4,4′-二异硫氰基芪-2,2′-二磺酸对内质网应激诱导心肌细胞损伤的影响

目的探讨氯离子通道阻滞剂4,4′二异硫氰基芪2,2′-二磺酸(DIDS)对衣霉素诱导心肌细胞损伤以及内质网应激标记性分子糖调节蛋白78(GRP78)和CCAAT增强子结合蛋白样同源蛋白(CHOP)的影响。方法取乳鼠心肌细胞培养后,应用衣霉素构建心肌细胞内质网应激模型,随机分为对照组、衣霉素组、衣霉素+DIDS组、DIDS组。应用100ng/ml衣霉素处理24、48、72、96 h;另用DIDS 25、50、75和100μmol/L处理72 h,噻唑蓝法检测心肌细胞存活率,Western blot法检测GRP 78和CHOP蛋白表达水平。结果与对照组比较,衣霉素组48、72和96 h心肌细胞存活率明显降低(P<0.05);与衣霉素组比较,DIDS 50、75和100μmol/L处理明显增加心肌细胞存活率(P<0.05)。与对照组比较,衣霉素组GRP78和CHOP蛋白表达水平明显升高(P<0.05);与衣霉素组比较,衣霉素+DIDS组GRP78和CHOP表达水平明显下调(P<0.05)。结论 DIDS可以抑制衣霉素诱导的大鼠心肌细胞损伤,其机制可能与拮抗GRP78和CHOP的表达,减轻内质网应激有关。     

环孢菌素A抑制内质网应激减轻β淀粉样蛋白_(25-35)对PC12细胞凋亡的研究

目的探讨环孢菌素A对β淀粉样蛋白_(25-35)(Aβ_(25-35))诱导PC12细胞凋亡的保护机制。方法 PC12细胞传代培养,分为对照组、Aβ_(25-35)组、Aβ_(25-35)+环孢菌素A组(环孢菌素A组)。Aβ_(25-35) 10μmol/L,环孢菌素A 20 -μmol/L。药物作用24 h后,采用流式细胞术检测细胞凋亡率,Western blot检测内质网应激蛋白钙网蛋白、半胱天冬氨酸酶12(caspase 12)活化片断的蛋白表达。结果与对照组细胞凋亡率(2.0±0.2)%比较,Aβ_(25-35)组细胞凋亡率(45.0±3.7)%明显升高,差异有统计学意义(P<0.05);而环孢菌素A组细胞凋亡率为(27.0±2.4)%,较Aβ_(25-35)组明显下降,差异有统计学意义(P<0.01)。Western blot显示,与对照组比较,Aβ25 35组钙网蛋白、caspase-12活化片断表达明显增加,差异有统计学意义(P<0.05);与Aβ_(25-35)组比较,环孢菌素A组钙网蛋白、caspase 12活化片断表达明显减少,差异有统计学意义(P<0.05)。结论环孢菌素A可通过抑制内质网应激相关蛋白表达,来减轻Aβ_(25-35)对PC12细胞的凋亡作用。     

4-苯基丁酸对高糖诱导的人晶状体上皮细胞发生上皮间质转化的影响

目的　探讨4-苯基丁酸（4-PBA）对高糖诱导的人晶状体上皮细胞发生上皮间质转化（EMT）的影响。方法　体外培养人晶状体上皮细胞系HLE-B3细胞,将培养的HLE-B3细胞分为正常对照组、高糖组和4-PBA预处理+高糖组。利用流式细胞术检测各组细胞内活性氧（ROS）的含量;利用Western blot检测各组细胞中内质网应激（ERS）相关通路分子的蛋白表达;采用免疫荧光染色观察各组细胞中上皮因子E-cadherin和间质因子α-SMA的表达;分别采用实时荧光定量PCR和Western blot检测各组细胞中EMT标志分子E-cadherin、α-SMA、Snail的 mRNA及蛋白表达。结果　高糖组细胞呈长梭形改变,丢失上皮细胞特性,类似纤维细胞的表型;4-PBA预处理+高糖组细胞形态不规则,相对于高糖组细胞,长梭形细胞数量较少。流式细胞术检测结果显示,高糖组细胞内ROS含量高于正常对照组,4-PBA预处理后ROS含量降低,差异均有统计学意义（均为P<0.01）。Western blot检测结果显示,高糖组细胞中ERS相关通路分子GRP78、CHOP、p-eIF2α的蛋白相对表达水平均高于正常对照组（均为P<0.05）;4-PBA预处理+高糖组细胞中GRP78、CHOP、p-eIF2α的蛋白相对表达水平均明显低于高糖组（均为P<0.05）。实时荧光定量PCR和Western blot检测结果显示,与正常对照组相比,高糖组细胞中E-cadherin mRNA及蛋白表达下调,α-SMA和Snail的mRNA及蛋白表达均上调（均为P<0.05）;与高糖组相比,4-PBA预处理+高糖组细胞中E-cadherin mRNA及蛋白的相对表达量均明显上调,α-SMA和Snail的mRNA及蛋白的相对表达量则与之相反（均为 P<0.05）。免疫荧光染色结果显示,高糖组细胞中E-cadherin荧光表达明显弱于正常对照组,4-PBA 预处理后荧光增强;α-SMA荧光表达则与之相反。结论　4-PBA通过抑制ERS相关通路分子的表达、减少ROS产生来抑制高糖诱导的晶状体上皮细胞发生EMT,从而对晶状体上皮细胞起到一定的保护作用。     

