异丙酚对小鼠海马脑片细胞外信号调节激酶ERK1/2磷酸化水平的影响

目的 观察异丙酚对小鼠海马脑片细胞外信号调节激酶ERK1／2磷酸化水平的影响。方法BALB／C小鼠，体重18～22g，雌雄不拘，迅速断头取脑，取海马用震动切片机切成450μm厚的脑片。量效实验随机将脑片分为空白对照组（C组）及不同浓度异丙酚组[10^-4mmol／L（P1组）、10^-5mmol／L（P2组）、10^-6mmol／L（P3组）、10^-7mmol／L（P4组）、10^-8mmol／L（P5组）、10^-8mmol／L（P6组）]；分别以不同浓度的异丙酚36℃恒温孵育海马脑片1h。时效实验应用5μmol／L异丙酚孵育脑片，分别于孵育即刻、1、2、5、7、9、12、15、30、60min取出脑片；取5μmol／L异丙酚孵育5min后的部分脑片，以人工脑脊液洗出异丙酚，分别于洗出2、4、7、10、25min取出脑片。应用SDS-PAGE和Westem blot生化技术及Gel Doc凝胶成像系统半定量检测小鼠海马p-ERKI／2水平。结果 异丙酚能降低ERK1／2磷酸化水平，降低呈时间、浓度依赖性（P〈0．05）。随异丙酚孵育5min后洗出时间延长，ERK1／2的磷酸化水平逐渐恢复，但洗出25min时仍明显低于药物处理前水平（P〈0．01）。结论 异丙酚能显著地抑制小鼠海马脑片细胞外信号调节激酶ERK1／2磷酸化水平。     

热休克蛋白90对大鼠缺氧心肌细胞丝氨酸苏氨酸蛋白激酶表达的影响

目的探讨内源性热休克蛋白90（HSP90）在缺氧心肌细胞丝氨酸苏氨酸蛋白激酶（AKT）相关信号通路中的作用。方法建立新生Wistar大鼠心肌细胞缺氧模型，将细胞分为正常组、缺氧组、加入HSP90特异性阻断剂格尔德霉素后再缺氧组（格尔德霉素＋缺氧组）。于缺氧后1、3、6、12、24、48h用噻唑蓝法检测心肌细胞的活力；缺氧24h，原位缺口末端标记法检测心肌细胞凋亡指数（AI）；缺氧1、3、6、12、24h，蛋白质印迹法检测大鼠心肌细胞中内源性HSP90及AKT表达水平。结果（1）缺氧24、48h，缺氧组、格尔德霉素＋缺氧组细胞活力均较正常组明显下降（P〈0．05）；格尔德霉素＋缺氧组细胞活力缺氧12h即开始明显下降，缺氧48h时明显低于缺氧组（P〈0．05）。（2）缺氧24h，缺氧组细胞AI为（10．7±1．2）％，明显高于正常组[（1．9±0．3）％．P〈0．05]；格尔德霉素＋缺氧组细胞AI为（26、3±5．3）％，明显高于缺氧组（P〈0．01）。（3）缺氧12h，缺氧组心肌细胞内源性HSP90及AKT表达水平高于正常组与格尔德霉素＋缺氧组；缺氧24h，缺氧组有所下降．格尔德霉素＋缺氧组则下降更明显。结论内源性HSP90对维持心肌细胞的活力有重要作用．缺氧心肌细胞AKT表达水平可受内源性HSP90表达水平的影响。     

耐药性癫(癎)患者颞叶中细胞周期依赖性蛋白激酶5的表达

目的 研究细胞周期依赖性蛋白激酶5(cyclin dependent kinases 5,CDK5)在耐药性癫(癎)患者颞叶中的表达,探索其在耐药性癫(癎)发病机制中的作用.方法 收集耐药性癫(癎)患者术后脑组织,用荧光定量PCR、免疫组化和Western blot 3种检测方法从基因和蛋白水平分别测定CDK5在耐药性癫(癎)患者颞叶中的表达,并与对照组进行比较.结果 荧光定量PCR发现CDK5 mRAN比对照组明显增加,免疫组化检测显示这种基因的蛋白表达产物主要分布在神经元轴突和胶质细胞中,Western blot检测在相对分子质量35 000处有一蛋白条带,并且可见实验组(颞叶和海马中分别为1.4293±0.1839和2.0733±0.4738)高于对照组(颞叶和海马中分别为0.9680±0.4147和1.403±0.6163,P＜0.05).结论 CDK5在耐药性癫(癎)患者颞叶中表达增强,提示他们可能参与了耐药性癫(癎)的形成.     

