电子线照射对小鼠胰岛素生长因子相关蛋白表达的影响

目的：探讨电子线对胰岛素生长因子-1家族蛋白表达的影响,为研究辐射对肝脏的早期损伤提供依据。方法：采用电子线照射小鼠,照射剂量为1、2、4 Gy,用Western blot法检测照射后12、48和72 h小鼠肝脏中胰岛素生长因子1受体（IGF-1R）、胰岛素生长因子结合蛋白1（IGFBP1）、胰岛素生长因子结合蛋白4（IGFBP4）和胰岛素生长因子1（IGF-1）蛋白表达水平的改变。结果：照射剂量为1 Gy时,与对照组相比,IGF-1蛋白在照射后48 h时表达增加,IGFBP1、IGFBP4和IGF1R蛋白的表达在照射后12、48和72 h均表达减少。照射剂量为2 Gy时,IGF-1和IGFBP4蛋白在照射后48 h时表达增加,其余时间点表达减少。IGFBP1蛋白在照射后12 h时表达增加,48和72 h时表达减少。IGF1R蛋白在照射后3个时间点均呈现为表达减少。照射剂量4 Gy时,肝脏中IGF-1、IGFBP1和IGFBP4蛋白均表达减少,IGF1R蛋白在照射后12 h时表达增加,48、72 h时均表达减少（均为P < 0.05）。结论：电子线照射后小鼠肝脏中胰岛素生长因子-1家族蛋白多以下调表达为主,与前期基因表达的结果相一致,有可能作为反映放射性工作人员早期肝脏损伤的生物标志物之一。     

阿魏酸对人脐静脉血管内皮细胞辐射损伤的保护作用及其机制

目的：研究阿魏酸对电离辐射所致人脐静脉血管内皮细胞（HUVEC）损伤的保护作用,及其对辐射敏感蛋白Thbd和γH2AX的影响,探讨阿魏酸的抗辐射作用与机制。方法：以4~16 Gy的⁶⁰Co γ射线照射HUVEC细胞,照后48 h以MTS法检测细胞活力,探索适宜照射剂量。以10 Gy γ射线照射HUVEC细胞,分别于照射后1、3、6、12、24和48 h提取细胞总蛋白,蛋白质印迹法检测蛋白Thbd和γH2AX的表达水平。并于照射前2 h,预防性给予细胞0.001~1 μmol/L的阿魏酸,照后48 h检测细胞活力、Thbd和γH2AX蛋白表达水平的改变。结果：与未照射组（0 Gy）比较,HUVEC受到10 Gy γ射线照射后48 h,细胞活力约降低30%（P < 0.05）。以此剂量照射,照后1 h Thbd约降至正常水平的0.6（P < 0.05）,照射后48 h γH2AX约升高至正常水平的5倍（P < 0.05）。与照射对照组比较,0.1和1 μmol/L 的阿魏酸作用后,受照HUVEC的细胞活力提高（P < 0.05）,Thbd蛋白表达升高（P < 0.05）,γH2AX蛋白表达降低（P < 0.05）。结论：0.1~1 μmol/L的阿魏酸可调节10 Gy γ射线照射后HUVEC的Thbd和γH2AX蛋白的表达水平,从而促进HUVEC增殖,提高其细胞活力,发挥抗辐射作用。     

X射线照射对人脐静脉内皮细胞焦亡的影响

目的：探讨X射线照射对人脐静脉内皮细胞（HUVEC）焦亡的影响。方法：单次10 Gy X射线照射HUVEC细胞,采用ELISA法检测照射后0~72 h HUVEC分泌细胞因子IL-1β、IL-6、TNF-α和IL-18的浓度,Western blot法检测照射后6~48 h HUVEC内Caspase-1的活化水平,采用透射电子显微镜检测照射后72 h HUVEC形态的变化。采用流式细胞术检测单次10 Gy以及多次小剂量（2.5 Gy/d,连续4 d）照射后HUVEC焦亡率的变化。结果：10 Gy X射线照射后0~72 h HUVEC中IL-1β、IL-6、TNF-α和IL-18浓度与对照组（0 Gy组）相比均明显升高（P<0.05）。10 Gy X射线照射后6~48 h细胞内Caspase-1活化水平升高（P<0.05）,细胞呈现体积增大肿胀状态。单次10 Gy照射组和多次小剂量照射组细胞焦亡率均明显高于空白对照组（P<0.01）;此外,单次10 Gy照射组细胞焦亡率较多次小剂量照射组细胞升高约1倍（P<0.01）。结论：X射线照射可引起人脐静脉内皮细胞HUVEC焦亡;在总剂量相同情况下,单次大剂量照射引起的HUVEC焦亡水平明显高于多次小剂量照射。     

