热休克蛋白在高温处理后鼠胚心脏的表达

目的：探讨热休克蛋白在高温处理后金黄地鼠胚心脏的表达变化。方法：将金黄地鼠孕鼠于受孕第8d置42℃水浴持续20min,定时剖腹取胎,制备石蜡切片。利用免疫组织化学(SABC法)染色,观察心脏正常发育和异常分化过程中热休克蛋白(HSP70和HSP90)的表达。结果：高温处理后8h,实验组鼠胚内皮细胞及心胶质HSP70和HSP90的表达较对照组明显增强;16h,表达最强;24h,仍较对照组强;48h,和对照组的表达无显著性差异。结论：高温诱导金黄地鼠胚心脏热休克蛋白表达增强,对心脏的异常发育具有保护作用。     

p-ezrin和p-p38在乳腺浸润性导管癌中的表达及临床意义

目的 Ezrin是细胞膜与细胞骨架连接蛋白,参与肿瘤细胞的侵袭和转移。本研究通过检测p-ezrin和p-p38在乳腺浸润性导管癌中的表达情况,探讨其与乳腺癌转移的关系和意义。方法采用免疫组织化学SP法检测80例乳腺浸润性导管癌组织切片中p-ezrin和p-p38的表达。结果 p-ezrin在33例无转移的癌组织中7例异常高表达(21.2%),47例有转移的癌组织中27例异常高表达(57.4%)。p-p38在无转移的癌组织中15例异常高表达(45.5%),而在有转移的癌组织中34例异常高表达(72.3%)。p-ezrin异常表达与p-p38异常表达正相关性(r=0.269,P=0.016)。结论 p-ezrin和p-p38与乳腺导管癌的浸润和转移密切相关,可以作为预测浸润性乳腺导管癌淋巴结转移的重要肿瘤标志物。     

抑制Rac1通过降低磷酸化p38MAPK提高1型糖尿病小鼠的心脏功能

目的:探讨抑制Rac1对1型糖尿病小鼠心肌细胞肥大、心脏功能的影响及其作用机制。方法:50只8周龄雄性C57小鼠随机分为对照组(control,n=10)、Rac1抑制剂NSC23766对照组(NSC,n=10)、1型糖尿病组(STZ,n=15)及NSC治疗组(STZ+NSC,n=15)。小鼠腹腔注射链脲佐菌素(STZ)建立1型糖尿病动物模型,血糖升高后给予小鼠腹腔注射NSC23766,实验于8周末结束,记录实验小鼠生存率、测量小鼠体重及左室重量并计算左室重量指数,运用超声心动图检测小鼠心脏功能,心肌组织进行HE染色结合图像分析软件定量心肌细胞大小,运用实时定量RT-PCR技术检测心肌组织心房利钠肽(ANP)、脑利尿肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA表达,以Westernblotting定量心肌组织磷酸化p38丝裂素活化蛋白激酶(pho-p38MAPK)表达。结果:抑制Rac1后:(1)糖尿病小鼠生存率提高、糖尿病小鼠左室重量指数降低(P0.01)、心脏左室射血分数(EF)增加、左室短轴缩短率增加(FS)(P0.01);(2)心肌细胞大小明显降低(P0.01);(3)心肌肥大相关基因ANP、BNP、β-MHC表达明显降低(P0.01);(4)心肌组织pho-p38MAPK表达明显降低(P0.01)。结论:抑制Rac1活性显著改善1型糖尿病小鼠心脏功能、降低心肌细胞肥大,其机制可能与明显降低心肌组织磷酸化p38MAPK密切相关。     

p38MAPK在小鼠早胚中的表达及其对胚泡发育的作用

目的观察p38丝裂原活化蛋白激酶(p38MAPK)在小鼠早胚中的表达及其作用,探讨p38MAPK与胚泡植入的相关关系。方法取小鼠胚胎的2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。用体外胚胎培养技术:将2细胞期小鼠早胚随机分组,接种到添加不同浓度p38MAPK特异性抑制剂SB220025的培养液内进行培养,台盼蓝染色观察p38MAPK特异性抑制剂对小鼠早胚发育的影响。结果 p38MAPK在小鼠胚胎发育各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。体外实验显示:抑制剂组早胚不能发育到胚泡阶段,抑制剂恢复实验发现抑制剂组胚卵仍具存活能力。结论 p38MAPK在小鼠胚胎发育过程中起作用,p38MAPK特异性抑制剂可抑制小鼠早胚的发育。     

