Exosome-transported IncRNA H19 regulates insulin-like growth factor-1 via the H19/let-7a/insulin-like growth factor-1 receptor axis in ischemic stroke

LncRNA(long non-coding RNA) H19 is a transcript of the H19 gene that is expressed during embryogenesis.We previously discove red a role for circular lncRNA H19 in the onset and prognosis of cerebral ischemic stroke.In this study,we used serum from patients with ischemic stroke,and mouse and cell culture models to elucidate the roles of plasma and neuronal exosomes in the regulatory effect of lncRNA H19 on insulin-like growth factor-1 and its mechanism in ischemic stroke,using western blotting,quantitative real-time polymerase chain reaction,and enzyme-linked immunosorbent assays.Plasma exosomal IncRNA H19 was negatively associated with blood levels of insulin-like growth factor-1 in samples from patients with cerebral ischemic stroke.In a mouse model,levels of exosomal IncRNA H19 were positively correlated with plasma and cerebral lncRNA H19.In a cell co-culture model,we confirmed that IncRNA H19 was transported from neuro ns to astrocytes by exosomes to induce downregulation of insulin-like growth factor-1 through the H19/let-7 a/insulin-like growth factor-1 receptor axis.This study provides the first evidence for the transpo rtation of IncRNA H19 by exosomes and the relationship between IncRNA H19 and insulinlike growth factor-1.

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor,and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.

Expression of insulin-like growth factor Ⅱ and its receptor in hepatocellular carcinogenesis

INTRODUCTIONInsulin-like growth factor Ⅱ(IGF-Ⅱ) is a mitogenic peptide of 74 kD and is mostly synthesized in fetal liver tissue .IGF-Ⅱ is believed to play an important role in fetal growth and development and is involved in cellular proliferation and differentiation[1-5]. Recently ,several researchers have reported increased expression of the IGF-Ⅱgene in human hepatocellular carcinoma (HCC) and adjacent non-cancerous liver tissues [6-10].

RNA干扰沉默IGF-Ⅰ R表达对非小细胞肺癌细胞生物学特性及化疗敏感性的影响

目的:评价RNA干扰(RNA interference,RNAi)技术,对人肺癌A549细胞系中胰岛素样生长因子类受体(IGF-Ⅰ R)表达的阻断效应,和IGF-Ⅰ R基因沉默后细胞增殖、凋亡等生物学特性及肿瘤细胞对化疗药物敏感性的改变。方法:应用U6启动子,介导DNA模板转录生成短发夹样RNA(shRNA),并转染人肺癌A549细胞株,从而产生IGF-Ⅰ R特异性小干扰RNA(siRNA),经RT-PCR和Western blot检测IGF-Ⅰ R表达的改变;联合应用化疗药物顺铂(DDP),通过MTT法和流式细胞技术等,观察细胞生长、细胞周期、细胞凋亡及DDP半数致死量(IC50)的变化。结果:IGF-Ⅰ R表达水平明显下降(抑制率高达89.8%),肿瘤细胞增殖能力明显减弱,细胞滞留于G0期的比例上升;DDP对肿瘤细胞24h、48h、72h的IC50均明显减少,IGF-Ⅰ R siRNA1组DDP作用72h的IC50为0.92 mg/L,明显低于control-siRNA组的3.77mg/L,0.5mg/L DDP联合IGF-Ⅰ R siRNA1作用48h后,A549细胞的生长抑制率达32.1%,明显高于control-siRNA组的18.9%,细胞凋亡率从27.8%上升至44.2%。结论:运用RNAi技术能有效抑制A549细胞IGF-Ⅰ R的表达,使细胞增殖能力减弱,化疗敏感性增加。