内质网应激在心肌缺血再灌注损伤中的作用

内质网应激（ERS）是细胞对内外环境变化的一种适应性反应,有助于细胞和生命个体的存活,但持续存在或过强时则最终诱发细胞凋亡。缺血,再灌注（I／R）时,钙超载及大量自由基的生成等因素诱导过度ERS导致组织损伤。缺血预处理及稳定Ca2＋稳态可激发适当的ERS,增强细胞耐受长时间缺血刺激的能力,延缓或减轻I／R造成的组织损伤。现综述ERS在缺血再灌注损伤（IRI）中的作用,并指出ERS已成为IRI防治的新靶点。     

Effect of endoplasmic reticulum stress on the activation of high glucose-induced monocytes

Objective To observe the effects of endoplasmic reticulum stress (ERS) on the activation of monocytes induced by high glucose and explore the underlying mechanism. Methods The monocyte cell line THP-1 was stimulated with high glucose, and then treated with molecular chaperone betaine. The levels of glucose regulation protein 78 (GRP78) and p-JNK, which were associated with ERS were detected by real-time PCR and Western blotting. The proliferation of the cell line was detected by MTT method. Transwell and immunofluorescence were applied to observe the chemotaxis and phenotype of cells respectively. Results The levels of GRP78 and p-JNK of THP-1 cells stimulated by high glucose were significantly increased compared with the normal control group (all P＜0.05). The proliferation and chemotactic were also enhanced (all P＜0.05). The number of cells in M1 phenotype was increased remarkably (P＜0.05). All the indexes above could be rescued by betaine. Conclusion The activation of THP-1 cells can be induced by high glucose through ERS, while molecular chaperone betaine can reverse the activation.     

光动力疗法治疗恶性肿瘤作用机制的研究进展

【摘要】 光动力疗法(PDT)是继手术、放化疗等传统治疗肿瘤手段外的一种新的抗肿瘤模式。PDT机制目前尚不完全清楚, 已知其利用肿瘤细胞高摄取光敏剂的特性, 使用相应波长的激光照射, 使光敏剂产生单线态氧或其他活性氧, 通过非细胞凋亡途径或直接高效诱导凋亡或导致肿瘤组织坏死杀死癌细胞。PDT也可损伤肿瘤组织的血管内皮细胞及介导自身免疫系统的激活。     

Mechanisms underlying the effect of an oral antihyperglycaemic agent glyburide on calcium ion (Ca2+) movement and its related cytotoxicity in prostate cancer cells

Glyburide is an agent commonly used to treat type 2 diabetes and also affects various physiological responses in different models. However, the effect of glyburide on Ca²⁺ movement and its related cytotoxicity in prostate cancer cells is unclear. This study examined whether glyburide altered Ca²⁺ signalling and viability in PC3 human prostate cancer cells and investigated those underlying mechanisms. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in suspended cells were measured by using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Glyburide at concentrations of 100–1000 μM induced [Ca²⁺]_i rises. Ca²⁺ removal reduced the signal by approximately 60%. In Ca²⁺-containing medium, glyburide-induced Ca²⁺ entry was inhibited by 60% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X), and modulators of store-operated Ca²⁺ channels (nifedipine, econazole and SKF96365). Furthermore, glyburide induced Mn²⁺ influx suggesting of Ca²⁺ entry. In Ca²⁺-free medium, inhibition of phospholipase C (PLC) with U73122 significantly inhibited glyburide-induced [Ca²⁺]_i rises. Treatment with the endoplasmic reticulum (ER) Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished glyburide-evoked [Ca²⁺]_i rises. Conversely, treatment with glyburide abolished BHQ-evoked [Ca²⁺]_i rises. Glyburide at 100–500 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, glyburide induced [Ca²⁺]_i rises by Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and Ca²⁺ release from the ER in a PLC-dependent manner. Glyburide also caused Ca²⁺-independent cell death. This study suggests that glyburide could serve as a potential agent for treatment of prostate cancer.