谷氨酰胺对人肺泡Ⅱ型上皮细胞热休克蛋白70表达的影响

目的评价谷氨酰胺对人肺泡Ⅱ型上皮细胞系A549细胞热休克蛋白（HSP）70表达的影响。方法取人肺泡Ⅱ型上皮细胞系A549细胞,在不含谷氨酰胺的DMEM培养液中孵育24h作为空白对照组（C组）,43℃孵育1h、37℃恢复4h作为阳性对照组（PC组）,不同浓度（2、4、8、12和16 mmol/L）谷氨酰胺的DMEM培养液中孵育24h作为不同浓度谷氨酰胺诱导组（Gln2组、Gln4组、Gln8组、Gln（12）组和Gln（16）组）,8mmol/L谷氨酰胺的DMEM培养液中孵育不同时间（1、2、6、12、24和48 h）作为不同时间谷氨酰胺诱导组（T1组、T2组、T3组、T4组、T5组和T6组）。分别采用RT-PCR和Western blot法检测HSP70 mRNA和蛋白的表达。结果与C组比较,PC组和不同浓度谷氨酰胺诱导组人肺泡Ⅱ型上皮细胞系A549细胞HSPTO mRNA和蛋白的表达均升高,Gln8组HSP70 mRNA和蛋白表达水平高于Gln2组、Gln4组、Gln（12）组和Gln（16）组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。与C组比较,PC组和不同时间谷氨酰胺诱导组HSP70 mRNA和蛋白的表达均升高,T5组HSP70 mRNA和蛋白表达水平高于T1组、T2组、T3组、T4组和T5组（P＜0．01）,而与PC组相比差异无统计学意义（P＞0．05）。结论谷氨酰胺可明显上调体外培养人肺泡Ⅱ型上皮细胞系A549细胞HSP70 mRNA和蛋白的表达,并呈浓度和时间依赖性。     

棉酚对睾丸支持细胞间隙连接蛋白表达的影响

目的:探讨药物棉酚对睾丸Sertoli细胞间隙连接蛋白Cx43表达的影响。方法:培养TM4睾丸Sertoli细胞,用1.25、2.5、5、10μmol/L的棉酚分别染毒细胞6、12、24、48 h。CCk-8试剂盒检测细胞毒性,应用细胞免疫荧光化学、RT-PCR检测Cx43在正常TM4细胞和在不同浓度及不同染毒时间的TM4细胞中的表达情况。结果:半定量RT-PCR及免疫荧光结果显示,正常TM4细胞中有较多的Cx43表达;棉酚染毒24 h后,Cx43 mRNA水平开始随时间增加而逐渐下降(P<0.05),且随剂量增加Cx43蛋白表达强度逐渐减弱(P<0.05)。结论:棉酚可抑制TM4细胞表达Cx43,可能是其抗生育的机制之一。     

Insulin potentiates expression of myocardial heat shock protein 70.

OBJECTIVE: Since insulin stimulates nitric oxide (NO) production and an increase in NO following heat shock is required for myocardial heat shock protein 70 (Hsp70) synthesis, we hypothesized that insulin would enhance myocardial Hsp70 synthesis by augmenting NO signaling. We examined whether a physiologic dose of insulin increased myocardial Hsp70 in unstressed and heat shock treated rats. METHODS: Adult male Sprague-Dawley rats were assigned to groups: (1) control, (2) insulin injected (200 microU/gm body weight), (3) heat shock treated (core body temperature 42 degrees C for 15 min), (4) heat shock and insulin treated, (5) L-nitroarginine methyl ester (L-NAME) and heat shock and insulin treated, (6) sodium nitroprusside (SNP) and heat shock and insulin treated. Six hours later, myocardial Hsp70 content and localization was analyzed. RESULTS: Hsp70 was increased in heat shock treated hearts (120.6+/-16.8 ng/mg protein, P < 0.001) vs. control (12.9+/-2.0 ng/mg protein), or insulin treated hearts (15.5+/-0.83 ng/mg protein). In addition, Hsp70 was increased in the heat shock and insulin treated hearts (164.4+/-7.53 ng/mg protein) compared to control, insulin only (P = 0.001) or heat shock only treated hearts (P = 0.01). L-NAME did not abolish the insulin induced increase in Hsp70 in heat shocked hearts (195.2+/-13.4 ng/mg protein, P = 0.21) and SNP did not further enhance Hsp70 in the insulin and heat shocked group (188.9+/-8.2 ng/mg protein, P = 0.71). Western analysis and confocal microscopy revealed a lowlevel expression of myocardial Hsp70 in response to insulin. Hsp70 was localized primarily in blood vessels after insulin or heat shock treatments. CONCLUSIONS: Insulin caused a low-level expression of myocardial Hsp70 and potentiated Hsp70 synthesis in response to heat shock. The ability of insulin to potentiate Hsp70 after heat shock is independent of NO signaling as it was not altered by either LNAME or SNP pretreatment. Blood vessels appear to be the primary site of Hsp70 after insulin or heat shock treatment.     