转化生长因子β型受体Ⅰ基因RNA干扰慢病毒载体的构建及其在人胚肺成纤维细胞中的表达

目的:构建转化生长因子β型受体Ⅰ(transforming growth factor beta receptorⅠ,TGFβRⅠ)基因RNA干扰(RNA interference,RNAi)慢病毒载体并在人胚肺成纤维细胞株MRC-5上鉴定其沉默效应。方法:构建4种针对人TGFβRⅠ基因不同干扰靶点的慢病毒干扰载体,PCR筛选阳性克隆,测序鉴定,将筛选出的4种有效重组慢病毒干扰载体和其它辅助质粒一起共转染293T细胞并测定病毒滴度。最后将4种重组的慢病毒干扰载体分别感染MRC-5细胞,采用Real-time PCR和Western blot检测它们对靶基因的沉默效率。结果:经双酶切鉴定和DNA测序,证实短发夹RNA正确插入慢病毒载体且插入的序列正确。4种重组慢病毒干扰载体经293T细胞成功包装后,其病毒滴度分别为:si RNA-1 6.37×107 TU/ml、si RNA-2 1.65×108 TU/ml、si RNA-3 4.50×108TU/ml、si RNA-4 2.31×108TU/ml,阴性对照组载体包装后的病毒滴度为:1.79×109 TU/ml。4种重组慢病毒干扰载体感染MRC-5细胞的效率均为93%左右。与正常对照组和阴性对照组比较,在感染4种重组慢病毒干扰载体(si RNA-1~4)后,MRC-5细胞内TGFβRⅠm RNA表达水平均明显下降(P=0.000),其中TGFβRⅠ-si RNA-2下降最明显,沉默效率最好。Western blot结果与q RT-PCR结果一致,4种重组慢病毒干扰载体均能使MRC-5细胞内TGFβRⅠ蛋白表达明显下降(P=0.000),且TGFβRⅠ-si RNA-2干扰效率最大。结论:筛选并构建了能有效沉默TGFβRⅠ基因的最佳RNAi重组慢病毒载体——TGFβR-si RNA-2,从而为后续研究奠定基础。

shRNA干预IGF-ⅠR活化对肝癌细胞增殖的抑制作用

目的:观察干扰胰岛素样生长因子-Ⅰ型受体(insulin-like growth factor-Ⅰreceptor,IGF-ⅠR)表达对肝癌(PLC/PRF/5及Bel-7404)细胞增殖、周期、凋亡的影响及联合抗癌、靶向药物抑制细胞增殖的协同作用.方法:设计与合成多条针对IGF-ⅠR序列的shRNA,插入pGPU6/GFP/Neo载体,构建、转染肝癌细胞株、筛选高效质粒,观察沉默IGF-ⅠR表达对肝癌细胞增殖的抑制作用与机制.结果:4对构建IGF-ⅠR-shRNA经筛选以s h R N A 4干扰效果最佳且具特异性;以shRNA4转染效率PLC/PRF/5细胞为71%和Bel-7404细胞为90%;在mRNA水平上抑制率前者为59.6%±2.8%,后者为54.9%±2.6%;蛋白水平上IGF-ⅠR表达均同步减少;转染72h后,PLC/PRF/5细胞增殖抑制率为63.87%±3.90%(t=19.244,P<0.001)及Bel-7404细胞为61.47%±1.70%(t=5.493,P<0.005),均呈时间依赖性,且增殖周期发生G1期阻滞,细胞周期蛋白(CyclinD1)表达受抑,细胞凋亡增加;shRNA4增加肝癌细胞对靶向药物索拉非尼及化疗药物奥沙利铂的敏感性.结论:下调IGF-ⅠR基因转录可抑制肝癌细胞增殖、诱导凋亡,改善肝癌细胞对靶向药物及化疗药物敏感性,提示IGF-ⅠR有望成为肝癌基因治疗的有效靶点.

雌、雄激素对鼠前列腺组织中胰岛素样生长因子Ⅰ及其受体mRNA表达的影响

探讨雌、雄激素对鼠前列腺组织中胰岛素样生长因子Ⅰ（IGF－Ⅰ）及其受体mRNA表达的影响。方法：采用地高辛标记探针的原位杂交技术并结合图象分析系统研究了正常组、去势组、雌激素组及去势加雄激素组鼠前列腺组织中IGF-Ⅰ、IGF－Ⅰ受体基因表达情况。结果：IGF－Ⅰ、IGF－Ⅰ受体mRNA在正常鼠前列腺组织中均有表达；去势鼠腹侧前列腺IGF－ⅠmRNA表达量较正常明显降低，而IGF-Ⅰ受体mRNA表达量较正常明显增加；外源性睾酮可以阻断去势所引起的IGF-Ⅰ基因表达变化；雌激素组鼠腹侧前列腺IGF－ⅠmRNA表达量较正常组明显升高，平均表达量是正常组表达量的1．9倍。结论：雄激素可以影响大鼠前列腺中IGF-Ⅰ、IGF－Ⅰ受体mRNA水平，雌激素可以上调鼠前列腺中IGF－ⅠmRNA表达。

