HMCES蛋白促进猪流行性腹泻病毒在Vero细胞中的复制

【目的】鉴定能够调控猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)复制的关键宿主蛋白。【方法】利用LC-MS/MS技术结合串联质谱标签(tandem mass tag,TMT)，分析PEDV感染Vero细胞36 h后和未感染组的蛋白组学差异。鉴定筛选了114个显著差异表达蛋白，其中宿主胚胎干细胞特异性5-羟甲基胞嘧啶结合蛋白(5-hydroxymethylcytosine binding,ES cell-specific protein,HMCES)显著上调。进一步构建HMCES真核表达质粒，通过蛋白免疫印迹和实时荧光定量PCR检测过表达HMCES对PEDV复制的影响；合成针对HMCES基因的特异性si RNA，利用Western blotting和RT-q PCR检测si RNA对HMCES表达的干扰效果及HMCES被干扰后对PEDV复制的影响。【结果】过表达HMCES能显著促进PEDV在Vero细胞中复制，并且复制水平随着HMCES的剂量递增呈现剂量依赖式增加；si RNA-341下调内源性HMCES表达进而抑制PEDV复制。【结论】H...     

RNA干扰对乙肝病毒基因的抑制作用

双链介导的遗传干涉的机制是1998年发现的。它通过双链RNA的介导特异性地降解相应序列从而导致转录后水平的基因沉默。RNA干扰作为后基因组时代的一种下调基因表达的工具已被广泛用于基因功能的研究以及疾病的治疗。利用小干扰RNA与乙肝病毒DNA通过共传染于HepG2肝癌细胞中使乙肝病毒基因沉默以达到抑制乙肝病毒复制作用。     

舞毒蛾Ldcht10基因序列的克隆及其功能分析

【目的】研究舞毒蛾Lymantria dispar几丁质酶基因的时空表达特性及其在蜕皮发育过程中的生物学功能,筛选在舞毒蛾发育过程中致死性的几丁质酶基因,为实现基于RNAi的舞毒蛾有效控制提供重要的基础数据。【方法】设计简并引物克隆舞毒蛾几丁质酶基因Ldcht10关键序列,通过使用实时荧光定量PCR方法检测该基因在舞毒蛾不同龄期与组织中的相对表达量,选取部分序列的双链RNA(ds RNA)研究该基因功能。【结果】本研究克隆了长度为2 057 bp的舞毒蛾几丁质酶基因Ldcht10序列;各组织与不同龄期RT-PCR结果表明Ldcht10的时空表达特性存在明显差异,Ldcht10在各个龄期均表达活跃且在前肠与后肠中的表达水平最高;Ldcht10的RNA干扰试验表明:注射Ldcht10 ds RNA后Ldcht10的表达受到极大抑制,该基因被沉默后24.3%的舞毒蛾幼虫因无法完成蜕皮或化蛹而死亡。【结论】舞毒蛾中几丁质酶基因Ldcht10在各个龄期与组织中的表达存在差异,且在舞毒蛾蜕皮过程中具有十分重要的生物学功能,该基因被沉默后部分舞毒蛾因无法完成蜕皮而导致死亡。     

RNA干扰ABCE1基因后可增加肺癌95-D/NCI-H446细胞的E-钙黏附蛋白表达并减低细胞侵袭力

研究ABCE1对肺癌(95-D和 NCI-H446)细胞的作用．使用RNA干扰技术,抑制ABCE1基因的表达,通过Western blot 分析及FACS检测,观察ABCE1基因对E-钙黏附蛋白在95-D/NCI-H446细胞表达的影响;运用transwell 侵袭实验,观察M95-D/ NCI-H446细胞侵袭力的变化．RNA干扰ABCE1基因后,实验组与对照组相比,在48 h后可显著抑制肺癌(95-D和 NCI-H446)细胞ABCE1蛋白的表达,同时,伴随E-钙黏附蛋白的高表达,以及细胞侵袭力的降低． ABCE1基因与E-钙黏附蛋白相关,抑制ABCE1基因可增加肺癌95-D/NCI-H446细胞的E-钙黏附蛋白的表达,减低细胞的侵袭力．     

