發(fā)布時間：2018-01-12 11:43

  本文關(guān)鍵詞：p38絲裂原活化蛋白激酶通路及其下游因子NF-κB對紫杉醇誘發(fā)細胞凋亡中γ-氨基丁酸B型受體表達變化的影響　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 紫杉醇 凋亡 p38MAPKs GABAB受體 NF-kB

【摘要】：目的:通過特異性給予p38絲裂原活化蛋白激酶(p38MAPKs)抑制劑(SB203580)和核因子k B(NF-k B)抑制劑(SN50)的方法,探討紫杉醇誘發(fā)細胞凋亡中p38MAPKs通路及其下游因子NF-k B對γ-氨基丁酸B型(GABAB)受體表達變化的影響。方法:SD大鼠新生24h,斷頭取腦,用含有木瓜蛋白酶(瑞士,Acros公司)的DMEM浸泡已經(jīng)分離好的海馬組織,放入培養(yǎng)箱30min。機械吹打后種植于多聚賴氨酸包被好的培養(yǎng)板中,6~8h更換含有雙抗(美國,Gibco公司)和人白細胞抗原(B27)(美國,Gibco公司)的Neurobasal(美國,Invitrogen公司)培養(yǎng)基。隨后,每間隔兩天更換細胞培養(yǎng)液(半量更換),并用光學(xué)顯微鏡觀察海馬神經(jīng)元生長情況,培養(yǎng)5天后用于實驗。選取原代培養(yǎng)5d、濃度約為1×106/ml的海馬細胞,給予不同濃度(0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L)紫杉醇,采用MTT法及流式細胞儀檢測法分別檢測海馬神經(jīng)元細胞抑制率及凋亡率變化,以確定紫杉醇誘發(fā)細胞凋亡的最適條件(最適濃度及處理時間),作為蛋白測定的實驗條件。憑據(jù)MTT比色法檢測所得實驗數(shù)據(jù),計算細胞抑制率=(1-實驗組AD值/對照組AD值)×100%,然后用改良寇氏公式(lg IC50=Xm-I(P-(3-Pm-Pn)/4))計算神經(jīng)元的半數(shù)抑制濃度(IC50),用IC50作為紫杉醇最適濃度用于進一步實驗。本實驗重復(fù)4次,觀察不同藥物濃度作用不同時間對神經(jīng)元抑制率的影響,計算其均數(shù);憑據(jù)流式細胞儀檢測法所測實驗數(shù)據(jù),以膜聯(lián)蛋白V(Annexin V)為橫坐標軸,PI為縱坐標軸,左上側(cè)的象限為機械性的損傷細胞;右上側(cè)的象限為晚期的凋亡細胞或者壞死細胞;左下側(cè)的象限為正常陰性的細胞;右下側(cè)的象限為早期的凋亡細胞。通過流式細胞儀檢測的凋亡率驗證MTT比色法檢計算的紫杉醇最適濃度的可靠性。另選取原代培養(yǎng)5d的海馬細胞隨機分為六組:對照組(C組),10μmol/L SB203580處理組(K1組),53μg/ml SN50處理組(K2組),紫杉醇最適濃度處理組(N組),10μmol/L SB203580+紫杉醇最適濃度混合組(K1+N組),53μg/ml SN50+紫杉醇最適濃度混合組(K2+N組)。培養(yǎng)時間為24h,保持各組加液量一致,采用蛋白質(zhì)印跡法(Western blot法)測定海馬神經(jīng)元細胞的GABAB受體及核因子k B(NF-k B)蛋白表達相關(guān)水平(n=5,x_±s)。結(jié)果:原代培養(yǎng)海馬神經(jīng)元起初小而圓、透亮并懸浮在細胞液中。24h后可通過光學(xué)顯微鏡觀察到大部分細胞為梭形并長出細長突起。3天后胞漿豐富,突起較前明顯增粗、增長。5天進入對數(shù)生長期,神經(jīng)元胞體豐滿,突起交織成更加稠密的網(wǎng)絡(luò)。紫杉醇抑制海馬神經(jīng)元活力是與時間和濃度交互相關(guān)的(F=9.127,P0.05)。在對凋亡率的分析中,由于右上象限中包含晚期凋亡細胞或者壞死細胞,所以本實驗中凋亡率的測定主要依據(jù)右下象限的早期凋亡細胞。1μmol/L紫杉醇處理24h誘發(fā)的細胞早期凋亡率為(48.63±5.76)%,為測定NF-k B及GABAB受體蛋白的實驗條件。蛋白表達結(jié)果:與C組相比,N組、K1+N組和K2+N組NF-k B與GABAB受體表達均明顯增高(P0.05),而K1組和K2組NF-k B與GABAB受體表達均降低(P0.05);與K1組相比,N組和K1+N組NF-k B和GABAB受體表達均增高(P0.05);與K2組相比,N組和K2+N組NF-k B和GABAB受體表達均增高(P0.05);與N組相比,K1+N組和K2+N組NF-k B和GABAB受體表達均降低(P0.05)。結(jié)論:在紫杉醇誘發(fā)細胞凋亡過程中,激活p38MAPKs通路可上調(diào)GABAB受體表達,而其下游因子NF-k B可能是其調(diào)控GABAB受體表達變化的關(guān)鍵靶點,NF-k B可通過下調(diào)GABAB受體蛋白表達抑制紫杉醇誘發(fā)細胞凋亡。
[Abstract]:Objective: through the specific administration of p38 mitogen activated protein kinase (p38MAPKs) inhibitor (SB203580) and K B (NF-k nuclear factor B) inhibitor (SN50) method of gamma aminobutyric acid type B p38MAPKs pathway and its downstream factor NF-k in apoptosis of B cells induced by taxol alcohol (GABAB) affect the expression of receptors. Methods: neonatal SD rats 24h, decapitated, with papain (Switzerland, Acros) has a good separation of DMEM immersion into the incubator hippocampus, 30min. mechanical percussion were seeded on polylysine coated culture plate, 6~8h replaced with double anti (America, Gibco company) and human leukocyte antigen (B27) (American Gibco company) Neurobasal (American Invitrogen company) medium. Then, every two days to replace cells (half replacement), and optical microscope was used to observe the growth of hippocampal neurons cultured for 5 days, for real Check. Select the primary culture of 5D concentration is about 1 x 106/ml cells in hippocampus, with different concentrations (0.01 mol/L, 0.1 mol/L, 1 mol/L, 10 mol/L) taxol, were determined by MTT assay and hippocampal neuron cell inhibition rate and apoptosis rate were detected by flow cytometry method to to determine the optimum condition of taxol induced apoptosis of cells (the optimal concentration and treatment time), as experimental conditions for protein determination. The detection of the experimental data according to MTT colorimetric method, calculate the cell inhibition rate (1- = AD value of experimental group / control group AD values) * 100%, and then use the improved type LG (Shigong Curtis IC50=Xm-I (P-) /4 (3-Pm-Pn)) half inhibitory concentration calculation neurons (IC50), using IC50 as the optimal concentration of paclitaxel for further experiments. The experiment was repeated 4 times, observe the different drug concentration at different time inhibition effects on neurons, calculate the mean number of credentials; flow cytometry 鎵，

本文編號：1414130