Melittin对非小细胞肺癌增殖、凋亡及PI3K/Akt信号通路的影响

目的探讨Melittin对非小细胞肺癌（NSCLC）细胞增殖、凋亡及PI3K/Akt信号通路的影响。方法分别采用0、10、20、50、100μmol/L Melittin处理NSCLC细胞株A549、SPC-A1及人肺上皮细胞株16HBE 24、48、72和96 h后,采用四甲基偶氮唑盐（MTT）比色法检测各细胞株的增殖抑制率变化,同时采用流式细胞术Annexin-FITC/PI双染法及PI单染法检测不同浓度Melittin处理24、48 h后的A549细胞凋亡及细胞周期情况,Western blotting检测不同浓度Melittin处理48 h后的A549细胞中PI3K/Akt信号通路相关蛋白（Akt和PTEN）及凋亡促进基因（caspase-9）的表达情况。结果在10~100μmol/L范围内,Melittin可呈剂量和时间依赖的方式提高A549、SPC-A1细胞的增殖抑制率,差异均有统计学意义（P〈0.05）,但对16HBE无细胞毒性作用（P〉0.05）;与0μmol/L比较,除10μmol/L Melittin处理24 h后的晚期凋亡率和G2/M期细胞比例无统计学差异（P〉0.05）,10~100μmol/L的早、晚期凋亡率、G0/G1期细胞比例及PTEN和caspase-9蛋白水平均升高,S期、G2/M期细胞比例及Akt水平均降低,以上差异有统计学意义（P〈0.05）,且10~100μmol/L范围内各浓度间的差异均有统计学意义（P〈0.05）。结论 Melittin可对NSCLC细胞有毒性作用,但对正常肺上皮细胞无影响,且可诱导A549细胞凋亡及细胞周期阻滞并抑制PI3K/Akt信号通路的激活。

蜂毒溶血肽(Melittin)cDNA的克隆及序列分析

为了克隆得到正确序列的蜂毒溶血肽 c DNA,对蜂毒中的蜂毒溶血肽进行了研究。实验从蜜蜂毒腺中提取总 RNA,通过 RT- PCR方法扩增得到了蜂毒溶血肽的 c DNA,扩增产物克隆到p T7Blu- T载体 ,转化大肠杆菌 JM1 0 9,DNA序列分析结果表明克隆得到的蜂毒溶血肽 c

rhuPAa-melittin在卵巢癌体内外的作用机制

目的利用流式细胞术检测不同浓度的rhu PAa-melittin作用于卵巢癌细胞的活性,进而初步探讨rhu PAa-melittin对卵巢癌细胞的抑制作用及体内抗瘤作用。方法利用不同浓度的rhu PAa-melittin蛋白分别作用于人卵巢癌细胞48 h,利用流式细胞术检测细胞周期及凋亡。rhu PAa-melittin蛋白作用于卵巢癌皮下移植瘤模型,观察移植瘤的生长情况。结果分别以0、4、8、16μg/m L浓度作用人卵巢癌细胞48 h,凋亡率分别为1.16%、3.83%、6.51%、10.2%。不同浓度下G0/G1期细胞无明显作用,主要作用于S期,阻止由S期进入G2/M期,从而抑制肿瘤生长。结论 rhu PAa-melittin在卵巢癌的体内外实验中有效抑制了肿瘤的生长。

牛乳铁蛋白活性肽（LactoferricinB）（3—17）与蜂毒肽（Melittins）（6—12）杂合多肽活性功能的测定

抗菌肽近年逐渐成为研究的热点。将牛乳铁蛋白活性多肽（LfcinB）与蜂毒肽（Melittins）核心功能区间，即LfcinB的3-17位的15个氨基酸与Melitins的6-12位的8个氨基酸，固相合成新的多肽分子Lfb15-Me8。经RT-HPLC和质谱检测合成多肽分子准确，纯度达到98％以上。经体外抑菌实验证明，所合成的新型抗菌肽分子对大肠杆菌ATCC25922、金黄色葡萄球菌ATCC25923、绿脓杆菌ATCC27853、酵母GS115、沙门氏菌ATCC12291的最小抑菌浓度（MIC）分别为16μg·mL^-1、32μg·mE-164μg·mL^-1、128μg·mL^-1、16μg·mL^-1，并在其抗菌浓度下并未表现溶血特性。获得了具有活性的新型多肽序列，为抗菌肽的研发奠定了新的研究内容。

