Evaluation of genetic response of mesenchymal stem cells to nanosecond pulsed electric fields by whole transcriptome sequencing

BACKGROUND Mesenchymal stem cells(MSCs)modulated by various exogenous signals have been applied extensively in regenerative medicine research.Notably,nanosecond pulsed electric fields(nsPEFs),characterized by short duration and high strength,significantly influence cell phenotypes and regulate MSCs differentiation via multiple pathways.Consequently,we used transcriptomics to study changes in messenger RNA(mRNA),long noncoding RNA(lncRNA),microRNA(miRNA),and circular RNA expression during nsPEFs application.AIM To explore gene expression profiles and potential transcriptional regulatory mechanisms in MSCs pretreated with nsPEFs.METHODS The impact of nsPEFs on the MSCs transcriptome was investigated through whole transcriptome sequencing.MSCs were pretreated with 5-pulse nsPEFs(100 ns at 10 kV/cm,1 Hz),followed by total RNA isolation.Each transcript was normalized by fragments per kilobase per million.Fold change and difference significance were applied to screen the differentially expressed genes(DEGs).Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were performed to elucidate gene functions,complemented by quantitative polymerase chain reaction verification.RESULTS In total,263 DEGs were discovered,with 92 upregulated and 171 downregulated.DEGs were predominantly enriched in epithelial cell proliferation,osteoblast differentiation,mesenchymal cell differentiation,nuclear division,and wound healing.Regarding cellular components,DEGs are primarily involved in condensed chromosome,chromosomal region,actin cytoskeleton,and kinetochore.From aspect of molecular functions,DEGs are mainly involved in glycosaminoglycan binding,integrin binding,nuclear steroid receptor activity,cytoskeletal motor activity,and steroid binding.Quantitative real-time polymerase chain reaction confirmed targeted transcript regulation.CONCLUSION Our systematic investigation of the wide-ranging transcriptional pattern modulated by nsPEFs revealed the differential expression of 263 mRNAs,2 miRNAs,and 65 lncRNAs.Our study demonstrates that nsPEFs may affect stem cells through several signaling pathways,which are involved in vesicular transport,calcium ion transport,cytoskeleton,and cell differentiation.

Tumor ablation with nanosecond pulsed electric fields

Liver cancer is one of the most malignant cancers. It is reported that 600 000 patients died from liver cancer every year. [1,2] Hepatocellular carcinoma (HCC) is a particular problem because symptoms are not evident until the disease has progressed and hepatitis B, which is prominent specific regions of Asia, is a common precursor of the disease. There are many liver cancer

Spatiotemporal characteristics of nanosecond pulsed discharge in an extremely asymmetric electric field at atmospheric pressure

In this paper,high resolution temporal-spatial diagnostics are employed to research the optical characteristics of nanosecond pulsed dielectric barrier discharge in needle-plate electrode configuration.Temporal-spatial distributions of discharge images,the emission intensities of optical emission spectra,and plasma vibrational and rotational temperatures are investigated.By analyzing the evolution of vibrational and rotational temperatures in space and time dimensions,the energy distribution and energy transfer process in plasma are also discussed.It is found that a diffuse structure with high density plasma concentrated in the region near the needle tip can be presented in nanosecond pulsed discharge,and an obvious energy transfer from electronic energy to vibration energy can be observed in each discharge pulse.

Study of the selective effect on cells induced by nanosecond pulsed electric field with the resistor-capacitor circuit model

A resistor-capacitor(RC)circuit model is proposed to study the effect of nanosecond pulsed electricfield on cells according to the structure and electrical parameters of cells.After a nanosecond step fieldhas been applied,the variation of voltages across cytomembrane and mitochondria membrane both in nor-mal and in malignant cells are studied with this model.The time for selectively targeting the mitochondriamembrane and malignant cell can be evaluated much easily with curves that show the variation of voltageacross each membrane with time.Ramp field is the typical field applied in electrobiology.The voltagesacross each membrane induced by ramp field are analyzed with this model.To selectively target the mito-chondria membrane,proper range of ramp slope is needed.It is relatively difficult to decide the range ofa slope to selectively affect the malignant cell.Under some conditions,such a range even does not exist.

