Small molecule deoxynyboquinone triggers alkylation and ubiquitination of Keap1 at Cys489 on Kelch domain for Nrf2 activation and inflammatory therapy

Activation of nuclear factor erythroid 2-related factor 2(Nrf2)by Kelch-like ECH-associated protein 1(Keap1)alkylation plays a central role in anti-inflammatory therapy.However,activators of Nrf2 through alkylation of Keap1-Kelch domain have not been identified.Deoxynyboquinone(DNQ)is a natural small molecule discovered from marine actinomycetes.The current study was designed to investigate the anti-inflammatory effects and molecular mechanisms of DNQ via alkylation of Keap1.DNQ exhibited significant anti-inflammatory properties both in vitro and in vivo.The pharmacophore responsible for the anti-inflammatory properties of DNQ was determined to be theα,β-unsaturated amides moieties by a chemical reaction between DNQ and N-acetylcysteine.DNQ exerted anti-inflammatory effects through activation of Nrf2/ARE pathway.Keap1 was demonstrated to be the direct target of DNQ and bound with DNQ through conjugate addition reaction involving alkylation.The specific alkylation site of DNQ on Keap1 for Nrf2 activation was elucidated with a synthesized probe in conjunction with liquid chromatography-tandem mass spectrometry.DNQ triggered the ubiquitination and subsequent degradation of Keap1 by alkylation of the cysteine residue 489(Cys489)on Keap1-Kelch domain,ultimately enabling the activation of Nrf2.Our findings revealed that DNQ exhibited potent anti-inflammatory capacity throughα,β-unsaturated amides moieties active group which specifically activated Nrf2 signal pathway via alkylation/ubiquitination of Keap1-Kelch domain,suggesting the potential values of targeting Cys489 on Keap1-Kelch domain by DNQ-like small molecules in inflammatory therapies.

SYNTHESIS OF POLYISOBUTYLENE WITH SEC-ARYLAMINO TERMINAL GROUP BY COMBINATION OF CATIONIC POLYMERIZATION WITH ALKYLATION

The highly reactive polyisobutylenes(PIBs) withα-double bonds(87.5 mol%) or tert-chloro(tert-Cl) groups(95 mol%) could be prepared via the cationic polymerization of isobutylene(IB) coinitiated by BF_3 or TiCl_4 respectively.The Friedel-Crafts alkylation of diphenylamine(DPA) with the highly reactive PIB withα-double bonds was further conducted under different conditions,such as at different alkylation temperature,in the mixed solvents of CH_2Cl_2/n-hexane with different solvent polarity and at DPA concentr...

Synthesis of spherical nano-ZSM-5 zeolite with intergranular mesoporous for alkylation of ethylbenzene with ethanol to produce m-diethylbenzene

Catalytic synthesis of m-diethylbenzene(m-DEB)through alkylation of ethylbenzene(EB)may be a promising alternative route in comparison with traditional rectification of mixed DEB,for which the top priority is to develop efficient and stable heterogeneous catalysts.Here,the spherical nano-ZSM-5 zeolite with abundant intergranular mesoporous is synthesized by the seed-mediated growth method for alkylation of EB with ethanol to produce m-DEB.The results show that the spherical nano-ZSM-5 zeolite exhibits better stability and higher alkylation activity at a lower temperature than those of commercial micropore ZSM-5.And then,the spherical nano-ZSM-5 is further modified by La_(2)O_(3) through acid treatment followed by immersion method.The acid treatment causes nano-ZSM-5 to exhibit the increased pore size but decreased the acid sites,and subsequent La_(2)O_(3) loading reintroduces the weak acid sites.As a result,the HNO_(3)-La_(2)O_(3)-modified catalyst exhibits a slight increase in EB conversion and DEB yield in comparison with unmodified one,and meanwhile,it still maintains high m-DEB selectivity.The catalyst after acid treatment achieves higher catalytic stability besides maintaining the high alkylation activity of EB with ethanol.The present study on the spherical nano-HZSM-5 zeolite and its modification catalyst with excellent alkylation ability provides new insights into the production of mDEB.

Tandem hydroalkylation and deoxygenation of lignin-derived phenolics to synthesize high-density fuels

Lignin is the most abundant naturally phenolic biomass,and the synthesis of high-performance renewable fuel from lignin has attracted significant attention.We propose the efficient synthesis of high-density fuels using simulated lignin cracked oil in tandem with hydroalkylation and deoxygenation reactions.First,we investigated the reaction pathway for the hydroalkylation of phenol,which competes with the hydrodeoxygenation form cyclohexane.And then,we investigated the effects of metal catalyst types,the loading amount of metallic,acid dosage,and reactant ratio on the reaction results.The phenol hydroalkylation and hydrodeoxygenation were balanced when 180℃ and 5 MPa H_(2)with the alkanes yield of 95%.By extending the substrate to other lignin-derived phenolics and simulated lignin cracked oil,we obtained the polycyclic alkane fuel with high density of 0.918 g·ml^(-1)and calorific value of41.2 MJ·L^(-1).Besides,the fuel has good low-temperature properties(viscosity of 9.3 mm^(2)·s^(-1)at 20℃ and freezing point below-55℃),which is expected to be used as jet fuel.This work provides a promising way for the easy and green production of high-density fuel directly from real lignin oil.

