Tet调控STGC3基因表达的CNE2细胞系裸鼠成瘤实验性研究

STGC3基因是一个新近克隆的肿瘤相关基因,前期的体外实验研究结果表明,STGC3基因在CNE2鼻咽癌细胞系高表达,可明显抑制CNE2的生长增殖.采用Tet/pTRE-STGC3/CNE2细胞系接种于裸鼠皮下,以强力霉素(Dox)诱导STGC3基因高表达,观测STGC3基因高表达对CNE2裸鼠体内成瘤的影响,并探讨其可能作用机制.运用RT-PCR、蛋白质印迹及免疫组织化学方法,分别从mRNA和蛋白质水平,分析瘤组织中STGC3基因的表达;用流式细胞仪检测移植瘤组织内肿瘤细胞的凋亡情况;用免疫组织化学方法,检测移植瘤组织凋亡相关蛋白Bcl-2和Bax的表达.研究结果显示,STGC3蛋白表达主要定位于细胞核内,Dox诱导STGC3基因在Tet/pTRE-STGC3/CNE2细胞系高表达,Tet/pTRE-STGC3/CNE2细胞系裸鼠体内成瘤受到明显抑制,与对照组比较,移植瘤成瘤时间晚、生长慢、肿块小、细胞凋亡率高,Bax蛋白表达增强,Bcl-2蛋白表达减少,差异有显著性意义(P<0.01).此体内实验研究结果与前期体外实验结果一致,进一步证明STGC3基因对肿瘤细胞生长具有抑制作用.     

Tet调控STGC3基因表达CNE2细胞系的建立及其功能初步研究

利用Tet-on调控系统,建立受强力霉素诱导STGC3基因表达的CNE2细胞系,为进一步研究STGC3的功能提供了一个理想的实验平台.先后将调控质粒pTet-on和反应质粒pTRE-STGC3转染入CNE2细胞,并用G418和潮霉素分别进行两轮筛选,运用RT-PCR选择对强力霉素诱导敏感的细胞克隆.用不同浓度强力霉素诱导CNE2/Tet/pTRE-STGC3细胞,RT-PCR检测STGC3的表达,确定强力霉素的最佳诱导浓度.采用此浓度的强力霉素分别诱导CNE2、CNE2/Tet/pTRE、CNE2/Tet/pTRE-STGC3三组细胞,测定细胞的生长曲线、克隆形成率和细胞周期分布.诱导STGC3基因高表达,CNE2细胞增殖速度显著减慢(P<0.05),克隆形成能力显著降低(P<0.01);流式细胞仪检测结果显示,瘤细胞群体中处于G0/G1期细胞数增加,细胞阻滞于G0/G1期.Tet调控STGC3基因表达CNE2细胞系的成功建立,为进一步研究STGC3基因的功能提供一个理想的细胞模型.     

NAG7基因转染对鼻咽癌细胞生长的影响

为了探讨鼻咽癌表达下调基因NAG7对鼻咽癌细胞系HNE1生长的影响, 构建了NAG7基因的真核表达载体pcDNA3.1(+)/NAG7, 并采用脂质体转染技术将真核重组体pcDNA3.1(+)/NAG7质粒和真核空载体pcDNA3.1(+)质粒分别导入HNE1细胞, 经G418筛选后获得稳定转染细胞克隆, RT-PCR和RNA印迹检测NAG7基因的表达, 并通过细胞生长曲线、裸鼠接种和流式细胞等方法对转染细胞的生物学行为进行检测.结果显示：转染NAG7基因后,基因表达增加,细胞生长倍增时间较空载体转染和HNE1明显延长,流式细胞技术检测表明,NAG7可延缓细胞由G0～G1期进入S期;裸鼠接种实验显示转染NAG7基因后的HNE1细胞致瘤性受到抑制.上述结果表明：NAG7基因转染后鼻咽癌细胞生长受到抑制,提示NAG7基因是一鼻咽癌相关的抑瘤基因候选者.     

STGC3基因缺失突变对CNE2细胞生长增殖能力的影响

为探讨鼻咽癌候选抑癌基因STGC3中层粘连蛋白G结构域(LG domain)对CNE2生长增殖能力的影响,应用基因定点突变技术将该结构域缺失,亚克隆至真核表达载体上,并将野生型及突变型STGC3稳定转染人鼻咽癌细胞系CNE2,检测其对CNE2细胞系表型的影响,包括测定稳转细胞系的生长曲线、细胞集落形成能力和细胞周期分布.研究发现,LGdomain的缺失明显降低了STGC3的肿瘤抑制活性,使受其稳定转染的细胞系恶性度显著增强,提示该结构域在STGC3发挥肿瘤抑制功能中起着重要作用.     