蜂毒肽Melittin对小鼠红细胞溶血效应及影响机制分析

蜂毒肽Melittin是一种具有高效抗细菌、真菌、肿瘤细胞等多种生物学活性的抗菌肽,然而其溶血活性限制了其发展为高效抗菌抗癌药物的应用.本文深入分析了Melittin对小鼠红细胞的作用机制,探讨影响Melittin溶血活性的细胞机制.结果表明,Melittin可以引起红细胞膜上形成空洞,抑制细胞内ATPase的活性.红细胞膜中糖蛋白或糖脂糖链中的唾液酸参与Melittin的相互作用,介导Melittin的溶血活性.质膜胆固醇也影响Melittin与红细胞膜相互作用,降低溶血活性.研究还发现,外源添加D-葡萄糖和D-蔗糖能显著抑制Melittin的溶血活性.研究结果为降低抗菌肽溶血性以及推动抗菌肽的药物应用提供了重要理论基础.

蜂毒肽突变体Melittin-K1抑制人肝癌细胞BEL-7402生长的作用

目的:为了提高蜂毒肽Melittin的抗肿瘤活性,我们设计出新型多肽并将其命名为Melittin-K1。并对多肽Melittin-K1抑制人肝癌细胞BEL-7402生长的作用进行研究。方法:采用CCK-8法检测细胞活力。扫描电镜拍照结合培养基上清中乳酸脱氢酶(lactic dehydrogenase,LDH)的表达水平来评价细胞膜的损伤水平。构建BEL-7402细胞裸鼠异种移植瘤模型来评价Melittin-K1、Melittin体内对肝癌生长的影响以及毒性。结果:与Melittin相比,Melittin-K1抑制BEL-7402细胞生长的作用更显著;与L02细胞相比,BEL-7402对Melittin-K1的杀伤作用更敏感;Melittin-K1体外抑制BEL-7402细胞生长呈时间、剂量依赖性。扫描电镜以及LDH检测结果均显示Melittin-K1处理组细胞膜的破损情况严重。BEL-7402细胞裸鼠皮下移植瘤模型结果表明,Melittin-K1体内显著抑制肿瘤生长,其抗肿瘤作用明显高于同剂量Melittin。Melittin-K1和Melittin体内毒性小,主要表现在,与模型组相比,体重曲线、肝功指标均未发生明显改变。结论:Melittin-K1能够明显抑制肝癌细胞BEL-7402的增殖,是一种极具潜力的抗肝癌药物。

Melittin induces human gastric cancer cell apoptosis via activation of mitochondrial pathway

AIM: To investigate the apoptotic effects of melittin on SGC-7901 cells via activation of the mitochondrial signaling pathway in vitro.METHODS: SGC-7901 cells were stimulated by melittin, and its effect on proliferation and apoptosis of was investigated by methyl thiazolyl tetrazolium assay, morphologic structure with transmission electron microscopy, annexin-V/propidium iodide double-staining assay, measuring mitochondrial membrane potential(MMP) levels, and analyzing reactive oxygen species(ROS) concentrations were analyzed by flow cytometry. Cytochrome C(Cyt C), apoptosis-inducing factor(AIF), endonuclease G(Endo G), second mitochondria-derived activator of caspases(Smac)/direct IAP binding protein with low isoelectric point(Diablo), and FAS were analyzed by western blot. The expression of caspase-3 and caspase-8 was measured using activity assay kits.RESULTS: Melittin was incubated at 1.0, 2.0, 4.0, or 6.0 μg/m L for 1, 2, 4, 6, or 8 h and showed a timeand concentration-dependent inhibition of SGC-7901 cell growth. Melittin induced SGC-7901 cell apoptosis, which was confirmed by typical morphological changes. Treatment with 4 μg/m L melittin induced early apoptosis of SGC-7901 cells, and the early apoptosis rates were 39.97% ± 3.19%, 59.27% ± 3.94%, and 71.50% ± 2.87% vs 32.63% ± 2.75% for 1, 2, and 4 h vs 0 h(n = 3, P < 0.05); the ROS levels were 616.53% ± 79.78%, 974.81% ± 102.40%, and 1330.94% ± 93.09% vs 603.74% ± 71.99%(n = 3, P < 0.05); the MMP values were 2.07 ± 0.05, 1.78 ± 0.29, and 1.16 ± 0.25 vs 2.55 ± 0.42(n = 3, P < 0.05); caspase-3 activity was significantly higher compared to the control(5492.3 ± 321.1, 6562.0 ± 381.3, and 8695.7 ± 449.1 vs 2330.0 ± 121.9), but the caspase activity of the non-tumor cell line L-O2 was not different from that of the control. With the addition of the caspase-3 inhibitor(Ac-DEVD-CHO), caspase-3 activity was significantly decreased compared to the control group(1067.0 ± 132.5 U/g vs 8695.7 ± 449.1 U/g). The expression of the Cyt C, Endo G, and AIF proteins in SGC-7901 cells was significantly higher than those in the control(P < 0.05), while the expression of the Smac/Diablo protein was significantly lower than the control group after melittin exposure(P < 0.01). Ac-DEVD-CHO did not, however, have any effect on the expression of caspase-8 and FAS in the SGC-7901 cells.CONCLUSION: Melittin can induce apoptosis of human gastric cancer(GC) cells through the mitochondria pathways, and it may be a potent agent in the treatment of human GC.