Pilot Test of Preparing 2-Alkylanthraquinone Using Alkylation-Oxidation Technology

To expedite the development of industrial technology for producing 2-alkylanthraquinone,a novel pilot test of alkylation-oxidation technology was conducted.The process mainly included anthracene alkylation,separation of anthracene and 2-alkylanthracene,oxidation of 2-alkylanthracene,and product purification.Optimal alkylation conditions yielded a 91.1%conversion of anthracene and a 71.73%selectivity for 2-alkylanthracene.To address the separation problem of anthracene and 2-alkylanthracene,solvent-assisted distillation technology was developed,resulting in a 98.9%purity of 2-alkylanthracene and a 91.82%separation yield.When the molar ratio of H2O_(2) to 2-alkylanthracene was 7:1,a 98.96%conversion of 2-alkylanthracene and a 99.94%selectivity for 2-alkylanthraquinone were achieved.A novel composition of 2-alkylanthraquinone,including 2-tert-butylanthraquinone,2-tert-amylanthraquinone,and 2-hexylanthraquinone,was developed.This composition could be effectively separated and purified through a combination of crystallization and washing processes.The elemental composition of the product met the existing standards,and its hydrogenation performance closely matched that of commercially available 2-tert-amylanthraquinone products.

Regulating^(*)COOH intermediate via amino alkylation engineering for exceptionally effective photocatalytic CO_(2) reduction

Photocatalytic reduction of CO_(2) into fuel represents a promising approach for achieving carbon neutrality,while realizing high selectivity in this process is challenging due to uncontrollable reaction intermediate and retarded desorption of target products.Engineering the interface microenvironment of catalysts has been proposed as a strategy to exert a significant influence on reaction outcomes,yet it remains a significant challenge.In this study,amino alkylation was successfully integrated into the melem unit of polymeric carbon nitrides(PCN),which could efficiently drive the photocatalytic CO_(2) reduction.Experimental characterization and theoretical calculations revealed that the introduction of amino alkylation lowers the energy barrier for CO_(2) reduction into^(*)COOH intermediate,transforming the adsorption of^(*)COOH intermediate from the endothermic to an exothermic process.Notably,the as-prepared materials demonstrated outstanding performance in photocatalytic CO_(2) reduction,yielding CO_(2)at a rate of 152.8μmol h^(-1) with a high selectivity of 95.4%and a quantum efficiency of 6.6%.

Preparation and Alkylation of Carbon Nanofibers from Polymethyl Methacrylate

Carbon nanofibers have revolutionized nanotechnology due to their potential applications in emerging frontiers of research and industrial sectors. This can be attributed to their superior properties such as higher mechanical strength, unique surface characteristics, and improved adherence that is transmitted into the polymer matrix to form a nanocomposite with improved properties. Polymethyl methacrylate is a common carbon source for the synthesis of carbon nanofibres of its high mechanical strength, thermal stability, and low moisture and water absorbing capacity that allows its products to have several applications. In this work, we report the successful electrospinning of carbon nanofibres from Poly methyl methacrylate and functionalizing the resulting carbon nanofibres. The functionalized carbon nanofibres were analyzed to determine their solubility/dispersion in selected organic solvents, then characterized using Fourier transform infra-red spectroscopy, Raman spectroscopy, scanning electron microscopy combined with Energy dispersive spectroscopy and Thermalgravimetric analysis.

In situ Alkylation of 4,4'-Bipyridine in Hydrothermal Synthesis of an Iodoplumbate Inorganic-organic Hybrid with Fluorescence

A novel 1-D iodoplumbate hybrid, 2（Pb3I9）^3＊·3（C14H18N2）^2＋ 1, has been hydrothermally synthesized and characterized by IR spectra, TGA and single-crystal X-ray diffraction. Complex 1 crystallizes in monoclinic, space group P21/c, with a = 12.057（2）, b = 14.024（3）, c = 24.742（5） A, β = 90.48（3）°, V = 4183.6（14） A^3, Z = 4, R= 0.0477 and wR = 0.0800. The title compound consists of 1-D chains （Pb3I9）n^3n＊ parallel to the [100] direction and stacks of alkylated 4,4＇-bipyridium cations running along the [011] direction. Each anionic chain is surrounded by six arrays of alkylated 4,4＇-bipyridium cations through extensive C-H…I atypical hydrogen-bonding interactions to afford an interesting 3-D supramolecular network. Significant fluorescent property of the compound was observed at room temperature.

