乌司他丁对大鼠脑缺血再灌注损伤后的保护作用及其机制研究

目的:探究乌司他丁在脑缺血再灌注损伤中的脑保护作用机制。方法:原代分离培养雄性SD大鼠脑皮质细胞,部分细胞经siRNA沉默HSP70基因。细胞先以无糖培养基在低氧条件下培养,12 h后复糖复氧模拟体外缺血再灌注损伤,并实施乌司他丁预处理干预,流式细胞术检测各组细胞的凋亡率,western-blotting检测Bcl-2,Bax,HSP70,JNK和p-JNK蛋白的表达。结果:与对照组比较,模型组脑组织细胞凋亡率明显增多(P0.05)、Bcl-2和Bax的表达量均有上调,Bcl-2/Bax的比值显著降低(P0.01)、HSP70的表达无显著变化;与模型组比较,乌司他丁处理组脑组织细胞凋亡率明显降低(P0.05)、Bax的表达量显著下调(P0.05),Bcl-2/Bax的比值显著上调(P0.05),HSP70的表达显著上调(P0.05),JNK的表达无显著变化、p-JNK则显著下调(P0.05)。HSP70沉默后乌司他丁的脑保护作用消失,对以上蛋白的表达无显著影响。结论:乌司他丁可能是通过上调HSP70表达进而抑制JNK信号转导通路对缺血再灌注引起的脑损伤起保护作用。     

HSP70在放线菌素D所致肺腺癌细胞增殖抑制和凋亡中的作用

为探讨热休克蛋白70(heatshockprotein70,HSP70)在肺腺癌中的表达及其作用,首先采用免疫印迹检测经临床支纤镜确诊并行手术切除的肺腺癌组织标本中HSP70的表达,结果显示在癌旁正常组织中HSP70的表达量显著低于其在癌组织中的表达量.其次,采用HSP70反义寡核苷酸阻断A549细胞中HSP70表达后,经MTT和Hoechst33258检测发现,HSP70下调能显著促进放线菌素D(actinomycinD,ActD)所致的A549细胞增殖抑制及凋亡,反义寡核苷酸处理组与正义或随机寡核苷酸处理组比较P值均小于0.05.进一步构建HSP70真核重组质粒并瞬时转染A549细胞后,能显著增加A549细胞中HSP70表达,同时采用MTT和流式细胞术及基因组DNA琼脂糖凝胶电泳检测发现,HSP70过表达能显著抵消ActD所致细胞增殖抑制及凋亡,转染HSP70重组质粒组与转染空载体组相比P值小于0.05.上述结果提示,HSP70在肺腺癌组织中高表达,高表达的HSP70在降低肺腺癌细胞对ActD的敏感性及促进癌细胞增殖与抑制凋亡等方面发挥了重要作用.     

人热休克蛋白70和热休克固有蛋白70的基因克隆和表达

目的:克隆人热休克蛋白70(HSP70)和热休克固有蛋白70(HSC70)基因,并在大肠杆茵中表达,获得重组蛋白.方法:用RT-PCR法从HepG2细胞中扩增HSP70及HSC70cDNA序列.测序后,将相应的cDNA插入pRSET-A表达载体,在大肠杆菌中表达,重组蛋白纯化后用SDS-PAGE及Western Blotting分析.结果:DNA序列结果显示.本研究所获得的HSP70及HSC70 cDNA序列与参考序列一致.将全长cDNA分别插入表达质粒后,转化BL21(DE3)细菌,在IPTG的诱导下,表达产物SDS-PAGE显示相应的分子量(70kDa)位置有明显的蛋白条带.Western Blotting结果证实了其为目的蛋白,经镍树脂柱纯化,获得了相应的重组多肽.结论:成功构建了原核表达重组质粒HSP70-pRSET-A和HSC70-pRSET-A,并获得了纯化的重组人HSP70和HSC70蛋白,为进一步研究这两种蛋白的结构、功能及临床应用奠定了基础.     

