大剂量顺铂所致大鼠急性肾衰过程中肠道细菌易位与肠内菌群比例变化的关系

目的研究大剂量顺铂(cisplatin,DDP)所致大鼠急性肾功能衰竭过程中肠道细菌易位与肠内菌群比例变化的关系。方法SD大鼠24只,雌雄各半,依体重随机分为DDP用药后24、48、72 h组和NS对照组,每组6只。10 mg/kg DDP单次腹腔内注射,等量生理盐水对照。随后观察并记录大鼠用药后的表现及体重变化情况;DDP用药后不同时间点分别取用药组大鼠和对照组大鼠,称重后内眦静脉取血,生化法检测血清尿素氮和肌酐的浓度;无菌条件下剖腹取回盲部淋巴结进行细菌培养;同时取十二指肠、空肠、回肠和结肠段内容物进行细菌涂片检查。结果大剂量DDP用药后6 h大鼠体重开始降低,用药48 h后出现腹泻症状并进行性加重。DDP用药后48 h大鼠出现血尿素氮、肌酐的比例显著升高,与对照组比较差异有非常显著性(P0.01)。DDP用药后24 h66%的大鼠回盲部淋巴结出现革兰阴性菌生长,用药后48 h革兰阴性菌生长的阳性率为83%,而对照组大鼠则无细菌生长。对照组大鼠十二指肠、空肠和回肠内主要以革兰阳性球菌为主,结肠内主要以革兰阴性杆菌和革兰阳性球菌为主。DDP用药后24 h,十二指肠、空肠、回肠和结肠内仍然以革兰阳性球菌为主;用药48 h后,空肠、回肠和结肠内革兰阴性杆菌所占比例逐渐增加。结论大剂量DDP可导致大鼠肠道细菌易位、空肠、回肠和结肠内菌群比例失衡,可能与大鼠内毒素血症及急性肾功能衰竭的发生、发展及恶化有关。     

利用CRISPRi技术沉默MRP1基因增强A549/DDP细胞对顺铂的敏感性*

目的: 采用CRISPRi技术沉默人肺癌A549/DDP细胞MRP1基因表达,观察细胞对顺铂敏感性的变化。方法: 采用生物信息学软件分析MRP1启动子序列,设计合成3对sgRNA干扰片段,定向克隆到pSPgRNA载体中,构建靶向MRP1的干扰表达载体,分别与dCas9表达载体共转染A549/DDP细胞。实验共分为5组,分别为A549/DDP细胞组,Scrambled组,sgRNA-MRP1-1组,sgRNA-MRP1-2组和sgRNA-MRP1-3组,每组设置3个复孔,处理48 h后进行后续实验。通过qRT-PCR和Western blot检测MRP1 mRNA和蛋白表达水平,MTT法检测细胞对药物的敏感性,激光共聚焦显微镜下观察细胞的形态变化。结果: 成功构建了sgRNA-MRP1-1、sgRNA-MRP1-2和sgRNA-MRP1-3 3种干扰载体,分别与dCas9表达载体共转染A549/DDP细胞后,均能显著降低细胞MRP1基因表达(P<0.01),其中sgRNA-MRP1-2干扰效果最好,MRP1 mRNA水平降低了72%,蛋白水平降低了53%。将sgRNA-MRP1-2转染细胞后,细胞对顺铂的敏感性显著增加,IC₅₀值由74.26±3.71降低至34.29±2.51,细胞形态由梭形变为椭圆形,染色质高度凝聚、边缘化,产生凋亡小体。结论: 成功构建3种靶向MRP1的干扰表达载体,均能有效沉默A549/DDP细胞中MRP1基因表达,可增强细胞对顺铂的敏感性。     

