p38丝裂原活化蛋白激酶通路及其下游因子NF-κB对紫杉醇诱发细胞凋亡中γ-氨基丁酸B型受体表达变化的影响

发布时间：2018-01-12 11:43

  本文关键词：p38丝裂原活化蛋白激酶通路及其下游因子NF-κB对紫杉醇诱发细胞凋亡中γ-氨基丁酸B型受体表达变化的影响　出处：《河北医科大学》2015年硕士论文　论文类型：学位论文

  更多相关文章： 紫杉醇 凋亡 p38MAPKs GABAB受体 NF-kB

【摘要】：目的:通过特异性给予p38丝裂原活化蛋白激酶(p38MAPKs)抑制剂(SB203580)和核因子k B(NF-k B)抑制剂(SN50)的方法,探讨紫杉醇诱发细胞凋亡中p38MAPKs通路及其下游因子NF-k B对γ-氨基丁酸B型(GABAB)受体表达变化的影响。方法:SD大鼠新生24h,断头取脑,用含有木瓜蛋白酶(瑞士,Acros公司)的DMEM浸泡已经分离好的海马组织,放入培养箱30min。机械吹打后种植于多聚赖氨酸包被好的培养板中,6~8h更换含有双抗(美国,Gibco公司)和人白细胞抗原(B27)(美国,Gibco公司)的Neurobasal(美国,Invitrogen公司)培养基。随后,每间隔两天更换细胞培养液(半量更换),并用光学显微镜观察海马神经元生长情况,培养5天后用于实验。选取原代培养5d、浓度约为1×106/ml的海马细胞,给予不同浓度(0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L)紫杉醇,采用MTT法及流式细胞仪检测法分别检测海马神经元细胞抑制率及凋亡率变化,以确定紫杉醇诱发细胞凋亡的最适条件(最适浓度及处理时间),作为蛋白测定的实验条件。凭据MTT比色法检测所得实验数据,计算细胞抑制率=(1-实验组AD值/对照组AD值)×100%,然后用改良寇氏公式(lg IC50=Xm-I(P-(3-Pm-Pn)/4))计算神经元的半数抑制浓度(IC50),用IC50作为紫杉醇最适浓度用于进一步实验。本实验重复4次,观察不同药物浓度作用不同时间对神经元抑制率的影响,计算其均数;凭据流式细胞仪检测法所测实验数据,以膜联蛋白V(Annexin V)为横坐标轴,PI为纵坐标轴,左上侧的象限为机械性的损伤细胞;右上侧的象限为晚期的凋亡细胞或者坏死细胞;左下侧的象限为正常阴性的细胞;右下侧的象限为早期的凋亡细胞。通过流式细胞仪检测的凋亡率验证MTT比色法检计算的紫杉醇最适浓度的可靠性。另选取原代培养5d的海马细胞随机分为六组:对照组(C组),10μmol/L SB203580处理组(K1组),53μg/ml SN50处理组(K2组),紫杉醇最适浓度处理组(N组),10μmol/L SB203580+紫杉醇最适浓度混合组(K1+N组),53μg/ml SN50+紫杉醇最适浓度混合组(K2+N组)。培养时间为24h,保持各组加液量一致,采用蛋白质印迹法(Western blot法)测定海马神经元细胞的GABAB受体及核因子k B(NF-k B)蛋白表达相关水平(n=5,x_±s)。结果:原代培养海马神经元起初小而圆、透亮并悬浮在细胞液中。24h后可通过光学显微镜观察到大部分细胞为梭形并长出细长突起。3天后胞浆丰富,突起较前明显增粗、增长。5天进入对数生长期,神经元胞体丰满,突起交织成更加稠密的网络。紫杉醇抑制海马神经元活力是与时间和浓度交互相关的(F=9.127,P0.05)。在对凋亡率的分析中,由于右上象限中包含晚期凋亡细胞或者坏死细胞,所以本实验中凋亡率的测定主要依据右下象限的早期凋亡细胞。1μmol/L紫杉醇处理24h诱发的细胞早期凋亡率为(48.63±5.76)%,为测定NF-k B及GABAB受体蛋白的实验条件。蛋白表达结果:与C组相比,N组、K1+N组和K2+N组NF-k B与GABAB受体表达均明显增高(P0.05),而K1组和K2组NF-k B与GABAB受体表达均降低(P0.05);与K1组相比,N组和K1+N组NF-k B和GABAB受体表达均增高(P0.05);与K2组相比,N组和K2+N组NF-k B和GABAB受体表达均增高(P0.05);与N组相比,K1+N组和K2+N组NF-k B和GABAB受体表达均降低(P0.05)。结论:在紫杉醇诱发细胞凋亡过程中,激活p38MAPKs通路可上调GABAB受体表达,而其下游因子NF-k B可能是其调控GABAB受体表达变化的关键靶点,NF-k B可通过下调GABAB受体蛋白表达抑制紫杉醇诱发细胞凋亡。
[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，

本文编号：1414130