吐温80合并温热诱导BGC-823胃癌细胞凋亡及对Hsp70,Bcl-2和Bax表达的影响

目的　观察膜活化剂吐温 80合并温热作用对BGC 82 3人胃癌细胞生长、凋亡及对Hsp70、Bcl 2、Bax表达的影响。方法　应用MTT法、荧光染色及DNA琼脂糖凝胶电泳 ,测定 0 1%吐温 80与 4 2℃ 10 0min合并作用对BGC 82 3细胞的抑制效应和诱导细胞凋亡的影响 ;应用免疫细胞化学染色 ,观察合并作用后不同时间 (0h、 8h、 16h、 2 4h)BGC 82 3细胞Hsp70、Bcl 2、Bax的表达改变。结果　①吐温 80合并 4 2℃温热作用对细胞具有明显的抑制效应 (P <0 0 0 1)。②合并作用后 2 4h ,可见大量肿瘤细胞凋亡。③合并作用可明显抑制BGC 82 3细胞Hsp70的表达 ;Bcl 2、Bax的表达均增加 ,且Bax的表达强于Bcl 2 ,并全部分布在胞浆。结论　吐温 80合并 4 2℃温热可通过诱导细胞凋亡来抑制肿瘤细胞生长 ,其对细胞Hsp70表达的抑制和对Bcl 2、Bax表达及分布的影响可能与凋亡的发生有关     

长江江豚TRAIL基因的克隆、体外表达及生物学功能分析

构建可溶性肿瘤坏死因子相关凋亡诱导配体(TRAIL)基因的表达体系,研究其蛋白表达产物对肿瘤细胞凋亡的影响,为以后江豚免疫系统的研究奠定基础。通过RT-PCR技术从江豚Neophocaena phoconoides血液总RNA中反转录扩增出肿瘤坏死因子相关凋亡诱导配体(简称fTRAIL)的全长cDNA序列,并将fTRAIL的胞外可溶性(简称fsTRAIL)片段连接入表达载体pET43.1a中,在大肠杆菌BL21(DE3)中表达并纯化,Western blotting对产物Nus-His-fsTRAIL蛋白进行鉴定。体外用MTT法、台盼蓝拒染法及流式细胞术检测Nus-His-fs TRAIL蛋白对Jurkat细胞和HeLa细胞的影响。成功构建了fTRAIL胞外可溶性片段(简称fsTRAIL)与pET43.1a组成的表达载体,并获得Nus-His-fsTRAIL蛋白。体外实验表明,Nus-His-fsTRAIL蛋白能够以剂量依赖的方式抑制Jurkat和HeLa细胞的增殖并诱导其凋亡。Nus-His-fsTRAIL表达产物具有对Jurkat和HeLa细胞体外抗肿瘤活性的作用。     

人骨保护素-热休克蛋白70融合蛋白在昆虫细胞中的表达和活性分析

目的:利用BaculoDirect杆状病毒表达系统融合表达人OPG功能片段p22-194和分枝杆菌HSP70 p111-125基因,并鉴定重组蛋白及其生物学活性。方法:将编码人OPG功能片段和分枝杆菌HSP70功能片段基因克隆至杆状病毒转座载体,将重组转座载体与BaculoDirectTM Linear DNA进行LR重组连接反应,构建出重组杆状病毒DNA,转染Sf9昆虫细胞,获得重组病毒。在Sf9细胞中进行表达,并对表达产物进行SDS-PAGE电泳、Western blotting分析,用Ni柱纯化。采用破骨细胞生成抑制试验和抑炎试验鉴定表达产物的生物学活性。结果:重组病毒在感染昆虫细胞后48h开始出现一相对分子质量为28 kDa大小的特异条带,感染后72～96 h蛋白量达到高峰。破骨细胞生成抑制实验及抑炎试验结果显示,重组蛋白能明显抑制破骨细胞的生长和分化,同时亦具有抑制炎症反应的作用。结论:利用杆状病毒表达系统在昆虫细胞中成功表达OPG-HSP70融合蛋白,该融合蛋白具有抑制破骨细胞生成和抑制炎症反应生物学活性。     

