本文選題：銅 + 腦微血管內(nèi)皮細胞��； 參考：《華南農(nóng)業(yè)大學》2016年碩士論文

【摘要】：銅是動物機體必需的微量元素之一。在大腦中,銅參與大量的生物進程,為機體發(fā)揮正常的生理功能扮演著重要的角色。但是,許多神經(jīng)中樞疾病,特別是神經(jīng)退行性疾病也與銅息息相關(guān)。為探究銅與大腦之間的密切關(guān)系,本試驗通過原代培養(yǎng)腦微血管內(nèi)皮細胞(brain microvascular endothelial cells,BMECs)建立體外模型,以30μM、60μM、120μM、180μM、240μM、300μM共6個不同濃度的CuCl2作用于BMECs 12h,從細胞毒性、claudin-1、-3、-5的蛋白水平、基因水平和細胞氧化應激相關(guān)的酶活水平幾個層面來評估不同濃度的銅對BMECs的影響。結(jié)果顯示:1.通過牛血清白蛋白梯度離心和Ⅱ型膠原酶消化的方法分離原代BMECs的方法得當,特異性抗原Ⅷ因子免疫熒光鑒定呈陽性,鈣粘蛋白CD144流式細胞術(shù)鑒定細胞純度高達99%以上。2.銅離子濃度的升高使BMECs的細胞活力呈現(xiàn)先上升后下降的趨勢,120μM銅離子濃度組的細胞活力在各個銅離子濃度組中居于高峰(63%)。胞外乳酸脫氫酶的活性測定結(jié)果與細胞活力的變化趨勢相一致。另外,BMECs對生長的條件與環(huán)境要求較高,不添加胎牛血清的陰性組也會使細胞活力大幅度下降。3.Claudin-1、-3、-5隨著銅離子濃度升高呈現(xiàn)出相似的蛋白表達變化趨勢,均是從低濃度銅離子組到中間濃度銅離子組呈現(xiàn)下調(diào),在180μM銅離子濃度組達到低峰,從中間濃度銅離子組到高濃度銅離子組又呈現(xiàn)上調(diào)。4.Claudin-1、-3、-5的mRNA相對表達量雖然相比于陰性組有著統(tǒng)計學上的差異,但是各個銅離子濃度組之間并沒有明顯的趨勢變化。5.在氧化應激的過程中,銅鋅-超氧化物歧化酶(CuZn-SOD)是BMECs的超氧化物歧化酶(SOD)主要分型,隨著銅離子濃度的升高,CuZn-SOD活性提高,但是銅負載可能存在一個臨界點,在120μM銅離子濃度組之后的Cu-SOD活性呈現(xiàn)下降。從整體上看,過氧化物酶(CAT)活性隨著銅離子濃度的提高而下降,在300μM銅離子濃度組降至最低點。誘導型一氧化氮合酶(iNOS)是BMECs的一氧化氮合酶(NOS)主要分型,隨著銅離子濃度的提高,iNOS的活性在120μM銅離子濃度組達到高峰,隨后又呈下降趨勢。結(jié)論:銅離子易對BMECs產(chǎn)生細胞毒性,但是可能存在最適濃度的銅離子能夠協(xié)助BMECs修復。銅離子能夠介導影響claudin-1、-3、-5的蛋白表達水平,且與濃度有密切關(guān)聯(lián)。銅離子會對BMECs造成氧化損傷。
[Abstract]:Copper is one of the essential trace elements in animals. In the brain, copper is involved in a large number of biological processes and plays an important role in the normal physiological function of the body. However, many neurological diseases, especially neurodegenerative diseases, are also associated with copper. In order to explore the close relationship between copper and brain, the model was established in vitro by primary culture of brain microvascular endothelial cells (BMECs) of brain microvascular endothelial cells. Six different concentrations of CuCl2 (30 渭 m ~ 60 渭 M ~ (60 渭 m), 180 渭 M ~ (20) and 240 渭 M ~ (300 渭 M) were used to treat BMECs for 12 h, and the protein levels of claudin-1 ~ (-1) ~ (-3) ~ (-5) were determined from the cytotoxic effect of 30 渭 m ~ 60 渭 M ~ (60 渭 m) ~ (60 渭 m) on BMECs for 12 h. Gene levels and enzyme activity levels associated with cellular oxidative stress were used to assess the effects of different concentrations of copper on BMECs. The result is 1: 1. The method of separation of primary BMECs by gradient centrifugation of bovine serum albumin and digestion of type 鈪，

本文編號：1932375