重组人p53腺病毒感染对携带不同p53状态胃癌细胞增殖和凋亡的影响

目的研究重组人p53腺病毒感染不同p53状态胃癌细胞对其p53蛋白表达、生长抑制率、细胞周期与凋亡率的影响。方法不同浓度重组人p53腺病毒感染3种不同p53状态胃癌细胞,即含野生型p53基因的细胞(wild-type)、含突变型p53基因的细胞(mutant-type)、含空载质粒即p53基因缺失的细胞(vector-cell)。48 h后,用Western blotting法检测p53蛋白在3种胃癌细胞中的表达;用MTT法测定重组人p53腺病毒感染3种胃癌细胞的生长抑制率,用流式细胞仪检测细胞周期分布和凋亡率。结果rAd-p53感染3种胃癌细胞48 h后p53蛋白表达阳性,对照组p53基因缺失的胃癌细胞无表达,对照组含野生型p53基因的细胞和含突变型p53基因的细胞弱表达。rAd-p53对3种胃癌细胞的生长抑制效应在一定的浓度范围内呈剂量依赖性,而与细胞内在的p53状态无关。含野生型p53基因的细胞、含突变型p53基因的细胞和p53基因缺失的细胞感染rAd-p53后诱导G2/M期阻滞与细胞凋亡率分别增加2.5、3.6、3.2倍。结论腺病毒介导p53基因感染3种不同p53状态胃癌细胞改变细胞内在的p53状态,p53蛋白表达、生长抑制率、细胞周期分布、凋亡率均与细胞内在的p53状态无关。     

弓形虫ROP18重组腺病毒载体的构建及其对神经干细胞C17.2凋亡的影响

目的 构建弓形虫棒状体蛋白ROP18重组腺病毒载体,研究其对神经干细胞C17.2凋亡的影响.方法 将弓形虫ROP18从PEGFP-G2-ROP18重组质粒上亚克隆至腺病毒载体pHBAd-MCMV-GFP,重组腺病毒载体pHBAd-MCMV-GFP- ROP18经PCR,测序鉴定正确后,与骨架质粒pHBAd-BHG共转染HEK293细胞进行包装,收获,扩增重组腺病毒并对其滴度进行测定.以MOI=70的重组腺病毒感染神经干细胞C17.2,转染不同时间荧光显微镜观察基因的表达情况.转染48 h用CCK-8法检测C17.2的增殖情况;转染24 h采用Annexin V-APC/PI双染流式细胞术检测细胞凋亡情况;Western blot法检测半胱天冬氨酸蛋白酶3(caspase-3)的蛋白水平.结果 重组ROP18过表达腺病毒在C17.2神经干细胞内能有效表达.转染48 h,与空载体对照组相比,C17.2细胞增殖活性明显降低(P<0.05);转染24 h,流式细胞术检测结果显示,ROP18基因转染组C17.2细胞凋亡率明显增加(P<0.001);Western blot检测显示caspase-3活性片段的表达明显增加(P<0.05).结论 成功构建了弓形虫ROP18重组腺病毒并在神经干细胞C17.2中表达,ROP18重组腺病毒能诱导C17.2神经干细胞凋亡.     

野生型p53基因转移对血管平滑肌细胞增殖的抑制作用

作者用正确构建的带野生型p53基因的复制缺陷型重组腺病毒感染体外培养的血管平滑肌细胞(VSMC),结果表明p53基因转移对VSMC的增殖具有明显的抑制作用;PCR扩增法证实了病毒感染的VSMC中含有外源性p53基因;半定量逆转录PCR方法显示有p53基因mRNA的表达;Western blot印迹转移证明有外源性p53蛋白的表达。进一步证明了作者构建的重组p53腺病毒载体的正确性和有效性,为高血压病、动脉粥样硬化、PTCA术后再狭窄等疾病的基因治疗提供了实验依据。     

