特异性小干扰RNA下调中期因子基因表达对膀胱癌细胞化疗敏感性的影响

目的 观察中期因子(MK)基因小干扰RNA(siRNA)对膀胱癌细胞化疗药物敏感性的影响.方法 设计合成3个MK siRNA,转染人膀胱癌RT4细胞株,定量RT-PCR方法筛选下调MK mRNA效果最好的siRNA;以该siRNA转染RT4细胞后分组:空白对照组(Con-A)、空载对照组(Con-B)、错配对照组及MK siRNA 3.125、6.25、12.50、25.00、50.00、100.00、200.00 nmol/L组,实时定量RT-PCR和蛋白质印迹方法检测各组细胞MK表达情况;噻唑盐法检测紫杉醇(PTX)对各组细胞生长的影响;通过检测caspase-3活性和TUNEL方法观察癌细胞凋亡情况.结果 3个siRNA均明显下调MK mRNA水平,以S3效果最好.PTX组、Con-A十PTX组、Con-B+PTX组、siRNA 12.50 nmol/L组、3.125 nmol/L+PTX组、siRNA 6.25 nmol/L+PTX组、siRNA 12.50nmol/L+PTX组肿瘤细胞抑制率分别为(18.21±0.36)%、(18.19±0.29)%、(17.89±0.33)%、(1.86±0.52)%、(32.56±0.53)%、(53.83±0.38)%、(78.95±0.55)%.TUNEL结果显示,ConA、Con-B、siRNA 3.125、6.25、12.50 nmol/L组凋亡指数分别为(1.81±0.36)%、(1.89±0.38)%、(5.56±0.58)%、(9.68±0.55)%和(15.25±0.56)%.结果 MK基因siRNA可增强膀胱癌细胞化疗敏感性,诱导凋亡是其重要机制之一.     

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。     

生存素反义核酸增加人结肠癌对泰素帝的化疗敏感性

目的探讨生存素(survivin)反义RNA真核表达质粒pcDNA3.0/survivin对泰素帝诱导人结肠癌多药耐药细胞株LOVO/Adr凋亡的影响。方法将已构建成功的survivin反义RNA真核表达质粒pcDNA3.0/survivin用脂质体瞬时转染LOVO/Adr细胞,以RT-PCR法检测质粒转染前后细胞survivinmRNA的变化,用MTT法和流式细胞术分别观察泰素帝对转染细胞的毒性作用和细胞凋亡变化。结果pcDNA3.0/survivin明显下调LOVO/Adr细胞survivinmRNA表达,MTT检测发现,泰素帝对转染pcDNA3.0/survivin、pcDNA3.0及未转染细胞抑制率分别为(37.3±2.9)%,(21.9±2.3)%和(21.1±1.9)%,前者与后两者间差异具有统计学意义(P<0.01);流式仪分析显示,各组细胞凋亡率分别为(28.7±1.7)%、(13.4±1.6)%与(14.3±1.8)%,前者与后两者间差异有统计学意义(P<0.01)。结论Survivin反义RNA真核表达质粒pcDNA3.0/survivin能下调LOVO/Adr细胞survivin基因表达,增加其对泰素帝的敏感性,为临床提高结肠癌疗效提供了一种新思路。     

Cold cardioplegic arrest enhances heat shock protein 70 in the heat-shocked rat heart