Ad-ING4-IL-24 双基因共表达对人肺腺癌化疗的增敏效应

研究ING4 (肿瘤生长抑制因子4) 和IL-24 (人白细胞介素24) 双基因共表达腺病毒载体 (Ad-ING4-IL-24) 对肺腺癌的化疗增敏效应及分子机制。将Ad-ING4-IL-24感染A549肺癌细胞及联合顺铂 (DDP) 化疗药物作用,RT-PCR和Western blotting检测ING4和IL-24基因在A549肺癌细胞中的转录和表达,MTT法、流式细胞仪 (FCM) 和 Hoechst 染色法检测Ad-ING4-IL-24联合DDP (联合组) 对A549肺癌细胞的生长抑制和凋亡效应。采用A549细胞株建立人肺腺癌裸鼠模型,然后瘤体局部注射干预用药,动态测量肿瘤体积,并计算瘤重抑瘤率,免疫组化检测ING4、IL-24、bax、bcl-2、VEGF等基因的表达。结果表明,Ad-ING4-IL-24感染A549肺癌细胞后可获得成功转录和表达,体外联合组能明显抑制A549肺癌细胞生长和诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。体内联合组同样能显著抑制肿瘤生长,瘤重抑瘤率达52.81％,免疫组化结果显示联合组能上调bax基因表达,同时下调bcl-2、VEGF等基因的表达。以上结果表明Ad-ING4-IL-24具有化疗增敏的作用,该作用机制可能与促进肿瘤细胞凋亡和抑制血管形成有关。     

PPAR-γ激动剂吡格列酮对大剂量顺铂所致小鼠肠道微生态失调的影响

利用大剂量顺铂（DDP）诱导小鼠急性肾功能衰竭模型,研究吡格列酮对DDP所致小鼠肠道微生态的影响及其机制。

昆明种小鼠,依性别、体重随机分组：DDP组、DDP+吡格列酮组、对照组,每组10只。每日观察小鼠的体重、记录粪便数量等,并进行粪便涂片检查。DDP用药后3 d,分别取各组小鼠称重后乙醚麻醉,内眦静脉取血;分别取空肠、回肠末段、升结肠上段内容物进行细菌涂片检查。生化法检测血清尿素氮（BUN）、尿酸（UA）、丙二醛（MDA）的水平,并对数据进行统计学分析。

DDP用药后3 d,小鼠回肠、结肠内G⁺菌与G⁻菌比值均显著低于对照组小鼠。吡格列酮可明显增加DDP用药后小鼠回肠、结肠内G⁺菌与G⁻菌比值。DDP组小鼠外周血BUN、UA和MDA水平明显高于对照组小鼠,差异具有统计学意义;DDP+吡格列酮组小鼠其水平明显低于DDP组小鼠。

大剂量DDP可导致小鼠肠内菌群比例失衡;吡格列酮可能通过抑制小鼠体内氧化应激、进而减轻DDP所致的小鼠肾损伤和肠内菌群失衡。

     

High-performance liquid chromatographic determination of glyoxal and methylglyoxal in urine by prederivatization to lumazinic rings using in serial fast scan fluorimetric and diode array detectors

Glyoxal and methylglyoxal are two important markers of oxidative stress and both are involved in the evaluation of several diseases. A new HPLC method for determining glyoxal and methylglyoxal in urine was developed. The method is based on the reaction of alpha-dialdehydes, glyoxal and methylglyoxal, with 5,6-diamino-2,4-hydroxypyrimidine sulfate in basic medium to form highly fluorescent lumazine derivatives. Creatinine was also included in the method even though it does not react with the reagent. The derivatives and creatinine are separated on a C(18) reversed-phase column with a mobile phase consisting of acetonitrile:citrate buffer, pH 6.0 (3:97 v/v). The flow rate was 1.0mLmin(-1) and the effluent was monitored photometrically at 250 nm for determination of creatinine and fluorimetrically at 500 nm (exciting at 330 nm) for determination of glyoxal and methylglyoxal derivatives. Recording time of the separation is less than 10 min. Determination of the analytes is performed in urine after incubation of the sample, with the reagent in alkaline medium, for 30 min at 60 degrees C. Urinary levels of glyoxal and methylglyoxal, expressed as glyoxal/creatinine and methylglyoxal/creatinine ratios, in healthy young women and men were determined. For women, values of 0.80+/-0.37 and 0.60+/-0.22 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. For men, values of 0.63+/-0.15 and 0.49+/-0.05 microg/mg of creatinine were found for glyoxal and methylglyoxal, respectively. These results were also related to the body mass index of each individual.     