特异性siRNA抑制胃癌细胞IGF-ⅠR表达的研究

目的:观察胰岛素样生长因子-Ⅰ受体(IGF-ⅠR)基因特异的siRNA对胃癌细胞体外增殖及凋亡的影响。方法:设计并体外合成2条靶向IGF-ⅠR的siRNAs,脂质体法瞬时转染MGC803细胞,Western Blot法检测IGF-ⅠR蛋白表达,MTT法检测细胞活力,细胞计数并描记生长曲线,流式细胞仪(FCM)检测凋亡。结果:转染后48h,Western Blot检测显示干扰组(siRNA2-L组、siRNA2-H组、siRNA1组)IGF-ⅠR蛋白抑制率分别为64.41%±4.11%、74.14%±6.15%、89.8%±4.10%;siRNA1组转染后第2～5天细胞活力逐渐减少(分别达49.9%±1.2%、45.9%±4.4%、39.1%±5.1%、29%±4.0%);同期生长曲线显示细胞数分别为对照组的65.58%±4.89%、55.59%±0.82%、44.18%±3.17%、21.15%±1.1%;但FCM检测各组细胞凋亡没有显著差异。结论:特异的siRNAs可显著抑制胃癌细胞中的IGF-ⅠR基因的表达,主要是通过抑制增生而不是增加凋亡导致的。

Inhibitory Effect of IGF1R siRNA on the growth of human liver cancer SMMC7721 cell xenograft in nude mice

Objective： To investigate the effect of insulin-like growth factor 1 receptor （IGF1R） small interfering RNA （siRNA） on the growth of human liver cancer SMMC7721 cell xenograft in nude mice. Methods： siRNA targeting IGF1R was designed, and plasmid SMMC7721-1GF1R-siRNA was constructed and transfected into SMMC7721 cells （SMMC7721-1GF1R-siRNA cells）; the cells transfected with SMMC7721-1GF1 R-mutation （SMMC7721-1GF1 R-mutation cells） were used as negative con- trol, and untransfected cells as empty control. Stable cell clones were screened by G418, and transplanted into nude mice to establish cancer xenograft. Tumor growth was monitored. Tumor morphology was observed with HE staining. The expression of IGF1R protein in tumor tissues was detected by Western blot. Microvessel density （MVD） in tumor tissues was detected by SP immunohistochemistry. Cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling （TUNEL） assay. Results： The tumor volume was significantly smaller in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （P 〈 0.05）. Necrosis and cell apoptosis were found in SMMC7721- IGF1R-siRNA group. The expression of IGF1R protein was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （P 〈 0.05）. MVD was significantly lower in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups （11.3 ± 4.4 vs. 36.7 ± 7.6 and 28.4 ±6.5, P 〈 0.05）. The apoptosis rate of tumor cells was significantly higher in SMMC7721-1GF1R-siRNA group than in SMMC7721-1GF1R-mutation and SMMC7721 groups [（50.2 ± 6.4）% vs. （5.4 ± 1.0）% or （6.0 ±2.1）%, P〈0.05]. Conclusion： IGF1R siRNA can inhibit the growth of SMMC7721 cell xenograft in nude mice.

RNA干扰TGF-β信号通路对人膀胱癌T24细胞迁移和侵袭能力的影响

目的探讨过表达的TGF-β1和作用于TGF-β受体Ⅰ的干扰RNA(TsiRNA)在体外对人膀胱癌T24细胞迁移、侵袭能力的影响。方法采用Transwell迁移试验、划痕试验以及Transwell侵袭实验观察过表达的TGF-β1和TsiRNA在体外对人膀胱癌T24细胞迁移、侵袭能力的影响,RT-PCR技术和蛋白免疫印迹法(Western blot)检测TGF-β1及TsiRNA处理后TGF-β受体Ⅰ基因与蛋白表达水平的变化。结果过表达的TGF-β1可以显著提高膀胱癌T24细胞的迁移、侵袭能力,而TsiRNA可以完全降低由TGF-β1引起的T24的迁移、侵袭能力的变化。结论膀胱癌细胞的迁移、侵袭能力与过表达的TGF-β1密切相关,TsiRNA可以降低T24细胞的迁移、侵袭能力。