BACKGROUND: Myocardial content of the 70-kd heat shock protein has been found to correlate with improved cardiac recovery after ischemia, but the mechanisms and conditions that regulate its level, particularly under clinical conditions, are unclear. The aim of this study was to assess the effect of hypothermic cardioplegic arrest and reperfusion on the expression of 70-kd heat shock protein in a protocol mimicking conditions of preservation for cardiac transplantation. METHODS: Heat-shocked and control hearts were subjected to 4 hours of cardioplegic arrest and global ischemia at 4 degrees C and then to 20 minutes of reperfusion. Hearts were freeze clamped at different time points-after 15 minutes of Langendorff perfusion, at the end of ischemia, and after 20 minutes of reperfusion, and analyzed for heat shock protein 70 content by Western blotting. Another set of hearts was subjected to 10 minutes of normothermic ischemia and 20 minutes of reperfusion followed by freeze clamping and analysis of heat shock protein 70 content as in cardioplegic arrest protocol. Cardiac function was measured by means of a left ventricular balloon at the end of reperfusion. RESULTS: Preischemic concentration of 70-kd heat shock protein was increased in heat-shocked hearts compared with control hearts. The content of 70-kd heat shock protein in heat-shocked hearts was further increased from 5.0 +/- 2.4 ng/microg at the end of ischemia to 11.0 +/- 4.9 ng/microg (n = 8, mean +/- SD; P <.05) at 20 minutes of reperfusion after cold cardioplegic arrest. No further rise in 70-kd heat shock protein of the heat-shocked hearts was observed after normothermic ischemia. Maximal developed pressure was 120.8 +/- 13.4 mm Hg in control hearts compared with 164.7 +/- 22.5 mm Hg in heat-shocked hearts (n = 5, mean +/- SD; P =.037) after cardioplegic arrest. By contrast, after normothermic ischemia, maximum developed pressure was 111.2 +/- 10.9 mm Hg in control hearts compared with 139.2 +/- 11.0 mm Hg in heat-shocked hearts (n = 4, mean +/- SD; P =.031). CONCLUSION: Hypothermic cardioplegic arrest but not short normothermic ischemia triggered a further increase in the level of 70-kd heat shock protein in heat-shocked rat hearts, which may enhance endogenous cardiac protection.     

In vitro chemosensitivity of J-82 human bladder cancer cells

Summary While chemotherapy offers a valuable adjunct to surgery in the management of intravesical bladder cancer, an accurate in vitro predictive test for chemosensitivity has yet to be developed. Drug sensitivity of the human bladder cancer cell line J-82 was assessed using monolayer, stem cell and [³H]thymidine incorporation assays. The 72-h monolayer assay provided a rapid reflection of in vitro drug sensitivity and when combined with the labeling index the results generally paralleled those obtained with the soft agar stem cell assay without the associated large commitment of time and labor. It is suggested that 72-h monolayer assay alone or in combination with [³H]thymidine labeling index may offer valuable insight into the chemotherapeutic response of bladder tumors.Supported in part by a grant from the Commission on Academic Excellence and a Cancer Research Fellowship from the University of Louisville     

谷氨酰胺诱导热休克蛋白70表达对高氧肺损伤的干预效果

目的观察谷氨酰胺诱导大鼠肺组织表达热休克蛋白70(HSP70)的作用及HSP70对高氧肺损伤的保护作用。方法健康清洁级雄性Wistar大鼠18只(体重252~286g),随机均分为谷氨酰胺组(G组)和对照组(C组):G组以0.75g/kg谷氨酰胺行腹腔注射,每天注射1次,连续3d;C组大鼠每天腹腔注射同等容积的生理盐水。于注射后第4天每组各取3只大鼠的肺组织,用免疫印迹法(Western blotting)检测HSP70表达,其余12只大鼠全部放入高氧环境(氧浓度95%)中继续喂养,观察在高氧环境下第6天两组大鼠肺组织形态学改变。结果注射谷氨酰胺后第4天,G组大鼠肺组织HSP70蛋白表达水平明显升高,其灰度值为20.34±2.26;C组HSP70蛋白表达水平较低,其灰度值为1.82±0.67,G组明显高于C组(P<0.05)。G组第6天肺部炎症表现为肺泡内极少量红细胞渗出;而C组病理改变为肺泡大小不等,肺泡腔变大,肺泡壁变薄,有肺大泡形成,肺泡内有出血和炎症细胞浸润。结论大鼠腹腔注射谷氨酰胺可明显增加肺组织HSP70表达,HSP70可减轻高氧肺损伤时肺组织炎性改变。     

热休克蛋白70 反义寡核苷酸对膀胱癌细胞系EJ细胞对丝裂霉素C敏感性的影响

目的　了解热休克蛋白 70 (HSP70 )反义寡核苷酸增强膀胱癌细胞系EJ细胞对丝裂霉素C (MMC)敏感性的作用。方法　用 10 μmol/LHSP70反义寡核苷酸封闭EJ细胞HSP70mRNA ,将 5 0μg/L的MMC与其共培养 ,采用逆转录 聚合酶链反应技术检测HSP70mRNA表达的降低情况 ,四甲基噻唑蓝试验和集落形成试验检测EJ细胞的生长情况。以正义寡核苷酸、无义寡核苷酸处理及未处理EJ细胞为对照。结果　经HSP70反义寡核苷酸处理的EJ细胞 ,HSP70mRNA的表达 (吸光度值为 132± 18)明显低于经正义、无义寡核苷酸处理的细胞 (吸光度值分别为 312± 2 3、32 5± 12 4 ,U值分别为95、10 1,均P <0 0 1) ;对MMC的敏感性 ,细胞生长抑制率、细胞集落抑制率分别为 (5 4 3± 12 3) %和(5 1 8± 12 6 ) % ,明显高于相应的正义 [(11 2± 3 6 ) %和 (13 4± 4 6 ) % ,U值分别为 86、98,均P <0 0 1]、反义寡核苷酸处理的细胞 [(9 6± 2 3) %和 (10 4± 3 0 ) % ,U值分别为 110、10 6 ,均P >0 0 1]。结论　HSP70反义寡核苷酸能增强膀胱癌细胞系EJ细胞对MMC的敏感性。     