NF-κB/p65特异的RNAi腺病毒载体的构建及其对p65表达的抑制效应

目的构建NF-κB p65亚基特异的RNA干扰（RNAi）腺病毒表达载体，并验证其对p65亚基的基因沉默效应。方法设计合成三对针对p65 mRNA不同位点的短发夹RNA（shRNA）编码序列，克隆到穿梭载体中，通过体外同源重组将短干扰RNA（siRNA）表达盒转移到腺病毒骨架质粒，构建RNAi腺病毒表达载体；在HEK293A细胞中包装并扩增病毒、空斑实验法进行病毒滴度测定；腺病毒感染人脐静脉内皮细胞株ECV304细胞，Western印迹法和免疫细胞化学法验证构建的RNAi腺病毒对p65蛋白表达的抑制效应。结果成功构建了NF-κB p65亚基特异的RNAi腺病毒表达载体，获得高滴度的腺病毒液；RNAi腺病毒感染ECV304细胞后可以高效抑制p65蛋白的表达，且对p65蛋白表达的抑制作用可持续6d以上。结论应用RNAi腺病毒表达载体能有效阻断目的基因的表达。     

融合表达白细胞介素10与卵清蛋白重组腺病毒口服变应原疫苗的构建及鉴定

目的构建免疫调节因子白细胞介素(IL)-10与模式变应原卵清蛋白(OVA)融合表达的重组腺病毒载体口服疫苗。方法直接合成鸡IL-10及OVA的DNA序列后,采用重叠PCR法进行连接。连接序列插入穿梭质粒p HBAd-MCMV的多克隆区,获得重组穿梭质粒p Ad IL-10-OVA。经测序鉴定后,p Ad IL-10-OVA与腺病毒骨架质粒p HBAd-BHG共转染293细胞,包装获得重组腺病毒Ad IL-10-OVA原代病毒,将原代病毒在293细胞中扩增到合适滴度。采用ELISA法检测细胞上清液中融合蛋白的表达水平。结果重组穿梭质粒插入目标序列经测序鉴定正确,重组腺病毒载体疫苗r Ad IL-10-OVA在293细胞中成功表达。结论成功构建了融合表达IL-10与OVA的重组腺病毒载体疫苗Ad IL-10-OVA,为后续动物实验验证其对变态反应性疾病的防治效应奠定了基础。     

缪刺对脑缺血大鼠细胞凋亡相关蛋白c-fos、p53表达的影响

目的探讨缪刺对脑缺血大鼠细胞凋亡相关蛋白c-fos、p53表达的影响。方法观察缪刺对脑缺血模型大鼠脑缺血后24 h、48h、72 h三个时间点大脑皮质细胞凋亡相关蛋白c-fos、p53表达的影响,并以免疫组化法检测。结果缪刺对脑缺血模型大鼠大脑皮质c-fos、p53蛋白表达有抑制作用。结论缪刺对大鼠局灶性脑缺血具有保护作用,可能与细胞凋亡相关蛋白c-fos、p53的表达有关。     

热休克蛋白70通过Bcl-2抑制氧化应激所致C2C12细胞凋亡

目的 探讨C2C12肌原细胞内热休克蛋白70对Bcl-2表达的影响及Bcl-2对热休克蛋白70抗细胞凋亡作用的影响.方法 应用Western Blotting观察Bcl-2在转染热休克蛋白70真核表达质粒(pcDNA3.1-HSP70)或其反义寡核苷酸C2C12肌原细胞中的表达;采用基因瞬间转染技术使热休克蛋白70过表达的C2C12肌原细胞内Bcl-2表达抑制,应用流式细胞术检测H2O2处理所致细胞凋亡的发生情况.结果 转染热休克蛋白70真核表达质粒的C2C12肌原细胞中,热休克蛋白70和Bcl-2的表达明显高于空载体转染组(P<0.01);而转染热休克蛋白70反义寡核苷酸后,热休克蛋白70和Bcl-2的表达明显低于随机寡核苷酸转染组 (P<0.01);热休克蛋白70过表达的C2C12肌原细胞分别转染Bcl-2反义寡核苷酸及随机寡核苷酸,0.5 mmol/L H2O2处理24 h,Bcl-2反义寡核苷酸转染组的细胞凋亡率明显高于随机寡核苷酸转染细胞组.结论 热休克蛋白70能上调C2C12肌原细胞内Bcl-2的表达;热休克蛋白70的抗细胞凋亡功能可能与其上调Bcl-2表达相关.     

