ResearchPad - basic-research https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Calciprotein particle inhibition explains magnesium-mediated protection against vascular calcification]]> https://www.researchpad.co/article/elastic_article_10780 Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation.MethodsVascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls.ResultsMg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P < 0.05) and CKD patients by 44 ± 13 min (P < 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups.ConclusionsOur results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification. ]]> <![CDATA[Exposure to biomass smoke, cigarettes, and alcohol modifies the association between <i>tumour necrosis factor</i> (<i>–308G/A</i>, –<i>238G/A</i>) polymorphisms and tuberculosis in Mexican carriers]]> https://www.researchpad.co/article/elastic_article_9499 Exposure to biomass smoke, cigarettes, alcohol, and the impairment of immunoregulation are considered to be risk factors for tuberculosis. Tumour necrosis factor (TNF) –308G/A and –238G/A gene polymorphisms have been associated with tuberculosis. However, the results remain inconsistent. The aim of this study was to determine the association between TNF polymorphisms and tuberculosis in the presence of biomass smoke, cigarettes, and alcohol in a Mexican population.Material and methodsTNF polymorphisms were determined in 118 tuberculosis patients and 223 controls. We performed a univariate, bivariate, stratified analysis. Odds ratios, confidence intervals, and p-values were calculated.ResultsOccupational biomass smoke exposure was associated with tuberculosis between the patients and controls (OR = 1.70, 95% CI: 1.08–2.70, p = 0.02). We also found an association of the –308A allele carriers between the patients and controls without exposure to occupational (p = 0.04, OR = 0.16, 95% CI: 0.01–0.92) and in-home (p = 0.02, OR = 0.14, 95% CI: 0.01–0.81) biomass smoke, as well as an association with alcohol (p = 0.01, OR = 0.24, 95% CI: 0.05–0.75). The haplotype analysis revealed an association of the –308A/238G haplotype between patients and nonconsanguineous controls without exposure to occupational (p = 0.02, OR = 0.12, 95% CI: 0.01–0.99) and in-home (p = 0.01, OR = 0.1, 95% CI: 0.01–0.9) biomass smoke, cigarette use (p = 0.04, OR = 0.28, 95% CI: 0.08–0.98), and alcohol (p = 0.02, OR = 0.22, 95% CI: 0.05–0.88) intake.ConclusionsThe TNF308A allele and the –308A/238G haplotype are associated with tuberculosis, as are exposure to biomass smoke, cigarettes, and alcohol. No association for the –238G/A polymorphism was found. Our results provide insight into a possible protective role of TNF polymorphisms in tuberculosis in our population. ]]> <![CDATA[Analysis of the