TAT-血红素氧合酶-1融合蛋白对原位肝移植术大鼠肝细胞凋亡的影响

目的 评价TAT-血红素氧合酶-1(TAT-HO-1)融合蛋白对原位肝移植术大鼠肝细胞凋亡的影响.方法 健康成年雄性SD大鼠,体重250 ～ 280 g,作为肝移植术的供体和受体.参照文献构建TAT-HO-1融合蛋白(1 mg/ml).采用二袖套法建立大鼠原位肝移植术模型.将受体大鼠随机分为2组(n=24):对照组(C组)和融合蛋白组(TAT-HO-1组).C组和TAT-HO-1组分别于肝移植术毕静脉注射生理盐水、TAT-HO-1融合蛋白10 ml/kg.于无肝期前即刻(T0)、肝脏移植术后1h、6h和12 h(T1～3)时,分别取动脉血样,检测血清谷丙转氨酶(ALT)活性及透明质酸(HA)浓度;取肝组织标本,采用免疫组化法检测HO-1表达水平,采用TUNEL法检测凋亡细胞,计算凋亡指数.结果 与C组比较,TAT-HO-1组T1-3时HO-21表达上调,血清ALT、HA水平及肝实质细胞和肝窦内皮细胞凋亡指数降低(P＜0.05).结论 大鼠原位肝移植术后静脉注射TAT-HO-1融合蛋白可转导进入移植肝脏细胞,通过减轻肝细胞和肝窦内皮细胞凋亡,对肝移植术后的肝脏功能发挥保护作用.     

转染血红素氧合酶—1基因减轻人肝细胞体外缺氧—复氧损伤

目的 探讨血红素氧合酶-1重组腺病毒载体(Ad-HO-1)体外转染人肝细胞的转染效果及其对肝细胞缺氧-复氧损伤的影响.方法 取人肝细胞系L-02细胞,滴加低温保存的Ad-HO-1,分别培养24 h、48 h和72 h(24 h组、48 h组和72 h组),并以加入空载体腺病毒共培养的L-02细胞为空白对照.采用逆转录聚合酶链反应法检测各组肝细胞HO-1 mRNA的表达水平;以间接免疫荧光标记法检测各组肝细胞的HO-1表达率;在倒置荧光显微镜下观察转染24 h和72 h的肝细胞中绿色荧光蛋白(EGFP)的表达.取空白对照组肝细胞(培养48 h)和48 h组肝细胞,缺氧培养4 h后再有氧培养8 h,采用四甲基偶氮唑盐法测定两组肝细胞存活率.结果 24 h组、48 h组和72 h组HO-1 mRNA表达水平明显高于空白对照组,且随着转染时间的延长,HO-1 mRNA的表达水平逐渐升高.空白对照组HO-1的表达率为2.0%,24 h组为29%,48 h组为85.6%,72 h组为84.6%.基因转染后24 h和72 h,可以观察到L-02细胞中EGFP的表达.经历缺氧-复氧实验后,空白对照组肝细胞的存活率为(37.7±3.5)%,48 h组肝细胞的存活率为(89.4±5.2)%,二者相比较,差异有统计学意义(P<0.01).结论 Ad-HO-1在体外能有效的转染人肝细胞;与未转染者相比,转染肝细胞的缺氧-复氧损伤程度较轻.     

丹参注射液对造影剂诱导的肾小管上皮细胞损伤的保护作用

目的：研究丹参注射液对造影剂碘海醇诱导的肾小管上皮细胞损伤的保护作用并对其作用机制进行探讨。方法：体外培养的人肾小管上皮细胞（HK-2细胞）,分为正常对照组、甘露醇对照组（与碘海醇渗透压相当）、造影剂对照组（碘海醇100mg/ml）、丹参防治组（碘海醇100mg/ml＋丹参注射液15mg/ml）。比色法测定细胞培养上清液LDH水平,MTT法测定细胞增殖活力,DCFH-DA荧光探针测定细胞内活性氧（ROS）,硫代巴比妥酸法测定细胞内MDA含量,黄嘌呤氧化酶法测定SOD活性,RT-PCR测定细胞内血红素加氧酶-1（HO-1）mRNA表达,Westernblot检测细胞内HO-1蛋白表达。结果：碘海醇100mg/ml可明显抑制HK-2细胞增殖、引起培养液中LDH水平上升、促使细胞内ROS和MDA含量增高而细胞内SOD活性下降,同时碘海醇可诱导HO-1mRNA和蛋白表达上调。加入丹参可明显降低培养液中LDH水平,促进细胞增殖,同时抑制细胞内ROS的产生,降低细胞内MDA含量,保护细胞内SOD活性,并使HO-1mRNA和蛋白表达进一步增强。结论：丹参对造影剂碘海醇所致肾小管上皮细胞具有明显的保护作用,其作用机制与其抗氧化能力及诱导HO-1表达有关。     