Recent progress in pulsed electric field ablation for liver cancer

The number of liver cancer patients is likely to continue to increase in the coming decades due to the aging of the population and changing risk factors.Traditional treatments cannot meet the needs of all patients.New treatment methods evolved from pulsed electric field ablation are expected to lead to breakthroughs in the treatment of liver cancer.This paper reviews the safety and efficacy of irreversible electroporation in clinical studies,the methods to detect and evaluate its ablation effect,the improvements in equipment and its antitumor effect,and animal and clinical trials on electrochemotherapy.We also summarize studies on the most novel nanosecond pulsed electric field ablation techniques in vitro and in vivo.These research results are certain to promote the progress of pulsed electric field in the treatment of liver cancer.

Discharge Characteristics of SF_6 in a Non-Uniform Electric Field Under Repetitive Nanosecond Pulses

The characteristics of high pressure sulphur hexafluoride（SF6） discharges in a highly non-uniform electric field under repetitive nanosecond pulses are investigated in this paper.The influencing factors on discharge process,such as gas pressure,pulse repetition frequency（PRF）,and number of applied pulses,are analyzed.Experimental results show that the corona intensity weakens with the increase of gas pressure and strengthens with the increase of PRF or number of applied pulses.Spark discharge images suggest that a shorter and thicker discharge plasma channel will lead to a larger discharge current.The number of applied pulses to breakdown descends with the increase of PRF and ascends with the rise of gas pressure.The reduced electric field（E/p） decreases with the increase of PRF in all circumstances.The experimental results provide significant supplements to the dielectric characteristics of strongly electronegative gases under repetitive nanosecond pulses.

Nanosecond pulsed electric fields prime mesenchymal stem cells to peptide ghrelin and enhance chondrogenesis and osteochondral defect repair in vivo

Mesenchymal stem cells(MSCs) are important cell sources in cartilage tissue development and homeostasis,and multiple strategies have been developed to improve MSCs chondrogenic differentiation with an aim of promoting cartilage regeneration.Here we report the effects of combining nanosecond pulsed electric fields(ns PEFs) followed by treatment with ghrelin(a hormone that stimulates release of growth hormone) to regulate chondrogenesis of MSCs.ns PEFs and ghrelin were observed to separately enhance the chondrogenesis of MSCs,and the effects were significantly enhanced when the bioelectric stimulation and hormone were combined,which in turn improved osteochondral tissue repair of these cells within Sprague Dawley rats.We further found that ns PEFs can prime MSCs to be more receptive to subsequent stimuli of differentiation by upregulated Oct4/Nanog and activated JNK signaling pathway.Ghrelin initiated chondrogenic differentiation by activation of ERK1/2 signaling pathway,and RNA-seq results indicated 243 genes were regulated,and JAK-STAT signaling pathway was involved.Interestingly,the sequential order of applying these two stimuli is critical,with ns PEFs pretreatment followed by ghrelin enhanced chondrogenesis of MSCs in vitro and subsequent cartilage regeneration in vivo,but not vice versa.This synergistic prochondrogenic effects provide us new insights and strategies for future cell-based therapies.

Improvement of Spatial Uniformity of Nanosecond-Pulse Diffuse Discharges in a Multi-Needle-to-Plane Gap

Large-scale non-thermal plasmas generated by nanosecond-pulse discharges have been used in various applications, including surface treatment, biomedical treatment, flow con- trol etc. In this paper, atmospheric-pressure diffuse discharge was produced by a homemade nanosecond-pulse generator with a full width at half maximum of 100 ns and a rise time of 70 ns. In order to increase the discharge area, multi-needle electrodes with a 3~3 array were designed. The electrical characteristics of the diffuse discharge array and optical images were investigated by the voltage-current waveforms and discharge images. The experimental results showed that the intensity of diffuse discharges in the center was significantly weaker than those at the margins, resulting in an inhomogeneous spatial uniformity in the diffuse discharge array. Simulation of the electric field showed that the inhomogeneous spatial uniformity was caused by the non-uniform distribution of the electric field in the diffuse discharge array. Moreover, the spatial uniformity of the diffuse discharge array could be improved by increasing the length of the needle in the centre of the array. Finally, the experimental results confirmed the simulation results, and the spatial uniformity of the nanosecond-pulse diffuse discharge array was significantly improved.