association between the XRCC2 rs3218536 polymorphism and ovarian cancer risk]]> https://www.researchpad.co/article/elastic_article_9491 Results conflict on the association between the XRCC2 rs3218536 polymorphism and ovarian cancer risk, despite wide-ranging investigations. This meta-analysis examines whether the XRCC2 rs3218536 polymorphism is associated with ovarian cancer risk.Material and methodsEligible case-control studies were searched in PubMed. We therefore performed a meta-analysis of 5,802 ovarian cancer cases and 9,390 controls from 7 articles published. The strength of association between XRCC2 rs3218536 polymorphism and ovarian cancer susceptibility was calculated using pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs).ResultsNo statistically significant associations between XRCC2 rs3218536 polymorphism and ovarian cancer risk were found in any genetic models. However, a significant relationship with ovarian cancer risk was discovered when the high quality studies were pooled in the meta-analysis (AA vs. GG: OR = 0.59, 95% CI: 0.37–0.94, p = 0.03; GA vs. GG: OR = 0.87, 95% CI: 0.78–0.96, p = 0.009; GA + AA vs. GG: OR = 0.85, 95% CI: 0.77–0.94, p = 0.003; AA vs. GG + GA: OR = 0.60, 95% CI: 0.38–0.95, p = 0.03).ConclusionsThis meta-analysis shows that the XRCC2 rs3218536 polymorphism was associated with ovarian cancer risk overall for high quality studies. Non-Caucasian groups and high quality studies should be further studied. ]]> <![CDATA[Design and Evaluation of a Rodent‐Specific Transcranial Magnetic Stimulation Coil: An <i>In Silico</i> and <i>In Vivo</i> Validation Study]]> https://www.researchpad.co/article/elastic_article_6861 Rodent models are fundamental in unraveling cellular and molecular mechanisms of transcranial magnetic stimulation (TMS)‐induced effects on the brain. However, proper translation of human TMS protocols to animal models have been restricted by the lack of rodent‐specific focal TMS coils.ObjectiveWe aimed to improve TMS focalization in rodent brain with a novel small, cooled, and rodent‐specific TMS coil.MethodsA rodent‐specific 25‐mm figure‐of‐eight TMS coil was developed. Stimulation focalization was simulated in silico for the rodent coil and a commercial human 50‐mm figure‐of‐eight TMS coil. Both coils were also compared in vivo by electromyography measurements of brachialis motor evoked potential (MEP) responses to TMS at different brain sites in anesthetized rats (n = 6). Focalization was determined from the coils' level of stimulation laterality. Differences in MEPs were statistically analyzed with repeated‐measures, within‐subjects, ANOVA.Results In silico simulation results deemed the human coil insufficient for unilateral stimulation of the rat motor cortex, whereas lateralized