TRAIL联合顺铂对小鼠移植型肝癌抑制作用及机制研究

目的探讨肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis inducing ligand,TRAIL)联合顺铂(cisplatin,DDP)对小鼠移植型肝癌的抑制作用及机制。方法将H22小鼠移植型肝癌模型随机分为生理盐水组、TRAIL组、TRAIL+DDP组和DDP组,称取瘤重并分析抑瘤率,Hoechst 33342荧光染色法检测细胞凋亡,免疫组织化学染色检测Caspase-3表达。结果与生理盐水组比较,TRAIL、DDP对小鼠移植型肝癌生长具有明显的抑制作用(P<0.05);TRAIL与DDP联合用药具有增效作用(P<0.05),可明显提高肝癌细胞的凋亡率(P<0.05)、上调Caspase-3表达(P<0.01)。结论 TRAIL与DDP联合用药对小鼠移植型肝癌生长具有协同抑制作用,其机制可能与其协同促进Caspase-3的表达有关。     

康莱特联合顺铂对宫颈癌SiHa细胞增殖和凋亡的影响

目的:通过康莱特联合顺铂对宫颈癌SiHa细胞增殖和凋亡的影响,探讨其作用机制。方法:体外培养宫颈癌Siha细胞,分别将康莱特(浓度为1,2,4,6,8 mg/mL),顺铂(浓度梯度为1.5,3,6,9,12μg/mL),单独作用于宫颈癌SiHa细胞,加药24h、48h用噻唑蓝(MTT法)检测细胞增殖情况。用流式细胞术检测康莱特组和顺铂组细胞24h凋亡率,选取合适的药物浓度(康莱特6 mg/mL,顺铂3μg/mL),进行联合用药,加药24h、48h用MTT法检测细胞增殖情况,用流式细胞术检测24h细胞凋亡率。结果:①MTT法显示加药后两组的24h、48h,宫颈癌SiHa细胞的抑制率均高于对照组(P0.05),并且在一定程度上呈浓度和时间依赖性。②联合用药时,细胞的抑制率和凋亡率要显著高于单独用药(P0.01)。结论:康莱特、顺铂单独或联合作用均能抑制SiHa细胞的增殖,促进其凋亡,且康莱特联合顺铂的作用要显著高于单独用药,康莱特与化疗药物联合使用可提高肿瘤细胞对化疗药物的敏感性。     

Neurological phenotype and reduced lifespan in heterozygous Tim23 knockout mice, the first mouse model of defective mitochondrial import

The Tim23 protein is the key component of the mitochondrial import machinery. It locates to the inner mitochondrial membrane and its own import is dependent on the DDP1/TIM13 complex. Mutations in human DDP1 cause the Mohr-Tranebjaerg syndrome (MTS/DFN-1; OMIM #304700), which is one of the two known human diseases of the mitochondrial protein import machinery. We created a Tim23 knockout mouse from a gene trap embryonic stem cell clone. Homozygous Tim23 mice were not viable. Heterozygous F1 mutants showed a 50% reduction of Tim23 protein in Western blot, a neurological phenotype and a markedly reduced life span. Haploinsufficiency of the Tim23 mutation underlines the critical role of the mitochondrial import machinery for maintaining mitochondrial function.     

Analysing the contribution of nucleic acids to the structure and properties of centric heterochromatin

A class of repetitive DNA sequences frequently found at centromeric regions are R/Y-satellites showing an asymmetric distribution of residues resulting in one strand being rich in purines (R-strand) while the complementary strand is pyrimidine-rich (Y-strand). The dodeca-satellite of Drosophila belongs to this class of centromeric satellites. In vitro, the dodeca-satellite forms altered DNA structures in which the R-strand forms very stable intramolecular fold-backs that are stabilised by the formation of tandem G · A mismatches. A single-stranded nucleic acids binding protein, DDP1, binds the unstructured dodeca-satellite Y-strand with high affinity. In polytene chromosomes, DDP1 associates with the heterochromatic chromocenter and, at the euchromatic chromosome arms, co-localises with HP1. DDP1 is a vigilin. Vigilins are highly conserved multi-KH-domain proteins. Scp160p, the vigilin from S. cerevisiae, is involved in the control of ploidy. DDP1 complements a scp160 deletion.     

