Tissue cross-reactivity (TCR) studies play an important role in the preclinical safety evaluation of monoclonal antibody (mAb) drugs.The objective of TCR studies is to find out off-target binding sites of mAbs,and provide valuable predictions for the toxicological evaluation and safety medication in vivo.According to the new drug application requirements of FDA,EMA and CFDA,TCR studies need to be carried out before Phase I clinic trails.As the origin of mAb drugs was transferred from murine antibodies to fully humanized antibodies in current years,immunohistochemical methods used in TCR studies were confronted with some new problems and challenges.Taking our own experiences and recent progress on TCR studies at home and abroad together,the authors summarized the recent exploration on technical difficulties of multipath system in TCR studies.This may provide valuable insight for further improving the quality of TCR studies and increase the predictive value of TCR studies for in vivo toxicological evaluation in China.

目的 评价氯胺酮的线粒体毒性,从而探讨氯胺酮可能的毒性机制.方法 HepG2细胞培养基中分别加入齐多夫定10～2000 μmol·L-1培养7d,或加入氯胺酮50～3000 μmol·L-1培养24 h,CCK8细胞计数法测定细胞存活率.HepG2细胞培养基中分别加入齐多夫定100 μmol· L-1培养7d后,或加入氯胺酮100和1200 μmol·L-1培养24 h,化学发光法检测胞内ATP合成水平,荧光探针法检测胞内钙离子浓度、活性氧及线粒体膜电位(△Ψm)的变化,同时设齐多夫定1000 μmol· L-1用于实时荧光定量PCR检测线粒体DNA(mtDNA)表达水平的变化.结果 与正常对照组相比,齐多夫定10～ 2000 μmol· L-1可以抑制细胞存活,IC50为100 μmol·L-1;氯胺酮1000～ 3000 μmol· L-1抑制细胞存活,IC50为1200 μmol·L-1.与正常对照组相比,齐多夫定100 μmol·L-1组线粒体ATP合成水平显著下降(P＜0.05),胞内钙离子浓度明显升高(P＜0.05),活性氧水平显著升高(P＜0.05),△Ψm显著下降(P＜0.05),而1000 μmol· L-1可以明显干扰mtDNA表达水平(P＜0.05).氯胺酮100 μmol· L-1组ATP合成水平明显下降(P＜0.05),其他指标均无显著性差异;氯胺酮1200 μmol·L-1组ATP合成水平显著下降(P＜0.01),活性氧水平和胞内钙离子浓度显著升高(P＜0.01),△Ψm显著下降(P＜0.05),mtDNA水平无明显改变.结论 氯胺酮可以通过干扰线粒体代谢功能而诱导线粒体损伤.

目的 评价马兜铃酸的线粒体毒性,从而探讨马兜铃酸的可能毒性机制.方法 HepG2细胞培养基中分别加入齐多夫定8 000～20 000 μmol/L,或加入马兜铃酸25～500μmol/L,均培养24 h,以CCK8细胞计数法测定细胞存活率.同时比较不同浓度齐多夫定(8 000、16 000和20 000μmol/L)和马兜铃酸(25、200和500 μmol/L)培养24 h的胞内ATP合成水平和胞内钙离子浓度、活性氧及线粒体膜通透性转换孔 (MPTP)变化,透射电镜观察线粒体超微结构.结果 齐多夫定8 000～20 000 μmol/L可抑制细胞存活,IC50为12 713μmol/L;马兜铃酸25～500 μmol/L可抑制细胞存活,IG0为214.6 μmol/L.与溶媒对照组(DMEM)相比,齐多夫定≥8 000 μmol/L细胞内活性氧水平显著升高(P＜0.01);≥16 000 μmol/L线粒体ATP显著下降(P＜0.01)、钙离子浓度明显升高(P＜0.01),并可见线粒体结构发生病理改变;20 000 μmol/L组MPTP开放水平显著升高(P＜ 0.01).与溶媒对照组(DMSO)相比,马兜铃酸≥25 μmol/L,ATP合成水平明显下降(P＜0.01)、MPTP开放水平显著升高(P＜0.01);≥200 μmol/L细胞内钙离子浓度明显升高(P＜0.01);500 μmol/L细胞内活性氧水平显著升高(P＜0.01)、并可见线粒体结构发生病变.结论 马兜铃酸可以通过干扰破坏线粒体代谢功能和结构而诱导线粒体损伤.