electrical field induction was projected attainable with the rodent coil. Cortical, in vivo MEP amplitude measurements from multiple points in each hemisphere, revealed unilateral activation of the contralateral brachialis muscle, in absence of ipsilateral brachialis activation, with both coils.ConclusionComputer simulations motivated the design of a smaller rodent‐specific TMS coil, but came short in explaining the capability of a larger commercial human coil to induce unilateral MEPs in vivo. Lateralized TMS, as demonstrated for both TMS coils, corroborates their use in translational rodent studies, to elucidate mechanisms of action of therapeutic TMS protocols. ]]> <![CDATA[Identification of protective T-cell antigens for smallpox vaccines]]> https://www.researchpad.co/article/N46220d90-9c2f-4820-a39c-9fab5e38332e E3L is an immediate-early protein of vaccinia virus (VV) that is detected within 0.5 h of infection, potentially before the many immune evasion genes of vaccinia can exert their protective effects. E3L is highly conserved among orthopoxviruses and hence could provide important protective T-cell epitopes that should be retained in any subunit or attenuated vaccine. We have therefore evaluated the immunogenicity of E3L in healthy VV-vaccinated donors.MethodsPeripheral blood mononuclear cells from healthy volunteers (n = 13) who had previously received a smallpox vaccine (Dryvax) were activated and expanded using overlapping E3L peptides and their function, specificity and antiviral activity was analyzed. E3L-specific T cells were expanded from 7 of 12 (58.3%) vaccinated healthy donors. Twenty-five percent of these produced CD8+ T-cell responses and 87.5% produced CD4+ T cells. We identified epitopes restricted by HLA-B35 and HLA-DR15.ResultsE3L-specific T cells killed peptide-loaded target cells as well as vaccinia-infected cells, but only CD8+ T cells could prevent the spread of infectious virus in virus inhibition assays. The epitopes recognized by E3L-specific T cells were shared with monkeypox, and although there was a single amino acid change in the variola epitope homolog, it was recognized by vaccinia-specific T-cells.ConclusionsIt might be important to include E3L in any deletion mutant or subunit vaccine and E3L could provide a useful antigen to monitor protective immunity in humans. ]]> <![CDATA[The Role of CD36 in Type 2 Diabetes Mellitus: β-Cell Dysfunction and Beyond]]> https://www.researchpad.co/article/N70c090c6-00ec-4aa2-99cf-4476607d4d84 Impaired β-cell function is the key pathophysiology of type 2 diabetes mellitus, and chronic exposure of nutrient excess could lead to this tragedy. For preserving β-cell function, it is essential to understand the cause and mechanisms about the progression of β-cells