression was 53 times higher than the control group, significant difference, namely (P0.01), 30 g/m L and 50 g/m in group L increased 26 and 16 times, significantly different (P0.05). Through the quantitative treatment of cadmium cells: the expression of STC-1 protein was significantly increased with the prolongation of time and increase more obviously, 2h group, 4H group, 8h group and 12h group compared with the control group, STC-1 protein upregulation of 1.6,17,26.1 and 26.2 times.2.h STC-1 recombinant protein on apoptosis induced by cadmium in common cells treated by quantitative cadmium with different concentrations of H recombinant protein STC-1: experimental group H STC-1 recombinant protein CO treatment compared with the control group, only quantitative cadmium treatment of Caspase-3, Bcl-2 and UCP-2 gene expression changes obviously. Caspase-3 showed the experimental group was reduced, which, after 4h, 0.5 M and 2 M group were down regulated 涓哄鐓х粍鐨，
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