纳秒脉冲电场消融治疗心房颤动的前景

导管消融已成为维持心房颤动患者窦性心律的主要治疗方法,肺静脉隔离是最常用的治疗策略。与应用于心房颤动消融的热能相比,不可逆电穿孔(Irreversible Electroporation,IRE)是一种非热替代方法,在细胞上施加高压场以诱导细胞膜发生改变,产生纳米孔,从而损害细胞的完整性和生存能力。纳秒脉冲电场消融(Nanosecond Pulsed Electric Field Ablation,nsPFA)具有通过IRE产生跨壁连续病变的能力,传输到心脏组织的热量较小,并且心肌细胞对电场有超高敏感性,因此,其具有安全性和有效性。本文总结了nsPFA治疗心房颤动的基本原理和当前的科学证据,并将其与最近的实践建议进行结合,讨论新兴技术和未来的发展方向。

纳秒脉冲电场肿瘤消融关键技术研究综述

癌症已成为威胁人类健康的主要疾病,传统治疗方法虽有疗效,但都有一定的禁忌证和副作用,而不可逆电穿孔作为一种新的局部物理治疗肿瘤技术,相对于传统的肿瘤治疗方式,具有独特的优势,可用于邻近血管、神经等重要器官和组织的治疗。临床研究表明,微秒级电脉冲产生的细胞不可逆电穿孔在消融肿瘤的同时会带来人体明显的肌肉收缩和心律失常现象,而纳秒脉冲消融肿瘤则通过改变脉冲发生器的硬件实现方式,提高电压,压缩脉宽,克服了上述缺点。本文介绍纳秒脉冲消融肿瘤技术的兴起与优点,综述国内外纳秒脉冲消融肿瘤的研究与临床应用现状,分析纳秒脉冲消融设备的迭代技术优势,从纳秒脉冲功率技术、纳秒脉冲电场分布、电场强度阈值和不同电脉冲参数消融肿瘤带来的不同量效关系等方面展望了纳秒脉冲消融肿瘤关键技术的最新动态及发展方向,并对纳秒脉冲消融肿瘤设备的临床应用进行了展望。

不可逆电穿孔肿瘤消融技术的过去和现在

不可逆电穿孔(Irreversible Electroporation,IRE)是在脉冲电场作用下,细胞膜脂质双分子层或生物组织产生不可逆亲水性孔道的现象,其生物效应为使细胞产生凋亡或死亡。2021年,国家药监局批准了2个国产IRE设备上市。至今,这个技术已经在国内多家医院进行了肝癌、胰腺癌、前列腺癌等的临床治疗应用,取得良好的治疗效果。本文综述了IRE肿瘤消融技术的国内外发展情况,从其发展脉络展望未来发展方向。

The latest research progress on minimally invasive treatments for hepatocellular carcinoma

Background:Hepatocellular carcinoma(HCC)is the fourth leading cause of cancer-related death worldwide.Due to the high prevalence of hepatitis B virus(HBV)infection in China,the incidence of HCC in China is high,and liver cirrhosis caused by chronic hepatitis also brings great challenges to treatment.This paper reviewed the latest research progress on minimally invasive treatments for HCC,including percutaneous thermal ablation and new nonthermal ablation techniques,and introduced the principles,advantages,and clinical applications of various therapeutic methods in detail.Data sources:The data of treatments for HCC were systematically collected from the Pub Med,Science Direct,American Chemical Society and Web of Science databases published in English,using“minimally invasive”and“hepatocellular carcinoma”or“liver cancer”as the keywords.Results:Percutaneous thermal ablation is still a first-line strategy for the minimally invasive treatment of HCC.The effect of microwave ablation(MWA)on downgrading treatment before liver transplantation is better than that of radiofrequency ablation(RFA),while RFA is more widely used in the clinical practice.High-intensity focused ultrasound(HIFU)is mainly used for the palliative treatment of advanced liver cancer.Electrochemotherapy(ECT)delivers chemotherapeutic drugs to the target cells while reducing the blood supply around HCC.Irreversible electroporation(IRE)uses a microsecond-pulsed electric field that induces apoptosis and necrosis and triggers a systemic immune response.The nanosecond pulsed electric field(ns PEF)has achieved a good response in the ablation of mice with HCC,but it has not been reported in China for the treatment of human HCC.Conclusions:A variety of minimally invasive treatments provide a sufficient survival advantage for HCC patients.Nonthermal ablation will lead to a new wave with its unique advantage of antitumor recurrence and metastasis.