结核杆菌抗原HSP65基因在大肠杆菌中的表达及纯化

目的 :为研制预防结核病疫苗 ,选取结核杆菌HSP65蛋白为免疫抗原 ,将结核分枝杆菌HSP65抗原基因在大肠杆菌中表达、纯化。方法 :将HSP65的全长cDNA插入到原核表达载体pGEX5T中 ,构建成pGEX5T HSP65重组质粒。将质粒转化到E .coli.K80 2细菌 ,用IPTG诱导HSP65表达 ,然后用亲和层析的方法进行纯化 ,最后用Western blot方法确认表达蛋白的特异性。结果 :获得了pGEX5T HSP65重组子 ,HSP65蛋白在k80 2菌中获得了表达 ,表达的蛋白条带大小约 86kDa ,与预期的结果相符。表达产物经亲和层析后获得了较单一的蛋白条带 ;表达及纯化的蛋白在纯化前后均可被结核病患者血清特异地识别。为进一步研究其在结核病诊断和防治中的应用打下了基础。     

热休克蛋白HSP70在LPS诱导急性肺损伤中的表达

目的检测内毒素诱导急性肺损伤中热休克蛋白70的表达状况,探讨HSP70在急性肺损伤中的作用机制。方法在LPS致Wistar大鼠急性肺损伤动物模型上,采用免疫组织化学(SABC法)和蛋白印迹实验研究各组动物肺组织中HSP70的表达情况。结果LPS处理后1h,大鼠的支气管粘膜上皮及肺泡上皮细胞HSP70的表达与正常对照组相比明显增多,2h的表达达到高峰,6h后与对照组水平一致。Western blot结果显示,LPS处理2h、4h时,HSP70的表达较对照组明显增强。结论LPS诱导的急性肺损伤中可引起支气管、细支气管和肺泡上皮细胞HSP70应激性表达,提示HSP70对肺损伤起保护作用。     

志贺毒素导向药物的初步研究

我们首次应用志贺毒素(Shiga毒素)A亚基基因(StxA)与IL2基因进行融合,并在二者之间插入IL2基因前10个氨基酸编码序列作为接头,构建了一种新的融合毒素StxA-IL2。所构建的融合毒素在大肠杆菌中获得了高效表达,表达量达到细菌总蛋白的15％～20％。经初步纯化后测活,结果表明StxA-IL2能特异地杀伤含中亲和力IL2受体的淋巴肉瘤细胞MLA114而对不含IL-2受体的HeLa细胞无明显杀伤作用。在此基础上,我们用白喉毒素跨膜     

蛋白激酶A抑制剂对HeLa细胞S期进程的影响

以同步化的HeLa细胞为实验材料,研究了蛋白激酶A(PKA)抑制剂对HeLa细胞S期进程的影响及其作用的分子机理.通过TdR双阻断法,获得了同步化的S期细胞,3H-TdR掺入实验表明PKA抑制剂typeⅢ(80mg/L)明显提高了S期3H-TdR的掺入水平,提示了PKA在S期进程中起阻抑作用.进一步实验表明,在PKA抑制剂typeⅢ作用下胸苷激酶(TK)活性和PCNA蛋白水平均有所提高,同时明显促进了CyclinA蛋白的表达,并抑制了周期负调因子p21蛋白的水平,但对CDK2表达几乎无影响.结果表明,PKA可通过作用于PCNA和引擎分子CyclinA的水平和通过影响p21的表达负调于S期进程.这可能是PKA负调HeLa细胞S期进程的分子机理之一.     

Inducible HSP70 Antagonizes IL-1β Cytocidal Effects through Inhibiting NF-kB Activation via Destabilizing TAK1 in HeLa Cells

Background

Despite several reports describing the HSP70-mediated cytoprotection against IL-1, the precise mechanism for this phenomenon remains to be determined.

Methods/Principal Findings

Here we used HeLa cells, a human epithelial carcinoma cell line, to evaluate the role of inducible HSP70 in response of IL-1β stimulation. We found that inducible HSP70 antagonized the cytotoxicity of IL-1β and improved the survival of HeLa cells. Further investigation demonstrated that increased expression level of inducible HSP70 reduced the complex of TAK1 and HSP90, and promoted the degradation of TAK1 protein via proteasome pathway. By overexpression and RNAi knockdown, we showed that inducible HSP70 modulated the NF-kB but not MAPKs signalings through influencing the stability of TAK1 protein in HeLa cells. Moreover, overexpression of HSP70 attenuated the production of iNOS upon IL-1β stimulation, validating that inducible HSP70 serves as a cytopretective factor to antagonize the cytocidal effects of IL-1β in HeLa cells.