整合素β3,p38MAPK及MMP-2在乳腺癌中存在功能性的联系

目的 研究整合素β3与配体Tenascin-c(TN-c)在乳腺癌组织中的表达.探讨其是否通过p38丝裂原活化蛋白激酶(p38mitogen-activated protein kinase,p38MAPK)激活基质金属蛋白酶-2(MMP-2).方法 本研究应用免疫组化sP方法检测了80乳腺浸润性导管癌中整合素β3及T...     

p38 MAPK在机械牵张致大鼠心脏TREK-1通道表达改变中的作用

目的：研究p38 MAPK信号途径在急性机械牵张致大鼠离体灌流心脏TREK-1通道表达改变中的作用。方法:30只雄性 Wistar大鼠随机分成3组,即对照组、机械牵张组和SB202190（p38 MAPK特异性抑制剂）组，每组10只。利用Langendorff灌流装置等建立离体灌流急性机械牵张的心脏模型；通过Western blotting方法检测各组大鼠左室的p38 MAPK、p-p38 MAPK及TREK-1通道的蛋白表达。结果:各组大鼠左室的p38 MAPK表达无明显差异（P>0.05）；机械牵张组左室p-p38 MAPK 及TREK-1通道的蛋白表达均明显高于对照组（P<0.01），而SB202190组的p-p38 MAPK及TREK-1蛋白表达与对照组比较无明显差异（P>0.05）且低于机械牵张组。结论:离体灌流心脏受机械刺激时p38 MAPK信号途径的激活可能介导了TREK-1通道的表达上调。心脏可能通过这种方式来改变自身电活动，从而减少心律失常发生，起到自身保护作用。     

丙酮酸通过抑制p38丝裂原激活蛋白激酶(p38MAPK)磷酸化减轻低氧对PC12细胞的损伤北大核心CSCD

目的观察丙酮酸对低氧诱导神经细胞损伤的保护作用及机制。方法采用丙酮酸处理PC12细胞,10 m L/L O2的低氧环境暴露12、24、48 h。MTT法观察细胞增殖情况,检测胱天蛋白酶3(caspase-3)活性观察细胞凋亡情况,ELISA检测白细胞介素1β(IL-1β)、IL-6、肿瘤坏死因子α(TNF-α)的水平,Western blot法检测p38丝裂原激活蛋白激酶(p38MAPK)、磷酸化的p38MAPK(p-p38MAPK)蛋白水平。结果低氧暴露24、48 h,抑制PC12细胞增殖,caspase-3活性增强,IL-1β、IL-6、TNF-α和p-p38MAPK水平增高;丙酮酸处理可显著增加PC12细胞增殖,减少细胞凋亡,降低IL-1β、IL-6、TNF-α水平,抑制p38MAPK的磷酸化水平。结论丙酮酸通过抑制p38MAPK的磷酸化而抑制低氧暴露引起PC12细胞炎症因子的表达,对低氧暴露导致的神经元损伤起保护作用。     

遗传性癫痫大鼠小脑中p-CaMKⅡ、p-ERK和p-p38蛋白表达的异常变化

目的研究遗传性癫痫大鼠(Tremor,TRM)与正常Wistar大鼠小脑中p-Ca MKII、p-ERK以及pp38蛋白含量的变化,进一步阐明癫痫发病机制。方法应用PCR技术,鉴定基因突变鼠(TRM),确定癫痫阳性大鼠。以Wistar大鼠作为正常对照模型,通过Western blot技术分别测定TRM及Wistar大鼠小脑内p-Ca MKII、pERK以及p-p38的蛋白表达量。结果与正常大鼠相比,TRM大鼠小脑p-Ca MKII蛋白和p-p38蛋白表达量升高,而pERK蛋白表达量没有明显变化。结论 TRM大鼠小脑内p-Ca MKII和p-p38蛋白表达上调,表明Ca MKII与MAPK信号通路可能参与癫痫的发病机制。     