IGF-Ⅰ对ERα阳性及阴性乳腺癌细胞蛋白质表达的影响

采用RNA干扰的方法,通过稳定转染表达靶向雌激素受体α的shRNA的载体,由ERα阳性的乳腺癌细胞系(MCF-7)构建出稳定ERα敲减细胞株,同时构建转染了阴性对照载体的细胞株用作对照。类胰岛素生长因子-I处理稳定雌激素受体α敲减细胞株以及对照细胞株,双向电泳技术分析两种细胞蛋白质组的差异,胶内酶解结合质谱技术鉴定差异表达的蛋白质,在选择的27个差异表达蛋白中,经检验最终确定了11种差异表达显著的蛋白。这是第一次用蛋白质组学方法研究雌激素受体α在IGF-Ⅰ/IGFR信号通路中的作用,研究结果为理解雌激素和IGF-Ⅰ这两个最重要的促乳腺癌分裂因子之间的相互作用提供了线索。

Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

INIRODUCTIONAccording to the therapeutic effect and strategy ofantisense RNA for hepatoccllular carcinoma(HCC),we have specifically synthesized partialcDNA of human insulin-like growth factor Ⅱ(IGF-Ⅱ)and constructed IGF-Ⅱ cDNA antisenseeukaryotic expression vector.The constructedvector was introduced into hepatoma cell lineSMMC-7721 to block the intrinsic IGF-Ⅱexpression.The biological behavior changes ofhepatoma cells were observed.All these

The role of endotoxin,TNF-α,and IL-6 in inducing the state of growth hormone insensitivity

AIM: Critical illnesses such as sepsis, trauma, and burns cause a growth hormone insensitivity, which leads to an increased negative nitrogen balance. Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-alpha, IL-6, and IL-1, which may play a very important role in inducing the growth hormone insensitivity. The objective of this current study was to investigate the role of endotoxin, TNF-alpha and IL-6 in inducing the growth hormone insensitivity at the receptor and post-receptor levels. METHODS: Spague-Dawley rats were injected with endotoxin, TNF-alpha, and IL-6, respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously. All rats were killed at different time points. The expression of IGF-I, GHR, SOCS-3 and beta-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay, the levels of TNF-alpha and IL-6 were detected by ELISA. RESULTS: There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection, however, liver IGF-I mRNA expression had been obviously down-regulated in endotoxemic rats. Liver GHR mRNA expression also had a predominant down-regulation after endotoxin injection. The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection, being decreased by 53% compared with control rats. For GHR mRNA expression, the lowest expression occurred at 8h and had a 81% decrease. Although SOCS-3 mRNA was weakly expressed in control rats, it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats. Exogenous GH could enhance IGF-I mRNA expression in control rats, but it did fail to prevent the decline in IGF-I mRNA expression in endotoxemic rats. Endotoxin stimulated the production of TNF-alpha and IL-6, and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression. The liver GHR mRNA expression was obviously down-regulated after TNF-alpha iv injection and had a 40% decrease at 8h, but the liver SOCS-3 mRNA expression was the 4.94 times up-regulation occurred at 40 min after IL-6 injection. CONCLUSION: The growth hormone insensitivity could be induced by LPS injection, which was associated with down-regulated GHR mRNA expression at receptor level and with up-regulated SOCS-3 mRNA expression at post-receptor level. The in vivo biological activities of LPS were mediated by TNF-alpha and IL-6 indirectly, and TNF-alpha and IL-6 may exert their effects on the receptor and post-receptor levels respectively.