酪氨酸激酶抑制剂对气管上皮细胞生长的影响

目的探讨酪氨酸激酶抑制剂(Tyrosine kinase inhibitor,TKI)对培养气管上皮细胞生长的影响.方法通过MMT法、3H-胸腺嘧啶(3H-TdR)掺入法及流式细胞计数,观察3种TKIs:Tyrphostin AG1478、Genistein(Sigma)及金转停对原代培养的大鼠气管上皮细胞增殖、周期及凋亡的影响,以及TKIs对表皮生长因子(EGF)的阻断作用.结果MTT法显示3种TKI均对气管上皮细胞的生长具有时间和剂量依赖性抑制作用,同时,TKI阻断EGF对气管上皮细胞生长的刺激作用,3种TKIs的作用无明显差异;1μmol/L的Tyrphostin AG1478、Genistein及金转停分别使气管上皮细胞的TdR掺入率降低18.3%、20.9%及19.7%,与MTT比色法结果一致.同时,Tyrphostin AG1478不仅加速气管上皮细胞的凋亡而且阻止细胞有丝分裂.TKIs可阻断表皮生长因子(EGF)对气管上皮细胞生长的刺激作用.结论TKIs不仅抑制对原代培养的大鼠气管上皮细胞的生长,加速其凋亡,而且可阻断EGF对气管上皮细胞生长的刺激作用,3种抑制剂的作用无明显差异.     

IL-2对垂体瘤细胞系RC-4B/C细胞分泌ACTH的影响

目的 :探讨白细胞介素 2 (IL 2 )对垂体瘤细胞系RC 4B/C细胞ACTH分泌的调控作用及其影响因素 .方法 :以放射免疫方法测定培养的垂体瘤细胞系RC 4B/C细胞的培养液中的ACTH浓度 .结果 :IL 2 (1× 10 4～ 5× 10 5U·L-1)促进RC 4B/C细胞分泌ACTH ;蛋白激酶A的抑制剂H 9(1μmol·L-1)和酪氨酸蛋白激酶的抑制剂tyrphostin2 3 (1μmol·L-1)均可显著性抑制IL 2的促ACTH分泌作用 .结论 :IL 2可促进RC 4B/C细胞分泌ACTH ,该作用与蛋白激酶A和酪氨酸蛋白激酶信号转导途径紧密相关     

p38丝裂原活化蛋白激酶在糖尿病大鼠神经病理性痛中的作用

目的探讨p38丝裂原活化蛋白激酶（p38MAPK）在链脲菌素诱导的糖尿病大鼠神经病理性痛中的作用。方法雌性Wistar大鼠31只，3月龄，体重180～220g，随机分为3组：对照组（C组，n=10）、糖尿病神经病理性痛组（D组，n=11）和p38MAPK抑制剂组（Ⅰ组，n=10）。D组、Ⅰ组单次腹腔注射链脲菌素65mg／kg制备糖尿病模型。糖尿病模型制备成功后，Ⅰ组尾静脉注射p38MAPK抑制剂SB203580 0．5mg／kg，1次／周，连续4周；C组和D组尾静脉注射等体积的生理盐水。给药4周后，测定机械缩足反应阈值（MWT）、左侧坐骨神经传导速率（NCV）、背根神经节（DRG）和脊髓的磷酸化p38MAPK水平。结果与C组比较，D组、Ⅰ组MWT下降，NCV减慢，伴有脱髓鞘现象，DRG和脊髓的磷酸化p38MAPK水平升高；与D组比较，Ⅰ组MWT升高，NCV增快，脱髓鞘程度减轻，DRG和脊髓的磷酸化p38MAPK水平下降。结论p38MAPK信号转导通路参与了糖尿病大鼠神经病理性痛的形成。     

Role of angiotensin Ⅱ and JAK2 signal pathway in transdifferentation of renal tubular cells in mice after acute ischemic followed by reperfusion

Objective To investigate the effect of angiotensin (Ang) Ⅱ and its Janns-activated kinase-2 (JAK2) signal pathway in transdifferentiation of renal tubular cells under the challenge of acute ischemic reperfusion injury.Methods Models of acute ischemic reperfusion injury were established and the level of local Ang Ⅱ ,a key element of renin-angiotensin system (RAS),in kidney was measured using radioimmunity technique.The expression of α-smooth muscle actin (α-SMA),a phenotype of mesenchymal cells,was detected by RT-PCR and inununohistochemistry methods.Renal tubule cells ( NRK-52E) were cultured with various concentration of Ang Ⅱ ,followed by blocking of PD123319,Ang U receptor 2 antagonist,and AG490,an inhibitor of JAK2 signal pathway.Results Ang 0 of kidney tissue increased immediately after acute ischemic-reperfusion injury,in time dependent fashion.Expression of α-SMA in renal tubule cells was found at 48 hours after ischemic-reperfusion injury and in NRK-52E cells treated by high concentration of Ang Ⅱ and was dose and time dependent.The peak of α-SMA expression was seen after 30 minute treatment at the dose of 10-9'mol/L,which was interrupted by both of PD123319 and AG490.Conclusions Transdifferentiarion of renal tubular epithelial cells occurs under acute ischemic- reperfusion injury.Local renin-angiotensin system may play a role in the transdifferentiation of TEC through AT2 receptor and its JAK2 signal pathway.     