MOI对PK-15细胞感染PPV后细胞因子mRNA转录时相的影响

【目的】更好地了解感染复数(MOI)对猪细小病毒(PPV)感染PK-15细胞后引起细胞因子的反应,探讨宿主-病毒之间的作用关系。【方法】运用荧光定量PCR技术,测定和分析3种感染复数对PPV感染PK-15细胞引起的病毒DNA量的变化和细胞因子IFN-、IRF-3、TNF-a和IL-18分泌水平。【结果】PPV感染PK-15细胞12 h病毒开始大量迅速增殖,感染48 h且MOI为1.0时达到最高峰;PPV感染后可引起PK-15细胞中IFN-、IRF-3、TNF-a和IL-18的表达量显著增加,其中IRF-3基因在感染后1 h且MOI为10.0时表达量达到967倍。【结论】细胞因子的分泌水平显著依赖感染复数和感染时间的交互作用。     

RNA干扰c-maf基因表达对多发性骨髓瘤细胞凋亡的影响及机制研究

目的:探讨RNA干扰c-maf基因表达对多发性骨髓瘤细胞凋亡的影响及机制。方法:将人骨髓瘤细胞株NCI-H929分为正常对照组、转染si RNA Control的阴性对照组和转染si RNA c-maf基因的沉默组,转染48 h后,提取细胞中的蛋白,Western blot检测各组细胞中c-maf的蛋白表达,流式细胞仪检测细胞凋亡情况,Western blot检测凋亡相关蛋白Cleaved caspase3及Wnt/β-catenin信号通路相关蛋白β-catenin和Cyclin D1蛋白表达。结果:阴性对照组c-maf的蛋白表达与正常对照组比较差异无统计学意义(P0.05),沉默组c-maf的蛋白表达显著低于正常对照组(P0.01),而阴性对照组c-maf的蛋白表达与正常对照组比较差异无统计学意义(P0.05)。与阴性对照组比较,沉默组的细胞凋亡率显著升高,Cleaved caspase3蛋白显著上调表达,β-catenin和Cyclin D1蛋白显著下调表达(P0.01)。结论:RNA干扰c-maf基因表达可诱导多发性骨髓瘤细胞凋亡,其机制可能与抑制Wnt/β-catenin信号通路有关。     

H1启动子siRNA载体的构建及应用

利用双链RNA(dsRNA)调控基因表达已经成为研究基因功能的有力工具。用人H1启动子构建了pBS／H1PS小干扰RNA(siRNA)表达载体,用于在哺乳动物细胞中产生特异性dsRNA转录产物。通过对293细胞中的PSMA7分子进行表达抑制,证明该siRNA载体能够有效产生针对靶基因的RNA干扰(RNAi)效应。     

犬细小病毒NS1 非结构蛋白可诱导细胞凋亡

【目的】研究犬细小病毒(Canine parvovirus,CPV)非结构蛋白NS1在CPV引起宿主细胞凋亡中的作用,初步探讨CPV引起细胞凋亡的机制。【方法】首先采用PCR方法从犬细小病毒基因组中扩增NS1编码基因,然后利用pcDNA3.1A质粒构建NS1真核表达载体pcDNA-NS1,并通过HEK293FT细胞瞬时表达NS1重组蛋白,用Western-blot检测以确定重组NS1蛋白能否在真核细胞中表达。然后用CPV感染和用pcDNA-NS1表达载体转染F81宿主细胞,通过AnnexinV/PI双染法检测磷脂酰丝氨酸外翻和通过化学发光法检测caspase-3/7活性,分析感染CPV或转染NS1基因对F81宿主细胞凋亡的影响。【结果】结果表明,本实验扩增的NS1基因序列与GenBank的序列一致,构建的表达载体结构正确,并能够介导NS1基因在真核细胞中表达。感染CPV和转染NS1基因均能诱导F81细胞膜磷脂酰丝氨酸外翻和明显提高细胞内caspase-3/7的活性,表明CPV和NS1蛋白均能引起细胞的凋亡。【结论】CPV诱导宿主细胞凋亡与其编码的NS1非结构蛋白有关。     