X射线对人肺癌A549细胞Bmi-1表达的影响

目的：研究X射线照射对人肺癌A549细胞株中Bmi-1 mRNA和蛋白表达的影响。方法：用不同剂量（0、2、4、6Gy）的X射线照射体外培养的人肺癌A549细胞株,分别采用实时荧光定量PCR和Western blot技术检测照射0、6、12、24、48和72 h后Bmi-1 mRNA和蛋白的表达水平。结果：与对照组比较,2、4、6 Gy X射线照射A549细胞后48 h内Bmi-1 mRNA表达升高,差异均有统计学意义（P〈0.05）;（2 Gy 48 h组除外）,2 Gy组照射后6 h、4 Gy组12 h、6 Gy组24 h升高最显著（P〈0.05）。照射后48 h内蛋白表达升高（4Gy除外）,48 h后蛋白表达逐渐下降,至72 h时接近未照射组水平。各时间点（除4 Gy 48 h和72 h外）的蛋白表达与对照组相比差异均有统计学意义（P〈0.05）。结论：在本实验条件下,2～6 Gy剂量X射线照射48 h内可使人肺癌A549细胞Bmi-1表达升高,之后表达逐渐降低。     

Y射线对小鼠胸腺淋巴细胞微丝形态及丝切蛋白磷酸化的影响

目的：通过观察γ射线诱导小鼠胸腺淋巴细胞的微丝形态变化和对丝切蛋白（cofilin）磷酸化的影响,探讨cofilin及相关蛋白在辐射损伤中的作用。方法：将36只BALB/c小鼠按对照组（0 h）,照射后3、6、9、12和24 h时间点随机分为6组,每组6只,除对照组外,其余各组均给予2 Gy γ射线诱导,根据cofilin蛋白表达规律,确定取材时间。另将24只BALB/c小鼠按γ射线诱导剂量随机分为未照射（0 Gy）组、2、6和10 Gy组共4组,照射3 h后取材,观察淋巴细胞微丝形态,进行cofilin蛋白1（cofilin 1）、磷酸化cofilin蛋白（p-cofilin）、LIM激酶1（LIMK1）、TES激酶2（TESK2）和Slingshot家族的磷酸酶2（SSH2）表达检测。结果：BALB/c小鼠在2 Gy γ射线照射3 h后胸腺淋巴细胞cofilin基因的mRNA和蛋白表达均显著下调（P < 0.05）,因此后续实验选择照后3 h为取材时间。通过对cofilin及相关蛋白表达的检测,发现随着γ射线照射剂量的增加,胸腺淋巴细胞cofilin的表达逐渐增加,与对照组相比,以10 Gy组增加最为明显（P < 0.05）;而p-cofilin的表达则减少,10 Gy组减少最为明显（P < 0.05）。LIMK1、TESK2和SSH2的表达在照射后无显著变化（P > 0.05）。结论：随着照射剂量的增加,更多的SSH2使p-cofilin的Ser3位点去磷酸化,胞质内cofilin的量增多,参与到微丝骨架组装的过程中,细胞的迁移能力增强。     

POLH mRNA作为辐射损伤分子生物标志物的可行性研究

目的:研究受γ射线照射后人外周血中POLH mRNA的表达变化,为确立POLH mRNA作为新的辐射损伤分子生物标志物提供实验依据。方法:采取3名健康成人的外周血,利用⁶⁰Co γ放射源分别对其进行0.5、1、2、4、6、10 Gy照射,在照射后2、4、8、12和24 h收集各个剂量组受试者全血,提取总RNA,利用TaqMan探针实时定量PCR法,检测POLH mRNA的相对表达量。另收集30名不同年龄段(男女各占一半)的健康人外周血作为本底组,检测POLH mRNA在不同个体间的表达差异。结果:照射后2 h,仅10 Gy照射组POLH mRNA表达增加,其余各照射组无显著变化;照射后4、8、12和24 h,在0~2 Gy范围内,POLH mRNA表达量随照射剂量的增加而增加,呈现明显的剂量依赖效应,大于2 Gy照射后增加趋势接近饱和。照射后4~12 h随时间的增加表达量也不断增加,于12 h达到峰值。在对健康人群本底表达水平的分析中发现,POLH mRNA表达水平不受性别、年龄影响,在不同个体间相对均一,波动范围小。结论:POLH mRNA可由为辐射诱导表达,在一定时间和剂量范围内呈现明显的剂量依赖关系,且在不同个体之间本底表达水平相对均一,具备作为辐射损伤分子生物标志物的潜力。     