大气压空气纳秒脉冲板-板放电中逃逸电子产生机理

经典放电理论(Townsend和流注理论)解释纳秒脉冲气体放电存在局限性,近年来基于高能电子逃逸的纳秒脉冲气体放电理论研究受到广泛关注.但是目前对大气压空气纳秒脉冲板-板放电中逃逸电子产生机理研究仍较少,严重阻碍了纳秒脉冲放电等离子体的应用发展.本文利用一维粒子模型,对幅值为20 kV的纳秒脉冲电压驱动下,间隙长为1 mm的板-板电极之间的大气压空气放电中逃逸电子的产生机理进行了数值模拟研究..结果表明,在空间电荷动力学行为的影响下,板-板电极之间出现了增强电场区域,使得电子可以满足电子逃逸判据而进入逃逸模式.此外,还观察到放电通道前逃逸电子的预电离效应导致了二次电子崩的产生,随着二次电子崩与放电通道不断汇聚,引导并加速了放电通道的发展,最终导致气隙击穿.本研究进一步揭示了纳秒脉冲板-板放电机理,拓展了纳秒脉冲气体放电基础理论,为纳秒脉冲放电等离子体的应用和发展开辟了新的机会.

纳秒脉冲电场局部消融小鼠三阴性乳腺癌的研究

目的三阴性乳腺癌(Triple Negative Breast Cancer,TNBC)是难治性的恶性肿瘤。纳秒脉冲电场(Nanosecond Pulsed Electric Field,nsPEF)是一种新型的局部消融方法,适用于浅表肿瘤的治疗。本研究旨在探究nsPEF消融小鼠TNBC的合适场强,并验证其治疗效果。方法分别利用0、20、30 kV/cm的场强进行小鼠4T1肿瘤消融细胞凋亡的研究,并探究免疫细胞浸润的情况。结果30 kV/cm场强组肿瘤完全消融率达54.5%,脉冲后0 h和24 h肿瘤细胞总体凋亡率分别为74.0%、34.8%。CD8^(+)和CD4^(+)T细胞主要位于对照组肿瘤边缘,脉冲后肿瘤内部浸润增加,特别是在消融边界区。结论30 kV/cm的场强可有效消融小鼠TNBC,nsPEF促进免疫细胞向肿瘤内部浸润。

纳秒脉冲电场治疗黑色素瘤的前景

作为一种新型的治疗手段,纳秒脉冲电场(Nanosecond Pulse Electric Field,nsPEF)在黑色素瘤治疗中显示出了良好的应用前景。本文综述了nsPEF治疗黑色素瘤在细胞实验、动物实验与人体实验方面的研究进展,指出了nsPEF可通过多种机制诱导黑色素瘤消融,并产生长期抗肿瘤记忆抑制其复发转移,同时具有高度靶向效果,不产生额外热损伤。这些独特优势对于黑色素瘤治疗具有广阔的临床应用前景,为临床治疗黑色素瘤提供了新的思路。

纳秒脉冲电场在运动系统中的应用前景

纳秒脉冲电场(Nanosecond Pulsed Electric Fields,nsPEF)是一种新兴的治疗技术,具有短脉冲宽度和高能量强度等特点。传统的高压脉冲电场已证实该项技术能够成功地应用于食品消毒保存、污水处理、生物育种及恶性肿瘤的电治疗等领域,具有极大的开发价值与应用前景。现在针对nsPEF在恶性肿瘤物理治疗中的研究也已非常深入,但在肌骨运动系统还相对有所不足。本研究从nsPEF在骨折、肌腱/韧带疾患及肌肉损伤中入手,进一步总结nsPEF的重要意义与应用前景,并对未来相关的研究进行展望。