failure. Glucotoxicity, lipotoxicity, and glucolipotoxicity have been suggested to be a major cause of β-cell dysfunction for decades, but not yet fully understood. Fatty acid translocase cluster determinant 36 (CD36), which is part of the free fatty acid (FFA) transporter system, has been identified in several tissues such as muscle, liver, and insulin-producing cells. Several studies have reported that induction of CD36 increases uptake of FFA in several cells, suggesting the functional interplay between glucose and FFA in terms of insulin secretion and oxidative metabolism. However, we do not currently know the regulating mechanism and physiological role of CD36 on glucolipotoxicity in pancreatic β-cells. Also, the downstream and upstream targets of CD36 related signaling have not been defined. In the present review, we will focus on the expression and function of CD36 related signaling in the pancreatic β-cells in response to hyperglycemia and hyperlipidemia (ceramide) along with the clinical studies on the association between CD36 and metabolic disorders.

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<![CDATA[Combination of Probiotics and <i>Salvia miltiorrhiza</i> Polysaccharide Alleviates Hepatic Steatosis via Gut Microbiota Modulation and Insulin Resistance Improvement in High Fat-Induced NAFLD Mice]]> https://www.researchpad.co/article/N5836c6f5-0d18-4abc-a4df-3b5cab9c68ce Nonalcoholic fatty liver disease (NAFLD) increases the risk of hepatocellular carcinoma, which is currently the leading cause of obesity-related cancer deaths in middle-aged men.MethodsProbiotics with lipid-lowering function were screened from the fecal microbiota of healthy adults. Polysaccharide from different sources was screened for improving insulin resistance. The combination of probiotics and Salvia miltiorrhiza polysaccharide (LBM) was investigated for alleviating hepatic steatosis.ResultsFirst, Bifidobacterium bifidum V (BbV) and Lactobacillus plantarum X (LpX) were obtained from the fecal microbiota of healthy adults. Second, to improve insulin resistance, a Salvia miltiorrhiza Bunge polysaccharide showing good performance in reducing insulin resistance was obtained. The liver total cholesterol (TC) and total triglyceride (TG) levels and the serum levels of free fatty acid, alanine transaminase, aspartate transaminase, low density lipoprotein cholesterol, TG, and TC can be significantly reduced through supplementation with LpX-BbV (LB) in NAFLD mice. Interestingly, the function of the probiotic LB can be enhanced by S. miltiorrhiza Bunge polysaccharide. Furthermore, the gut microbiota was modulated by LpX-BbV+S. miltiorrhiza Bunge polysaccharide (LBM). The lipopolysaccharide concentration of the LBM group was decreased by 73.6% compared to the NAFLD