3D-QSAR Study of Melittin and Amoebapore Analogues by CoMFA and CoMSIA Methods

Peptides are one of the indispensable substances in life. The use of computer aided drug design(CADD) methods to design peptides and peptiodmimetics can short the design cycle, save research funding, improve the level of whole research to a large extent and guide the discovery of new drugs. In this paper, Melittin and amoebapore three-dimensional quantitative structureactivity relationship(3D-QSAR) models were established by using comparative molecular field analysis(CoMFA) and comparative molecular similarity indices analysis(CoMSIA) method. The result shows that, the correlation coefficient(q^2) was 0.583 and non-cross-validation correlation coefficient(r^2) was 0.972 for the melittin CoMFA model. The q^2 and r^2 were 0.630 and 0.995 for the best CoMSIA model, 0.645 and 0.993 for the amoebapore CoMFA model, and 0.738 and 0.996 for the best CoMSIA model. The statistical parameters demonstrated that the CoMFA and CoMSIA models had both good predictive ability and high statistical stability, and can provide theoretical basis for designing new high activity polypeptide drugs.

基于Mel声谱图与改进SEResNet的鱼类行为识别

养殖环境中饲料投放、水流变化等刺激源导致鱼类声音分辨难,使行为识别准确率不高,为解决上述问题,提出基于Mel声谱图(Mel spectrogram)与改进SEResNet的鱼类行为识别模型TAP-SEResNet。首先针对鱼类行为声音频率波动大、特征差异小,造成特征提取难的问题,采用高分辨率、特征表示较好的Mel声谱图以捕捉鱼类声音的频谱特征。其次针对鱼类声音特征关键信息易丢失的难题,提出在SEResNet模型中融合时序聚合池化层(Temporal Aggregated Pooling,TAP),提取池化区域的最大值和平均值,保留鱼类行为更多细粒度声音特征,提高识别准确率。为验证所提模型的有效性,分别设计了消融试验和模型性能对比试验,试验结果显示:TAP-SEResNet相比SEResNet在不降低检测速度的条件下准确率提升了3.23%;相比PANNS-CNN14、ECAPA-TDNN及MFCC+ResNet等先进声音识别模型,TAP-SEResNet在准确率上分别提升了5.32%、2.80%和1.64%。所提模型有助于养殖过程中对鱼类行为实现精准监测,对精准养殖具有重要的推动作用。

基于Mel-GADF与ConvNeXt-T的变压器铁心松动故障诊断方法

为解决传统梅尔(Mel)时频谱图对变压器铁心松动程度识别率较低的问题,提出一种基于梅尔-格拉姆角差场(Mel-GADF)时频谱图与ConvNeXt-T网络相结合的变压器铁心松动故障诊断模型。将变压器声纹信号生成Mel时频谱图,同时将原始声纹数据经过格拉姆角场(GAF)变换得到格拉姆角和场(GASF)与GADF这2种时频谱图;生成Mel-GASF与Mel-GADF这2种特征融合的时频谱图来弥补Mel时频谱图的低频缺失问题;将3种时频谱图放入ConvNeXt-T网络进行训练对比,选出效果最佳的诊断模型。以型号为S13-M-200/10的变压器为对象进行空载试验,对不同铁心松动程度下的声纹信号进行分析,分析结果表明,将Mel-GADF作为特征时频谱图结合ConvNeXt-T网络,可将测试集准确率从传统Mel时频谱图的98.273%提升至99.500%,提升了1.227个百分点。

Promoting effect of ethanol on dewetting transition in the confined region of melittin tetramer

To study the influence of ethanol molecules on the melittin tetramer folding,we investigated the dewetting transition of the melittin tetramer immersed in pure water and 8%aqueous ethanol solution(mass fraction) by the molecular dynamics simulations.We found that the marked dewetting transitions occurred inside a nanoscale channel of the melittin tetramer both in pure water and in aqueous ethanol solution.Also,ethanol molecules promoted this dewetting transition.We attributed this promoting effect to ethanol molecules which prefer to locate at the liquidvapor interface and decrease the liquid-vapor surface energy.The results provide insight into the effect of ethanol on the water dewetting phenomena.