涎液化糖链抗原-6、C-反应蛋白/白蛋白比值联合改良临床-影像-病理诊断评分在间质性肺疾病严重程度和预后评估中的应用

目的:探讨涎液化糖链抗原-6(KL-6)、C-反应蛋白/白蛋白比值(CAR)联合改良临床-影像-病理诊断(CRP)评分在间质性肺疾病(ILD)严重程度和预后评估中的应用。方法:选取间质性肺疾病患者167例,根据病情严重程度分为轻度组(n=106)和重度组(n=61),根据预后情况分为预后良好组(n=122)和预后不良组(n=45),另选103例同期健康体检者为对照组。比较不同病情严重程度患者血清KL-6水平、CAR以及改良CRP评分,多因素Logistic分析ILD患者预后不良的危险因素,分析KL-6、CAR联合改良CRP评分在间质性肺疾病严重程度和预后评估中的应用价值。结果:与对照组比较,轻度组和重度组患者血清KL-6水平、CAR、改良CRP评分均显著升高(均P<0.05),与轻度组比较,重度组患者血清KL-6水平、CAR、改良CRP评分均显著升高(均P<0.05)。两组年龄、性别、病程、吸烟史、饮酒史、高血压史、慢性阻塞性肺疾病(COPD)合并情况无统计学差异(均P>0.05),预后不良组患者肺功能异常患者占比、血清KL-6水平、CAR、改良CRP评分明显高于预后良好组(均P<0.05)。肺功能异常、高血清KL-6水平、高CAR、高改良CRP评分是间质性肺疾病患者预后不良的危险因素(均P<0.05)。血清KL-6、CAR联合改良CRP评分诊断重度间质性肺疾病的灵敏度、特异度明显高于三者独立检查(均P<0.05)。血清KL-6、CAR联合改良CRP评分预测间质性肺疾病患者预后不良的灵敏度、特异度明显高于三者独立检查(均P<0.05)。结论:KL-6、CAR联合改良CRP评分在重度ILD患者的严重程度诊断中具有较高准确度,且对ILD患者的预后也具有较高的预测价值。

Alkylation of toluene with tert-butyl alcohol over HPW-modified Hβ zeolite

An Hβ-supported heteropoly acid （H3PW12O40 （HPW）/Hβ） catalyst was successfully prepared by wetness impregnation, and investigated in the alkylation of toluene with tert-butyl alcohol for the synthesis of 4-tert-butyltoluene （PTBT）. X-ray diffraction, scanning electron microscopy, transmis- sion electron microscopy, fourier-transform infrared spectroscopy, inductively coupled plas- ma-optical emission spectrometry, the brunauer emmett teller （BET） method, tempera- ture-programmed NH3 desorption, and pyridine adsorption infrared spectroscopy were used to characterize the catalyst. The results showed that loading HPW on Hβ effectively increased the B acidity and decreased the pore size of Hβ. The B acidity of HPW/Hβ was 142.97 μmol/g, which is 69.74% higher than that of Hβ （84.23 μmol/g）. The catalytic activity of the HPW/Hβ catalyst was much better than that of the parent Hβ zeolite because of its high B acidity. The toluene conversion over HPW/Hβ reached 73.1%, which is much higher than that achieved with Hβ （54.0%）. When HPW was loaded on Hβ, the BET surface area of Hβ decreased from 492.5 to 379.6 m2/g, accompa- nied by a significant decrease in the pore size from 3.90 to 3.17 nm. Shape selectivity can therefore play an important role and increase the product selectivity of the HPW/Hβ catalyst compared with that of the parent Hβ. PTBT （kinetic diameter 0.58 nm） can easily diffuse through the narrowed pores of HPW/Hβ, but 3-tert-butyltoluene （kinetic diameter 0.65 nm） diffusion is restricted because of steric hindrance in these narrow pores. This results in high PTBT selectivity over HPW/Hβ （around 81%）. The HPW/Hβ catalyst gave a stable catalytic performance in reusability tests.