group. Ultimately, the mRNA concentrations of the proinflammatory cytokines (tumor necrosis factor α, interleukin 1β [IL-1β], and IL-6) decreased with LB and LBM treatment.ConclusionThe results of this this study indicate that the LBM combination can be used as a therapeutic for ameliorating NAFLD via modulating the gut microbiota and improving insulin resistance. ]]> <![CDATA[Higher Plasma Sclerostin and Lower Wnt Signaling Gene Expression in White Adipose Tissue of Prediabetic South Asian Men Compared with White Caucasian Men]]> https://www.researchpad.co/article/N4cc073e9-0754-4e79-a510-ee46e38351f7 South Asians generally have an unfavourable metabolic phenotype compared with white Caucasians, including central obesity and insulin resistance. The Wnt protein family interacts with insulin signaling, and impaired Wnt signaling is associated with adiposity and type 2 diabetes mellitus. We aimed to investigate Wnt signaling in relation to insulin signaling in South Asians compared with white Caucasians.MethodsTen Dutch South Asian men with prediabetes and overweight or obesity and 10 matched Dutch white Caucasians were included. Blood samples were assayed for the Wnt inhibitor sclerostin. Subcutaneous white adipose tissue (WAT) and skeletal muscle biopsies were assayed for Wnt and insulin signaling gene expression with quantitative reverse transcription polymerase chain reaction (Clinicaltrials.gov NCT02291458).ResultsPlasma sclerostin was markedly higher in South Asians compared with white Caucasians (+65%, P<0.01). Additionally, expression of multiple Wnt signaling genes and key insulin signaling genes were lower in WAT in South Asians compared with white Caucasians. Moreover, in WAT in both ethnicities, Wnt signaling gene expression strongly positively correlated with insulin signaling gene expression. In skeletal muscle, WNT10B expression in South Asians was lower, but expression of other Wnt signaling and insulin signaling genes was comparable between ethnicities. Wnt and insulin signaling gene expression also positively correlated in skeletal muscle, albeit less pronounced.ConclusionSouth Asian men with overweight or obesity and prediabetes have higher plasma sclerostin and lower Wnt signaling gene expression in WAT compared with white Caucasians. We interpret that reduced Wnt signaling could contribute to impaired insulin signaling in South Asians. ]]> <![CDATA[Histone Deacetylase 9: Its Role in the Pathogenesis of Diabetes and Other Chronic Diseases]]> https://www.researchpad.co/article/N27a051f2-26af-49c0-93e0-45befc03c099 As a member of the class IIa histone deacetylases (HDACs), HDAC9 catalyzes the deacetylation of histones and transcription factors, commonly leading to the suppression of gene transcription. The activity of HDAC9 is