Comparative study on the immunogenicity between Hsp70 DNA vaccine and Hsp65 DNA vaccine in humanMycobacterium tuberculosis

Summary The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in humanMycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index (SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0.01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0.01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0.05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P< 0.05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying. This project was supported by a grant from National Natural Sciences Foundation of China (No. 39870663).     

Expression of Hsp27 and Hsp70 in lymphocytes and plasma in healthy workers and coal miners with lung cancer

In coal mines, main occupational hazard is coal-mine dust, which can cause health problem including coal workers' pneumoconiosis and lung cancer. Some heat shock proteins (Hsps) have been reported as an acute response to a wide variety of stressful stimuli. Whether Hsps protect against chronic environmental coal-mine dust over years is unknown. It is also interesting to know that whether the expression of Hsp27 and Hsp70 proteins as a marker for exposure is associated risk of lung cancer among coal miners. ...     

姜黄素减轻沙土鼠海马CA1区脑缺血再灌注损伤与热休克蛋白表达变化的关系

目的:探讨姜黄素对缺血再灌注沙土鼠脑海马CA1区热休克蛋白(Hsp)基因表达的影响及意义.方法:采用沙土鼠双侧颈总动脉阻断缺血再灌注损伤模型.随机分为假手术组(SH)、脑缺血再灌注组(IR)、姜黄素组(CU)、溶剂对照组(SC);每组据再灌注时间点不同又分6 h、1 d、3 d、5 d及7 d 5个亚组,每组6只动物.在预定时间点行开阔法行为学检查,以TUNEL法行海马CA1区凋亡细胞检测,免疫组化ABC法测定Hsp70、Hsp27基因表达产物Hsp70及Hsp27蛋白在海马CA1区的动态变化.结果:姜黄素可显著减少沙土鼠探索活动及海马CA1区凋亡锥体细胞数量(与IR组相比,P＜0.01),进一步诱导Hsp70蛋白表达并抑制Hsp27蛋白的表达(与IR组相比,P＜0.01).结论:姜黄素对脑缺血再灌注损伤有保护作用,调控热休克基因hsp70和hsp27的表达可能是其作用机制之一.     

Hsp90特异性抑制剂对宫颈癌细胞存活机制的探讨

目的:研究Hsp90的特异性抑制剂格尔德霉素(GA)在宫颈癌Hela细胞存活中的作用,并对其机制进行探讨。方法:培养人宫颈癌细胞株Hela,MTT法检测GA作用后细胞抑制率,RT-PCR方法检测GA作用后Bcl-2 mRNA的表达。结果:MTT法显示GA对Hela细胞有生长抑制作用,用药浓度越高,抑制率越高。不同浓度的GA处理宫颈癌Hela细胞后,Bcl-2 mRNA呈剂量依赖性降低,10μmol/L的GA抑制作用最强。10μmol/L的GA处理Hela细胞不同时间点后,Bcl-2 mRNA的表达呈时间依赖性降低。结论:在宫颈癌Hela细胞应用特异性Hsp90抑制剂GA,能够抑制宫颈癌细胞的生长,并通过抑制Bcl-2来促进其凋亡,Hsp90可以成为宫颈癌治疗中的一个新靶点     

人HSP70真核载体的构建及其在胆囊癌细胞中的表达

目的构建真核表达载体pcDNA3．0-Hsp70，并在胆囊癌细胞SGC996中进行表达。方法构建真核表达载体pcDNA3．0-Hsp70，经EcoRⅠ及BarnHⅡ双酶切鉴定并测序；通过脂质体法转染SGC996胆囊癌细胞，经G418抗性筛选获得稳定表达细胞株；用流式细胞术检测其表达情况。结果酶切电泳和DNA测序证实载体构建正确；体外转染入SGC996细胞中后，可见转染细胞有Hsp70蛋白的表达。结论成功构建了pcDNA3．0-Hsp70真核表达载体，为研究Hsp70在肿瘤免治疗中的作用奠定了基础。     