采用混合策略联合优化的模糊C-均值聚类信息熵点云简化算法

针对传统聚类算法处理点云简化问题时精度低、耗时长且易丢失特征信息等问题,提出了一种基于动态精英自适应混合策略的鹈鹕算法(DEAMPOA)与加权熵法联合优化的模糊C-均值聚类(FCM)信息熵点云简化算法。采用动态自适应种群混合策略,同时融合了精英反向化思路,显著提升了鹈鹕优化算法(POA)的收敛趋势和全局寻优能力,提高了寻找FCM最优聚类中心的成功率;利用DEAMPOA结合加权熵法对FCM进行优化,提高鲁棒性的同时增强了搜索精度,得到较好的聚类结果;在8种UCI标准数据集上与4种算法对比进行聚类性能评估实验,验证了所提方法综合性能优越;将所提方法与信息熵融合,并应用在三维点云KITTI数据集简化中。实验结果表明:与包围框简化法、随机采样简化法和特征选择简化法对比,所提方法全局误差简化前后点集之间平均欧式距离(MED)指标分别降低了2.25%、6.93%、5.74%,点云简化效果最优且运行速度满足要求。

抑制miRNA-376c-3p对肾透明细胞癌生长的影响

目的探究miRNA-376c-3对肾透明细胞癌(ccRCC)中肿瘤细胞生长的影响。方法以实时荧光定量PCR(RT-PCR)检测人ccRCC细胞系786-O、Caki-1、SN12-PM6、ACHN及正常人近段肾小管上皮细胞系HKC中miRNA-376c-3p的表达。体外培养786-O细胞,随机分为对照组、miR-376c-3p inhibitor组(转染miR-376c-3p inhibitor)、miR-376c-3p mimics组(转染miR-376c-3p mimics)、阴性对照组(转染miR-376c-3p阴性对照),分组转染后以RT-PCR检测4组细胞miRNA-376c-3p表达;以Ki67免疫荧光染色、集落生成实验检测4组786-O细胞增殖;以TUNEL染色检测4组786-O细胞凋亡;以免疫荧光染色检测4组786-O细胞凋亡相关蛋白Bax、Bcl-2表达并比较其Bax/Bcl-2。于右腋窝皮下接种上述4组786-O细胞建立其裸鼠移植瘤模型,3周后测定其肿瘤体积与质量;以Ki67免疫荧光染色、TUNEL染色分别检测4组裸鼠肿瘤细胞增殖与凋亡。结果与HKC细胞比较,ccRCC细胞系786-O、Caki-1、SN12-PM6、ACHN中miR-376c-3p表达降低(P<0.05)。与对照组比较,miR-376c-3p inhibitor组细胞增殖率与集落形成率、肿瘤体积与质量、裸鼠肿瘤组织Ki67阳性比升高(P<0.05),细胞miR-376c-3p相对表达、Bax/Bcl-2与凋亡率、裸鼠肿瘤组织TUNEL阳性比降低(P<0.05);miR-376c-3p mimics组细胞增殖率与集落形成率、肿瘤体积与质量、裸鼠肿瘤组织Ki67阳性比降低(P<0.05),细胞miR-376c-3p相对表达、Bax/Bcl-2与凋亡率、裸鼠肿瘤组织TUNEL阳性比升高(P<0.05);阴性对照组各指标无明显变化(P>0.05)。结论抑制miRNA-376c-3p可削弱ccRCC细胞增殖及集落生成活性,促进其凋亡,并在裸鼠体内抑制其移植瘤生长。

2型糖尿病患者血清hsa-miR-30c-5p水平表达对微血管并发症的预测价值研究

目的探讨2型糖尿病(type 2 diabetes mellitus,T2DM)患者血清人微小核糖核酸(homo sapiensmicroRNA,hsa-miR)-30c-5p表达对微血管并发症的预测价值。方法选取2021年5月~2022年9月巴中市中心医院收治的T2DM患者205例为糖尿病组,并根据患者微血管并发症情况将糖尿病组进一步分为并发组(n=124)和未并发组(n=81),另选取同期健康体检者205例为对照组,均采用逆转录-聚合酶链反应(reverse transcriptionpolymerasechain reaction,RT-PCR)检测受试者血清hsa-miR-30c-5p表达并进行比较。采用多因素Logistic回归分析影响微血管并发症的因素,绘制受试者工作特征(receiver operating characteristic,ROC)曲线预测血清hsa-miR-30c-5p表达对T2DM患者发生微血管并发症的价值。结果并发组、未并发组的血清hsa-miR-30c-5p表达分别为0.58±0.06,0.72±0.08,均低于对照组(0.89±0.21),差异具有统计学意义(t=16.038,7.079,均P=0.001);并发组低于未并发组,差异具有统计学意义(t=14.289,P=0.001)。糖尿病病程[OR(95%CI):3.873(2.976~4.770)]、尿酸[OR(95%CI):2.125(1.211~3.040)]、糖化血红蛋白[OR(95%CI):2.680(1.745~3.616)]均为T2DM患者发生微血管并发症的独立危险因素(均P<0.05),葡萄糖目标范围内时间[OR(95%CI):0.491(0.135~0.846)]、血清hsa-miR-30c-5p[OR(95%CI):0.532(0.146~0.817)]均为T2DM患者发生微血管并发症的保护因素(均P<0.05)。血清hsa-miR-30c-5p表达预测T2DM患者发生微血管并发症的敏感度、特异度、曲线下面积(95%置信区间)分别为81.45%,85.19%,0.802(0.741~0.854)。结论T2DM患者血清hsa-miR-30c-5p表达异常降低,且血清hsa-miR-30c-5p为T2DM患者发生微血管并发症的保护因素,对T2DM患者发生微血管并发症具有一定的预测价值。