干扰素诱导的跨膜蛋白1在宫颈鳞癌中的表达及其生物学作用

目的:探讨干扰素诱导的跨膜蛋白1(IFITM1)在宫颈鳞癌中的表达及其生物学作用。方法:通过免疫组化、RT-PCR和Western blot检测宫颈鳞癌组织和癌旁组织中IFITM1的表达。使用靶向IFITM1的si RNA(si-IFITM1组)和高表达IFITM1基因的重组pcDNA3.1质粒(pcDNA3.1-si-IFITM1组)转染Si Ha细胞下调或上调IFITM1的表达。通过伤口愈合实验、Transwells迁移实验和基质胶侵袭实验检测细胞迁移和侵袭能力。细胞计数试剂盒-8 (CCK-8)检测细胞增殖;流式细胞术测定细胞凋亡。Western blot检测PTEN、PI3K和AKT的表达。通过对BALB/c Nude裸鼠接种Si Ha细胞来考察IFITM1在体外对肿瘤生长的影响。结果:癌组织中IFITM1的表达水平明显高于癌旁组织(P<0.05)。与对照组比较,si-IFITM1组的迁移和侵袭能力明显增强,而pcDNA3.1-si-IFITM1组明显降低(P<0.05)。细胞转染48 h和72 h后,与对照组比较,si-IFITM1组的细胞增殖明显增强,而pcDNA3.1-si-IFITM1组明显减弱(P<0.05)。与对照组比较,si-IFITM1组的细胞凋亡率明显降低,而pcDNA3.1-si-IFITM1组明显升高(P<0.05)。与对照组比较,si-IFITM1组的PTEN被下调,而PI3K和AKT被上调(P<0.05);pcDNA3.1-si-IFITM1组的PTEN的被上调,而PI3K和AKT被下调(P<0.05)。与对照组比较,si-IFITM1组裸鼠的肿瘤体积显著增大,而pcDNA3.1-si-IFITM1组显著减小(P<0.05)。结论:IFITM1过表达抑制人宫颈鳞癌细胞Si Ha的生长和转移能力,并在体外抑制肿瘤的形成,从而发挥抗癌作用。IFITM1可能通过调控PTEN/PI3K/AKT信号通路发挥抗癌作用。     

LMTK2基因沉默抑制人上皮性卵巢癌细胞生长和转移的作用机制

摘要 目的：探讨狐猴酪氨酸激酶2（LMTK2）基因沉默对人上皮性卵巢癌(EOC)细胞生长和转移的抑制作用及其可能的机制。方法：通过RT-qPCR和Western-blot检测了人正常卵巢上皮细胞IOSE80和人上皮性卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的表达,使用Lipofectamine 3000转染试剂将LMTK2的短发夹RNA（shRNA）、阴性对照shRNA、LMTK2过表达重组pcDNA3.1质粒或阴性对照质粒转染到SKOV3细胞中,并分为LMTK2-shRNA组、NC-shRNA组、LMTK2-pcDNA3.1组或NC-pcDNA3.1组。另外,使用PI3K/Akt抑制剂LY294002处理SKOV3细胞1 h。通过CCK-8法测定细胞增殖,Annexin V-FITC/PI染色法测定细胞凋亡,划痕实验评价细胞迁移,Transwell实验评价细胞侵袭。对BALB/c雌性裸鼠皮下注射转染NC-shRNA或LMTK2-shRNA的SKOV3细胞建立体内移植瘤模型,并记录接种28 d内的肿瘤体积。结果：与人正常卵巢上皮细胞IOSE80相比,卵巢癌细胞系（SKOV3、ES2、OVCAR-3和HEY）中LMTK2的mRNA和蛋白表达水平均显著升高,其中SKOV3的LMTK2 mRNA和蛋白表达水平最高（P<0.05）。与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞活力、相对迁移面积、侵袭细胞数均显著降低,而细胞凋亡率显著升高（P<0.05）。此外,与NC-shRNA组相比,LMTK2-shRNA组SKOV3细胞中Bax的蛋白表达水平显著升高,而Bcl-2、MMP2、MMP9、p-Akt的蛋白表达水平显著降低（P<0.05）。LY294002处理逆转了上调LMTK2对SKOV3细胞生长和转移的影响（P<0.05）。在接种第21天和28天时,与NC-shRNA组相比,LMTK2-shRNA组裸鼠的肿瘤体积显著降低（P<0.05）。结论：LMTK2基因沉默通过抑制PI3K/Akt信号通路降低了人上皮性卵巢癌细胞的生长和转移能力。     