干扰LRP16基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及机制研究

目的:探究干扰人白血病相关蛋白16(LRP16)基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及其相关机制。方法:采用Real-time PCR和蛋白免疫印迹(WB)检测LRP16在敏感组(SKOV3细胞)、耐药组(SKOV3/DDP细胞)、LRP16干扰组(SKOV3/DDP细胞-稳定转染LRP16 shRNA质粒)和NC组(即阴性对照组,SKOV3/DDP细胞-稳定转染阴性对照质粒)细胞中的表达情况;MTT试验检测LRP16对SKOV3/DDP细胞耐药性的影响;彗星试验检测LRP16对顺铂(DDP)诱导DNA损伤的影响;流式细胞术(FCM)试验检测细胞凋亡变化;WB试验检测PTEN、p-Akt和NF-κB蛋白表达水平。结果:LRP16干扰组细胞的耐药指数(RI)值(3.19±0.21)显著低于耐药组细胞(6.84±0.37)(P0.05)。DDP(25μmol/L)处理24 h后,LRP16干扰组DNA损伤的细胞百分比、细胞凋亡百分比均显著低于耐药组和NC组(P均0.05),而耐药组和NC组比较差异无统计学意义(P0.05);LRP16干扰组细胞PTEN蛋白相对表达量高于耐药组和NC组(P均0.05),而p-Akt和NF-κB相对表达量低于耐药组和NC组(P均0.05)。结论:干扰LRP16基因表达可逆转卵巢癌耐药SKOV3/DDP细胞的耐药性,PTEN/Akt/NF-κB可能是其中的关键信号通路。     

间充质干细胞联合铂类对肝癌大鼠抗癌效果及对免疫功能、细胞凋亡的影响

摘要 目的：探讨骨髓间充质干细胞（bone marrow mesenchymal stem cells,BMSCs）联合顺铂（cis-diamminodichloroplatinum Ⅱ dichloride,DDP）对肝癌大鼠抗癌效果及其免疫功能和细胞凋亡水平的影响。方法：将40只雄性Wistar大鼠随机分为空白对照组、模型组、DDP组和联合组,各10只。除空白对照组外,其余均采用二乙基亚硝胺饲喂法建立肝癌模型。造模成功后,空白对照组和模型组大鼠静腹腔注射生理盐水并经尾静脉注射DMEM培养液,DDP组大鼠经腹腔注射DDP溶液并经尾静脉注射DMEM培养液,联合组大鼠经腹腔注射DDP溶液并经尾静脉注射BMSCs悬液。移植BMSCs两周后处死大鼠,称量肝脏质量和体质量,采用酶联免疫吸附法检测各组大鼠外周血中白细胞介素-2（interleukin-2,IL-2）、IL-8、肿瘤坏死因子α（tumor necrosis factor α,TNF-α）、血管内皮生长因子（vascular endothelial growth factor,VEGF）和低氧诱导因子-1α（hypoxia-inducible factor-1α,HIF-1α）水平,采用TUNEL技术检测肝细胞凋亡指数。结果：（1）模型组肝脏质量、肝脏质量/体质量均显著大于其他各组,体质量显著小于其他各组（P<0.05）。联合组肝脏质量、肝脏质量/体质量均显著显著小于模型组和DDP组,体质量显著大于模型组和DDP组（P<0.05）。（2）DDP组和联合组大鼠治疗后血清IL-2水平显著升高,IL-8和TNF-α水平显著降低（P<0.05）。联合组大鼠治疗后血清IL-2水平显著高于DDP组,IL-8和TNF-α水平显著低于DDP组（P<0.05）。（3）DDP组和联合组大鼠治疗后血清VEGF和HIF-1α水平显著降低,且显著低于模型组（P<0.05）。联合组大鼠治疗后血清VEGF和HIF-1α水平显著低于DDP组（P<0.05）。（4）联合组和DDP组肝癌细胞凋亡指数显著高于模型组,且联合组也显著高于DDP组（P<0.05）。结论：BMSCs联合DDP能够显著改善肝癌大鼠免疫功能,抑制肿瘤血管生成,促进肝癌细胞凋亡,效果优于DDP单药。     

CircFAT1 Promotes Lung Adenocarcinoma Progression by Sequestering miR-7 from Repressing IRS2-ERK-mediated CCND1 Expression