PEP-1-血红素加氧酶-1融合蛋白转导对大鼠H9c2心肌细胞缺氧复氧损伤的影响

目的 探讨PEP-1-血红素加氧酶(HO)-1融合蛋白转导对大鼠H9c2心肌细胞缺氧复氧损伤的影响.方法 构建含人HO-1基因的原核表达质粒pETl5b-PEP1-hHO-1,质粒转化后诱导目的 蛋白PEP-1-HO-1表达.用含15%胎牛血清高糖DMEM培养基培养H9c2心肌细胞,随机分为4组(n=4):正常对照组(C组)常规培养;缺氧复氧组(H/R组)细胞缺氧22 h,复氧8 h;低浓度融合蛋白组(L-HO组)缺氧前用终浓度为1.0 μmol/L PEP-1-HO-1融合蛋白孵育细胞;高浓度融合蛋白组(H-HO组)缺氧前即刻用终浓度为2.0 μmol/L PEP-1-HO-1融合蛋白孵育细胞.复氧结束后收集细胞及培养液上清,采用2,4-二硝基苯肼显色法检测培养液乳酸脱氢酶(LDH)活性,硫代巴比妥酸比色法检测细胞MDA含量,黄嘌呤氧化酶法检测细胞SOD活性.结果 与C组比较,H/R组、L-HO组、H-HO组心肌细胞SOD活性降低,MDA含量升高,培养液LDH活性升高(P＜0.05);与H/R组比较,L-HO组和H-HO组心肌细胞SOD活性升高,MDA含量降低,培养液LDH活性降低(P＜0.05);与L-HO组比较,H-HO组心肌细胞SOD活性升高,MDA含量降低,培养液LDH活性降低(P＜0.05).结论 PEP-1-HO-1融合蛋白转导入大鼠H9c2心肌细胞可减轻细胞缺氧复氧损伤.     

血红素加氧酶-1预处理对大鼠肺泡Ⅱ型上皮细胞氧化损伤的影响

目的 评价血红素加氧酶-1(HO-1)预处理对大鼠肺泡Ⅱ型上皮细胞氧化损伤的影响.方法 成年健康雄性SD大鼠,体重180～220 g,原代培养肺泡Ⅱ型上皮细胞,经鉴定后随机分为6组(n=8):对照组(C组),不给予任何药物,继续培养5 h;H2 O2组,加入0-5 mmol/L H2 O2,孵育3 h;不同浓度HO-1预处理组(H1-4组),分别加入0.01、0.10、1.00、10.00μmol/L HO-1孵育2 h,然后加入0.5 mmol/L H2 O2,孵育3 h.孵育结束后,于倒置相差显微镜下观察细胞形态,并进行肺泡Ⅱ型上皮细胞计数,测定细胞活力.结果 C组、H2～4组肺泡Ⅱ型上皮细胞大部分贴壁,呈圆形,胞质均匀,胞浆内含颗粒状物质;而H2 O2组和H1组肺泡Ⅱ型上皮细胞内可见反光增强的空泡,上清液中有较多的细胞碎片.与C组比较,H2O2组和H1组细胞计数和细胞活力降低(P＜0.05),H2～4组细胞计数和细胞活力差异无统计学意义(P＞0.05);与H2O2组和H1组比较,H2～4组细胞计数和细胞活力升高(P＜0.05);H2O2组和H1组间,H2～4组间细胞计数和细胞活力差异无统计学意义(P＞0.05).结论 0.10～10.00μmol/L HO-1预处理可减轻大鼠肺泡Ⅱ型上皮细胞氧化损伤.     

