Involvement of alpha1beta1 integrin in insulin-like growth factor-1-mediated protection of PC12 neuronal processes from tumor necrosis factor-alpha-induced injury.
Involvement of alpha1beta1 integrin in insulin-like growth factor-1-mediated protection of PC12 neuronal processes from tumor necrosis factor-alpha-induced injury.
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α1β1 整合素参与胰岛素样生长因子-1 介导的 PC12 神经元过程免受肿瘤坏死因子-α 诱导损伤的保护。
DOI：
10.1002/jnr.20712
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发表时间：
2006
期刊：
影响因子：
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通讯作者：
Reiss,Krzysztof
中科院分区：
文献类型：
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作者：
Wang,JinYing;Grabacka,Maja;Marcinkiewicz,Cezary;Staniszewska,Izabella;Peruzzi,Francesca;Khalili,Kamel;Amini,Shohreh;Reiss,Krzysztof
材料和方法细胞培养先前报道了PC 12大鼠嗜铬细胞瘤（ATCC No. CRL-1721）的培养条件（Wang等人，2003年），并进行了一些修改。为了维持细胞增殖，将PC 12细胞在胶原蛋白I（Sigma，St. Louis，MO）包被的培养皿上在补充有50 U/ml青霉素、50 ng/ml链霉素、10%马血清和5%胎牛血清（FBS）的Dulbecco改良Eagle培养基（DMEM; Gibco BRL，Grand Island，NY）中培养。为了诱导神经元分化，将PC 12细胞铺在胶原IV（Sigma）包被的培养皿上，并用无血清培养基（SFM）中的20 ng/ml神经生长因子（NGF; Invitrogen，卡尔斯巴德，CA）处理，所述无血清培养基由DMEM、0.1%牛血清白蛋白（BSA，级分V，用于细胞培养; Sigma）和2 mM L-谷氨酰胺（Gibco）组成。对于长期实验，SFM补充有1%FBS。神经元过程开始出现在第一个24小时内治疗后，并保存为完全分化的神经元过程中的NGF的情况下，几个星期。通过在用新霉素抗性pGR 15表达载体进行逆转录病毒转导后收集混合的PC 12细胞群来产生PC 12/IGF-1 R细胞，其中wt IGF-1 R cDNA被克隆到基于MSCV的逆转录病毒表达载体中（Romano et al.，2001年）。使亲本PC 12细胞和用空逆转录病毒载体（PC 12/EV）或pGR 15（PC 12/IGF-1 R）转导的PC 12在NGF存在下分化5天。将培养基更换为新鲜的SFM或SFM+1%FBS，并且用以下物质处理分化的细胞：50 ng/ml TNF α（BD Biosciences，Palo Alto，CA）、50 ng/ml IGF-1（Invitrogen）、obtustatin（200 ng/ml; Marcinkiewicz等人，2003; Kisiel等人，2004）和放线菌酮（CHX; Sigma），浓度范围为0.1 - 1 μg/ml。神经元样突起的测量值取自来自所选显微镜视野的一系列照片，并反映了特定显微镜视野中神经元样突起的平均长度，如前所述（Wang等人，2003年）。使用Image J（ij-130）软件收集测量值并计算处理后指定时间点的神经元突起平均长度的变化。凋亡DNA链断裂的检测根据制造商的推荐用原位死亡检测试剂盒（Roche，Indianapolis，IN）通过TUNEL测定标记。在配备有一组荧光素激发-发射滤光片的荧光显微镜下评价DNA链断裂。还通过使用抗半胱天冬酶3兔多克隆抗体评估分化的神经元中细胞凋亡的存在，所述抗体识别半胱天冬酶3的裂解（活性）片段（18 kD）（BD Pharmingen，San Diego，CA）。
MATERIALS AND METHODSCell Culture Culture conditions for PC12 rat pheochromocytoma (ATCC No. CRL-1721) were previously reported (Wang et al., 2003) and were used with some modifications. To maintain cell proliferation, PC12 cells were cultured on collagen I (Sigma, St. Louis, MO)-coated dishes in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 50 U/ml penicillin, 50 ng/ml streptomycin, 10% horse serum, and 5% fetal bovine serum (FBS). To induce neuronal differentiation, PC12 cells were plated on collagen IV (Sigma)-coated dishes and treated with 20 ng/ml of nerve growth factor (NGF; Invitrogen, Carlsbad, CA) in serum-free medium (SFM), which consists of DMEM, 0.1% bovine serum albumin (BSA, fraction V, for cell culture; Sigma), and 2 mM L-glutamine (Gibco). For long-term experiments, SFM was supplemented with 1% FBS. Neuronal processes began to appear within first 24 hr following the treatment and were preserved as fully differentiated neuronal processes for several weeks in the absence of NGF. PC12/IGF-1R cells were generated by collecting a mixed population of PC12 cells after retroviral transduction with neomycinresistant pGR15 expression vector in which wt IGF-1R cDNA was cloned into MSCV-based retroviral expression vector (Romano et al., 2001). Parental PC12 cells and PC12 transduced with the empty retroviral vector (PC12/EV) or pGR15 (PC12/IGF-1R) were allowed to differentiate in the presence of NGF for 5 days. The medium was changed to fresh SFM or SFM+ 1% FBS, and the differentiated cells were treated with: 50 ng/ml TNFa (BD Biosciences, Palo Alto, CA), 50 ng/ml IGF-1 (Invitrogen), obtustatin (200 ng/ml; Marcinkiewicz et al., 2003; Kisiel et al., 2004), and cycloheximide (CHX; Sigma) at concentrations ranging from 0.1 to 1 μg/ml. The measurements of neuron-like processes were taken from a series of photographs from the selected microscopic fields and reflect an average length of neuron-like processes in a particular microscopic field, as previously described (Wang et al., 2003). The Image J (ij-130) software was utilized to collect measurements and to calculate changes in the average length of neuronal processes at indicated time points after treatment.Detection of Apoptosis DNA strand breaks were labeled by TUNEL assay with the In Situ Death Detection kit according to the manufacturer’s recommendations (Roche, Indianapolis, IN). DNA strand breaks were evaluated under a fluorescent microscope equipped with a set of excitation–emission filters for fluorescein. The presence of apoptosis in differentiated neurons was also evaluated by using anticaspase 3 rabbit polyclonal antibody, which recognizes a cleaved (active) fragment (18 kD) of caspase 3 (BD Pharmingen, San Diego, CA).