热休克蛋白70在心理应激诱发的C3H/HeJ雌鼠乳腺癌中的表达

目的探讨热休克蛋白70(Heat shock protein70,Hsp70)在心理应激诱发的乳腺癌中的表达、作用及可能的机制.方法动物随机分为实验组(21只)和对照组(14只),对实验组小鼠给予饮水冲突和限制应激,以建立心理应激的动物模型,然后比较实验组和对照组的乳腺癌发病率、皮质酮水平和Hsp70的表达水平.结果 (1)实验组的乳腺癌发病率(71.4%)明显高于对照组的发病率(21.4%)(P<0.05);(2)实验组血浆皮质酮水平(161.51±162.82)μg/ml明显高于对照组(10.63±8.37) μg/ml(P<0.01);(3)实验组Hsp70的阳性细胞数目(12.47±11.63)显著高于对照组(3.16±1.77)(P<0.01).结论心理应激促进了Hsp70在C3H/HeJ雌鼠乳腺组织中的表达, Hsp70的过度表达和乳腺癌的发生密切相关.     

结核分枝杆菌Hsp65基因的分子克隆和表达

[摘要】 目的 对结核分枝杆菌的分泌蛋白Hsp65基因进行克隆、表达和纯化,为结核病的诊断、重组疫苗的应用提供理论基础。方法 用PCR方法从结核分枝杆菌H37Rv基因组中克隆了了Hsp65基因,将其链接到pMD18-T载体中,序列测定正确后,将完整的Hsp65基因克隆到Pet28a载体并在大肠杆菌BL21中诱导表达,表达产物经SDS-PAGE进行鉴定,并通过亲和层析对蛋白进行纯化。结果 克隆的Hsp65基因序列与GenBank公布的序列有高度同源性,表达蛋白相对分子量为65 KD,并能够纯化出单一的目的蛋白。结论 成功克隆了Hsp65基因,实现了该基因的表达和纯化,为其在结核病诊断研究中的应用奠定了基础。     

青蒿琥酯对肺纤维化大鼠热休克蛋白47表达的影响

目的：探讨青蒿琥酯对肺纤维化大鼠热休克蛋白47（Hsp47）表达的影响。方法雄性 SD 大鼠30只,分为对照组、模型组和青蒿琥酯干预组（简称干预组）。对照组一次性气管内滴入生理盐水约0．4 mL,模型组和干预组一次性气管内滴入博来霉素5 mg／kg。对照组和模型组次日每天腹腔注射生理盐水1．0 mL。干预组每天腹腔注射青蒿琥酯10 mg／100 g。记录0、14、28 d 大鼠的体质量。各组大鼠于第28天处死,计算肺系数,取肺组织进行苏木精-伊红（HE）染色、Masson 染色、羟脯氨酸检测和Hsp47、Ⅰ型胶原 RT-PCR 和 Western blot 检测。结果（1）对照组大鼠体质量增长明显高于其他两组（P ＜0．05）。（2）肺系数：模型组[（12．31±1．89）mg／g]＞干预组[（8．54±0．67）mg／g]＞对照组[（4．81±0．38）mg／g],P ＜0．05。（3）Ashcroft 评分：模型组[（5．70±1．09）分]＞对照组[（3．08±0．56）分],模型组[（5．70±1．09）分]＞干预组[（4．01±1．25）分],P ＜0．05;对照组和干预组差异无统计学意义（P ＞0．05）。（4）羟脯氨酸含量：模型组（620．33±66．16）μg／g＞对照组（379．00±35．51）μg／g,模型组（620．33±66．16）μg／g＞干预组（429．00±36．51）μg／g,P ＜0．05;对照组和干预组间差异无统计意义（P ＞0．05）。（5）Hsp47 mR-NA：模型组高于对照组,模型组和干预组比较差异无统计意义（P ＞0．05）。Ⅰ型胶原 mRNA：模型组＞干预组＞对照组（P ＜0．05）。（6）Hsp47蛋白表达水平模型组明显高于对照组和干预组。结论青蒿琥酯可能通过下调 Hsp47表达水平来抑制胶原的合成进而减轻肺纤维化。     

Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

There are 3million deaths perannum worldwidedue to tuberculosis,and AIDS is compounding theproblem.A better vaccine than the live mycobacteri-um currently in use,Bacillus Calmette- Guerin( BCG) ,is needed.Mycobacterium heat shock pro-tein ( HSP) can not only enhance the immune re-sponse,but also keep the antigen characteristics ofmycobacterium itself.Our cooperator,Silva fromLowrie group of British National Medical Academy,cloned the mycobacterium Hsp65 gene into a eukary-otic expressi…     

喉癌中Hsp70、p53、PCNA的表达及意义

目的 探讨热休克蛋白70（Hsp70)在喉癌中的表达及临床意义。方法 采用免疫组化ABC法检测Hsp70、p53蛋白及增殖细胞核抗原（PCNA）在喉癌组织中的表达情况。结果 分化差的喉癌组织中Hsp70表达水平明显增高（P＜0．01）;Hsp70与p53在喉癌组织中的表达呈密切相关性（P＜0．01）;Hsp70与PCNA增殖指数的表达呈明显相关（P＜0．001）,提示Hsp70的高表达可能是喉癌细胞高度增殖的结果;有淋巴结转移的喉癌组织中,Hsp70表达水平显著高于无淋巴结转移的癌组织（P＜0．05）。结论 Hsp70与p53蛋白在喉癌的演变过程中可能具有协同作用;Hsp70在喉癌癌变过程中起重要作用。     

HSP70诱饵载体的构建及其在酵母双杂交系统中自激活作用的检测

目的：构建用于酵母双杂交系统的“诱饵”载体pGBKT7-Hsp70，并检测其是否具有自激活作用。方法：用PCR的方法从质粒pGBKT7-Hsp70中扩增出Hsp70基因片段，引入酶切位点EcoRⅠ，BamHⅠ，测序正确后，将Hsp70基因克隆至载体pGBKT7中，并检测pGBKT7-Hsp70在MATHMAK－ER GAL4 Two-Hybrid System3中的自激活作用。结果：成功构建了“诱饵”载体pGBKT7-Hsp70，并证明其在酵母双杂交系统中无自激活作用。结论：pGBKT7-Hsp70可以用于酵母双杂交系统中，寻找与Hsp70相互作用的分子。     

RNA干扰Hsp70基因表达抑制胃癌细胞增殖

目的 构建Hsp70的短发夹状RNA(shRNA),检测其在胃癌细胞株SGC-7901中对Hsp70基因表达的抑制作用以及对肿瘤细胞增殖的影响.方法 根据Hsp70编码基因,设计并合成2对反向重复序列,同时设计1对与目的 基因不相匹配的重复序列作为对照组分别定向克隆至载体pTZU6+1的U6转录启动子下,构建重组质粒phspl-siRNA,phsp2-siRNA及对照组质粒phsp3-siRNA,分别转染胃癌细胞株SGC-7901,采用RT-PCR和Western blot检测phsp-siRNA对Hsp70基因表达的抑制作用.利用AlamarBlue法检测转染后对胃癌细胞生长的影响.结果 酶切电泳分析和DNA测序证实重组质粒phsp-siRNA构建成功;证实phsp1-siRNA和phsp2-siRNA对内源性Hsp70基因的表达有明显的抑制作用,同时phsp1-siRNA和phsp2-siRNA转染组细胞的生长较对照组受到明显抑制.结论 构建的重组质粒phsp1-siRNA和phsp2-siRNA能特异而有效抑制内源性Hsp70基凶在SGC-7901细胞中的表达,并对细胞生长有明显抑制作用.     