乳腺癌改良根治术后常规放疗剂量学分析

目的 分析乳腺癌改良根治术后常规二维放疗模式下胸壁及锁骨上区剂量分布和内乳区非计划性受量。方法 回顾分析2015-2016年间20例改良根治术后放疗的女性乳腺癌患者资料,左右乳腺癌各10例。放疗范围为患侧胸壁和锁骨上下区,处方剂量43.5Gy (2.9 Gy/次)。胸壁采用单前野电子线照射,锁骨上下野予以6MV X线单前野照射。同时比较锁骨上下采用前后对穿野照射时的剂量分布。结果 锁骨上单前野照射中85%患者接受了D90≥90%处方剂量,前后对穿野中所有患者均达到了D90≥90%处方剂量(39.15Gy, EQD2≥45Gy),胸壁单前电子线野D90中位数为35.38Gy。非计划性内乳区域照射的平均剂量中位数为13.65Gy。体重指数小患者锁骨上、胸壁的D90更高(P=0.039、0.347)。结论 锁骨上下单前X线野能满足绝大多数受量≥90%的处方剂量照射,而前后对穿野在满足所有人受量需求时不增加正常组织受量。单前电子线野胸壁剂量分布不佳,内乳区有一定的非计划性照射,但剂量有限。体重指数是影响剂量分布的因素。     

放射诱导神经元细胞发生RIP3介导的程序性坏死

目的 观察PC12细胞X线照射后有无程序性坏死,检测受体相互作用蛋白3(RIP3)的表达变化及其对程序性坏死的调控作用。方法 PC12细胞经不同剂量照射后在不同时间点通过乳酸脱氢酶(LDH)释放检测其坏死。程序性坏死特异性抑制剂Nec-1预处理后,通过LDH检测细胞坏死变化。免疫印迹(WB)检测照射干预后RIP3的表达情况。RIP3特异性抑制剂GSK’872预处理后通过LDH检测细胞坏死变化。通过WB筛选RIP3敲低效果最佳的转染序列。将细胞分为对照组、照射组、溶媒对照组、空载对照组和预处理组,采用WB、免疫荧光染色、MTT、LDH和AnnexV-FITC/PI流式检测分析。结果 细胞经4 Gy照射后培养3 h细胞坏死程度相对最大,RIP3蛋白表达增加。Nec-1、GSK’872和RIP3基因敲低预处理后,细胞坏死减少。结论 4 Gy照射可诱导PC12细胞出现程序性坏死,照射后培养3 h作用最明显;RIP3参与放射诱导PC12细胞的程序性坏死过程,并且发挥重要的正向调控作用。     

单次大剂量电子线照射SD大鼠胃损伤观察

目的 观察成年雄性SD大鼠经单次大剂量电子线照射胃后的损伤变化,为胃癌术中放疗提供动物实验依据。方法 将38只SD大鼠随机分为对照组与实验组,利用专利技术“小动物放射线精确照射试验台”对实验组大鼠胃进行单次6 MeV 20 Gy照射,照射后不同天数评估大鼠一般情况,观察胃的病理损伤及体重变化。结果 SD大鼠胃受照射后14 d时,镜下观察损伤最重,28 d时已逐渐修复伴腺体萎缩,56 d时胃损伤已纤维素性修复。与对照组相比实验组第1周内体重下降(P<0.05),第2周体重开始上升,但幅度低于对照组(P<0.05),第6周时体重与对照组相近(P>0.05)。结论 单次6 MeV电子线 20 Gy照射SD大鼠胃后损伤最重在2周内,但无穿孔发生,8周内可修复恢复完全正常。     

