聚乙二醇单修饰重组人粒细胞集落刺激因子对大鼠血液学及骨髓毒性的研究

目的：研究聚乙二醇单修饰重组人粒细胞集落刺激因子（PEG-rhG-CSF）对SD大鼠血液学和骨髓造血的影响,为其应用于临床治疗提供资料。方法：将SD大鼠随机分为3个实验组和1个对照组,分别sc不同剂量PEG-rhG-CSF和溶媒对照品,每周给药3次,连续给药3个月,恢复期2周。在给药和恢复期结束时进行血液学和骨髓检查,并做t-检验统计分析。结果：给药结束后,高中低剂量组血液白细胞计数显著升高（升高了7.8～28.2倍）,中性粒细胞计数明显增加（增加了2.5～3.9倍）,网织红细胞计数显著增加,淋巴细胞、嗜酸性粒和红细胞计数显著降低;同时,高剂量组血红蛋白量和血小板计数显著降低,高、中剂量红细胞比容显著增加;此外,高中低剂量骨髓红细胞系比例、中幼及晚幼红细胞比例显著降低,而成熟粒细胞比例显著升高（升高了29.1％～79.1％）,粒／红（M／E）比值也显著升高（最高增加1.3倍）。上述改变在停药2周后,均恢复到正常水平,与对照组比较均无显著性差异。结论：PEG-rhG-CSF具有明显的促进造血功能,临床可用于治疗肿瘤化疗引起的中性粒细胞减少症。     

磷脂酰肌醇激酶-3小干扰RNA真核表达载体的构建和鉴定

目的：构建和鉴定靶向磷脂酰肌醇激酶-3（PI3K） 亚单位编码基因PI3Kcb （catalytic,beta polypeptide）基因的小干扰RNA真核表达载体.方法：根据siRNA设计原则,在PI3Kcb mRNA序列中选择1个特异性靶序列,体外合成对应发卡样DNA片段,经退火后,将其定向克隆入siRNA载体pDC316-EGFP-U6,重组后获得siRNA载体质粒pDC316-PI3Kcb siRNA-EGFP,采用PCR检测和DNA测序以保证siRNA的方向和序列正确.结果：得到阳性重组克隆pDC316-PI3Kcb siRNA,经过PCR鉴定和测序,结果正确.结论：成功构建靶向PI3Kcb基因的siRNA载体pDC316-PI3Kcb siRNA-EGFP,为PI3Kcb的基因沉默研究奠定了可靠的基础.     

HSV-1基因转移载体在荷瘤裸鼠体内的生物分布研究

目的 研究HSV-1基因转移栽体在BALB/c荷瘤裸鼠体内的生物分布.方法 BALB/c裸鼠注射人乳腺癌MCF-7细胞制备肿瘤模型,瘤体内注射1×107pfu HSV-1,每2天给药1次,共给药3次.分别于给药后24h、5d和17d采集组织脏器,采用Taqman实时荧光定量PCR法,检测不同组织脏器中HSV-1基因拷贝数.结果 成功制备裸鼠肿瘤模型.在不同时间点,肿瘤中检测到的病毒载体最多,且随着时间延长不断增加;淋巴结、背根神经节和性腺中也有较高水平的病毒载体;其他组织器官中病毒载体量较少.结论 HSV-1能为基因转移提供有效、安全的载体平台,并能用于构建溶瘤基因治疗产品.     

全人抗EGFR单抗PUM200与不同种属EGFR的结合特征

目的:建立全人抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单抗PUM200临床前研究相关种属动物的筛选方法。方法:使用流式细胞术,细胞免疫荧光的方法分析PUM200与人外阴鳞状细胞癌A431及人结肠癌SW620细胞表面EGFR特异性的结合。使用免疫组织化学及直接免疫荧光的方法比较PUM200与食蟹猴、恒河猴、SD大鼠、Wistar大鼠及Balb/c小鼠皮肤组织EGFR特异性结合。结果:PUM200与A431细胞结合呈阳性,与SW620细胞不结合。与食蟹猴、恒河猴皮肤呈阳性结合,与SD大鼠、Wistar大鼠及Balb/c小鼠的皮肤结合反应呈阴性。结论:PUM200与表达EGFR的人癌细胞、非人灵长类动物皮肤特异性结合,与啮齿类动物皮肤不结合。     