rhuPAa-melittin的生物活性检测

目的利用高纯度的rhuPAa-melittin进行人成纤维细胞和卵巢癌细胞进行活性检测,探讨rhuPAa-melittin对卵巢癌治疗作用及对正常细胞毒性作用。方法利用高纯度的rhuPAa-melittin蛋白分别作用于人成纤维细胞和人卵巢癌细胞SKOV3 48 h,在490 nm波长处测定其吸光度,以反映活细胞的数量并间接反映rhuPAa-melittin对培养中细胞的生长抑制作用。结果人卵巢癌细胞SKOV3组,在4μg/mL时对细胞抑制率已经达到了66.59%,在16μg/mL时,细胞已经全部死亡,差异有统计学意义(P<0.05)。而人成纤维细胞组,在4μg/mL时其对细胞的抑制率仅为达到7.3%,8μg/mL时抑制率为12.3%,16μg/mL时抑制率为22.0%。结论 rhuPAa-melittin对人卵巢癌细胞SKOV3细胞有着明显的抑制杀伤作用,而对于人成纤维细胞的抑制作用不明显。

Expression and Purification of the Melittin Gene from Polistes hebraeus by the GST Gene Fusion System

A cDNA encoding melittin from a wasp (Polistes hebraeus) was amplified and cloned into the GST fusion expression vector pGEX-4T-2. The expressed protein appeared on the SDS-PAGE profiles with an about 29 kDa band. Western blotting proved that the recombinant protein is the fusion protein of GST-PhM. The expression conditions of GST-PhM fusion protein for E. coli BL21 transformant were optimized. Thin layer scanning on the SDS-PAGE profiles showed that the expressed target protein had accumulated up to about 10%～12% of total protein of bacterial cells under the optimized expression condition. Purified and recovered recombinant melittin of Polistes hebraeus showed bioactivity in activating rabbit platelets to aggregate.

Real-time Analysis of the Interaction between Calmodulin and Melittin by SPR Spectroscopy

The dynamic interaction process of calmodulin with an immobilized peptide melittin was investigated in real time by surface plasmon resonance spectroscopy, and dissociation constant of the complex was calculated to be 3.3710-6 mol/L.

Low molecular fucoidan and its macromolecular complex with bee venom melittin

Low molecular weight (LMW) fucoidan, obtained by free radical depolymerization of high molecular polysaccharide extract of brown algae Hizikia fusiformis, was complexed with HPLC purified bee venom melittin. Water soluble form of the LMW fucoidan – melittin complex shows increased anti-inflammatory activity, inhibiting the production of nitric oxide in murine macrophage cell line Raw 264.7. The LMW fucoidan:melitin complex obtained in this study showed good biological activities, resulting in 2-fold reduction of the melittin toxicity. The fucoidan: melittin macromolecular complex obtained should be useful in future therapeutic applications.

Anti-angiogenesis effect of melittin on Mock/MHCC97-H cells by the regulation of cathepsin S in vivo

Objective： To study the anti-angiogenesis effect of melittin on human hepatoma Mock/MHCC97-H cells by regulatingthe expression of cathepsin S （CatS） in vivo. Methods： Models of in situ transplantation tumor of Mock/MHCC97-Hcells and silencing cathepsin shRNA-CatS/ MHCC97-H cells in nude mice were established. The model mice wererandomly divided into four groups. In the A1 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells andtreated with melittin. In the A2 group, the mice were inoculated with shRNA-CatS/MHCC97-H cells and treated withsaline. In the B1 group, the mice were inoculated with Mock/MHCC97-H cells and treated with melittin. In the B2 group,the mice were inoculated with Mock/MHCC97-H cells and treated with saline. The A1 and B1 group were injected withmelittin （80 mg/kg） intraperitoneally every day. The A2 and B2 group were injected with 0.2 mL normal salineintraperitoneally every day. After administration for 25 days, the animals were sacrificed. The tumor size and weight innude mice in each group were recorded. The expression of CD34 protein in the xenograft tumor tissues was detected byimmunohistochemistry. The expression of Cat S, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 andp-ERK1/2 proteins were detected by western blot. Results： The B1 group had significantly smaller tumor volumes andlower tumor weights than the B2 group （both P 〈 0.001）. There was no significant difference between the A1 group andA2 group in tumor volumes and weights. The number of CD34-positive microvessels in the B2 group was significantlyhigher than that in the A2 group （P 〈 0.001）. The number of CD34-positive microvessels in the B1 group wassignificantly lesser than that in the A1 group （P 〈 0.001）. Most strikingly, in the model featuring inoculation ofMock/MHCC97-H cells, CatS, VEGF-A, p-VEGFR2, Ras, Raf, p-Raf, MEK1, p-MEK1, ERK1/2 and p-ERK1/2expression were inhibited when treated with melittin. However, in the model featuring the inoculation ofshRNA-CatS/MHCC97-H cells, no such effects were observed with similar treatments. Conclusion： Melittin can inhibitthe growth of tumors and angiogenesis by blocking the CatS-VEGf-A signaling pathway.