量子点标记共培养睾丸体细胞体外培养观察

目的 初步验证量子点能否对共培养睾丸体细胞进行标记,为睾丸体细胞的体内外研究提供一种细胞损伤小,且简便、有效的标记手段.方法 取雄性Wistar大鼠的睾丸组织小块,胶原酶消化,差速贴壁法收集睾丸体细胞,取第一代睾丸体细胞以0.25%胰酶蛋白酶-0.02%EDTA消化,制备单细胞悬液,与量子点共同孵育2h后贴壁及传代培养,共聚焦荧光显微镜动态观察细胞生长、增殖情况.结果 相差显微镜下观察,标记细胞形态无明显改变,荧光显微镜下可见摄取量子散在分布于细胞内,主要集中于细胞核周围,分裂细胞及增殖活跃细胞摄取量明显高于老化细胞.传代后仍可见细胞内的量子点分布,荧光强度未见明显衰减.结论 量子点能够被共培养睾丸体细胞摄取,且摄取速度快,荧光显微镜下易于观察.量子点标记法对共培养细胞损伤小,操作简便易行,是共培养睾丸体细胞标记的理想选择之一.     

IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.     

IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.     

IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.     

IRX1真核表达载体的构建及在胃癌细胞SGC-7901中的定位

目的 构建pEGFP-IRX1真核表达载体,体外转导至SGC-7901胃癌细胞株,观察IRX1基因转染后蛋白表达与细胞内定位.方法 PCR扩增IRX1全长1443 bp编码序列,将PCR扩增产物连接人pGEM-Teasy载体,经测序确认序列无误后,亚克隆入pEGFP-N1载体,构建pEG-FP-IRX1真核表达载体.采用Lipofectamine 2000转染SGC-7901胃癌细胞株,在荧光显微镜下观察外源性IRX1基因导入后在胃癌细胞内的表达与亚细胞定位.结果 成功构建有绿色荧光蛋白融合的IRX1真核表达载体,外源性IRX1基因在胃癌SGC-7901细胞中表达成功,在荧光显微镜下IRX1表达蛋白定位于细胞核内.结论 pEGFP-IRX1真核表达载体构建成功,并可在细胞内表达,这将为IRX1在胃癌内的功能研究奠定基础.     

HIV-1反式激活蛋白-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期肝细胞凋亡的影响

Objective To study the effects of TAT-HO-1 fusion protein,HIV-1 transactiviting protein (TAT) and heme oxygenase-1 (HO-1),on hepatic cell apoptosis of rat donors in cold storage stage.Methods Forty-eight male SD rats were randomly divided into two groups.Rat livers were flushed and preserved with 4℃ HTK solution containing(group P) or uncontaining(group C) 50 mg/L of TAT-HO-1.The preserved solution and hepatic tissue were collected at 0,6,12,18 h of cold storage stage.TAT-HO-1 transducing into liver,alanine aminotransferase(ALT) level in preserved solution,hyaluronic acid(HA) level and the expression of caspase-3 in hepatic tissue,and the apoptotic index (AI) of hepatocytes and sinusoidal endothelial cells (SECs) were measured or detected.Results ALT level in preserved solution,HA level and the expression of caspase-3 in hepatic tissue,and the AI of hepatocytes and SECs increased time-dependently in cold storage stage in both groups (P＜0.05),with lower increasing extent in group P than that in group C (P＜0.05) at 6h,12h and 18h of cold storage stage.A stronger accumulation of HO-1 staining was also detected at the same time-points in group P than that in group C(P＜0.05).Conclusion TAT-HO-1 may transduce efficiently into rat livers,exerting protective effects on both hepatocytes and SECs during cold storage stage.Protein transduction technology may be a novel therapeutic means to reduce donor liver injury in preservation period for transplantion.     