regulated transcriptionally and post-translationally. HDAC9 is known to play an essential role in regulating myocyte and adipocyte differentiation and cardiac muscle development. Also, recent studies have suggested that HDAC9 is involved in the pathogenesis of chronic diseases, including cardiovascular diseases, osteoporosis, autoimmune disease, cancer, obesity, insulin resistance, and liver fibrosis. HDAC9 modulates the expression of genes related to the pathogenesis of chronic diseases by altering chromatin structure in their promotor region or reducing the transcriptional activity of their respective transcription factors. This review summarizes the current knowledge of the regulation of HDAC9 expression and activity. Also, the roles of HDAC9 in the pathogenesis of chronic diseases are discussed, along with potential underlying mechanisms.

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<![CDATA[Toppgene筛选肺腺癌候选疾病基因]]> https://www.researchpad.co/article/5b5b887c463d7e1effa460f0 肺腺癌是危害人类健康最主要的肺癌类型之一,其发生机制仍不清楚。本研究利用生物信息学方法,筛选新的肺腺癌候选基因,为揭示肺腺癌发病机制提供依据。方法从GEO数据库中获得GSE10072和GSE7670两个数据集,然后利用dchip软件进行差异表达基因分析,将其获得的差异基因定义为“检测基因集”(test gene set);采用genecard和Fable文献挖掘已知肺腺癌疾病基因,并将其定义为“训练基因集”(train gene set);最后,利用Toppgene筛选肺腺癌候选基因,并通过荧光定量PCR对其获得的部分基因进行验证。结果获得一个含344个基因的“检测基因集”和含277个基因的“训练基因集”。采用Toppgene共获得36个候选疾病基因,其中21个基因则在肿瘤方面的研究几无报道。荧光定量PCR实验研究发现,CD36PMAIP1FABP4三个基因在A549细胞中均为下调表达,与芯片数据一致。结论Toppgene可发现新的肺腺癌候选疾病基因,为下一步发现特异性肺腺癌致病基因提供理论依据。 ]]> <![CDATA[吉西他滨调整方案治疗晚期非小细胞肺癌的Ⅱ期临床试验]]> https://www.researchpad.co/article/5b5b86f7463d7e1effa460e9 吉西他滨与铂类的联合化疗是晚期非小细胞肺癌(non-small cell lung cancer, NSCLC)最常用的治疗方案。通常3周方案中的吉西他滨需间隔1周给药。为提高依从性,本研究将吉西他滨第8天给药时间调整为第5天,并评价调整方案一线治疗晚期NSCLC的疗效及安全性。方法2007年10月-2009年10月共入组83例晚期NSCLC患者,采用吉西他滨1, 000 mg/m2-1, 250 mg/m2第1天、第5天静脉滴注30 min,联合顺铂75 mg/m2,或联合卡铂(AUC=5)第1天静滴,每21天为1周期,每例至少完成2周期治疗后评价疗效,观察毒性反应及无进展生存期和总生存期。结果83例患者的客观有效率为37.3%,中位无进展生存期和中位生存期分别为6.1个月和15.0个月,1年、2年生存率分别为57.8%与16.2%。调整方案的主要不良反应为血液学毒性与胃肠道反应,Ⅲ度-Ⅳ度白细胞、血红蛋白、血小板减少发生率分别为26.5%、10.8%、7.2%,联合顺铂治疗组Ⅲ度-Ⅳ度胃肠道反应发生率为27.5%。无治疗相关死亡。结论吉西他滨联合铂类5天调整方案一线治疗晚期NSCLC疗效肯定,毒副反应可耐受,值得进一步开展随机对照研究。 ]]> <![CDATA[体外诱导建立NCI-H2228/Crizotinib耐药细胞株的方法学探讨及鉴定分析]]> https://www.researchpad.co/article/5b5b86bf463d7e1effa460e8 小分子靶向药物发生耐药的机制及寻找克服耐药的手段是目前提高临床疗效需要迫切解决的问题。本研究探讨采用不同方法建立对Crizotinib耐药的非小细胞肺癌NCI-H2228/Crizotinib细胞株的可行性及鉴定分析,为深入研究Crizotinib耐药发生的机制并寻找克服耐药的手段提供实验基础和理论依据。方法采用逐步增加药物浓度和化学诱变剂处理NCI-H2228细胞,诱导细胞对Crizotinib耐药。MTT法检测亲本细胞和耐药细胞的50%抑制浓度(50% inhibitory concentration, IC50)和群体倍增时间。RT-PCR和Western blot实验检测棘皮动物微管相关蛋白样4-间变性淋巴瘤激酶(echinoderm microtubule-associated protein like 4-anaplastic lymph kinase, EML4-ALK)基因表达。对耐药细胞和亲本细胞的EML4-ALK基因全长测序并对比分析发生耐药的机制。结果逐步增加药物浓度的方法耗时过长,细胞恢复生长缓慢,不能有效诱导NCI-H2228细胞对Crizotinib耐药;化学诱变剂ENU可以在短时间内诱导NCI-H2228细胞对Crizotinib耐药[IC50=(3.810±1.100)μmol/L,P=0.002, 9,vs亲本细胞]。