基因组编辑对酿酒酵母DNA的损伤作用及修复响应

【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。     

沉默BCAT1对肺癌细胞增殖、迁移及侵袭能力的影响

目的:研究BCAT1在肺癌细胞A549的增殖、迁移及侵袭能力中的作用。方法:通过小干扰RNA(si RNA)沉默A549细胞中BCAT1的表达,细胞分为对照组(Con)、BCAT1基因沉默组(si RNA-BCAT1)和si RNA阴性对照组(si RNA-NC)。利用Western blot检测si RNA对BCAT1的沉默效果;划痕愈合实验检测沉默BCAT1后A549细胞迁移能力的改变;Transwell小室侵袭实验检测沉默BCAT1后A549细胞侵袭能力的变化;MTT实验检测沉默BCAT1对A549细胞增殖能力的影响。结果:与Con组相比,si RNA-BCAT1组的BCAT1蛋白表达明显降低(P0.05),细胞划痕愈合率明显降低(P0.05),能够穿膜的细胞数明显减少(P0.05),而Con组和si RNA-BCAT1组细胞的增殖能力比较差异无明显统计学意义(P0.05)。结论:沉默BCAT1抑制A549细胞的迁移和侵袭能力,而对其增殖能力无影响。     

Effects of mycoplasma contamination on phenotypic expression of mitochondrial mutants in human cells.

HeLa cells sensitive to the mitochondrial protein synthesis inhibitors erythromycin (ERY) and chloramphenicol (CAP) and HeLa variants resistant to the effects of these drugs were purposefully infected with drug-sensitive and -resistant mycoplasma strains. Mycoplasma hyorhinis and the ERY-resistant strain of Mycoplasma orale, MO-ERYr, did not influence the growth of HeLa and ERY-resistant ERY2301 cells in the presence or absence of ERY. M. hyorhinis also did not affect the growth of HeLa and CAP-resistant Cap-2 cells in the presence or absence of CAP. However, both HeLa and Cap-2 cells infected with the CAP-resistant strain of M. hyorhinis, MH-CAPr, were more sensitive to the cytotoxic effect of CAP. This may be due to the glucose dependence of the cells, which was compromised by the increased utilization of glucose by MH-CAPr in these infected cell cultures. In vitro protein synthesis by isolated mitochondria was significantly altered by mycoplasma infection of the various cell lines. A substantial number of mycoplasmas copurified with the mitochondria, resulting in up to a sevenfold increase in the incorporation of [3H]leucine into the trichloroacetic acid-insoluble material. More importantly, the apparent drug sensitivity or resistance of mitochondrial preparations from mycoplasma-infected cells reflected the drug sensitivity or resistance of the contaminating mycoplasmas. These results illustrate the hazards in interpreting mitochondrial protein synthesis data derived from mycoplasma-infected cell lines, particularly putative mitochondrially encoded mutants resistant to inhibitors of mitochondrial protein synthesis.     

Rapid tissue culture method for detection of Mycoplasma hyorhinis

An easy, rapid tissue culture method for detection of Mycoplasma hyorhinis is described. The organism induces morphological changes in mink S + L - cells. This effect was not observed in eight other animal cell lines infected by M. hyorhinis and it did not occur in the mink cells infected with six other strains of mycoplasma. This cell system should be useful in research laboratories which do not have other standard techniques available for monitoring the presence of M. hyorhinis.     

The RNA-binding protein CUG-BP1 increases survivin expression in oesophageal cancer cells through enhanced mRNA stability

In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 μM) and deoxyuridine (Km=578 μM), it prefers uridine (Km=92 μM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.     

Impact of Mycoplasma hyorhinis infection on L-arginine metabolism: differential regulation of the human and murine iNOS gene

Infection with mycoplasma is a common problem in cell cultures, with Mycoplasma hyorhinis being the predominant species. Here we investigate the effect of M. hyorhinis infection on L-arginine metabolism, with focus on iNOS-mediated NO synthesis in murine keratinocytes and the human colon cancer cell line DLD-1. iNOS and arginase are L-arginine-metabolizing enzymes involved in the regulation of inflammatory processes, with NO contributing to innate immunity. In murine cells, M. hyorhinis infection enhances cytokine-induced iNOS expression and augments iNOS activity, whereas in the absence of cytokines it causes de novo induction of iNOS mRNA without subsequent translation into iNOS protein. In turn, arginase-1 mRNA expression is diminished in M. hyorhinis-infected murine keratinocytes, resulting in decreased arginase activity. One of the underlying upstream mechanisms is NF-kappaB activation. In contrast, in human cells neither iNOS mRNA nor protein expression is affected by M. hyorhinis infection, but NO synthesis is enhanced, which may be caused by increased L-arginine import. This demonstrates that infection with M. hyorhinis leads to different effects on gene regulation of the murine and human iNOS gene. Our study underlines the importance of routine checking of cell cultures for mycoplasma contamination, particularly in studies on NO-mediated effects or inflammatory processes.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Reiterated DNA sequences defining genomic diversity within the species Mycoplasma hyorhinis