PEP-1-SOD1融合蛋白转导入大鼠肝脏组织的能力及酶活性

目的研究PEP．1-SOD1融合蛋白转导入大鼠肝脏组织的能力及其酶活性。方法实验分为SOD1组和PEP1-SOD1组，分别经尾静脉注射500μgSOD1和500μg PEP-1-SOD1融合蛋白入大鼠体内，于0．5，1，2，4，8和24h时间点取肝脏（n=6，每组，每个时间点）。免疫荧光技术检测PEP-1-SOD1的转导能力。黄嘌呤氧化酶法测定肝脏组织SOD1活性。结果SOD1蛋白不能进入肝脏组织内。PEP-1-SOD1可转导入肝脏组织内，SOD1活性于注射后0．5h开始升高，1h达高峰，持续存在达24h，两组各个时间点比较，差异有统计学意义（P〈0．01）。SOD1组内各时间点比较差异无统计学意义（P〉0．05）。结论PEP-1-SOD1以天然酶活性形式转导入肝脏组织，为进一步的动物模型实验及临床疾病进行蛋白质治疗提供理论基础。     

TAT-血红素氧合酶-1融合蛋白对大鼠供肝冷保存期炎性反应及肝功能的影响

目的 探讨HIV-1反式激活蛋白-血红素氧合酶-1(TAT-HO-1)融合蛋白对大鼠供肝冷保存期炎性反应及肝功能的影响.方法 成年健康雄性SD大鼠48只,体重250～300 g,开腹游离腹主动脉并结扎,于左右髂总动脉分叉处向心端穿刺腹主动脉,切断肝上下腔静脉,随机灌注4℃HTK液(C组,U=24)或4℃含TAT-HO-1融合蛋白50 μg/ml的HTK液(P组,n=24),至流出灌洗液清亮后停止灌注,取肝脏,置于相应保存液中冷保存.分别于冷保存即刻、6、12和18 h时取保存液并切取肝组织.采用全自动生化分析仪测定保存液谷草转氨酶(AST)、谷丙转氨酶(ALT)及乳酸脱氢酶(LDH)活性,免疫组化法检测肿瘤坏死因子α(TNF-α)的表达,光镜下观察肝组织形态学.结果 随冷保存时间延长,两组保存液AST、ALT及LDH活性升高,TNF-α表达上调(P<0.05);与C组比较,冷保存6、12、18 h时P组保存液AST、ALT及LDH活性降低,TNF-α表达下调(P<0.05).结论 TAT-HO-1融合蛋白可抑制大鼠供肝冷保存期的炎性反应,对肝功能具有一定保护作用.     

Transduced PEP-1–heme oxygenase-1 fusion protein protects against intestinal ischemia/reperfusion injury

Background

Heme oxygenase-1 (HO-1) has been shown to have antioxidant and anti-apoptotic properties. The present study transduced HO-1 protein into intestinal tissues using PEP-1, a cell-penetrating peptide, and investigated its potentiality in prevention against intestinal ischemia/reperfusion (I/R) injury.

Materials and methods

PEP-1-HO-1 fusion protein was administered intravenously to explore the time and dose characteristics through measuring serum HO-1 levels. Twenty-four male Sprague-Dawley rats were randomly divided into three groups: sham, intestinal I/R (II/R), II/R + PEP-1-HO-1 fusion protein (HO). The model was established by occluding the superior mesenteric artery for 45 min followed by 120 min reperfusion. In HO group, PEP-1-HO-1 was administered intravenously 30 min before ischemia, whereas animals in sham and II/R groups received the equal volume of physiological saline. After the experiment, the intestines were harvested for determination of histologic injury, wet/dry ratio, enzyme activity, apoptosis, and His-probe protein (one part of PEP-1-HO-1).

Results

Levels of serum HO-1 were dose- and time-dependent manner after intravenous injection of PEP-1-HO-1. I/R caused deterioration of histologic characteristics and increases in histologic injury scoring, wet/dry ratio, myeloperoxidase activity, malondialdehyde, and intestinal apoptosis. These changes were also accompanied by a decrease in superoxide dismutase activity (P < 0.05). PEP-1-HO-1 treatment significantly reversed these changes (P < 0.05). Furthermore, His-probe protein expression was only detected in PEP-1-HO-1–treated animals.

Conclusion

Treatment of PEP-1-HO-1 attenuates intestinal I/R injury, which might be attributable to its antioxidant and anti-apoptotic roles of HO-1.     

Heme oxygenase-1 mediates cytoprotection against nitric oxide-induced cytotoxicity via the cGMP pathway in human pulp cells.