miR-26a通过HGF/c-Met通路调控乳腺癌血管生成及分子机制

目的:研究miR-26a通过肝细胞生长因子(HGF)/细胞间质上皮转化(c-Met)通路调控乳腺癌血管生成及分子机制。方法:选择深圳市宝安区松岗人民医院2021年1月~2022年3月收治的乳腺癌患者40例,采集所有受试者的乳腺癌组织以及癌旁正常组织,比较不同乳腺组织织miR-26a、血管内皮生长因子A(VEGFA)mRNA相对表达量以及微血管密度(MVD)情况。此外,通过LipofectamineTM2000脂质体法将miR-26a模拟物以及抑制物分别转染至乳腺癌细胞中,记作miR-26a转染组、miR-26a抑制组,并将未进行任何处理的乳腺癌细胞作为阴性对照组。采用PCR方法检测HGF/c-Met通路相关蛋白及其下游靶基因磷脂酰肌醇激酶3(PI3K)、蛋白酶B(AKT)、雷帕霉素靶蛋白(mTOR)表达情况。结果:乳腺癌组织中miR-26a相对表达量相较于癌旁正常组织更低,而VEGFA相对表达量与MVD相较于癌旁正常组织更高,差异均有统计学意义(P<0.05)。miR-26a抑制组HGF、p-c-Met/c-MET表达水平相较于阴性对照组以及miR-26a转染组均更高,而miR-26a转染组HGF、p-c-Met/c-MET表达水平相较于阴性对照组更低,差异均有统计学意义(P<0.05)。miR-26a抑制组PI3K、AKT、mTOR mRNA相对表达量相较于阴性对照组以及miR-26a转染组更高,而miR-26a转染组PI3K、AKT、mTOR mRNA相对表达量相较于阴性对照组更低,差异均有统计学意义(均P<0.05)。结论:miR-26a通过抑制HGF/c-Met通路,降低PI3K、AKT、mTOR表达,抑制乳腺癌的发生及发展。

冠心病经皮冠状动脉介入术治疗前后患者血清肺炎衣原体抗体IgA、超敏C-反应蛋白、血管生成素-2变化及预后预测价值研究

目的:探讨冠心病(CHD)经皮冠状动脉介入术治疗前后患者血清肺炎衣原体抗体IgA(CP-IgA)、超敏C-反应蛋白(hs-CRP)及血管生成素-2(Ang-2)变化及预后预测价值。方法:选取CHD患者135例为观察组,根据患者预后情况分为预后良好组(119例)及预后不良组(16例)。另选取同期体检健康者135例为对照组。比较观察组和对照组、观察组PCI前后以及两个亚组血清CP-IgA、hs-CRP及Ang-2水平。分析血清CP-IgA、hs-CRP及Ang-2与PCI术后CHD患者预后的相关性。分析血清CP-IgA、 hs-CRP及Ang-2对PCI术后CHD患者预后的预测价值。结果:观察组血清CP-IgA、hs-CRP及Ang-2水平高于对照组(均P<0.05)。观察组患者PCI术后血清CP-IgA及Ang-2水平较PCI术前降低,血清hs-CRP水平较PCI术前升高(均P<0.05)。预后良好组患者血清CP-IgA、hs-CRP及Ang-2水平低于预后不良组(均P<0.05)。血清CP-IgA、hs-CRP及Ang-2与PCI术后CHD患者预后呈正相关(均P<0.05)。血清CP-IgA、hs-CRP及Ang-2单独预测PCI术后CHD患者预后的曲线下面积(AUC)小于三项联合(均P<0.05)。结论:CHD患者血清CP-IgA、hs-CRP及Ang-2水平升高,PCI术后血清CP-IgA和Ang-2水平降低,hs-CRP水平升高。预后不良患者血清CP-IgA、hs-CRP及Ang-2水平升高,且三者联合对PCI术后CHD患者预后的预测价值较高。