Our understanding of coding gene functions in lung cancer leads to the development of multiple generations of targeted drugs. Noncoding RNAs, including circular RNAs (circRNAs), have been demonstrated to play a vital role in tumorigenesis. Uncovering the functions of circRNAs in tumorigenesis and their underlying regulatory mechanisms may shed new light on the development of novel diagnostic and therapeutic strategies for human cancer. Here we report the important role of circFAT1 in lung adenocarcinoma (LUAD) progression and the potential impact of circFAT1 on LUAD treatment. We found that circFAT1 was one of the top expressed circRNAs in A549 cells by circRNA-seq and was significantly upregulated in human LUAD tissues. Multiple cellular assays with A549 and PC9 LAUD cell lines under both gain-of-function and loss-of-function conditions demonstrated that circFAT1 promoted proliferation of LUAD cells in vitro and in vivo. At molecular level, circFAT1 sequestered miR-7 to upregulate IRS2, which in turn regulated downstream ERK1/2 phosphorylation and CCND1 expression, ultimately promoting tumor progression. In addition, we showed that DDP treatment was much more effective in circFAT1 knockdown tumor cells in vitro and in a xenograft tumor model. Our results indicate that circFAT1 promote tumorigenesis in LUAD through sequestering miR-7, consequently upregulating IRS2-ERK1/2-mediated CCND1 expression, and can be a valuable therapeutic target and an important parameter for precision treatment in LUAD patients.     

不同化疗方案对晚期非小细胞肺癌患者骨髓抑制及免疫功能的影响

目的:探讨不同化疗方案对晚期非小细胞肺癌患者骨髓抑制及免疫功能的影响。方法:选取2011年1月至2013年12月收治的130例晚期非小细胞肺癌患者,按照知情同意原则随机分为三组:NP(长春瑞滨+顺铂)组45例、GP(吉西他滨+顺铂)组43例、TP(紫杉醇+顺铂)组42例,分别于化疗前及化疗2个周期后检测患者的骨髓抑制及免疫水平。结果:三种化疗方案进行治疗后骨髓抑制水平由高到低排列为GP组、TP组、NP组,差异有统计学意义(P0.05);GP组血小板减少发生率高于其他两组,TP组白细胞下降发生率高于其他两组,差异有统计学意义(P0.05);三组患者化疗后的免疫功能指标均较化疗前低,差异有统计学意义(P0.05)。三种化疗方案进行治疗后免疫功能抑制水平由高到低排列为GP组、TP组、NP组,差异有统计学意义(P0.05)。结论:GP组患者血小板下降更明显,TP组白细胞下降更为明显,NP组对骨髓抑制及免疫功能抑制较缓和,更适于老年人,因此临床选择化疗方案时要综合考虑患者骨髓状况、免疫功能情况及年龄等。     

Functional and mutational characterization of human MIA40 acting during import into the mitochondrial intermembrane space

A first component involved in import into the mitochondrial intermembrane space, named Mia40, has been described recently in yeast. Here, we identified the human MIA40 as a novel and ubiquitously expressed component of human mitochondria. It belongs to a novel protein family whose members share six highly conserved cysteine residues constituting a -CXC-CX9C-CX9C- motif. Human MIA40 is significantly smaller than the fungal protein and lacks the N-terminal extension including a transmembrane region and mitochondrial targeting signal. It forms soluble complexes within the intermembrane space of human mitochondria. Depletion of MIA40 in human cells by RNA interference specifically affected steady-state levels of small and cysteine-containing intermembrane space proteins like DDP1 and TIM10A, suggesting that MIA40 acts along the import pathway into the intermembrane space. Studies on the in vivo redox state of human MIA40 demonstrated that it contains intramolecular disulfide bonds. Thiol-trapping assays revealed the co-existence of different oxidation states of human MIA40 within the cell. Furthermore, we show that the twin -CX9C- motif is specifically required for import and stability of MIA40 in mitochondria. Partial mutation of this motif affects stable accumulation of MIA40 in the intermembrane space, whereas mutation of all cysteine residues in this motif inhibits import in mitochondria. Taken together, we conclude that the biogenesis and function of MIA40 in the mitochondrial intermembrane space is dependent on redox processes involving conserved cysteine residues.     

A peculiar toxicity manifested by platinum(II)amines in rats: Gastric distension after intraperitoneal administration

Metoclopramide i.p. reduces the gastric distension consistently seen in rats given cisplatin i.p. at effective immunosuppressant doses (6 mg/kg). Many other immunosuppressant/oncolytic platinum amines also engendered gastric distension but certain dimers and 1,2-cyclohexanediaminoplatinum (II) compounds did not. This phenomenon appears to be due to paralysis of gastric emptying (without appetite suppression).