鼻咽癌相关新基因NPCEDRG表达对CNE2细胞生长的影响

为研究鼻咽癌相关新基因NPCEDRG的功能,探讨其对鼻咽癌细胞生长特性的影响,利用Tet-on调控系统,建立受强力霉素(deoxycycline,Dox)诱导NPCEDRG基因表达的CNE2细胞系.运用RT-PCR选择背景表达低、诱导活性高的细胞克隆,以不同浓度Dox诱导CNE2/Tet/TRE-NPCEDRG细胞,确定Dox的最佳诱导浓度.借助形态学观察、细胞生长曲线、软琼脂克隆形成试验和流式细胞仪分析等方法,对Dox诱导NPCEDRG高表达后CNE2细胞的生物学行为进行了检测.结果显示,NPCEDRG高表达后CNE2细胞增殖速度显著减慢(P<0.05),克隆形成能力显著降低(P<0.01),瘤细胞群体中处于G0/G1期细胞数增加,S期细胞数减少,细胞阻滞于G0/G1期.Tet调控NPCEDRG基因表达CNE2细胞系成功建立,恢复NPCEDRG表达能部分逆转CNE2的恶性表型,证明NPCEDRG是一个鼻咽癌相关的抑瘤基因.     

人结肠癌SW480细胞裸鼠皮下移植瘤DcR3基因的RNA干扰及其作用

目的:观察DcR3基因小干扰RNA(siRNA)对人结肠癌SW480细胞裸鼠皮下移植瘤DcR3基因表达的影响。方法:建立结肠癌SW480细胞裸鼠皮下移植瘤模型,瘤体注射脂质体与DcR3siRNA混合物,转染DcR3siRNA,免疫组织化学及RT-PCR检测观察DcR3基因的表达。结果:建立了结肠癌SW480细胞裸鼠皮下移植瘤模型;治疗后,治疗组移植瘤明显减小,空白对照组、阴性对照组肿瘤体积显著大于治疗组(P<0.01);各组肿瘤组织中DcR3基因均有不同程度的表达,治疗组表达程度明显低于阴性对照组及空白对照组(RT-PCRP<0.05,免疫组化P<0.01)。结论:人结肠癌SW480细胞在裸鼠皮下有良好的成瘤性;脂质体与DcR3siRNA混合物可特异性抑制结肠癌裸鼠皮下移植瘤内DcR3基因的表达。     

RNA干扰NRP2基因表达对胃癌细胞SGC7901裸鼠移植瘤的抑制作用

目的:探讨特异性抑制NRP2基因表达对人胃癌细胞SGC7901裸鼠移植瘤生长及淋巴管新生的影响。方法:采用小RNA干扰方法,构建shNRP2质粒,稳定转染入SGC7901细胞株,Westen blot检测转染前后NRP2蛋白的表达。建立人胃癌细胞SGC7901裸鼠移植瘤模型,随机分为shNRP2组(实验组)、shCon组(HK阴性对照组)和正常对照组,观察移植瘤的生长情况。6周后处死裸鼠,免疫组化检测NRP2蛋白的表达及微淋巴管密度(Micro-vessel density,MLD)。结果:成功构建shNRP2质粒,与另两组比较,shNRP2组细胞NRP2蛋白表达明显降低,且移植瘤组织生长、NRP2表达及MLD明显受抑制。结论:抑制NRP2基因的表达,可以抑制胃癌裸鼠移植瘤的生长及淋巴管的形成,NRP2基因有可能成为一个潜在的胃癌生物治疗靶点。     

过表达SIRT3抑制裸鼠异位移植人肝细胞癌的生长及其机制研究

该文旨在研究人肝细胞癌异位移植瘤裸鼠模型中沉默信息调节因子3(silent information regulator 3,SIRT3)对肝细胞癌生长的影响及其机制。建立稳定过表达SIRT3和pc DNA3.1的SK-Hep-1细胞株;将稳定过表达SIRT3和pc DNA3.1的细胞悬液分别注射入裸鼠皮下,实时监测两组移植瘤的生长,25 d后剥离出移植瘤并称重;免疫组织化学检测移植瘤中SIRT3、Ki67的表达水平;应用定量逆转录PCR(q RT-PCR)筛选SIRT3影响移植瘤生长的下游靶向分子,Western blot检测下游靶向分子Bax的表达量以及移植瘤中cleaved-PARP(poly ADP-ribose polymerase)的表达水平。结果显示,过表达SIRT3组移植瘤的体积和重量都小于pc DNA3.1组;过表达SIRT3组移植瘤中Ki67的表达水平较pc DNA3.1组降低;过表达SIRT3上调Bax的m RNA和蛋白质水平并促进PARP的剪切。该文结果提示,SIRT3可能通过Bax凋亡信号通路抑制人肝细胞癌异位移植瘤的生长。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.