BDNF对全脑放疗后NFAT3/c4介导的信号通路相关因子作用研究

目的 观察不同剂量电离辐射对NFAT3/c4信号通路表达的影响以及外源性的BDNF对该通路的改善作用。方法 选用1个月龄SD大鼠分成4个组分别接受单次0、2、10、20 Gy照射,照后3 d进行双侧海马立体定向注射外源性的BDNF。后取脑部海马组织进行蛋白印迹法及PCR技术观察NFAT3/c4相关蛋白表达情况。结果 蛋白印迹法及PCR结果表明电离辐射后NFAT3/c4的表达水平明显下降并呈剂量及时间依赖性。IR+BDNF组较单纯照射组相比,随着照射剂量增加及时间延长NFAT3/c4和CaN表达水平明显升高。结论 全脑放疗抑制了CaN/NFAT3/c4信号通路,外源性BDNF能促进NFAT依赖的转录,从而改善认知功能的下降。     

Expressions of insulin-like growth factor receptor-1 and insulin-like growth factor binding protein 3 in advanced non-small-cell lung cancer

BackgroundThe insulin-like growth factor (IGF) pathway plays an important role in cell proliferation, differentiation, and apoptosis, and IGF induces those effects mainly through IGF receptor-1 (IGF-1R). The activities of IGF are strictly regulated by a family of IGF binding proteins (IGFBP), especially IGFBP3, a major serum carrier protein for IGF.Patients and MethodsBetween January 2006 and February 2009, in our hospital, 191 patients were histologically diagnosed as having non–small-cell lung cancer (NSCLC), and 74 patients were treated by chemotherapy alone. We examined immunohistochemical expression of both IGF-1R and IGFBP3 in 68 patients who were definitively diagnosed as having adenocarcinoma or squamous cell carcinoma among the 74 patients.ResultsThe clinical characteristics of the included patients were as follows: median age was 68 years (range, 29-86 years); men vs. women, 40 vs. 28; stage III vs. IV, 18 vs. 50; performance status 0-1 vs. 2-4, 58 vs. 10; smoker vs. non-smoker, 44 vs. 24; and squamous cell carcinoma vs. adenocarcinoma, 13 vs. 55. Expression of IGF-1R and IGFBP3 was observed in 37 (54%) and 11 patients (16%), respectively. IGF-1R expression was detected more frequently in patients with squamous cell carcinoma (100%) than in patients with adenocarcinoma (44%) (P < .001), although IGFBP3 expression was not significantly associated with any clinical variables. Among all factors, including IGF-1R and IGFBP3 expression, IGF-1R was significantly associated with response to chemotherapy (P = .028) and performance status was significantly associated with overall survival (P < .001).ConclusionsHigh sensitivity of IGF-1R to squamous cell carcinoma (100%) in this study and another study encourages the use of IGF-1R antibody in the pathologic diagnosis between squamous cell and non-squamous cell carcinoma when using small biopsy specimens.     

Transferrin reverses the anti-invasive activity of human prostate cancer cells that overexpress sema3E

In vitro invasion and adhesion of stably semaphorin (sema) 3E-transfected PC-3 prostate cancer cells were determined in the presence and absence of transferrin. Invasion and adhesion decreased compared to untransfected cells; however, transferrin reversed the effects. Transferrin differentially regulated E-cadherin and beta-catenin in these cells. Insulin growth factor 3 (IGFBP3) negated the invasive and adhesive effects of transferrin. Transferrin increased binding of insulin growth factor (IGF)-1 to the activated IGF-1 receptor, and IGF-1 mimicked the invasive and adhesive effects of transferrin. These data suggest that transferrin modulates sema3E-transfected cells through an IGFBP3/IGF-1-dependent pathway, in part, by regulation of adhesion proteins.     

Expression Characteristics of Proteins of the Insulin-like Growth Factor Axis in Non-small Cell Lung Cancer Patients with Preexisting Type 2 Diabetes Mellitus