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。     

Immunolocalization of heat shock protein 70 in bovine spermatozoa

Heat shock protein 70 (HSP70) is part of a superfamily of molecular chaperones, which protect cells from chemical and heat shock. The objectives of this study were to determine the presence of HSP70 in bovine spermatozoa and its subcellular localization during different stages of spermatogenesis. Analysis of sperm proteins by Western blotting using a monoclonal antibody to the inducible form of HSP70 revealed a single immunoreactive band with an estimated molecular weight of 70 kDa in samples from 18 of 18 bulls. Using immunofluorescence microscopy and the same antibody, HSP70 was localized to the cytoplasm of prophase spermatocytes and elongating spermatids, to cytoplasmic droplets of caput epididymal spermatozoa, and to cytoplasmic droplets, acrosome, post-acrosomal region and middle piece of corpus and cauda epididymal spermatozoa. The pattern of distribution changed in freshly ejaculated spermatozoa as HSP70 was detected on the acrosome only. During capacitation and acrosome reaction, HSP70 was once again redistributed, and was localized to the equatorial segment, post-acrosomal region and middle piece. Thus, HSP70 is present in the spermatozoa of mature bulls and redistribution of the protein occurs during capacitation and the acrosome reaction.     

Expression of heat shock protein 70 mRNA in polymorphonuclear cells responding to surgical stress

Purpose. This study was performed to investigate the expression of heat shock protein (HSP) 70 mRNA in polymorphonuclear neustrophils (PMN) as a possible new biomarker for surgical stress. Methods. The HSP70 mRNA in PMN of 10 patients who underwent lobectomy was evaluated by Northern blot analysis. Their leukocyte counts, including white blood cells (WBC) and PMN, plasma cortisol levels, and plasma interleukin-6 (IL-6) levels, were obtained by cell counting, radioimmunoassay, and enzyme-linked immunosorbent assay, respectively. Results. The level of HSP70 mRNA in PMN slightly increased at the end of surgery and showed a significant increase 6 h after surgery. It promptly decreased at 24 h postoperatively and returned to the basal preanesthetic level 48 h after surgery. On the other hand, WBC/PMN counts, plasma cortisol, and IL-6 significantly increased at the end of surgery. WBC/PMN counts remained at increased levels until 48 h postoperatively. Cortisol peaked at 6 h postoperatively and gradually decreased. IL-6 reached a maximum at 1 h postoperatively, then tapered down to its basal level at 48 h postoperatively. Conclusion. Expression of HSP70 mRNA in PMN that is induced after thoracic surgery appears to be a promising candidate as a marker for evaluating surgical stress. Received for publication on August 31, 1998; accepted on March 10, 1999     

丙戊酸钠增强免疫细胞对人膀胱癌细胞的杀伤作用

目的:探讨丙戊酸钠(VPA)对膀胱癌细胞MICA表达的影响以及所产生的肿瘤免疫作用,以期为防治膀胱癌复发和浸润提供新的治疗方法。方法:采用不同浓度VPA处理人膀胱尿路上皮癌T24细胞后,用半定量RT-PCR和流式细胞术检测癌细胞中MICA mRNA和蛋白表达。用乳酸脱氢酶法检测外周血单个核细胞(PBMCs)对经VPA处理的T24细胞的杀伤作用。结果:VPA从mRNA和蛋白水平诱导T24细胞表达MICA,并增强T24细胞对PBMCs细胞毒作用的敏感性。结论:VPA能增强膀胱癌对免疫细胞杀伤作用的敏感性,可作为防治膀胱癌的辅助药物。     