卡介苗菌株Hsp16.3基因打靶载体的构建及鉴定

目的 构建卡介苗菌株Hsp16.3基因打靶载体.方法 体外培养卡片介苗菌株并提取基因组DNA,扩增Hsp16.3基因两侧序列5′-Hsp16.3和3'-Hsp16.3,分别插入到pKO质粒载体预定位点中构建Hsp16.3基因置换型打靶载体,筛选并进行PCR、双酶切鉴定及测序鉴定.结果 构建的卡介苗菌株Hsp16.3基因打靶载体,经PCR、双酶切鉴定及测序证实pKO质粒中的2个插入片段为所需目的基因.结论 成功构建出卡介苗菌株Hsp 16.3基因打靶载体.     

Temperature-induced stress on growth and expression of hsp in freshwater alga Scenedesmus quadricauda

Objective To investigate the impact of various levels of sublethal temperature （26℃, 31 ℃, 33℃, 36℃, and 39℃） on growth and heat shock protein （hsp） expression in freshwater green alga Scenedesmus quadricauda. Methods Impact of selected levels of temperature on growth rate （based on optical density）, population count, chlorophyll-a and biomass of the alga was evaluated in artificial growth medium for 19 days. To determine the induction of hsp in the alga, it was exposed to selected temperature levels for 3 h and further kept for 6 h at culturing condition at 26℃. Induction of hsp was confirmed by immuno-detection followed by SDS-polyacrylamide gel electrophoresis. Results The selected growth parameters such as growth rate, population count, chlorophyll-a and biomass were reduced significantly （P〈0.001） at 39℃. However, hsp 70 expression was observed only at 39℃. Conclusion Temperature up to 36℃ may be considered as the limit of safe exposure for thermal, stress for the alga Scenedesmus quadricauda.     

正畸力作用下大鼠牙髓组织中热休克蛋白70的表达及意义

目的检测正畸力作用下大鼠牙髓组织中热休克蛋白70（Hsp70）的表达和分布。探讨其在正畸移动中的作用和机制。方法建立大鼠正畸牙移动模型,分别在受力1、15、30d处死,制备左侧上颌第一磨牙及其周围牙槽骨的石蜡标本,采用免疫组织化学法检测大鼠正畸牙移动过程中牙髓组织中Hsp70的表达情况。结果正常对照组、正畸加力1d组、正畸加力15d组和正畸加力30d组牙髓组织中Hsp70表达量分别为402±003、28．13±0．23、10．02±0．08、5．23±0．42,正畸加力1d组大白鼠牙髓组织中Hsp70表达量显著高于正常对照组（P〈0．01）,正畸加力15d组和正畸加力30d组与正常对照组的差异无统计学意义（P〉0．05）。结论Hsp70参与了正畸牙移动,并且保护了牙髓组织。     

梅毒螺旋体Hsp40蛋白的生物信息学分析及基因克隆

目的分析梅毒螺旋体(Treponema pallidum,Tp)Hsp40蛋白的生物学特性并克隆其基因。方法通过GenBank查到Hsp40蛋白的序列,利用生物信息学软件分析其生物学特性。根据Tp Hsp40基因序列设计特异性引物,利用PCR扩增Hsp40基因,并把该DNA片段连接到pMD19-T载体,转化大肠杆菌后挑取阳性克隆采用PCR和DNA测序进行验证。结果 Tp Hsp40基因能够编码374个氨基酸的蛋白质,其分子量为40.1 k Da,无信号肽,呈亲水性。Swissmodel预测得到Tp Hsp40的三级结构与嗜热杆菌(Thermus thermophilus)Hsp40结构最接近。从Tp基因组中扩增得到1125 bp的Tp Hsp40基因并将其连接到pMD19-T载体。结论本文预测了梅毒螺旋体毒力因子Hsp40蛋白的结构和克隆得到Tp Hsp40基因。     