p38γMAPK蛋白在结肠癌组织中的表达及临床意义

目的探讨人结肠癌中p38γMAPK蛋白的表达及其临床意义。方法应用免疫组织化学法检测54例结肠癌组织、近癌旁组织、癌周正常组织和15例腺瘤息肉组织中p38γ蛋白的表达情况。结果p38γ蛋白的表达主要定位于胞质中,仅少量在胞核中表达。p38γ蛋白在结肠癌组织、癌旁组织、癌周正常组织中的高表达率分别为75.93%、51.85%、37.04%,在结肠腺瘤息肉组织中高表达率为33.33%。p38γ蛋白在结肠癌中的表达明显高于癌旁组织、癌周正常组织和腺瘤息肉组织,有统计学意义（P〈0.01）。p38γ的表达与Duke分期,组织分化程度及有无淋巴结转移有显著差异（P〈0.01）,p38γ的表达与年龄、性别、肿瘤大小、肿瘤位置无明显相关（P〉0.05）。结论结肠癌组织中p38γ蛋白处于过度表达状态,与结肠癌的发生、发展和转移密切相关。     

高温致金黄地鼠神经管畸形的上调表达基因筛选及其鉴定

目的:筛选高温致金黄地鼠神经管畸形的上调表达基因,探讨其分子生物学机制。方法:通过抑制性消减杂交技术获得消减产物,转化大肠杆菌DH5α,构建高温致金黄地鼠胚胎神经管畸形的消减cDNA文库;联合蓝白斑和菌落PCR方法筛选含插入片段的阳性克隆,进行测序和同源性分析,并经Northern杂交技术检测基因的表达。结果:成功构建高温致金黄地鼠胚胎神经管畸形的消减cDNA文库,其中2个克隆插入片段的基因序列与小鼠磷酸甘油酸酯激酶(Pgk-1)同源。Northern杂交证实该基因在高温致畸胚胎神经管的表达较其在同龄正常胚胎神经管明显升高。结论:Pgk-1的上调表达可能与高温致神经管畸形密切相关。     

p38 MAPK在大鼠梗阻性肾病肾组织中的表达及可能作用

目的：探讨p38 MAPK在大鼠梗阻性肾病模型肾组织中的表达及其在肾脏纤维化发病机制中的作用。方法：采用单侧输尿管结扎制造梗阻性肾病模型，分别于造模后1 h、3 h、6 h、12 h、1 d、3 d、5 d、7 d、14 d、21 d、28 d取肾组织，应用免疫沉淀结合特异性底物磷酸化法测定肾组织p38 MAPK活性，用免疫组织化学法检测磷酸化p38 MAPK在肾组织中的表达；用免疫组织化学及原位杂交方法检测肾组织TGFβ₁ mRNA和蛋白水平的表达。结果：正常对照组大鼠肾组织有一定的p38 MAPK基础活性（吸光度A值）(0.22±0.06)。UUO术后1 h，p38 MAPK即被激活（0.45±0.14 vs 对照组，P<0.05），12 h时达第一个高峰（0.91±0.07 vs 对照组，P<0.01），此后活性逐渐下降，3 d后又进行性升高，7 d达到第二个高峰（0.93±0.06 vs 对照组，P<0.01），此后又缓慢下降，28 d降至最低水平(0.12±0.02)。而TGFβ₁的表达在UUO术后1 h、3 h、6 h、12 h及24 h无明显增加，第3 d有明显增加（13.55%±6.33% vs 对照组，P＜0.05），第7 d达高峰（26.78%±8.77% vs 对照组，P<0.01），第28 d表达最少。梗阻肾肾组织p38 MAPK的激活明显早于TGFβ₁表达且p38 MAPK早期活性水平与TGFβ₁的表达水平呈正相关；梗阻肾高表达的TGFβ₁与肾组织p38MAPK的后期活性的高低显著相关（r=0.84, P<0.01）。结论：p38 MAPK在大鼠梗阻性肾病肾组织中的活性明显增高，可能介导梗阻肾肾组织TGFβ₁的表达，并可能参与TGFβ₁诱导的肾间质纤维化的过程。     