丙氨酰-谷氨酰胺双肽对肿瘤术后患者热休克蛋白70及肿瘤坏死因子的影响

目的 观察丙氨酰-谷氨酰胺双肽对肿瘤手术患者热休克蛋白、炎症反应及免疫状态的影响。方法 接受胸、腹部肿瘤切除手术患者116例进入研究，采用双盲设计，研究组58例接受丙氨酰-谷氨酰胺双肽补充的全胃肠外营养，对照组58例接受一般全胃肠外营养。7d后观察热休克蛋白（HSP）-70、肿瘤坏死因子（TNF）-α及CIM／CD8变化。结果 两组患者研究期间均未出现死亡及过敏等副反应，丙氨酰-谷氨酰胺组7d后TNF—α明显降低，CIM／CD8比率明显增多，HSP-70表达明显增高，差异有统计学意义（P〈0．05）；而对照组7d前后无明显变化。结论 肿瘤手术患者术后使用丙氨酰-谷氨酰胺能增加HSP-70表达，减轻患者炎症反应，并改善患者免疫功能状态，对患者恢复有益。     

Induction of heat shock protein 70 inhibits ischemic renal injury

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-β (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.     

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.     

Kinetics of heat shock protein 70 synthesis in the human heart after cold cardioplegic arrest.

OBJECTIVE: Protection of the myocardium against ischemia/reperfusion injury is a major challenge in cardiac surgery and cardiology. A cardioprotective role of heat shock proteins (Hsp), in particular Hsp 70, against ischemia has been demonstrated. A prerequisite for clinical exploitation of high Hsp 70 levels in the heart during ischemia is the determination of the efficacy and the kinetics of cardiac Hsp synthesis in vivo. METHODS: We examined Hsp 70 and other immediate early genes, that are induced by cardioplegia and reperfusion, in right atrial biopsies taken from 15 patients during coronary artery bypass grafting. Specimens were obtained before cardioplegia and after ending of reperfusion and subsequently studied by immunohistochemistry and Western blot analyses. RESULTS: Overall Hsp 70 increased 2.0+/-1.1-fold (P<0.01) in the nucleus as well as in the cytosol of myocytes and endothelial cells during open-heart surgery. As determined by comparison to a dilution series of recombinant protein, Hsp 70 levels amounted up to 6 per thousand of total cellular protein. The increase of Hsp 70 correlated well with the duration of cardioplegia and reperfusion (P<0.005) showing a markedly accelerated increase at periods longer than 2 h. Further, the immediate early gene c-Fos also increased 2.4+/-2.2-fold during open-heart surgery (P<0.05), whereas other members of the Hsp family, like Hsp 27 and Hsp 90, showed no significant changes in protein levels during cardioplegia and reperfusion. CONCLUSIONS: These findings demonstrate that protein levels of Hsp 70 in the myocardium increase to significant amounts within few hours after induction. The optimum time point for induction of Hsp 70 appears to be at least 2 h before open-heart surgery.     

Mild electrical stimulation with heat stimulation increase heat shock protein 70 in articular chondrocyte

The objective of this study is to investigate the effects of mild electrical stimulation (MES) and heat stress (HS) on heat shock protein 70 (HSP70), that protects chondrocytes and enhances cartilage matrix metabolism, in chondrocyte and articular cartilage. Rabbit articular chondrocytes were treated with MES and/or HS. The safeness was assessed by LDH assay and morphology. HSP70 protein, ubiquitinated proteins and HSP70 mRNA were examined by Western blotting and real‐time PCR. Rat knee joints were treated with MES and/or HS. HSP70 protein, ubiquitinated proteins, HSP70 mRNA and proteoglycan core protein (PG) mRNA in articular cartilage were investigated. In vitro, HS increased HSP70 mRNA and HSP70 protein. MES augmented ubiquitinated protein and HSP70 protein, but not HSP70 mRNA. MES + HS raised HSP70 mRNA and ubiquitinated protein, and significantly increased HSP70 protein. In vivo, HS and MES + HS treatment augmented HSP70 mRNA. HS modestly augmented HSP70 protein. MES + HS significantly increased HSP70 protein and ubiquitinated proteins. PG mRNA was markedly raised by MES + HS. This study demonstrated that MES, in combination with HS, increases HSP70 protein in chondrocytes and articular cartilage, and promotes cartilage matrix metabolism in articular cartilage. MES in combination with HS can be a novel physical therapy for osteoarthritis by inducing HSP70 in articular cartilage. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 894–900, 2013