川芎嗪预先给药对胎鼠海马神经细胞缺氧/复氧时c-fos和HSP70表达的影响

目的 探讨川芎嗪预先给药对胎鼠海马神经细胞缺氧/复氧时c-fos和热休克蛋白70(HSP70)表达的影响.方法 胎鼠海马神经细胞培养鉴定后,随机分为5组(n=24):正常对照组(C组)、缺氧/复氧损伤组(A/R组)、不同浓度川芎嗪预先给药组(L组、M组和H组).C组不制备缺氧/复氧模型;A/R组、L组、M组和H组制备缺氧/复氧模型;L组、M组和H组加入川芎嗪,终浓度分别为60、200和800μg/ml,孵育1 h后制备缺氧/复氧模型.缺氧/复氧模型制备方法:海马神经细胞置入90%N2-10%CO2培养箱中孵育2 h诱导缺氧,然后放入37 ℃、5%CO2培养箱中复氧24 h.处理结束后测定海马神经细胞凋率、细胞活力、c-fos和HSP70的表达水平.结果 与C组比较,A/R组、L组和H组海马神经细胞活力降低,细胞凋亡率升高(P＜0.01);与A/R组比较,L组、M组和H组海马神经细胞活力升高,细胞凋亡率降低,c-fos表达下调,HSP70表达上调(P＜0.05);与L组比较,M组和H组海马神经细胞活力升高,细胞凋亡率降低,c-fos表达下调,HSP70表达上调(P＜0.05);与M组比较,H组细胞活力下降,细胞凋亡率升高,c-fos表达上调,HSP70表达下调(P＜0.01).结论川芎嗪预先给药抑制胎鼠海马神经细胞缺氧/复氧时细胞凋亡的机制可能与下调c-fos表达,上调HSP70表达有关.     

Differential expression of heat shock proteins 70-1 and 70-2 mRNA after ischemia-reperfusion injury of rat kidney

Ischemia-reperfusion injury in the kidney is known to cause induction of the inducible form of the 70 kDa heat shock protein HSP70i (or HSP72). However, knowledge of the expressional regulation of the two coding genes for HSP70i –HSP70-1 gene and HSP70-2 gene – is very limited. We investigated the time course of HSP70-1 and -2 mRNA expression and its relation to cellular ATP levels in the renal cortex after different periods of unilateral warm renal ischemia (10–60 min) and reperfusion (up to 60 min) in 10-week-old male Wistar rats. Immediately after ischemia there was a significant induction of both HSP70i genes. While HSP70-1 expression constantly increased (up to 4-fold) during reperfusion, even to a higher extent with prolongation of ischemia, HSP70-2 mRNA – which was generally expressed at a far lower level than HSP70-1 mRNA – was strongly induced (3-fold) during reperfusion only after brief periods (10 min) of ischemia. Cellular ATP levels rapidly dropped to 5% with ischemia and the pattern of recovery during reperfusion significantly depended on the duration of the ischemic period, thus showing a good relation with the heat shock (protein) gene expression. We conclude that HSP70-2 is the more sensitive gene with a lower activation threshold by mild injury, while the HSP70-1 gene mediates the major response of heat shock protein induction after severe injury. Received: 16 November 1998 / Accepted: 11 March 1999     

热休克蛋白70 、P53和P-糖蛋白在膀胱癌中的表达

目的：研究热休克蛋白70（HSP70）、P53和P－糖蛋白（P－gp)在膀胱癌中表达的临床意义。方法：应用免疫组织化学方法检测40例膀胱移行细胞癌标本中HSP70、P53和P－gp表达,并分析其与膀胱癌生物学行为的关系。结果：HSP70、P53和P－gp的阳性率分别为55.0%、47.5%和57.5%,其中G1期阳性率分别为46.2%、38.5%、38.5%,G2期阳性率为52.9%、39.8%、47.1%,G3期阳性率为70.0%、80.0%、60.0%。24例初发肿瘤标本阳性率分别为37.5%、41.7%、33.3%,16例化疗后复发肿瘤标本阳性率分别为75.3%、81.3%、68.8%。检测结果提示HSP70、P53和P－gp随肿瘤级别增高呈表达增强的趋势,复发组三种蛋白的阳性率显著增高（P＜0.05)。结论：这三种蛋白的异常表达可能是膀胱肿瘤腔内化疗失败的重要原因。     

缺血预处理对犬脊髓损伤及热休克蛋白70表达影响的研究

目的评价缺血预处理对犬脊髓损伤及热休克蛋白70表达的影响。方法41条杂种犬随机分成假手术组6只、预处理组21只、对照组14只。预处理组主动脉阻断6min后开放6min,反复2次,之后阻断35min;对照组主动脉阻断35min。术后进行神经功能评分,检测脊髓组织中热休克蛋白70表达。结果在再灌注后6h、24h预处理组热休克蛋白70于胞质和胞核均有表达,且强于对照组;而且神经功能评分预处理组高于对照组。在再灌注后7d预处理组神经功能评分无明显改变,且仍见热休克蛋白70表达。结论缺血预处理可以增加脊髓的缺血耐受;热休克蛋白70在胞质和胞核中表达可能在缺血耐受中起到一定的作用。