Role of p38 mitogen-activated protein kinase pathway on heart failure in the infant rat after burn injury

We examined the hypothesis that post-burn activation of the p38 mitogen-activated protein kinase (MAPK) pathway is one aspect of the signalling cascade culminating in post-burn secretion of tumour necrosis factor (TNF)-alpha which contributes to post-burn myocardial apoptosis. Studies were designed to determine the time course of the induction of p38MAPK, TNF-alpha and myocardial apoptosis after burn injury. Our quantitative bacterial culture data demonstrated that viable bacteria reached the heart, and Western blotting data identified the increase in the phosphorylation of p38MAPK at an early time after burn. The peak incidence of myocardial apoptosis was also seen at an early time after burn. The expression of TNF-alpha mRNA, infiltrated neutrophils and serum creatine phosphokinase myocardial band data peaked at a late time after burn. FR167653, a specific inhibitor of p38MAPK, prevented the induction of myocardial apoptosis, TNF-alpha expression and myocardial injury after burn. Presumably, the bacterial LPS-induced activation of p38MAPK pathway occurring at an early time after burn induced the subsequent myocardial apoptosis. The p38MAPK-induced activation of pro-inflammatory cytokine appeared to promote the degenerative myocardial injury at a late time after burn. Our present data provided evidence for the hypothesis that the p38MAPK pathway controls both myocardial apoptosis and the pro-inflammatory mediator.     

磷酸化细胞周期素依赖性激酶在局灶性脑缺血大鼠神经细胞中的表达

目的:观察磷酸化细胞周期素依赖激酶在大鼠局灶性脑缺血后神经元和星形胶质细胞的表达。方法:采用大脑中动脉阻塞（MCAO）方法制作大鼠局灶性脑缺血模型,应用免疫荧光技术检测缺血后1 d、3 d、7 d、14 d大鼠缺血侧病灶周围神经元和星形胶质细胞中磷酸化周期素依赖激酶2（CDK2）、磷酸化细胞分裂周期激酶2（CDC2）的表达。结果:在神经元中,与正常对照组相比,缺血后1 d、3 d组磷酸化CDK2的表达量明显增加（P<0.05）,磷酸化CDC2在正常组及缺血组均无明显表达;在星形胶质细胞中,缺血后1 d、3 d、7 d均出现明显的磷酸化CDK2、CDC2的表达,与对照组相比有显著差异（P<0.05）。结论:大鼠局灶性脑缺血后早期部分神经元再次进入细胞周期,细胞周期调控机制可能参与了大鼠局灶性脑缺血后神经元的凋亡及星形胶质细胞的分裂增殖。     

小鼠胚胎心脏发育期T型钙离子通道的表达

目的 检测哺乳动物心脏中T型钙离子通道α1亚基在小鼠胚胎心脏发育过程中表达部位和时间的分布特征。 方法 对小鼠胚胎心脏发育关键时期（Ｅ9.5至胚胎E18.5共9d）的胚胎进行连续切片,用RT-PCR和免疫组织化学的方法对α1G和α1H蛋白进行阳性检测。 结果 α1H从胚胎期10.5d左右开始表达并一直延续到发育晚期（E18.5）,而α1G则从E11.5d延续到E18.5;免疫组织化学的结果显示,T型钙离子通道主要在心肌层以及左右传导束支稳定表达,而在心内膜垫以及心室肌小梁的心内膜内皮和间充质细胞中不表达。 结论 T型钙离子通道对于心肌细胞以及心脏传导组织的形成起到了一定的作用。     

胶原诱导性关节炎大鼠滑膜组织中磷酸化p38与JNK通路及痹肿消汤的影响

背景：有研究报道滑膜细胞信号传导异常是类风湿关节炎发病的重要机制之一。  目的：观察胶原诱导性关节炎大鼠中丝裂原活化蛋白激酶通路中磷酸化p38和磷酸化JNK的活化及痹肿消汤的影响。 方法：将72只SD大鼠随机分为正常组、模型组、痹肿消汤组。模型组及痹肿消汤组大鼠足趾注射胶原蛋白乳剂制备胶原诱导性关节炎模型大鼠,10 d后加强免疫,初次免疫后第14天开始给痹肿消汤组大鼠每天痹肿消汤灌胃,模型组蒸馏水灌胃,正常组自由饮水。 结果与结论：Western blot 检测结果显示,胶原诱导性关节炎大鼠中磷酸化p38及JNK的表达较正常组显著增高(P < 0.05)。与模型组相比,痹肿消汤组能下调磷酸化p38及JNK蛋白的表达(P < 0.05)。说明痹肿消汤可抑制类风湿关节炎中磷酸化p38及JNK的高表达。     

p38 mitogen-activated protein kinase activity commits embryonic stem cells to either neurogenesis or cardiomyogenesis