Hsp70.1在冬凌草甲素处理的肝癌细胞中的作用研究

目的 研究Hsp70.1在冬凌草甲素处理后的肝癌细胞HepG2中的表达情况及其与冬凌草甲素抗肿瘤活性的关系。方法 采用蛋白印迹法及实时荧光定量PCR技术观察Hsp70.1在冬凌草甲素处理的肝癌细胞HepG2的表达变化,并采用RNA干扰技术观察抑制Hsp70.1表达后对冬凌草甲素抗肝癌效果的影响。结果 冬凌草甲素作用12 h后,HepG2细胞的Hsp70.1蛋白及RNA表达均增加。Hsp70.1 短发夹RNA（shRNA）转染抑制Hsp70.1表达后,与对照组相比,Hsp70.1 shRNA转染组、冬凌草甲素处理组或两者联合应用组的HepG2细胞的存活率均明显降低。 结论Hsp70.1可促进HepG2细胞的生存,对HepG2细胞和冬凌草甲素处理的HepG2细胞具有保护作用。     

结核杆菌Hsp65和hGM-CSF双顺反子表达质粒的构建与表达

目的:应用分子生物学技术,构建pIHsp65GM双顺反子真核表达质粒,并在真核细胞中表达.方法:以结核分枝杆菌H37Rv株基因组为模板经聚合酶链反应(PCR)扩增出Hsp65基因,同时从质粒pORF-hGM-CSF中扩增出基因佐剂hGM-CSF,分别克隆到真核表达载体pIRES的多克隆位点A(MCSA)和B(MCSB)中,构建pIHsp65GM真核表达质粒,并转染HepG-2细胞中表达和检测.结果:酶切鉴定提示插入的基因片段大小分别为1.64 kb和0.47 kb,测序分析表明克隆的Hsp65和hGM-CSF序列与GenBank上公布序列完全一致;免疫组织化学方法检测到表达Hsp65的阳性细胞;ELISA方法检测到pIHsp65GM转染组细胞培养上清中hGM-CSF的表达,与空载体对照组比较差异有统计学意义(P＜0.01).结论:成功构建和表达了pIHsp65GM质粒,为研制优于卡介苗的新型抗结核病DNA疫苗奠定基础.     

In vitro study on role of Hsp70 expression in DNA damage of human embryonic lung cells exposed to Benzo[a]pyrene

Objective Benzo[a]pyrene (B[a]P), a ubiquitous environmental pollutant, is a potent procarcinogen and mutagen that can elicit tumors, leading to malignancy. Heat shock proteins (Hsp) have been shown to protect cells against damages caused by various stresses including exposure to numerous chemicals. Whether Hsps, or more specifically Hsp70, are involved in repair of B[a]P-induced DNA damage is currently unknown. Methods We assessed the potential role of the inducible form of Hsp70 in B[a]P-induced DNA damage of human embryonic lung (HEL) cells using immunoblot and the comet assay (i.e., the single cell gel electrophoresis assay). Results Exposure to B[a]P induced a dose-dependent decrease in the level of Hsp70, but a dose-dependent -increase in DNA damage both in untreated (control) HEL cells and in cells preconditioned by a heat treatment. Heat preconditioning prior to B[a]P exposure potentiated the effect of B[a]P at a low dose (10μmol/L), but appeared to be protective at higher doses. There was a negative correlation between Hsp70 level and DNA damage in the non-preheated as well as in the preconditioned cells.Conclusion These data suggest that exposure of HEL cells to B[a]P may induce a dose-dependent reduction in the levels of the inducible Hsp70. The detailed mechanisms for the reduction of Hsp70 levels by B[a]P and the role of Hsp70 in DNA damage under different concentrations of B[a]P remains to be determined.     

基于hsp65基因的PCR技术在检测临床标本中结核杆菌的应用

目的:评价基于hsp65的多聚酶链反应(PCR)在快速检测结核分枝杆菌(MTB)中的应用。方法:从80例临床标本(68份痰标本,10份脑脊液,2份活检标本)中提取MTB基因DNA,设计针对hsp65基因的特异性引物,PCR扩增hsp65基因后用于琼脂糖凝胶电泳观察,同时选择部分阳性产物进行测序。结果:本方法的敏感性为86.44%,特异性为90.48%,阳性和阴性预测值分别为96.23%和70.37%。结论:基于hsp65的PCR能有效区分结核分枝杆菌和非结核分枝杆菌,是一种简单有效的方法。