Study on the Cloning, Expression, and Bioactivity of Recombinant Chicken IGF-Ⅰ

The DNA sequence encoding the chicken＇s insulin-like growth factor Ⅰ （IGF-Ⅰ） was amplified with the reverse transcription polymerase chain reaction （RT-PCR）, which was then cloned into vector pMD18-T and sequenced. The sequencing result showed that there was 100% homology among the documented sequences and the sequence reported in this article, which was successfully inserted into the expressing plasmid pRLC and was highly expressed in E.coli. The Tricine-SDS- PAGE result showed that the cloned recombinant protein was expressed in the form of inclusion bodies in the E.coli cell with molecular weight of 7.6 kD and was amount to 23% of the whole protein in the E.coli cell. Western blotting indicated that recombinant protein had the antigenicity of IGF- Ⅰ. The inclusion bodies were subsequently dissolved in 7 M guanidine chloride and renatured with dilution in refolding buffer containing 0.5 M arginine. To obtain pure protein, the renatured chicken IGF- Ⅰ was desalting by Hiprep 26/10 and purified by Hiprep Sephacryl S-200 chromatography. The biological activities of IGF- Ⅰ product were assayed in NIH 3T3 cells and osteoblastic cells of embryonic chicken by using MTT method. The results show that the expressed IGF- Ⅰ can obviously stimulate NIH3T3 cells and osteoblastic cells to proliferate at the concentration ranging from 100, 200, 400 to 800 ng mL^-1, suggesting that the protein has its biological activities.

胰岛素样生长因子Ⅰ受体mRNA与实验性糖尿病视网膜病变的初步研究

目的 探讨胰岛素样生长因子Ⅰ受体 (IGF ⅠR)mRNA在糖尿病视网膜病变 (DR)发病中的作用。方法 复制糖尿病大鼠模型 ,分别于成模后 3个月和 6个月时经光镜和透穿电镜观察视网膜的形态学改变。应用原位杂交技术对IGF ⅠRmRNA在视网膜上的表达情况进行动态观察与分析。结果 (1)IGF ⅠRmRNA在正常大鼠视网膜节细胞层及内核层均有表达 (约占 5 %) ;(2 )糖尿病大鼠视网膜内IGF ⅠRmRNA表达显著增强 ,3个月病程表达量约占 15 %,6个月病程表达量约占 18%;(3)经胰岛素治疗的糖尿病大鼠视网膜上IGF ⅠRmRNA表达明显少于相同病程模型组。3个月治疗组表达量约占 8%,6个月治疗组表达量约占 10 %。结论 IGF ⅠRmRNA随糖尿病大鼠病程延长、病情加重 ,表达量增加 ,随胰岛素治疗 ,表达量减少 ,IGF ⅠRmRNA表达量的变化很可能是DR发生、发展的重要因素。

进行期银屑病患者皮损中胰岛素样生长因子Ⅰ受体蛋白与mRNA的表达

目的探讨胰岛素样生长因子Ⅰ受体（IGFIR）和进行期银屑病的关系。方法应用免疫组织化学技术，逆转录酶聚合酶链反应和原位杂交分别检测15例银屑病患者和10例正常人皮肤组织块中IGFIR蛋白与mRNA的表达。结果IGFIR蛋白与mRNA表达在银屑病患者基底层和棘细胞层，IGFIR蛋白与mRNA相对积分光密度值在正常对照组与银屑病组间存在极显著性差异（P＜0．01）。结论进行期银屑病患者皮损中表达增加的IGFIR蛋白和mRNA可能介导了角质形成细胞的增殖与分化不全。

Blockage of IGF-1R signaling sensitizes urinary bladder cancer cells to mitomycin-mediated cytotoxicity

A major problem which is poorly understood in the management of bladder cancer is low sensitivity to chemotherapy and high recurrence after transurethral resection. Insulin-like growth factor 1 receptor (IGF-1R) signaling plays a very important role in progression, invasion and metastasis of bladder cancer cells. In this study, we investigated whether IGF-1R was involved in the growth stimulating activity and drug resistance of bladder cancer cells. The results showed: The mRNAs of IGF-1, IGF-2 and IGF-1R were strongly expressed in serum-free cultured T24 cell line, whereas normal urothelial cells did not express these factors/receptors or only in trace levels; T24 cell responded far better to growth stimulation by IGF-1 than did normal urothelial cells; blockage of IGF1R by antisense oligodeoxynucleotide (ODN) significantly inhibited the growth of T24 cell and enhanced sensitivity and apoptosis of T24 cells to mitomycin (MMC). These results suggested that blockage of IGF-IR signaling might potentially contribute to the treatment of bladder cancer cells which are insensitive to chemotherapy.