Mouse embryonic stem (ES) cells can be differentiated, in vitro into a variety of cell types including cardiac cells and neurons. This process is strictly controlled by the potent morphogen retinoic acid (RA). At a concentration of 10(-7) M, RA induces ES cell differentiation into neurons and, conversely, inhibits cardiomyogenesis. We found that p38 mitogen-activated protein kinase (p38MAPK) activity peaked spontaneously, between day 3 and day 5, during ES cell differentiation and that RA completely inhibited this peak of activity. In contrast to wild-type cells, which required RA treatment, p38alpha(-/-) ES cells differentiated spontaneously into neurons and did not form cardiomyocytes. Moreover, inhibition of the peak of p38MAPK activity by a specific inhibitor, PD169316, committed ES cells into the neuronal lineage and blocked cardiomyogenesis. By genetic and biochemical approaches, we demonstrate that, in two different ES cell lines, the control of p38MAPK activity constitutes an early switch, committing ES cells into either neurogenesis (p38 off) or cardiomyogenesis (p38 on).     

Chromosome-specific telomeric associations in Chinese hamster embryonic cells

Telomeric associations (TAs) represent an important cytogenetic marker of human tumor cells. It has been thought that the primary cause of TAs is telomere shortening. However, we report here a surprising aspect of telomere maintenance in primary Chinese hamster embryonic (CHE) cells: relatively high frequencies of TAs in spite of normal telomere length. These TAs are present in both interphase and metaphase cells, suggesting that metaphase TAs may be relics of interphase chromosome organization. In addition, some TAs observed here are chromosome-specific and recurrent in at least three consecutive cell cycles in two different CHE cell strains. In spite of relatively high frequencies of TAs, none of the CHE strains show chromosome instability resulting from breakage-fusion-bridge cycles, as would be expected from tumor cell studies. It appears that TAs in CHE cells may be reversible events. These results are discussed in light of current understanding of telomere biology.     

In situ demonstration of phosphorylated c-jun and p38 MAP kinase in epidermal keratinocytes following ultraviolet B irradiation of human skin

Ultraviolet B (UVB) irradiation is known to induce activation of cellular stress response pathways in cultured cells or intact human skin, as demonstrated by phosphorylation of MAP kinase family members and up- or down-stream targets, using biochemical assays. This study demonstrates by immunohistochemistry that low-dose UVB irradiation of normal human skin induces rapid and reversible phosphorylation of c-jun (a target of c-jun N-terminal kinase) and p38 mitogen activated protein kinase (p38 MAP kinase). Phosphorylation was maximal at 4-8 h and returned to normal levels at 48 h after irradiation. Nuclear localization of these phosphorylated substrates was found using antisera against the epitope containing the phosphorylated serine-73 of c-jun, and the dually phosphorylated epitope (threonine-180 and tyrosine-182) of p38 MAP kinase. Nearly all epidermal cells were positive for c-jun phosphorylation, whereas p38 phosphorylation was seen predominantly in the differentiated layers. In contrast to the massive activation of c-jun and p38, only a small population of the suprabasal cells showed nuclear translocation of nuclear factor kappa B (NFkappaB), and a few scattered cells became apoptotic, as determined by TUNEL (TdT mediated dUTP nick end labelling) staining. The expression of involucrin and skin-derived anti-leukoproteinase (SKALP)/elafin, two genes putatively under control of the c-jun and p38 pathways, was found to be increased. These findings establish the first cellular localization of UVB-induced protein phosphorylation of stress response proteins in human epidermis, thereby providing a link between cellular activation and gene expression in defined cell populations.