Genomic restriction fragments isolated from Mycoplasma hyorhinis and Mycoplasma hyopneumoniae were shown by DNA hybridization and nucleotide sequence analyses to contain sequences common to these two species, as well as another porcine-derived mycoplasma, Mycoplasma flocculare. Intraspecies hybridization experiments using these fragments as probes indicated that the sequence is highly redundant in several strains of M. hyorhinis, but that there is diversity in the sizes of restriction fragments detected among these strains. In contrast, repetition of the sequence was limited in M. hyopneumoniae and M. flocculare, and no homologies to this repeated element were apparent in mycoplasma species isolated from animal hosts other than the swine. The reiterated sequence may reflect intraspecies genomic diversification in M. hyorhinis and its selective presence in otherwise unrelated species raises the possibility that it has been horizontally transmitted between these organisms.     

Mycoplasmal infection of insect cell cultures

Twenty-five cell cultures of three insect orders from eight laboratories were tested for mycoplasmal infection. Acholeplasma laidlawii was detected in one culture, an incidence of 4.0%. A. laidlawii, Mycoplasma orale, M. arginini, but not M. hyorhinis, could establish infections of drosophila Dm-1 cell cultures at 25 degrees C. In prospective studies, drosophila Dm-1 cultures were intentionally infected with broth-propagated A. laidlawii and M. hyorhinis. M. hyorhinis did not grow and was eliminated from the Dm-1 cultures during consecutive passages. A. laidlawii grew without obvious cytopathic effects during six weekly passages; titers of over 10(7) CFU/ml were recorded at Passages 2 and 5 (p2 and p5). Minimal cell culture infectious doses were also determined during these studies. 0.1 milliliter cell samples were inoculated into Leighton tubes containing either fresh M1A culture medium or 3T6 indicator cells in McCoy's 5a medium. After 4 d of incubation at 25 and 37 degrees C, respectively, the cover slips were stained by DNA fluorochrome Hoechst 33258 (A. laidlawii) or by specific fluorescein-conjugated antiserum (M. hyorhinis). At p2 with both mycoplasma species, the procedure using M1A medium and incubation at 25 degrees C without 3T6 cells was inferior to indicator cells. In five of six experiments at least a two-log higher titer of mycoplasmas was needed to be detected with M1A and 25 degrees C. At p5 no difference could be found. Uridine phosphorylase assays of Dm-1 cultures infected with A. laidlawii, M. hyorhinis, M. orale, and M. arginini gave clearly positive results only with A. laidlawii. The ratio of incorporated uridine to incorporated uracil method yielded false positives with two drosophila cell lines. Suggestions for assay of mycoplasmas in invertebrate cell cultures are given.     

Mycoplasma nucleases able to induce internucleosomal DNA degradation in cultured cells possess many characteristics of eukaryotic apoptotic nucleases

It was previously shown (Paddenberg et al (1996) Eur J Cell Biol 69, 105 - 119) that cells of established lines like NIH3T3 fibroblasts and the human pancreatic adenocarcinoma PaTu 8902 line only degrade their chromatin at internucleosomal sites after an apoptotic stimulus when infected with Mycoplasma hyorhinis. In order to distinguish mycoplasma nucleases (Mr 47 - 54 kDa) from already described eukaryotic apoptotic enzymes, the mycoplasma nucleases were partially purified from serum-free culture supernatants and further characterized. Here we demonstrate directly that the enriched mycoplasma nucleases were able to fragment the DNA of nuclease-negative substrate nuclei at internucleosomal sites. The DNA degradation was accompanied by morphological changes typical of apoptosis like chromatin condensation and margination followed by shrinkage of the nuclei. The biochemical characterization revealed that the mycoplasma nucleases had a neutral to weakly basic pH-optimum. They required both calcium and magnesium in the mM range for maximal activation and were inhibited by zinc chloride, EGTA and EDTA. In two dimensional zymograms they migrated as three spots with isoelectic points between 8.1 and 9.5. They were not inhibited by monomeric actin. Our data also demonstrate that nuclear extracts prepared from nuclei isolated from Mycoplasma hyorhinis infected cells contained the mycoplasma nuclease activities leading to their internucleosomal DNA-degradation after incubation in the presence of calcium and magnesium.