miR-34c-5p靶向poFUT1对胎盘血管形成的影响

目的:探讨miR-34c-5p与poFUT1的靶向关系及对胎盘血管形成的影响。方法:利用ENCORI数据库预测miR-34c-5p与poFUT1的结合位点,采用双荧光素酶报告实验验证。随机选取2023年4至10月在郑州大学第三附属医院住院的胎儿生长受限(FGR)孕妇20例(FGR组),选择同期正常孕妇20例为对照。采用实时荧光定量PCR法检测两组胎盘组织中miR-34c-5p和poFUT1 mRNA的表达。取脐静脉内皮细胞,分组转染miR-34c-5p模拟物及其对照(NC)、miR-34c-5p抑制物及其NC、si-poFUT1 NC、si-poFUT1、si-poFUT1+miR-34c-5p抑制物,采用CCK-8法、Transwell实验以及成管实验检测细胞转染24、48、72、96 h后的增殖能力,转染48 h后的侵袭能力和成管能力。结果:FGR组和对照组胎盘组织中miR-34c-5p表达水平分别为(1.57±0.39)、(1.09±0.18),poFUT1 mRNA表达水平分别为(0.51±0.17)、(1.06±0.13),FGR组胎盘组织中miR-34c-5p表达水平高于对照组,poFUT1 mRNA表达水平低于对照组(P<0.05)。与miR-34c-5p模拟物NC组比较,模拟物组侵袭细胞数和管腔节点数减少,细胞增殖活性降低(P<0.05)。与miR-34c-5p抑制物NC组比较,抑制物组侵袭细胞数和管腔节点数增加,细胞增殖活性升高(P<0.05)。与si-poFUT1 NC组相比,si-poFUT1组侵袭细胞数和管腔节点数减少,细胞增殖活性降低(P<0.05),而si-poFUT1+miR-34c-5p抑制物组上述变化较si-poFUT1组部分逆转(P<0.05)。结论:miR-34c-5p可能通过调控poFUT1影响血管内皮细胞功能,从而影响胎盘的血管形成,参与FGR的发生。

PKC-α介导的线粒体功能障碍在妊娠糖尿病致胚胎神经管缺陷中的机制研究

目的:探讨蛋白激酶C-α(PKC-α)介导的线粒体功能障碍在妊娠糖尿病致胚胎神经管缺陷(NTD)中的作用。方法:将雌性C57BL/6J小鼠随机分为对照组、NTD组、NTD+sh-PKC-α组,每组6只。除对照组外,NTD组、NTD+sh-PKC-α组建立糖尿病胚胎病小鼠模型。NTD+sh-PKC-α组分别在胚胎第1天(E1)、第4天(E4)和第8天(E8)时,经尾静脉向小鼠注射100μL携带sh-PKC-α的慢病毒(1×10^(8) TU/mL)。在第11.5天(E11.5)时,从子宫中解剖出胚胎进行分析。采用透射电子显微镜观察神经管中线粒体。采用蛋白免疫印迹法(Western Blot)分析胚胎神经管中PKC-α、抗微管相关蛋白1轻链3(LC3)和抗溶酶体相关膜蛋白2(LAMP2)蛋白表达。体外构建PKC-α敲低C17.2小鼠神经干细胞,并在低(5 mmol/L)或高(25 mmol/L)葡萄糖条件下培养,采用免疫荧光染色检测细胞自噬体变化情况。结果:在NTD组中,胚胎神经管中线粒体肿胀,嵴消失。与对照组相比,NTD组胚胎神经管中LC3Ⅱ和LAMP2蛋白表达降低(P<0.05),PKC-α蛋白表达上调(P<0.01)。通过沉默小鼠胚胎神经管中PKC-α表达,胚胎中LC3Ⅱ和LAMP2蛋白表达增加(P<0.05)。此外,NTD+sh-PKC-α组小鼠胚胎中的NTD发生率低于NTD组(P<0.01)。在高糖条件下,C17.2神经干细胞中LC3Ⅱ、LAMP2蛋白表达水平和Cyto-ID染色点的数量均低于正常葡萄糖条件下水平(P<0.05)。PKC-α蛋白敲低增加了高糖条件下的LC3Ⅱ、LAMP2蛋白表达水平和Cyto-ID染色点的数量(P<0.05)。结论:PKC-α介导的线粒体功能障碍与母体糖尿病诱导的NTD胚胎相关,通过沉默母体PKC-α减弱了神经管自噬抑制导致的NTD形成。