目的:评价DPP-Ⅳ抑制剂类口服降糖药A化合物的线粒体毒性,从而探讨A化合物的可能毒性机制.方法:HepG2细胞培养基中分别加入曲格列酮50～300μmol/L,培养24h,或加入A化合物溶液100～300 μmol/L,培养24h,CCK8细胞计数法测定细胞存活率.HepG2细胞培养基中分别加入曲格列酮100、200和225 μmol/L培养24 h,或加入A化合物溶液100、150和200μmol/L培养24h,化学发光法检测胞内ATP合成水平,荧光探针法检测胞内钙离子浓度、活性氧、线粒体膜电位(△ψm)变化,透射电镜观察线粒体超微结构.结果:与溶媒对照组相比,曲格列酮50～300 μmol/L可以抑制细胞存活率,IC50为178μmol/;A化合物100～300 μmol/L对细胞存活率有抑制作用,IC50为159 μmol/L.与溶媒对照组相比,曲格列酮≥200 μmol/L时导致线粒体ATP显著下降(P＜0.01)、△ψm显著性下降(P＜0.01),≥100μmol/L时可致活性氧水平和钙离子浓度显著升高(P＜ 0.05或P＜ 0.01).A化合物≥100 μmol/L时ATP合成水平显著降低、钙离子浓度显著升高(P＜0.01),≥150μmol/L时活性氧水平明显升高(P＜0.01),≥200μ,mol/L时△ψm显著性下降(P＜0.05),并可导致线粒体结构发生病变.结论:DPP-Ⅳ抑制剂类口服降糖药A化合物可以通过干扰线粒体代谢功能和结构而诱导线粒体损伤.

OBJECTIVE To investigate the time and dose relation of new urine bio markers in rat model of acute kidney injury induced by genta mycin (GM)to search for more sensitive,noninvasive and specific markers than traditional approaches to monitor nephrotoxicity.METHODS SD Rats were im treated with GM5,20,80 mg·kg -1 or saline once daily.Rats were randomly divided into 20 subgroups:treated for 1 ,3,7,14 d and 14 d followed with 28 d recovery period.Ten rats per group (5 rats per sex)were scarified at 24 h after the last dosing or the end of recovery period.Blood sa mples were col-lected for blood urea nitrogen (BUN)and creatinine(CRE)analysis.Urine was collected at each nec-ropsy for urine protein by dry che mistry method,for kidney injury molecule-1 (KIM-1 )analysis by ELISA, and for β2-microglobulin (β2-MG)analysis by ELISA.Kidneys were obtained for histological exa mination after HE stains.RESULTS Positive protein(3 +)was noted for several fe male animals treated for 7 or 14 d at 80 mg·kg -1 and the tendency of recovery were noted at the end of recovery period.Co mpared with those in saline control group treated for 7 d,the seru m BUN and CRE levels for fe males and the CRE level for males were significantly increased at 80 mg·kg -1 (P <0.05),and the BUN level showed the tendency of increase for males at 80 mg·kg -1 (P >0.05).When treated for 14 d,the seru m BUN and CRE levels for fe males and males at 80 mg·kg -1 and the seru m CRE level for fe males at 20 mg·kg -1 were significantly increased when compared with those in saline control group(P <0.05). The seru m BUN and CRE recovered to base line for all animals treated for 14 d followed by a 28 d recov-ery period.Histopathological observation of kidney tissues indicated that focal tubule dilatation was noted for animals treated for 3 d at 20 and 80 mg·kg -1 ,infla mmatory cell infiltration and focal tubule dilatation were noted at 20 mg·kg -1 and focal renal tubular epithelial cell degeneration,infla mmatory cell infiltra-tion,focal casts (lightly)were noted at 80 mg·kg -1 for animals treated for 7 or 14 d.For animals treated for 14 d followed by a 28 d recovery period,only basophilic tubules and renal casts were noted at 80 mg·kg -1 .New urine bio markers determination indicated that KIM-1 level was significantly increased at 20 and 80 mg·kg -1 for animals treated for 3,7 or 14 d when compared with that in saline control group (P<0.05).For animals treated for 14 d followed by a 28 d recovery period,the KIM-1 level was still significantly higher than saline control group for males and fe males at 80 mg·kg -1 and males at 20 mg·kg -1 (P <0.05 ),but there was evidence for reversal.The β2-MG level was significantly increased at 80 mg·kg -1 for animals treated for 3 d(P<0.05),at 20 or 80 mg·kg -1 for animals treated for 7 or 14 d(P<0.05 or P<0.01 ),when compared with that in the saline control group.For animals treated for 14 d followed by a 28 d recovery period,the β2-MG level was still significantly higher than saline control group for males and fe males at 80 mg·kg -1 and fe males at 20 mg·kg -1 (P <0.05),but there was also evidence for reversal.CONCLUSION Urine KIM-1 and β2-GM are more sensitive and specific markers for early diagnosis of kidney injury induced by GM when compared with the traditional approaches to monitor nephrotoxicity.