耐药细胞EML4-ALK基因发生点突变的频率高于亲本细胞。结论化学诱变剂诱导细胞耐药操作简便,可有效缩短实验流程,为深入研究耐药发生机制,寻找克服靶向药物耐药的手段提供了前期技术方法和实验依据。 ]]> <![CDATA[吉非替尼耐药的人肺腺癌HCC-827/GR细胞乙醛脱氢酶亚型表达分析]]> https://www.researchpad.co/article/5c053697d5eed0c4848be2fd 肿瘤的复发和耐药是肿瘤患者死亡的主要原因。乙醛脱氢酶(acetaldehyde dehydrogenase, ALDH)家族与肿瘤细胞的增殖、迁移、侵袭和耐药密切相关,且ALDH不同亚型基因在不同肿瘤细胞中有差异性表达。本实验旨在分析对吉非替尼耐药的人肺腺癌细胞HCC-827/GR ALDH亚型的表达。方法利用人肺腺癌细胞系HCC-827制备吉非替尼耐药细胞株HCC-827/GR;利用流式检测HCC-827及HCC-827/GR中ALDH的表达;采用MTT法检测ALDH抑制剂二乙氨基苯甲醛(diethyllaminaldehyde, DEAB)处理前后HCC-827/GR细胞的增殖能力和对吉非替尼的敏感性;利用qRT-PCR检测HCC-827与HCC-827/GR细胞中ALDH各亚型在mRNA水平的表达。结果与HCC-827细胞相比,ALDH在吉非替尼耐药的细胞株HCC-827/GR的阳性率增加;经100 μmol/L DEAB处理后,HCC-827/GR细胞增殖能力下降;与HCC-827细胞相比,ALDH1A1和ALDH1L1在HCC-827/GR细胞中mRNA表达水平增高;ALDH3B2表达降低。结论ALDH具有检测吉非替尼耐药的人肺腺癌细胞的标志分子的潜力,其中ALDH1A1可能参与人肺腺癌细胞对吉非替尼耐药性的形成过程。 ]]> <![CDATA[器官特异性转移肺癌细胞株的筛选及建立]]> https://www.researchpad.co/article/5c0532c9d5eed0c4848b5d5b 肺癌转移是肺癌的恶性标志和特征,也是肺癌病人治疗失败和死亡的主要原因。肺癌转移具有器官特异性,最常转移的部位是淋巴结、大脑、骨、肝脏和肾上腺。本研究的目的是应用我们实验室的人高转移大细胞肺癌细胞株L9981,筛选鉴定出具有器官特异性转移的肺癌亚代细胞株,为进一步研究肺癌细胞器官特异转移提供科学可靠的细胞模型。方法通过裸鼠实验,将母系细胞株L9981-Luc皮下接种,每周一次动物活体成像观察肺癌器官转移情况,数周后构建出具有肺、脊柱、纵隔淋巴结和大脑等器官特异性转移的小鼠模型;处死裸鼠,切取肺、脊柱、纵隔淋巴结和大脑器官肺癌转移瘤进行原代培养,构建具有器官靶向特异性转移潜能的肺癌亚代细胞株;将第一代器官特异性转移肺癌细胞株接种裸鼠皮下,再次构建肺癌器官特异性转移小鼠模型;通过反复多次将肺癌器官特异性转移瘤构建肺癌细胞株,再裸鼠接种,最终获得具有肺、脊柱、纵隔淋巴结和大脑器官特异性转移的肺癌细胞株。结果经过裸鼠反复接种,动物活体成像、动物体内反复筛选鉴定,成功构建了4株分别特异性转移到肺、脊柱、纵隔淋巴结和大脑的器官特异性转移肺癌细胞株,分别命名为L9981-LuM、L9981- BoM、L9981-LnM和L9981-BrM。结论成功构建出具有肺、纵隔淋巴结、脊柱和大脑特异性转移的人大细胞肺癌细 胞模型,为进一步研究肺癌器官特异转移的分子机制、信号调控途径,以及未来研究和开发抑制或/和阻断肺癌转移的分子靶向药物提供了可靠的细胞模型。 ]]> <![CDATA[具有不同酶活性并可抵抗特异shRNA降解的nm23-H1真核表达载体的构建和表达]]> https://www.researchpad.co/article/5c0532bfd5eed0c4848b5c4f 已有的研究证明nm23-H1基因是一个重要的肿瘤转移抑制基因,但其抑制肿瘤转移的生化机理尚不完全清楚。Nm23-H1基因结构和功能异常与肿瘤的侵袭转移有密切关系。我们前期已构建了nm23-H1的短发夹RNA(short hairpin RNA, shRNA)载体以及可抵抗此shRNA降解的nm23-H1的cDNA的表达载体,在此基础上我们欲应用基因定点突变技术构建具有不同酶活性并能抵抗此shRNA降解的nm23-H1cDNA真核表达载体,并通过恢复实验验证其表达,为进一步研究肿瘤抑制基因nm23-H1的分子机制提供理论基础和实验依据。方法以pcDNA3.1(+)-shRNA-resistant-nm23-H1质粒为突变模板,应用重叠延伸PCR方法引入nm23-H1基因四个单点突变和一个联合位点突变,并将突变基因片段克隆到真核表达载体pcDNA3.1Hygro(+)。将突变质粒转染人肺腺癌细胞株A549/nm23-H1-shRNA(稳定沉默nm23-H1基因),利用Western blot技术验证不同突变体nm23-H1蛋白的表达。结果成功构建了shRNA抵抗的nm23-H1S44A、nm23-H1P96S、nm23-H1H118F、nm23-H1S120G、nm23-H1P96S-S120G五个突变型真核表达载体,经DNA序列分析突变的碱基序列与实验设计完全一致,经Western blot验证nm23-H1蛋白表达正常。结论成功构建了五个具有不同突变位点的shRNA抵抗的nm23-H1基因真核表达载体,并且突变蛋白质nm23-H1表达正常,同时也表明重叠延伸PCR技术是一种高效、便捷、经济的DNA定点突变方法。 ]]> <![CDATA[<sup>99m</sup>Tc标记T7肽及其在裸鼠非小细胞肺癌模型体内的生物分布研究]]> https://www.researchpad.co/article/5c0532bbd5eed0c4848b5be5 肺癌是一种死亡率极高的恶性肿瘤,针对肺癌的早期诊断和治疗有着重要的意义和价值,本研究旨在探讨羰基锝法标记的肿瘤抑素T7肽用作裸鼠肺癌早期显像剂的初步研究。方法采用羰基锝法标记T7肽,薄层色谱法检测99mTc-T7的放化纯度与稳定性,丙酮作展开系统。测定99mTc-T7与NCI-H157细胞的亲和力。研究99mTc-T7于0.5 h、1 h、2 h、4 h、8 h在荷人非小细胞肺腺癌裸鼠体内的生物分布特性,并计算肿瘤(T)与非肿瘤组织(NT)放射性比值。结果99mTc对T7肽标记率高,放化纯度达90%以上,不需进一步纯化,体外稳定性好。99mTc-T7与NCI-H157细胞的平衡解离常数为196.1 nM。