miR-520c-3p/CXCL8信号轴在慢性髓系白血病细胞红系分化中的调控作用

目的:探讨miR-520c-3p/CXCL8信号轴在慢性髓系白血病(CML)细胞红系分化中的作用及其机制。方法:用氯化血红素诱导人CML细胞系K562细胞红系分化,逆转录实时荧光定量PCR(RT-qPCR)法检测CXCL8 mRNA的表达水平。用不同浓度CXCL8(0、20、40、60、80和100 ng/mL)处理K562细胞72 h,RT-qPCR实验检测γ-globin mRNA的表达情况,据此分析CXCL8对K562细胞红系分化的影响。将miR-520c-3p模拟物和inhibitor转染到K562细胞以增强或抑制miR-520c-3p的功能,RT-qPCR法检测γ-globin mRNA的表达水平,分析miR-520c-3p对K562细胞红系分化的影响。分别采用Western blot和RT-qPCR实验检测CXCL8 mRNA和蛋白的表达水平,观察miR-520c-3p对CXCL8表达的影响。细胞分为实验组(共转染miR-520c-3p模拟物与CXCL8 3′-UTR)和对照组(共转染模拟物对照与CXCL8 3′-UTR),用双荧光素酶报告基因实验检测miR-520c-3p是否靶向CXCL8的3′-UTR。结果:与对照组相比,氯化血红素诱导分化处理后,K562细胞中CXCL8 mRNA的表达水平升高(P<0.01)。与未处理组相比,不同剂量CXCL8处理组K562细胞中γ-globin mRNA的表达水平均明显升高(P<0.01)。与阴性对照组细胞相比,miR-520c-3p模拟物转染组K562细胞中γ-globin mRNA的表达水平、CXCL8 mRNA和蛋白表达水平均下降(均为P<0.01);miR-520c-3p抑制物转染组细胞中γ-globin mRNA表达水平、CXCL8 mRNA和蛋白表达水平均上升(均为P<0.01)。双荧光素酶报告基因实验检测发现,与对照组相比,实验组的相对荧光素酶活性明显下降(P<0.01)。结论:本研究揭示了miR-520c-3p/CXCL8信号轴在CML细胞红系分化中的关键作用。miR-520c-3p通过靶向调控CXCL8的表达,在K562细胞的红系分化过程中发挥抑制效应。

超敏C-反应蛋白、血清降钙素原及白细胞计数在新生儿感染性疾病中的早期诊断价值

目的研究超敏C-反应蛋白(hs-CRP)、血清降钙素原(PCT)及白细胞(WBC)计数在新生儿感染性疾病的早期诊断价值,为降低临床抗生素使用率及新生儿病死率等提供科学依据。方法将2021年1月—2021年12月本院收治的62例患有感染性疾病的新生儿为病例组,同期同科室收住的50例患新生儿非感染性疾病的病例为对照组,在入院第1天和第7天分别采静脉血对比两组患儿hs-CRP、PCT和WBC的差异,计算hs-CRP、PCT和WBC灵敏度、特异度并绘制ROC曲线。结果入院时病例组hs-CRP、PCT和WBC计数均高于对照组,差异具有统计学意义(P<0.001);入院7天时,病例组hs-CRP、PCT均明显下降,与对照组差异无统计学意义(P>0.05),WBC计数虽较入院时明显下降,但仍较对照组高,差异具有统计学意义(P<0.05);hs-CRP、PCT、WBC计数和hs-CRP+PCT在诊断新生儿感染性疾病的ROC曲线下面积分别是0.954、0.962、0.732和0.985。Hs-CRP+PCT的约登指数最高,曲线下面积最大,其次是PCT、hs-CRP、WBC,差异均具有统计学意义(P<0.05)。结论hs-CRP、PCT和WBC计数在新生儿患有感染性疾病的早期均具有一定的诊断价值,hs-CRP联合PCT有助于早期判断是否使用抗生素。