OBJECTIVE: This study examined the effects of exogenous nitric oxide (NO) on human pulp cells and the involvement of cyclic 3',5'-monophosphate (cGMP) in pulpal protection induced by heme oxygenase-1 (HO-1) against NO-induced cytotoxicity. STUDY DESIGN: This study investigated cytotoxicity and HO-1 induction in pulp cells induced by the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP), by using Western blotting and a cell viability assay. It also investigated whether HO-1 contributes to the cytoprotective effect against the cytotoxicity caused by NO and the relationship between HO-1 and cGMP in the signaling pathway. RESULTS: S-nitroso-N-acetyl-D,L-penicillamine decreased cell viability, but increased HO-1 expression in a concentration- and time-dependent manner in human pulp cells. NO-induced cytotoxicity was inhibited in the presence of hemin (inducer of HO-1), whereas it was enhanced in the presence of zinc protoporphyrin IX (ZnPP IX, HO-1 inhibitor); therefore, the NO-induced cytotoxicity was correlated with HO-1 expression. Pretreatment with a membrane-permeable cGMP analog, 8-bromo-cGMP, restored cell death and enhanced the HO-1 protein expression induced by SNAP. By contrast, 1 mM SNAP inhibited guanylate cyclase in pulp cells pretreated with 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (ODQ), resulting in marked cytotoxicity. CONCLUSION: These findings of a link between HO-1, regulated via the cGMP system and NO-induced cytotoxicity in human pulp cells, suggest a protective role for HO-1 in pulpal inflammation.     

Characterization of human T lymphocytes engineered to express interleukin-15 and herpes simplex virus-thymidine kinase

Introduction

Preclinical studies have demonstrated that tumor-reactive T cells expressing the interleukin (IL)-15 transgene had enhanced activity. Gene therapy strategies using IL-15 should include a safety mechanism in anticipation of possible adverse effects because IL-15 overexpression has been implicated in autoimmune disorders and may be involved in the pathogenesis of some leukemias. We developed a retroviral vector carrying both IL-15 and the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene and characterized its application in the transduction of human T lymphocytes.

Methods

A retroviral vector carrying IL-15 and HSV-TK genes was optimized for the transduction of human T lymphocytes. IL-15 production was measured by enzyme-linked immunosorbent assay. Thymidine incorporation and cell viability assays were used to assess the efficacy of the HSV-TK suicide gene. Genetically modified tumor-infiltrating lymphocytes (TILs) were assayed for survival after withdrawal from exogenous IL-2. The activity and specificity of retrovirally transduced TILs were assessed using tumor coculture assays.

Results

Human T cells transduced with the IL-15 HSV-TK vector exhibited thymidine uptake in the absence of exogenous cytokine support and survived in culture for up to 80 d without IL-2. IL-15 HSV-TK–transduced T cells were efficiently killed by ganciclovir at concentrations as low as 0.1 μM. TILs transduced with the IL-15 HSV-TK vector retained specific recognition of HLA-A2+, MART1+ melanomas, even after withdrawal of IL-2.

Conclusions

Human T lymphocytes genetically modified with the IL-15 HSV-TK retroviral vector retained the ability to recognize tumor antigen while gaining the ability to secrete IL-15 and prolong their own survival. IL-15 HSV-TK–transduced T cells expressed HSV-TK and could be efficiently eliminated by ganciclovir.     

载阿霉素聚氰基丙烯酸正丁酯纳米粒对L-02细胞的毒性研究

目的:检验载阿霉素聚氰基丙烯酸正丁酯纳米粒(ADM-PBCA-NP)对人肝细胞株L-02细胞的毒性。方法:体外培养人肝细胞株L-02，检测各组不同纳米粒浓度培养上清液中乳酸脱氢酶（LDH）的活性；用噻唑蓝（MTT）法检测L-02在与ADM-PBCA-NP，阿霉素（ADM）和空白纳米粒(PBCA-NP)共同培养的条件下对肝细胞的毒性，通过对不同浓度PBCA-NP的溶血试验测定其生物相容性。结果:MTT结果显示，ADM组，ADM-NP组，PBCA-NP组在 10-6 mol/L的浓度范围内，细胞毒性均为1级，即对细胞无害。各组肝细胞L-02培养上清液中LDH活性的测定无统计学差异。结论:在10-6 mol/L浓度范围内，ADM-NP和PBCA-NP对肝细胞L-02无明显毒性作用；在一定的浓度范围内细胞相容性良好，不会引起溶血。