發(fā)布時間：2018-02-26 23:09

  本文關(guān)鍵詞： 斯鈣素1 鎘 牛腎細(xì)胞 細(xì)胞凋亡 活性氧　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：斯鈣素1(STC-1)是一種糖蛋白激素,在哺乳動物體內(nèi)的多種器官組織都有分布與表達(dá),屬于多效因子。STC-1可以通過腸道與腎臟調(diào)節(jié)鈣磷代謝。在腎臟中,主要是通過抑制鈣的吸收,同時增加磷的重吸收來調(diào)節(jié)鈣磷穩(wěn)態(tài)。在多種細(xì)胞組織中的研究表明,STC-1還具有抗凋亡的作用,分子機(jī)制尚存在爭議。鎘是一種有蓄積性毒性的重金屬元素。人的鎘中毒已經(jīng)受到普遍關(guān)注,然而對哺乳動物的鎘中毒研究還很缺乏。與人相比,哺乳動物的鎘中毒具有高耐受性和隱蔽性的特點(diǎn)。鎘的主要蓄積部位是動物腎臟并引起腎臟損傷。因此,研究鎘作用細(xì)胞后,STC-1在其中的相關(guān)作用,為STC-1功能研究提供參考,另外,研究STC-1對鎘誘導(dǎo)牛腎細(xì)胞(MDBK細(xì)胞)的作用還可以有助于保障哺乳動物健康和規(guī)范動物食品安全檢測。本實(shí)驗(yàn)對牛腎細(xì)胞(MDBK細(xì)胞)進(jìn)行培養(yǎng),在細(xì)胞對數(shù)生長期添加氯化鎘處理,通過熒光定量PCR與Western-Blot方法對STC-1的基因與蛋白的表達(dá)量進(jìn)行測定。通過添加定量氯化鎘和不同濃度的人斯鈣素(h STC-1)重組蛋白處理細(xì)胞,檢測凋亡基因Caspase3、Bcl-2和解偶聯(lián)蛋白2(UCP-2)的m RNA表達(dá)量變化,流式細(xì)胞儀檢測細(xì)胞凋亡率和活性氧(ROS)產(chǎn)生量,結(jié)果如下:1.鎘對牛腎細(xì)胞STC-1 m RNA和蛋白表達(dá)的影響通過不同濃度的鎘處理細(xì)胞:STC-1基因有不同程度的上調(diào)并具有明顯的劑量與時間依賴性。2h后,STC-1表現(xiàn)為上調(diào)顯著,其中,20μg/m L鎘誘導(dǎo)細(xì)胞8h后,與對照組相比,STC-1基因表達(dá)量是對照組的53倍,差異極顯著,即(P0.01),30μg/m L和50μg/m L組上調(diào)26和16倍,差異顯著(P0.05)。通過定量鎘處理細(xì)胞:STC-1蛋白表達(dá)量上調(diào)明顯并隨時間延長上調(diào)越明顯,2h組、4h組、8h組和12h組與對照組相比STC-1蛋白上調(diào)1.6、17、26.1和26.2倍。2.h STC-1重組蛋白對鎘誘導(dǎo)的細(xì)胞凋亡過程的影響通過定量鎘與不同濃度h STC-1重組蛋白共同處理細(xì)胞:有h STC-1重組蛋白共同處理的實(shí)驗(yàn)組與只有定量鎘處理的對照組相比,Caspase-3、Bcl-2和UCP-2基因有明顯的表達(dá)量變化。實(shí)驗(yàn)組的Caspase-3表現(xiàn)為下調(diào),其中,4h后,0.5μM和2μM組基因下調(diào)為對照組的0.11和0.18倍,差異顯著,即(P0.05),8h后,0.5μM和2μM組基因下調(diào)為對照組的0.13和0.24倍,差異顯著,即(P0.05)。實(shí)驗(yàn)組的Bcl-2表現(xiàn)為上調(diào),其中,2h后,0.5μM組基因上調(diào)為對照組的3.2倍,差異顯著,即(P0.05),4h后,0.5μM組基因上調(diào)為對照組的2.4倍,差異顯著,即(P0.05)。實(shí)驗(yàn)組的UCP-2表現(xiàn)為上調(diào),2μM組基因上調(diào)為對照組的1.9倍,差異顯著,即(P0.05),8h后,0.5μM組基因上調(diào)為對照組的3倍,差異顯著,即(P0.05)。因此,h STC-1重組蛋白在鎘誘導(dǎo)細(xì)胞凋亡過程中,早期上調(diào)Bcl-2和UCP-2明顯,晚期下調(diào)Caspase-3明顯,作用濃度為0.5μM-2μM。通過定量鎘與不同濃度h STC-1重組蛋白共同處理細(xì)胞:0.5、2、10μM h STC-1重組蛋白處理的實(shí)驗(yàn)組的早期凋亡率分別為8.39%、4.23%和4.64%,細(xì)胞ROS產(chǎn)生量的平均值分別為9.88、3.53和8.58,與對照組早期凋亡率13.95%和ROS產(chǎn)生量的平均值19.8相比,說明h STC-1重組蛋白可以明顯抑制凋亡與ROS產(chǎn)生,作用濃度2μM-10μM。結(jié)論:鎘誘導(dǎo)細(xì)胞凋亡中STC-1基因蛋白表達(dá)上調(diào)顯著,提示可以參考STC-1表達(dá)量來判斷鎘的蓄積情況。h STC-1重組蛋白可以有效抑制鎘誘導(dǎo)的細(xì)胞凋亡,分子機(jī)制包括凋亡早期Bcl-2表達(dá)和UCP-2基因的表達(dá),凋亡晚期時抑制促凋亡基因Caspase-3表達(dá)并抑制ROS的產(chǎn)生。本實(shí)驗(yàn)為進(jìn)一步研究STC-1生物功能提供參照。
[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，

本文編號：1540071