心脏型脂肪酸结合蛋白对急性心肌梗死早期诊断价值的研究

选取115例疑诊急性心肌梗死（AMI）的急性胸痛患者,并按胸痛时间分为〈3h和3～6h两个时段,分别进行心脏型脂肪酸结合蛋白（H—FABP）和传统心肌损伤标记物肌钙蛋白T（cTnT）、肌酸激酶同工酶（CK—MB）及肌红蛋白（MYO）检测,比较不同时段检测结果对AMI早期诊断的敏感性及特异性。结果显示,胸痛发生≤6h检测H—FABP对AMI诊断的敏感性显著高于cTnT、CK—MB、MYO（P值分别为0．026、0．005、0．048）;H—FABP的特异性低于cTnT、CK-MB（P〉0．05）。ST段抬高的心肌梗死组与非ST段抬高的心肌梗死组间H—FABP敏感性分别为81．6％、78．1％（P=0．095）。无论胸痛发生〈3h还是3-6h,H—FABP联合cTnT检测对AMI诊断的敏感性均优于cTnT、CK—MB及MYO联合（P值分别为0．005、0．028）。提示,H—FABP在AMI患者早期、快速诊断中具有较高的敏感性,但特异性较差,对早期诊断有参考价值。     

大鼠尿中酶类参考值检测

目的调查正常大鼠尿液样本中酶类参考值范围。方法采集95只正常SD大鼠的4 h尿液,用生化仪检测尿液中的N-乙酰基-β-D氨基葡萄糖酶(NAG)、碱性磷酸酶(ALP)、谷氨酰氨基转移酶(γ-GT)和乳酸脱氢酶(LDH)的值,参考范围以95%百分位上界表示。结果参考值范围为,雌性:NAG<5.3 U/L,ALP<110.8 U/L,γ-GT<753.0 U/L,LDH<32.6 U/L;雄性:NAG<27.3 U/L,ALP<329.0 U/L,γ-GT<769.8 U/L,LDH<40.0 U/L。以/g Cre为单位的参考值范围,雌性:NAG<28.9 U/g,ALP<723.5 U/g,γ-GT<1763.2 U/g,LDH<113.1 U/g;雄性:NAG<84.3 U/g,ALP<680.2 U/g,γ-GT<2522.3 U/g,LDH<121.8 U/g。结论尿中酶类参考值范围为药物肾毒性评价提供了背景数据。     

食管癌高发区60181例患者及父母、同胞食管癌和贲门癌患病情况调查

目的通过调查食管癌高发区60 181例患者及父母、同胞食管癌和贲门癌患病情况,了解夫妻不同患病类型(双发、单发和正常)与子女发病关系,加深对食管癌遗传倾向性的认识。方法研究对象来自我们实验室食管癌和贲门癌资料库,采用卡方检验和Logistic回归方法,比较夫妻不同患病类型的发生率与其子女食管癌和贲门癌的患病风险。结果与正常夫妻相比,夫妻均为食管癌和/或贲门癌患者的子女≥2人患食管癌和贲门癌的风险升高近12倍(13.79%,1.32%,P<0.05,OR=11.867,95%CI=9.026～15.601),单发(妻)患者的子女≥2人患癌风险升高近10倍(11.93%,1.32%,P<0.05,OR=10.042,95%CI=8.357～12.067),单发(夫)患者的子女≥2人患癌风险升高近9倍(10.63%,1.32%,P<0.05,OR=8.817,95%CI=7.419～10.479)。结论夫妻癌和单发(夫/妻)癌其子女≥2人患食管癌或贲门癌的几率明显高于正常夫妻的子女,提示了遗传因素对食管癌和/或贲门癌癌变具有重要的影响。     

Corn oil as a vehicle in drug development exerts a dose‐dependent effect on gene expression profiles in rat thymus