曲古抑菌素A抑制乳腺癌细胞MCF-7生长增殖及促进凋亡的机制

目的 探讨曲古抑菌素A(TSA)对乳腺癌细胞系MCF-7细胞生长增殖、凋亡及p21、p53表达的影响.方法 分别以不同浓度的TSA处理MCF-7细胞,采用MTT比色法测定细胞增殖活性;用流式细胞仪检测细胞凋亡率;用RT-PCR方法检测细胞p21、p53mRNA的表达水平;用Western印迹法检测MCF-7细胞的p21、p53蛋白表达.结果 TSA作用后,各组细胞均出现显著的生长抑制作用,存活率明显降低,并呈剂量依赖性.流式细胞仪分析,在100 ～ 400 nmol/L范围内凋亡率随TSA浓度的升高而升高(P＜0.01).经不同浓度TSA处理的细胞p21mRNA和蛋白表达水平明显上调(P＜0.01);、而p53mRNA和蛋白表达水平则没有明显变化.结论 TSA显著抑制MCF-7细胞生长,促进凋亡可能与TSA维持p53的稳定表达,从而促进其下游因子p21的表达有关.     

腺病毒adv-hmepe质粒构建与转染表达优化

目的构建人细胞外基质磷酸糖蛋白(hmepe)基因的重组腺病毒p Yr-ads-4-h MEPE真核表达载体,并优化其转染条件。方法构建腺病毒包装质粒p Yr-ads-4-h MEPE,在HEK293细胞中包装形成腺病毒颗粒,转染前列腺上皮细胞株RWPE1,同时转染空质粒作为对照细胞株。设置腺病毒重组质粒梯度检测最佳转染浓度,免疫印迹检测转染细胞株中MEPE蛋白表达水平,确定转染效果。共聚焦显微镜检测不同条件下的转化率。结果经酶切与测序验证,p Yr-ads-4-h MEPE重组质粒构建成功,免疫印迹分析验证转染r Ad-4-h MEPE的RWPE1细胞株中MEPE蛋白表达水平上调,而转染相应空载体的细胞株中MEPE蛋白的表达水平与野生型细胞株一致,说明腺病毒质粒构建成功可在真核细胞表达,共聚焦检测分析质粒浓度对转染效率可信度较好。结论成功构建hmepe真核表达载体,重组腺病毒转染RWPE1细胞最佳融合比例1.25∶1 000,即质粒量为1.25μl加入1 ml培养液中转染效率最佳。     

p16INK4α重表达对人食管癌细胞系EC109生长的抑制作用

目的探讨用腺病毒介导野生型的p16基因在食管癌细胞系表达,研究重表达野生型p16基因对食管癌细胞系EC109生长的抑制作用,为寻找食管癌致癌机制的研究及其基因治疗提供理论基础.方法用基因重组技术,将pcDNA3-p16中的野生型p16基因用Kpn I/BamH I进行双酶切,克隆入腺病毒表达载体pAdCMV中,将重组质粒pad-CMV-p16与腺病毒质粒JM17用Lipofectamime 2000共转染293细胞,产生重组腺病毒.用重组腺病毒感染人食管癌细胞系EC109,在感染后的不同时段,用免疫荧光、打点杂交方法检测p16基因在细胞中的表达用MTT法及流式细胞仪观察p16基因的表达对食管癌细胞株生长的影响.结果经酶切鉴定野生型p16基因克隆入腺病毒表达载体中,与JM17共转染293细胞后,可产生具有感染活性的重组腺病毒,用重组腺病毒感染食管癌细胞株EC109细胞后,可抑制食管癌细胞的生长,同对照组相比其最大抑制率可达52.7%.Multipcycle分析软件分析对细胞周期影响表明,处于G0～G1期的细胞为41%～63%感染后的细胞经Dot blot和Westernblot杂交证实,有外源的p16mRNA及蛋白的表达.结论重组腺病毒可将野生型p16基因导入人食管癌细胞系EC109中,野生型p16基因在p16基因功能缺失的细胞中重表达能抑制癌细胞恶性生长.p16基因功能缺失是食管癌致癌因素之一     