Background: Preexisting type 2 diabetes mellitus (T2DM) affects the prognosis and mortality of patients withsome cancers. Insulin like growth factor (IGF) and insulin receptor (IR) signaling axes play important roles in bothcancer and diabetes development. We aimed to explore the expression characteristics of proteins in IGF/IR axisin non-small cell lung cancer (NSCLC) cases with preexisting T2DM. Methods: Fifty-five NSCLC patients withpreexisting T2DM were retrospectively included and matched by 55 NSCLC without diabetes at a 1:1 ratio. Theexpression of proteins in IGF/IR axis was detected by immunohistochemical staining. Clinicopathological datawere collected to analyze their relationship with the protein expression. Results: Both IGF 1 receptor (IGF-1R)and insulin receptor substrate 2 (IRS-2) showed higher expression in the NSCLC with T2DM group, comparedwith those without T2DM. The high expression of IGF-1R and IRS-2 were found to be negatively associated withlymph node metastases and T staging in the T2DM group, respectively, and IRS-2 expression was also found morein the subgroup whose T2DM duration was more than 4 years. No difference was detected in the expression ofIRS-1, IGF-1, IGF-2, IGFBP3, IR and mTOR between groups with or without T2DM. Conclusion: Our studyfound higher expression of IGF-1R and IRS-2 proteins in NSCLC patients with preexisting T2DM, and thatthere was an association with early stage NSCLC, which suggested that IGF signaling may play an importantearly event in development of NSCLC associated with diabetes.     

Metformin Down-regulates Endometrial Carcinoma Cell Secretion of IGF-1 and Expression of IGF-1R

As metformin can inhibit endometrial carcinoma (EC) cell growth and the insulin growth factor (IGF)system is active in EC, the question of whether t can regulate endometrial carcinoma cell secretion of IGF-1or expression of IGF-1 receptor (IGF-1R) is of interest. In this study, serum IGF-1 levels in EC patients werefound to be comparable with that in the non EC patients (p>0.05). However, the IGF-1 level in the medium ofcultured cells after treatment with metformin was decreased (p<0.05). IGF-1R was highly expressed in humanendometrial carcinoma paraffin sections, but IGF-1R and phosphor-protein kinase B/protein kinase B (p-Akt/Akt) expression was down-regulated after metformin treatment (p<0.05). In summary, metformin can reduce thesecretion of IGF-1 by Ishikawa and JEC EC cell lines and their expression of IGF-1R to deactivate downstreamsignaling involving the PI-3K/Akt pathway to inhibit endometrial carcinoma cell growth.     

The Insulin-Like Growth Factor Pathway in Lung Cancer

The insulin-like growth factor (IGF) pathway is involved in the normal control of fetal development, tissue growth, and metabolism. Two distinct ligands (insulin-like growth factor-1 [IGF-1] and IGF-2) plus insulin, and two receptors (insulin-like growth factor receptor-1 [IGF-1R] and the insulin receptor) capable of both homo- and heteropolymerization mediate the actions of this pathway. Cellular functions of IGF-regulated signaling are influenced by the expression of a variety of receptor docking proteins, including four different insulin receptor substrate proteins. Downstream signaling is primarily through the phosphatidylinositol-3 kinase-Akt pathway and the mitogen-activated protein kinase pathway, resulting in increased cell proliferation and apoptosis inhibition. Ligand-driven activation is influenced by upstream endocrine factors (particularly for IGF-1), imprinting (for IGF-2), by multiple circulating and tissue-based IGF-binding proteins/proteases, and by the expression of the IGF-2 clearance receptor (IGF-2R). Deregulation of IGF signaling has been described in several cancer types, including both small cell and non-small cell lung cancer. A number of IGF receptor inhibitors, including monoclonal antibodies and small molecule inhibitors are currently undergoing testing in clinical trials as both monotherapy, and in combination with chemotherapy, or with other targeted agents. Preliminary results from a randomized phase II trial of an anti-IGF-1R monoclonal antibody in combination with carboplatin/paclitaxel already suggest a potential efficacy benefit from targeting this pathway in the first line advanced non-small cell lung cancer setting.     

The Role of IGF‐1R in Pediatric Malignancies

The insulin‐like growth factor (IGF) family consists of ligands (IGF‐I, IGF‐II, insulin), several receptors (including IGF‐1R), and six binding proteins (IGFBP‐1 through IGFBP‐6). Members of this family regulate key cellular activities and they also play an important role in the development and progression of both adult and childhood cancers. Binding of a ligand to the receptor leads to its activation, followed by signal transduction along several pathways. In some childhood malignancies, IGF‐1R can be activated by endocrine, autocrine, or paracrine mechanisms. Although mutations in IGF‐1R have not been identified, this signaling pathway is upregulated in many childhood cancers. These findings have led to the development of a host of IGF‐1R signaling modulators that are currently being tested in clinical trials. This review explores the role of IGF‐1R in a range of childhood malignancies.