N-乙酰半胱氨酸对脂多糖诱导的小鼠肝MAPK磷酸化的影响

目的： 探讨N-乙酰半胱氨酸（NAC）对脂多糖（LPS）诱导的肝MAPK磷酸化的影响。方法: 雄性昆明种小鼠54只随机分为对照组(n=6)：0.9 % NaCl 0.2 mL ip；LPS组（n=24）：LPS 5 mg ip；NAC+LPS组（n=24）：NAC 150 mg·kg-1·d-1ip，连续3 d；第3 d NAC灌胃后1 h时，LPS 5 mg ip。将小鼠分别在注射LPS或生理盐水后0.5 h、1 h、2 h和6 h时，在戊巴比妥钠麻醉下开腹取肝，测定肝MDA和还原型谷胱甘肽（GSH）含量；Western blotting方法测定肝脏MEK1/2、ERK1/2、p38MAPK磷酸化水平，放免法测定肝TNF-α含量。结果: NAC预处理使肝MDA含量明显下降，使肝GSH含量升高。NAC预处理显著抑制了LPS所致的肝MEK1/2、ERK1/2、p38MAPK磷酸化，同时使肝TNF-α水平显著降低。结论: 在LPS诱导的急性肝损伤过程中，活性氧（ROS）在激活MAPK信号转导中起重要作用。NAC通过其抗氧化作用部分抑制了LPS诱导的MAPK磷酸化，使TNF-α生成减少，从而发挥抗损伤作用。     

转化生长因子α,β1对大鼠肺泡Ⅱ型细胞生长的影响

目的 探讨转化生长因子α和β１对肺泡Ⅱ型细胞增生的影响及其作用机制。方法 采用原代培养的成年大鼠肺泡Ⅱ型细胞,加入ＴＧＦα、ＴＧＦβ１作用４８小时,测定肺泡Ⅱ型细＾３Ｈ－ＴｄＲ掺入量和细胞数量,并用斑点杂交、原位杂交和免疫组化方法检测细胞内细胞周期蛋白Ｄ１、细胞周期依赖性激酶４ ｍＲＮＡ和蛋白的表达。结果 随ＴＧＦα浓度递增,肺泡Ⅱ型细胞＾３Ｈ－ＴｄＲ掺入量和细胞数量均逐渐增加,呈量效正相关;ＴＧ     

IGF-I and vanadate stimulate Na/Pi-cotransport in OK cells by increasing type II Na/Pi-cotransporter protein stability

 Insulin-like growth factor (IGF)-I and vanadate increase Na-dependent phosphate (Na/P_i) cotransport in opossum kidney (OK) cells. To gain more information about the mechanisms by which IGF-I and vanadate stimulate Na/P_i-cotransport, we measured type II Na/P_i-cotransporter (NaPi-4) protein abundance by Western blot analysis and investigated the effects of protein synthesis and tyrosine kinase inhibitors. The key findings in the present studies are as follows. First, incubation in IGF-I (10^–8 M) and/or vanadate (10^–3 M) for 3 h led to a non-additive 1.4-fold increase in Na/P_i-cotransport activity which was paralleled by a 1.5- to 2-fold increase in NaPi-4 protein. Second, actinomycin D did not abolish the increase in Na/P_i-cotransport and cycloheximide did not prevent the IGF-I-induced increase in Na/P_i-cotransport and NaPi-4 protein. Third, among the protein kinase inhibitors tested, only staurosporine substantially reduced the stimulation of Na/P_i-cotransport. In conclusion, the stimulatory effect of IGF-I on Na/P_i-cotransport is paralleled by an increased expression of NaPi-4 protein that is independent of protein synthesis and therefore results from increased protein stability. The observation that IGF-I and/or vanadate lead to similar increases in Na/P_i-cotransport and NaPi-4 protein abundance provides further evidence that the stimulation of Na/P_i-cotransport by IGF-I and vanadate involves protein tyrosine phosphorylation of the same signalling molecules. Received: 1 May 1998 / Received after revision: 25 August 1998 / Accepted: 1 September 1998