The purpose of this study was to investigate the effects of corn oil (CO), which is widely used as a vehicle for water‐insoluble agents in drug development, on the gene expression profiles in rat thymus with microarray technique. Female Wistar rats were administered daily with normal saline (NS) and CO 2, 5 and 10 ml kg^?1 per day for 14 days, respectively. Then, the thymus samples of rats were collected for microarray test and histopathology examination. CD4⁺ and CD8⁺ lymphocytes in peripheral blood were also numerated to assess the effects on lymphocyte subpopulations. The microarray data showed that the numbers of differentially expressed genes in the 2, 5 and 10 ml kg^?1 CO groups were 0, 40 and 458, respectively, compared with the NS control group. The altered genes were mainly associated with immune response, cellular response to organic cyclic substance and regulation of fatty acid β‐oxidation. However, no abnormal changes in thymus weight, CD4⁺ and CD8⁺ lymphocytes counts and histopathological examination were observed in the three CO groups. These data showed that 10 ml kg^?1 CO, the usually recommended dosing volume as a vehicle in drug safety assessment, caused obvious dysregulated genes in rat thymus. Our study suggests that the appropriate dosing volume of CO gavage as a vehicle for water‐insoluble agents in drug development should be 2 ml kg^?1 per day, if agent effects on thymus will be assessed in gene levels. Copyright © 2012 John Wiley & Sons, Ltd.     

BUT－6010的抗肿瘤活性实验研究

目的研究BUT-6010对肿瘤的抑制作用。方法体外抗肿瘤实验：选取9种人源肿瘤细胞株,并将其分为BUT-6010组、顺铂阳性对照组、细胞对照组和培养基空白对照组,MTS法测定抑制率和IC50值;体内抗肿瘤实验：实验分为BUT-6010 10mg／kg组、BUT-6010 20mg／kg组、BUT-6010 30mg／kg组、顺铂阳性对照组（2．5mg／kg）和模型组。通过对S180荷瘤小鼠瘤重的研究,观察BUT-6010的体内抗肿瘤作用。结果BUT-6010对9种人癌细胞株的IC50均小于顺铂。顺铂（2．5mg／kg）的抑瘤率达51．8％（P〈0．05）,BUT-6010（30mg／kg）抑瘤率为52．8％（P〈0．05）。结论BUT-6010（30mg／kg）的安全性与有效性同顺铂（2．5mg/kg）相当。     

Development of genetically engineered CD4+ and CD8+ T cells expressing TCRs specific for a M. tuberculosis 38-kDa antigen

Cell-mediated immunity is critical to the clearance of Mycobacterium tuberculosis due to the primarily intracellular niche of this pathogen. Adoptive transfer of M. tuberculosis-specific effector T cells has been shown to confer immunity to M. tuberculosis-infected recipients resulting in M. tuberculosis clearance. However, it is difficult to generate sufficient numbers of M. tuberculosis antigen-specific T cells in a short time. Recent studies have developed T cell receptor (TCR) gene-modified T cells that allow for the rapid generation of large numbers of antigen-specific T cells. Many TCRs that target various tumor and viral antigens have now been isolated and shown to have functional activity. Nevertheless, TCRs specific for intracellular bacterial antigens (including M. tuberculosis antigens) have yet to be isolated and their functionality confirmed. We isolated M. tuberculosis 38-kDa antigen-specific HLA class I and class II-restricted TCRs and modified the TCR gene C regions by substituting nine amino acids with their murine TCR homologs (minimal murinization). Results showed that both wild-type and minimal murinized TCR genes were successfully cloned into retroviral vectors and transduced into primary CD4⁺ and CD8⁺ T cells and displayed anti-M. tuberculosis activity. As expected, minimal murinized TCRs displayed higher cell surface expression levels and stronger anti-M. tuberculosis activity than wild-type TCRs. To the best of our knowledge, this is the first report describing TCRs targeting M. tuberculosis antigens and this investigation provides the basis for future TCR gene-based immunotherapies that can be designed for the treatment of immunocompromised M. tuberculosis-infected patients.