纳秒脉冲电场刺激促进人真皮成纤维细胞成骨的实验研究

目的 探究超短纳秒脉冲电场(Nanosecond Pulsed Electric Field,nsPEF)是否能够促进成纤维细胞向成骨细胞的表型转化。方法 用0、5 kV/cm的场强刺激成纤维细胞,观察其在普通培养基和三向分化诱导培养基中生长情况,蛋白印迹测定细胞内调控生长及分化Hippo通路下游信号分子水平,并通过体内注射细胞-凝胶复合体进行裸鼠体内成骨实验。结果 5kV/cm nsPEF促进了成纤维细胞的增殖,并促进了成骨、成软骨分化,但对成脂分化影响不显著。低能量脉冲电场刺激抑制了Hippo通路下游各信号分子磷酸化水平,促进了裸鼠体内成骨,灰度分析显示,YAP1及LATS1的磷酸化蛋白与非磷酸化蛋白比值在5 kV/cm的脉冲电场刺激后明显降低,提示HIPPO信号通路受到抑制(P<0.05)。结论 低强度nsPEF刺激有效促进了成纤维细胞的增殖和表型转化,并在裸鼠模型上证实了可行性和有效性。

纳秒脉冲电场在肝脏肿瘤消融中的研究进展

纳秒脉冲电场(Nanosecond Pused Electric Field,nsPEF)作为一种新型的实体肿瘤消融物理因子,在肝癌治疗中受到广泛关注,目前已进展到临床研究阶段。大量研究表明,nsPEF不仅能够诱导肝癌细胞凋亡、消融肝脏肿瘤,还能激发机体的先天和适应性免疫反应,抑制肝脏肿瘤的复发和转移。本文对nsPEF在肝脏肿瘤消融中的研究进展进行综述,并对尚需研究和探讨的问题进行展望,以期进一步推动nsPEF肝脏肿瘤消融的应用。

纳秒脉冲电场技术的皮肤恶性肿瘤应用前景

皮肤恶性肿瘤主要包括基底细胞癌、皮肤鳞状细胞癌和恶性黑色素瘤。对于皮肤恶性肿瘤的治疗主要包括根治性手术和全身治疗。但是手术造成的周围健康组织损伤和全身治疗的副作用在一定程度上影响了患者的预后。纳秒脉冲电场(Nanosecond Pulsed Electric Field,nsPEF)是一种新兴的电脉冲消融技术,其具有微创、非热能、非药物、几乎不产生瘢痕等优点。在之前的研究中已经证实了nsPEF能够有效地诱导肿瘤细胞凋亡,并且能够激发机体强大的免疫反应。但是目前国内对nsPEF的相关理论知之甚少,本文旨在概述国内外nsPEF在皮肤恶性肿瘤中的应用情况,为nsPEF应用临床提供参考。

纳秒脉冲电场通过破坏线粒体诱导心肌消融研究

目的通过纳秒脉冲电场(Nanosecond Pulsed Electric Field,nsPEF)对体内外心肌细胞的实验研究,探讨其对细胞凋亡及对心脏活动的影响,并通过转录组研究进一步探讨机制。方法利用0~20 kV/cm场强对体外H9c2心肌细胞进行各50次电击的消融试验,确定能实现有效细胞凋亡的参数,进一步在此参数下进行小鼠心肌消融及转录组研究。结果15 kV/cm场强+50次电击体外心肌细胞在0、24、48 h的凋亡率分别为62.3%、78.8%、80.6%,可见消融区域线粒体膜结构明显破坏,诱导心肌细胞进入凋亡调控,导致心肌细胞释放细胞色素c;小鼠心肌完全消融率达76.2%。结论15 kV/cm场强+50次电击通过破坏线粒体诱导心肌凋亡并最终实现消融区域心肌细胞坏死性凋亡,且不影响心脏周围的其他组织,可作为心房颤动(Atrial Fibrillation,AF)消融的有效手段。