miR-30c-5p对前列腺癌细胞增殖、迁移和侵袭的抑制作用及其机制

目的:探讨微小RNA(miR)-30c-5p对人前列腺癌细胞(LNCap)增殖、迁移和侵袭的影响,并阐明其可能的机制。方法:LNCap细胞根据转染质粒不同分为LNCap组(无转染质粒)、miR-30c-5p mimic组(转染miR-30c-5p mimic)、mimic NC组(转染miR-30c-5p mimic NC)、sh-DNA损伤诱导转录因子4(DDIT4)组(转染sh-DDIT4)、sh-NC组(转染sh-DDIT4 NC)、miR-30c-5p mimic+pc-DNA3.1-NC组(共转染miR-30c-5p mimic和pc DNA3.1-DDIT4空载质粒)和miR-30c-5p mimic+pc-DNA3.1-DDIT4组(共转染miR-30c-5pmimic和pc-DNA3.1-DDIT4过表达质粒),RWPE-1细胞正常培养。实时荧光定量PCR(RT-qPCR)法检测各组细胞中miR-30c-5p和DDIT4mRNA表达水平,Western blotting法检测各组细胞中DDIT4蛋白表达水平,CCK-8法检测各组LNCap细胞增殖率,Transwell小室实验检测各组LNCap细胞侵袭细胞数,划痕实验检测各组LNCap细胞划痕愈合率,双荧光素酶报告基因实验验证miR-30c-5p与DDIT4的靶向关系。体内裸鼠成瘤实验,18只雄性BALB/c裸鼠随机分为空白组、agomiR-NC组(转染agomiR-30c-5p NC)和agomiR-30c-5p组(转染agomiR-30c-5p),每组6只。agomiR-NC组和agomiR-30c-5p组裸鼠皮下注射LNCap细胞,空白组裸鼠注射等量生理盐水,检测各组小鼠肿瘤体积。HE染色观察各组小鼠前列腺癌组织形态表现,RT-qPCR法和免疫荧光染色法检测各组小鼠前列腺癌组织中miR-30c-5p和DDIT4 mRNA表达水平及DDIT4蛋白荧光强度。结果:体外前列腺癌细胞实验,与人正常前列腺上皮RWPE-1细胞比较,前列腺癌LNCap细胞中miR-30c-5p表达水平明显降低(P<0.01),DDIT4 mRNA和蛋白表达水平明显升高(P<0.05或P<0.01);转染48 h后,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞中miR-30c-5p表达水平明显升高(P<0.01)。与LNCap组和sh-NC组比较,sh-DDIT4组LNCap细胞中DDIT4 mRNA表达水平明显降低(P<0.01);与miR-30c-5p mimic组和miR-30c-5p mimic+pcDNA3.1 NC组比较,miR-30c-5p mimic+pc-DNA3.1-DDIT4组LNCap细胞中miR-30c-5p表达水平明显降低(P<0.01);与miR-30c-5p mimic组和miR-30c-5p mimic+pcDNA3.1 NC组比较,miR-30c-5p mimic+pc-DNA3.1-DDIT4组LNCap细胞中DDIT4 mRNA表达水平明显升高(P<0.01);与miR-30c-5pmimic组和miR-30c-5pmimic+pcDNA3.1NC组比较,miR-30c-5pmimic+pc-DNA3.1-DDIT4组LNCap细胞中DDIT4蛋白表达水平明显升高(P<0.05)。CCK-8法检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞增殖率明显降低(P<0.01);与LNCap组和sh-NC组比较,sh-DDIT4组LNCap细胞增殖率明显降低(P<0.01);与miR-30c-5p mimic组和miR-30c-5pmimic+pcDNA3.1NC组比较,miR-30c-5pmimic+pc-DNA3.1-DDIT4组LNCap细胞增殖率明显升高(P<0.01)。Transwell小室实验检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞侵袭细胞数明显减少(P<0.01);与LNCap组和sh-NC组比较,sh-DDIT4组LNCap细胞侵袭细胞数明显减少(P<0.01);与miR-30c-5pmimic组和miR-30c-5p mimic+pcDNA3.1 NC组比较,miR-30c-5p mimic+pc-DNA3.1-DDIT4组LNCap细胞侵袭细胞数明显增加(P<0.01)。划痕实验检测,与LNCap组和mimic NC组比较,miR-30c-5p mimic组LNCap细胞划痕愈合率明显降低(P<0.01);与LNCap组和sh-NC组比较,sh-DDIT4组细胞划痕愈合率明显降低(P<0.01);与miR-30c-5p mimic组和miR-30c-5p mimic+pcDNA3.1 NC组比较,miR-30c-5p mimic+pc-DNA3.1-DDIT4组细胞划痕愈合率明显升高(P<0.01)。双荧光素酶实验检测,与共转染WT-DDIT4和mimic NC的LNCap细胞比较,共转染WT-DDIT4和miR-30c-5p mimic的LNCap细胞中荧光素酶活性明显降低(P<0.01)。体内裸鼠成瘤实验,细胞注射后第3、6、9、12和15天,与空白组和agomiR-NC组比较,agomiR-30c-5p组裸鼠肿瘤体积明显减小(P<0.05)。HE染色观察,空白组和agomiR-NC组小鼠前列腺癌组织中细胞核增大,核仁突出且畸形;agomiR-30c-5p组小鼠前列腺癌组织部分区域细胞排列整齐,细胞核恢复正常形态。RT-qPCR法和免疫荧光染色法检测,与agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中miR-30c-5p表达水平明显升高(P<0.01);与空白组和agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中DDIT4 mRNA表达水平明显降低(P<0.01);DDIT4蛋白主要在细胞质表达,与空白组和agomiR-NC组比较,agomiR-30c-5p组小鼠前列腺癌组织中DDIT4蛋白荧光强度明显降低(P<0.01)。结论:miR-30c-5p在前列腺癌LNCap细胞中表达水平明显降低,其可通过靶向下调DDIT4抑制前列腺癌细胞增殖、迁移和侵袭,从而参与前列腺癌的发生发展。