我国自从2007年发布致癌试验的要求原则以来,已经走过了6年多的时间。然而,致癌试验在我国实际的药物评价工作中,并没有大规模的展开。一方面,由于需要6个月以上甚至更长(国际上致癌试验一般试验周期为24个月)的动物给药时间,对动物的质量(高质量的动物避免自发疾病对试验结果的影响)、动物房的环境、动物福利以及操作人员的技术要求(比如避免连续24个月经口给药中发生的操作失误)比较高;另一方面,长时间的试验周期,对新开发的药品来说,2年以上的等待时间需要有力的资金保障。因此,目前我国开展的致癌试验并不多。而在国际上,比如日本,已经进行了20年以上,上百个致癌试验,积累了大量的长期饲育动物中自发背景病变的数据,通过统计学分析而建立自发性病变高危器官的数据库。在致癌实验的动物(尤其是大鼠的品系)选择中,针对不同的药物可以更好地更合理地挑选动物品种、品系和性别(如果需要)。在组织病理学诊断和分析中,更准确地判断肿瘤和药物的关系,综合评价药物对动物的致癌影响。目前,在本中心(国家上海新药安全评价研究中心),已经完成了1个长达24个月240例SD大鼠模拟致癌试验的研究。本实验是本中心首次实行致癌试验,以单纯饲育动物为主,观察实验动物在实验条件下长时间饲育所出现的一般症状、体重变化、摄食量变化、应激状态等等,作为本中心致癌实验的自发病变的背景资料,建立自发背景病变数据库。其中存在自发肿瘤的分类、以及合并诊断等,由于啮齿类动物中一些肿瘤形成过程是系统性的,例如淋巴瘤和白血病。这些肿瘤应该被作为一个整体记录,还是根据发生的器官分别诊断,不同的结果对于肿瘤的统计学和生物学具有很大的意义;发生在同一器官组织或者不同器官组织的、形态学相似的肿瘤的发生率能否合并,这个问题很显然可以影响对化合物致癌性的评价,试验结果说明化合物的致癌性不是简单的全或无的现象。以上这些结果和经验都为将来进行药物致癌性试验研究提供参考和依据。

OBJECTIVE To explore the changes and role of plasma microRNA(miR-122)in non-clini-cal evaluation of drug-induced liver injury.METHODS Male SD rats were ig given acetaminophen(APAP)0(control),625 and 1250mg·kg-1.At 1.5,3,6,12,24,36 and 96h after administration,blood samples were collected.miR-103,as a suitable endogenous reference gene for plasma microRNAs quanti-fication in rats with acetaminophen-induced hepatotoxicity,was employed to detect the expression of miR-122 in plasma samples with real-time quantitative reverse transcription PCR(RT-qPCR).The blood levels of glu-tamic pyruric transminase(GPT)and glutamic oxaloacetic trnasminase(GOT)were detected and patho-logical changes in liver tissue were observed.RESULTS Histopathological examination showed that,compared with normal control group,there was no significant difference at 1.5 and 3h after rats were ig given APAP 625 and 1250mg·kg-1.Vacuolated hepatocytes and congestion in sinusoids were observed in zoneⅢat 6 and 12h after administration,while at 24h centrilobular necrosis was prominent in APAP 625 and 1250mg·kg-1 groups.Compared with normal control group,GPT and GOT in serum significantly in-creased at 12 and 24h in APAP 625 and 1250mg·kg-1 groups,and GPT activity more than doubled in APAP 1250mg·kg-1 group.Compared with normal control group,plasma miR-122 increased by 5.2-fold at 6h after administration in APAP 625mg·kg-1 and 3.6-fold at 1.5h after administration in APAP 1250 mg·kg-1.The level of plasma miR-122 continued to elevate but returned to normal at 36h in APAP 625 mg·kg-1 group and at 96h in APAP 1250mg·kg-1 group,respectively.The course of plasma miR-122 levels displayed similar kinetics to GPT and GOT.CONCLUSION miR-122 Expression in plasma may be-come an ideal hematologic molecular marker for early detection of drug-induced liver injury in pre-clinical and clinical evaluation.

酪氨酸激酶抑制剂(TKI)作为近几年倍受关注的抗肿瘤药物,其临床疗效及患者耐受性得到了普遍认可,同时此类药物的不良反应较传统细胞毒抗肿瘤药物更少。随着研究的深入,TKI的某些特殊的毒性作用逐渐引起人们的关注,其中有些甚至是致死性的,有些毒性作用机制目前尚无法得以解释。本文就其动物试验中发现的受损的正常组织器官毒性作用表现及部分可能的相关机制作一综述。

上海市老干部大学电气分校合唱班上课的那天早上,天公不作美,下起了倾盆大雨.这并没有阻挡老同志们求学的热情和脚步,大家冒雨从四面八方赶到学校.顾不得喘口气、歇歇脚,顾不得拧干湿漉漉的衣衫,立刻精神抖擞地投入到"和谐中国"的铿锵旋律中.