阻塞性腺样体和扁桃体肥大与胰岛素样生长因子系统

阻塞性腺样体和扁桃体肥大可能引起儿童生长发育异常,其病理生理机制尚不明确。本文就血清胰岛素样生长因子-1(insulin-likegrowthfactorI,IGF-1)/胰岛素样生长因子结合蛋白-3(insulin-likegrowthfactor-bindingprotein3,IGFBP-3)概况及其在阻塞性腺样体和扁桃体肥大患儿生长激素分泌异常诊断和疗效判断方面的意义进行综述。

Protective effect of exogenous IGF-I on the intestinal mucosal barrier in rats with severe acute pancreatitis

Severe acute pancreatitis （SAP） can result in intestinal mucosal barrier （IMB） dysfunction. This study was undertaken to demonstrate the effect of IGF-I on the intestinal mucosal barrier in rats with SAP and its possible mechanisms. Seventy-two male Wistar rats were randomly divided into three groups： a sham operation （SO group, n=24）, a SAP group not treated with IGF-I （SAP group, n=24）, and a SAP group treated with IGF-I （IGF-I group, n=24）. SAP was induced in the rats by injecting 5.0% sodium taurocholate into the biliary-pancreatic duct. The SO rats were given an infusion of normal saline instead. The rats in the IGF-I group underwent the SAP procedure and were given a subcutaneous injection of IGF-I at 30 minutes before the operation and at 3 hours after the operation. Eight rats in each group were sacrificed at 6, 12 and 24 hours after operation. Apoptosis of mucosal cells in the small intestine was determined by TUNEL. The levels of endotoxin and DAO and serum amylase were also measured. Pathologic changes in the small intestine were monitored. Changes of bax and bcl-2 mRNA expression in the small intestine were determined by reverse transcription polymerase chain reaction （RT-PCR）. The levels of serum amylase were lower in the IGF-I group than in the SAP group at all three time points （P〈0.05）. The levels of endotoxin in the IGF-I group were higher than those in the SAP group at 6 hours, but lower in the IGF-I group than in the SAP group at 12 and 24 hours （P〈0.05）. The levels of diamine oxidase were higher in the IGF-I group at 6 hours but lower than those in the SAP group at 12 and 24 hours. The pathological score of the small intestine was lower in the IGF-I group than in the SAP group, and the difference was statistically significant at 12 and 24 hours. The pathologic changes observed under electron microscopy were better in the IGF-I group than those in the SAP group. The apoptosis index of intestinal epithelial cells was significantly decreased in the IGF-I group compared with the SAP group. Compared with the SO group, the mRNA expression levels of bax were increased at each time point in the SAP group, and were significantly decreased in the IGF-I group as compared with the SAP group at each time point （P〈0.05）. The expression levels of bcl-2 were weak and not different between the SO group and the SAP group （P〉0.05）. They were significantly increased in the IGF-I group versus the SO and SAP groups （P〈0.05）. The ratio of bax and bcl-2 mRNA expression levels at each time point in the SAP group were significantly higher than those in the SO group, but they were obviously decreased in the IGF-I group. Exogenous IGF-I seems to protect mucosal cells in the small intestine against SAP-induced apoptosis and could alleviate SAP-induced injury of the intestinal mucosa. The underlying mechanisms include enhanced mRNA expression of bcl-2 and inhibition of bax mRNA expression.

ALTERATION IN ENTEROCYTE GENE EXPRESSION MAY EXPLAIN STRUCTURAL AND FUNCTIONAL CHANGES FOLLOWING GLUTAMINE SUPPLEMENTED PARENTERAL NUTRITION

Following extensive bowel resection, the intestinal tract undergoes a variety of adaptive responses to enhance bowel function. The purpose of this study was to determine the effect of glutamine-supplemented parenteral nutrition on mucosal cellularity and gut function. In addition, enterocyte gene expression of two relevant systems was also characterized and related to the structural and functional changes that occurred. Male Wistar rats underwent a 60% small bowel resection and jugular vein catheterization and were randomized into two groups. The control group (n = 10) received a standard intravenous nutritional solution and the study group (n = 10) received a similar solution but enriched with alanylglutamine dipeptide. After 7 days blood was taken for amino acid analysis, and bowel was harvested to determine mucosal morphology and expression of mucosal cell glutaminase and IGF-I mRNA. Mesentery lymphnodes were cultured to determine the presence of bacteria and thus access bacteria translocation. Serum glutamine concentration and mucosal architecture were maintained in the study group compared to the controls. Seventy percent of lymphnodes were cultured positive in control vs. only 20% in the study group (P