99mTc-T7在裸鼠体内主要通过内脏器官代谢,血液清除较快,在肿瘤部位有一定程度聚集,肿瘤/肌肉比值随时间延长而增加,可达到5以上,在4 h-8 h之间取值较为理想。99mTc-T7在肺内存在一过性聚集。结论99mTc-T7制备方法简便,标记率高,稳定性好,并能在肺癌肿瘤部位聚集,有望用作肺癌SPECT/CT显像剂。 ]]> <![CDATA[肺癌细胞中miR-182启动子甲基化状态研究]]> https://www.researchpad.co/article/5c053091d5eed0c4848b0f5a 已有的研究证明MiR-182的异常调控与恶性肿瘤的发生发展密切相关,本研究旨在探讨肺癌细胞中miR-182启动子的甲基化状态对miR-182表达的影响。方法荧光定量PCR检测肺癌细胞中miR-182表达水平,甲基化特异性PCR检测各细胞株中miR-182启动子区的甲基化状态,并通过测序进行验证。DNA甲基转移酶抑制剂5’-Aza-dC处理后检测各肺癌细胞株中miR-182表达变化。结果MiR-182在不同肺癌细胞株的表达水平不同,其中,在高转移性肺癌细胞株如A549和L9981中相对呈低表达;而在低转移性细胞株95C则相对呈高表达。MSP及测序分析显示多株肺癌细胞株中miR-182启动子区域存在DNA甲基化,其中A549细胞甲基化程度最高。在5'-氮杂-脱氧胞苷酸(5’-Aza-dC)作用下,A549细胞及其他肺癌细胞中miR-182表达水平均明显升高。结论在肺癌细胞中miR-182启动子区域存在异常甲基化,miR-182的表达受DNA甲基化的调控。MiR-182的甲基化在肺癌中的作用尚需进一步研究。 ]]> <![CDATA[胸腔内注射重组人血管内皮抑素对裸鼠恶性胸腔积液的治疗作用]]> https://www.researchpad.co/article/5c05308fd5eed0c4848b0f0d 恶性胸腔积液(malignant pleural effusion, MPE)临床预后不佳,胸腔内抗血管治疗可能对恶性胸腔积液具有治疗作用,本研究旨在探讨胸腔内注射重组人血管内皮抑素、顺铂、重组人血管内皮抑素联合顺铂对裸鼠恶性胸腔积液的治疗作用。方法BALB/c裸鼠胸膜腔内注射Lewis肺癌细胞(Lewis lung cancer cell, LCC)构建恶性胸腔积液模型,造模后分别胸腔内注射重组人血管内皮抑素(E)、顺铂(P)以及重组人血管内皮抑素联合顺铂(EP)并分析各组裸鼠胸腔积液量、胸膜肿瘤微血管密度(micro vessel density, MVD)以及血管生成、凋亡相关基因的表达变化。结果重组人血管内皮抑素及重组人血管内皮抑素联合顺铂胸腔内注射可以使裸鼠MPE量减少,且与裸鼠胸腔肿瘤组织MVD下降呈正相关;且重组人血管内皮抑素及重组人血管内皮抑素联合顺铂胸腔内注射后,MPE裸鼠胸腔肿瘤组织血管内皮生长因子(Vescular epidermal growth factor-α, VEGF-α)表达下降、低氧诱导因子-α(hypoxia induced factor-1, HIF1-α)表达升高。结论胸腔内注射LLC细胞可成功制作裸鼠MPE模型。重组人血管内皮抑素裸鼠胸膜腔内注射对MPE裸鼠具有治疗作用,其治疗作用可能是通过下调VEGF-α,抑制肿瘤新生血管生成,下调微血管密度而达成的。 ]]> <![CDATA[硫利达嗪对肺癌PC9细胞的杀伤效应及其机制]]> https://www.researchpad.co/article/5c05307ad5eed0c4848b0cce 硫利达嗪作为一种吩噻嗪类抗精神疾病药物,近期研究显示其在体外可抑制多种肿瘤细胞的增殖,但对肺癌的作用尚未见报道。本实验以PC9细胞株为研究对象,旨在观察硫利达嗪对其杀伤效应以及探讨其可能的作用机制。方法不同浓度的硫利达嗪作用PC9细胞后,四甲基偶氮唑蓝(methyl thiazolyltetrazolium, MTT)法检测细胞增殖率,流式细胞术检测细胞周期及细胞凋亡率,Western blot检测周期相关蛋白CyclinD1及凋亡相关蛋白Caspase-3、Caspase-8、Caspase-9、Bcl-2、Bax、Bcl-xl表达水平。结果硫利达嗪可显著抑制PC9细胞的增殖,其抑制作用呈时间和浓度依赖性。流式结果显示:随着硫利达嗪药物浓度的增高,细胞发生不同程度的G0/G1期阻滞,细胞凋亡率明显增高。各实验组与对照组比较,差异有统计学意义(P < 0.05)。Western blot结果显示:与对照组比较,实验组CyclinD1、Bcl-2、Bcl-xl表达水平明显下调(P < 0.01),Bax表达水平明显上调(P < 0.01),Caspase-3、Caspase-8、Caspase-9活性显著增加(P < 0.01)。结论硫利达嗪可显著抑制PC9细胞的增殖,其机制可能与其激活Caspase内外源性凋亡途径,下调CyclinD1、Bcl-2、Bcl-xl,上调Bax有关。 ]]> <![CDATA[PEPT2 mRNA在肺纤维化大鼠肺组织中的表达]]> https://www.researchpad.co/article/5c05306bd5eed0c4848b0b46 肺纤维化是肺癌放化疗后的常见病理改变,是阻碍药物转运到肺部的关键因素之一,肽转运载体已经成为合理设计肽和肽类药物的靶标,本研究旨在探讨肽转运载体2(peptide transporter 2, PEPT2)mRNA在肺纤维化大鼠肺组织中的表达。方法健康SD大鼠50只,随机分为5组。博莱霉素(bleomycin, BLM)7 d、14 d、28 d组:气管内一次性滴入博莱霉素溶液复制肺纤维化大鼠模型,分别于给药后7 d、14 d和28 d放血处死;生理盐水组滴入等量生理盐水,于14 d放血处死;正常组不做任何处理。各组取肺组织,光镜观察组织病理变化;检测样本羟脯氨酸含量;半定量RT-PCR检测肺组织PEPT2 mRNA表达。结果BLM 7 d组大鼠肺组织呈急性炎症性改变,无纤维增生;BLM 14 d组和28 d组大鼠肺组织均有纤维化改变,以28 d组最为明显。BLM 7 d组肺组织羟脯氨酸含量与正常对照组和生理盐水组相比无统计学差异(P > 0.05);14 d组和28 d组大鼠肺组织羟脯氨酸含量均高于正常对照组和生理盐水组(P < 0.05)。各组肺组织PEPT2 mRNA的相对表达量无统计学差异(P > 0.05)。结论PEPT2 mRNA在博莱霉素致肺纤维化大鼠肺组织表达水平无明显变化,PEPT2可能是设计肺纤维化的新型肽类药物靶标之一。 ]]>