rhuPAa-melittin发酵条件及大规模纯化方法

目的 通过rhuPAa-melittin在80 L发酵罐中的发酵表达,优化发酵条件及改进适合大规模纯化的方法,获得纯化的目的蛋白.方法 采用实验制备的rhuPAa-melittin重组质粒,通过电转化法将rhuPAa-melittin-pPICZαC载体转入毕赤酵母（Pichia pastoris）中,用PCR法扩增和检测rhuPAa-melittin基因在毕赤酵母基因组中的整合.利用毕赤酵母进行小量发酵,摸索最佳表达时间、pH及最佳的纯化方法.通过rhuPAa-melittin在80 L发酵罐中的发酵表达,优化发酵条件及改进适合大规模纯化的方法.结果 rhuPAa-melittin发酵条件在温度为28℃～30℃,FM21培养基中添加0.5％的蛋白胨,甲醇最终的流加速度控制在9 mL/（h.L）,初始发酵液体积、DO值（溶解氧）在25％～ 30％,发酵时间为12h,pH在6.0～7.0时获得高产量、高纯度、高收率的蛋白.结论 获得产量约350 mg/L,纯度可达96％以上,收率可达65％左右的rhuPAa-melittin蛋白.

基于Mel光谱数据增强和ResNet网络的滚动轴承故障诊断模型

针对工业数据采集困难和稀疏数据特征提取能力有限等问题,提出一种基于Mel光谱数据增强和ResNet网络的滚动轴承故障诊断模型。为了增加训练样本数据的多样性,引入了Mel光谱数据增强技术,提高模型的泛化能力,使其能够更好地适应各种滚动轴承故障情况。通过ResNet网络残差单元的堆叠深度捕捉复杂的故障特征信息,进而有效识别滚动轴承的故障模式。最后,对实验现场采集的滚动轴承故障数据进行验证评估。结果表明:所提模型在增强数据集上的诊断准确率高达99.83%,比原始数据集准确率提高了1.39%;与其他方法相比,该模型在准确性和鲁棒性方面都取得了显著改进,能更准确地识别滚动轴承的不同故障类型。

High-sensitive state perception method for inverter-fed machine turn insulation based on FrFT-Mel

Amidst the swift advancement of new power systems and electric vehicles,inverter-fed machines have progressively materialized as a pivotal apparatus for efficient energy conversion.Stator winding turn insulation failure is the root cause of inverter-fed machine breakdown.The online monitoring of turn insulation health can detect potential safety risks promptly,but faces the challenge of weak characteristics of turn insulation degradation.This study proposes an innovative method to evaluate the turn insulation state of inverter-fed machines by utilizing the fractional Fourier transform with a Mel filter(FrFT-Mel).First,the sensitivity of the high-frequency(HF)switching oscillation current to variations in turn insulation was analyzed within the fractional domain.Subsequently,an improved Mel filter is introduced,and its structure and parameters are specifically designed based on the features intrinsic to the common-mode impedance resonance point of the electrical machine.Finally,an evaluation index was proposed for the turn insulation state of inverter-fed machines.Experimental results on a 3kW permanent magnet synchronous machine(PMSM)demonstrate that the proposed FrFT-Mel method significantly enhances the sensitivity of turn insulation state perception by approximately five times,compared to the traditional Fourier transform method.

基于Mel频谱特征的水声信号分类研究

为解决传统的时域、频域特征不足以完全表征水声信号非线性、非高斯的问题,文章以海洋环境下采集的水声信号为对象,选用Mel频谱特征作为水声信号特征,提取了四类水声信号的Mel频谱特征制作数据集,分别用SVM分类器、BP神经网络分类器以及VGG16卷积神经网络分类器进行训练与分类测试,以验证水声信号识别能力。结果表明,三种分类器模型对四类水声信号的平均分类精度均可达89%以上,证明Mel频谱特征能够有效表达水声信号特征的能力。而BP神经网络分类器可获得优于SVM的分类精度,卷积神经网络的分类精度则高出BP神经网络6.0%,其平均分类准确率为97.5%,因此,基于VGG16卷积神经网络构建的分类器具有更好的识别能力。