Contrasting effects of HSP72 expression on apoptosis in human umbilical vein endothelial cells and an angiogenic cell line, ECV304

The effect of overexpression of heat shock protein (HSP)72 on apoptosis induced by different stimuli in human umbilical vein endothelial cells (HUVECs) and the angiogenic cell line, ECV304, was studied. Transient overexpression of HSP72 was achieved using an adenoviral vector (Advhsp72) and apoptosis was induced by heat shock, tumour necrosis factor (TNF)-alpha with cycloheximide (CHX), lipopolysaccharide (LPS) with TNF-alpha and verocytotoxin (VT). Apoptosis induced by heat shock was reduced by HSP72 expression. However, HSP72 expression in HUVECs increased apoptosis induced by TNF-alpha/CHX, LPS and VT measured by flow cytometric analysis of propidium iodide (PI)-stained permeabilized cells. In contrast, apoptosis in ECV304 induced by the same stimuli was reduced by HSP72 expression. No difference was seen in cells transduced with a control adenoviral vector expressing beta-galactosidase. These data imply that induction of HSP72 in cells modulates responses to apoptotic stimuli, but that the nature of the response varies with the cell type. However, it is clear that in situations where apoptosis may be part of a pathological process, HSP72 induction, for example by reperfusion injury, may exacerbate the process.     

Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer. METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting. RESULTS: The number of viable cells decreased in a dose-and time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10μmol/L for 48 h or 8μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G_1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h. CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.     

过表达HSP20的慢病毒载体对H2O2诱导的大鼠心肌细胞凋亡的影响

目的构建过表达大鼠热休克蛋白20(HSP20)基因慢病毒载体,探讨其对H2O2诱导的大鼠H9C2心肌细胞凋亡的影响。方法构建大鼠HSP20基因pHIV-HSP20过表达质粒慢病毒,与包装质粒psPAX2、pMD2G共转染293FT细胞,检测其转染效率;包装慢病毒并转染H9C2心肌细胞;72 h后观察其转染效率,RT-PCR法检测细胞HSP20 mRNA表达,CCK-8、Hochest33258染色法检测H2O2诱导后H9C2心肌细胞凋亡情况。结果成功构建了HSP20慢病毒过表达载体,经293FT细胞包装后,其对H9C2细胞72 h的转染效率为95%;与慢性病毒组比较,H9C2细胞转染72 h后HSP20 mRNA水平显著升高,细胞活力下降,心肌细胞凋亡率明显降低(P均<0.01)。结论过表达HSP20能显著抑制H2O2诱导的H9C2心肌细胞凋亡。     

热休克蛋白70心肌保护的研究进展

热休克蛋白是细胞在应激条件下产生的一类高度保守的蛋白质,通过稳定细胞内变性的蛋白质;减轻细胞内的离子紊乱;保护血管内皮细胞的功能;干扰应激所启动的细胞凋亡程序等方面来发挥心肌缺血/再灌注损伤的保护作用。现对热休克蛋白及其心肌缺血/再灌注损伤的保护作用及机制进行综述。     

Heat Shock Preconditioning Induces Protein Carbonylation and Alters Antioxidant Protection in Superficially Injured Guinea Pig Gastric Mucosa In Vitro