Conclusions/Significance

Our observations provide evidence for a novel signaling mechanism involving HSP70, TAK1, and NF-κB in the response of IL-1β cytocidal effects. This research also provides insight into mechanisms by which HSP70 exerts its cytoprotective action upon toxic stimuli in tumor cells.     

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.     

HSP_(70)基因表达对高粱育性的影响(英文)

高粱细胞质雄性不育系３１９７Ａ（３Ａ）在常温条件下是不育的（Ｆｉｇｓ．１１＆２）,经热激（４５℃）诱导不同程度地恢复了育性（Ｆｉｇｓ．１３＆４）,为研究其不育机理提供了线索。热激２ｈ后,３Ａ中即可产生一类线粒体热激蛋白（ＨＳＰｓ）。其中,分子量为７０ｋＤ的ＨＳＰ７０含量最高,也最为稳定。不过,３Ａ中ＨＳＰｓ的稳定性弱于保持系３１９７Ｂ（３Ｂ）（Ｆｉｇ．２,Ｐａｎｅｌｓ１～４）。放线菌素Ｄ抑制ＨＳＰｓ的合成,而氯霉素无此作用（Ｆｉｇ．２,Ｐａｎｅｌｓ５＆６）,表明：ＨＳＰｓ是由核基因编码、在细胞质中合成、再跨膜转运到线粒体中的。３Ａ幼穗经热激后,线粒体的总蛋白量猛增了２．７倍（Ｆｉｇ．３）,达到３Ｂ的水平,育性亦变为可育的。Ｆｉｇ．４表明：ＨＳＰ７０反义链ｃＤＮＡ（Ｒ１）能进入到３Ｂ花药细胞中,并与靶ＲＮＡ（ＨＳＣ７０ｍＲＮＡ）结合,而对照、正义链ｃＤＮＡ（Ｄ）链无此反应。由此、再增加一个通用保守序列的反义链ｃＤＮＡ（Ｒ２）、共两个探针（Ｒ１、Ｒ２）,可以检测到：３Ａ在常温下没有能力合成ＨＳＣ７０ｍＲＮＡ（Ｆｉｇ．５）,而在热激条件下,转变为有能力（Ｆｉｇ．６）。启示：３Ａ在热激条件下由不育转变为可育     

Exogenous heat shock protein HSP70 reduces response of human neuroblastoma cells to lipopolysaccharide

The effect of exogenous heat shock protein HSP70 and lipopolysaccharide (LPS) on the production of reactive oxygen species (ROS), TNFα secretion, and mRNA expression by human neuroblastoma SK-N-SH cells. It was shown that exogenous HSP70 protects neuroblastoma cells from the action of LPS. The protection mechanism of HSP70 includes a reduction in the production of ROS and TNFα and a decrease in the expression of TLR4 and IL-1β mRNA in SK-N-SH cells induced by LPS.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.     

Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70.     

The endogenous danger signals HSP70 and MICA cooperate in the activation of cytotoxic effector functions of NK cells

Although natural killer (NK) cells are often described as first line defence against infected or malignant cells which act without the need of prior activation, it is known now that the NK cell activity is tightly regulated by other cells and soluble factors. We show here that the stress‐inducible heat shock protein (HSP) 70 activates human NK cells to kill target cells expressing major histocompatibility complex class I chain‐related molecule A (MICA) in a natural killer group 2 member D (NKG2D‐) dependent manner. The HSP70‐derived peptide TKD (TKDNNLLGRFELSG) was able to replace the full‐length HSP70 and to exert the same function. Interestingly, the expression of the cytotoxic effector protease granzyme B in NK cells was increased after TKD stimulation. When MICA and MICB expression was induced in human tumour cells by a histone deacetylase inhibitor and NK cells were activated by HSP70 or TKD, both treatments jointly improved the killing of the tumour cells. Thus, the synergistic activity of two stress‐inducible immunological danger signals, HSP70 and MICA/B, leads to activation and enhanced cytotoxicity of human NK cells against tumour cells.