According to our previous studies, heat shock preconditioning of gastric mucosa requires modulation of protein synthesis and eicosanoid pathways to induce protection against superficial injury. This may be caused by heat shock–induced oxidative stress. We studied the effect of heat shock preconditioning with normothermic recovery on redox status in superficially injured (1.25 mmol NaCl for 5 min) Ussing chamber perfused guinea pig gastric mucosa allowed to recover for 3 hr after injury. Protein oxidation, lipid peroxidation, level of superoxide dismutase, level of heat shock protein 72 (HSP72), and level of oxygen radical absorbance capacity were measured. Superficial injury increased lipid peroxidation. Heat shock preconditioning decreased oxygen radical absorbance capacity and increased protein carbonyl and HSP72 levels, but inhibited electrophysiologic recovery. Exposure to indomethacin and arachidonic acid (AA) partially abolished this pro-oxidative and inhibitory effect on recovery, but maintained HSP72 levels and decreased protein carbonyls, lipid peroxidation, and oxygen radical absorbance capacity. In conclusion, superficial injury increased lipid peroxidation. Heat shock preconditioning alone induced oxidative stress via indomethacin- and AA-sensitive mechanisms. The development of optimal cytoprotective strategy may therefore require control of oxidative stress and modulation of the eicosanoid pathways.     

The remnant liver dysfunction after 84% hepatectomy in dogs

BACKGROUND/AIMS: We have been investigating the mechanism of remnant liver dysfunction after extensive hepatectomy in a canine model since 1990. This study focused on the role of heat shock protein and hepatocyte apoptosis. METHODOLOGY: Adult mongrel dogs were randomly divided into 3 groups: Group 1, sham operation; Group 2, 70% hepatectomy; and Group 3, 84% hepatectomy. Heat shock protein and hepatocyte apoptosis after hepatectomy were examined by using isolated hepatocytes and Kupffer cells. RESULTS: Heat shock protein significantly increased in Groups 2 and 3, but rose much higher in Group 3. Examination of pure hepatocyte culture showed no apoptosis in Group 2, but significant apoptosis occurred in Group 3. In co-cultures of hepatocytes and Kupffer cells, induction of apoptosis in Group 2 was mild, but it increased earlier and reached very high levels in Group 3. The TNF-alpha level in co-culture supernatant was significantly higher in Group 3 than Group 2. CONCLUSIONS: After extensive (84%) hepatectomy, apoptosis signal transduction predominates over anti-apoptosis signal transduction, despite high expression of heat shock protein in the remnant liver. Accordingly, the cytotoxic mechanism overcomes the cytoprotective mechanism, leading to significant induction of hepatocyte apoptosis and severe liver damage.     

Limited heat-shock protein 72 induction in Caco-2 cells by L-glutamine.

BACKGROUND/AIMS: L-glutamine (L-gln) is a conditionally essential amino acid which is consumed by certain tissues like the intestine in large amounts as energy source during critical illness. Apart from nutritive action, recent data suggest a link to heat-shock protein (hsp) induction. We investigated the effect of L-gln on hsp72 expression in the intestinal cell line Caco-2 under basal and heat-shock conditions and compared it with related amino acids. METHODS: Total cellular protein was extracted and separated by SDS-PAGE. Immunoblots were performed with anti-hsp72 followed by chemiluminescence detection and densitometric scanning. RESULTS: Following heat shock, hsp72 protein expression increased by 72 and 53% at 2 and 4 mmol/l L-gln, respectively, compared to heat shock alone (p < 0.05). Under basal conditions a limited increase occurred only at 8 mmol/l L-gln (p < 0.01). Levels remained unaffected when related amino acids including alanine, glutamate or glycine were supplemented under basal and heat-shock conditions. Similarly, the nonmetabolizable glutamine analogue DON or the toxic metabolite L-pyroglutamate did not induce hsp72. CONCLUSION: We conclude that the glutamine-mediated effect may be specifically attributed to the metabolic action of L-gln.