中国人肠道产甲酸草酸杆菌frc基因的分离及其在真核细胞中的表达

目的：探讨中国人肠道产甲酸草酸杆菌甲酰辅酶A转移酶基因（frc）的分离、克隆和鉴定。方法：提取中国人肠道产甲酸草酸杆菌的基因组DNA，利用PCR技术扩增frc基因片段并克隆入真核表达载体pEGFP—C1，重组质粒命名为pEGFP—frc，通过限制性内切酶酶切电泳和测序鉴定插入片段。将pEGFP—frc通过脂质体转染293细胞，通过逆转录PCR（RT—PCR）和蛋白印迹（Western blot）分别从mRNA和蛋白水平检测frc基因在真核细胞中的表达。结果：中国人肠道产甲酸草酸杆菌fre基因全长1287bp，存在53个碱基和4个氨基酸残基的变异，与GenBank中的frc基因的碱基序列的同源性为95．88％，氨基酸残基序列的同源性为99．07％。转染293细胞后24-72h，可观察到明亮的绿色荧光，RT—PCR和Western blot分别从mRNA和蛋白水平上检测到frc基因在真核细胞中的表达。结论：中国人肠道产甲酸草酸杆菌中可以分离出frc基因；中国人肠道产甲酸草酸杆菌frc基因存在一定的变异；frc基因可在真核细胞293细胞中表达。     

产甲酸草酸杆菌FRC基因慢病毒表达载体构建及鉴定

产甲酸草酸杆菌(Oxalobacter formigenes,Ox.F)是一种寄居于脊椎动物胃肠道、依赖分解肠道草酸生存的革兰阴性菌[12].我们采用基因克隆重组技术从前期分离培养的中国人肠道Ox.F[3]中克隆了草酸分解关键酶--甲酰辅酶A转移酶(FCoAT)基因FRC,并成功构建重组慢病毒表达载体pLenti6.3-FRC-IRES-EGFP,为高草酸尿症基因治疗提供基础.     

中国人肠道产甲酸草酸杆菌oxc基因的克隆及其在真核细胞中的表达

目的 观察中国人肠道产甲酸草酸杆菌(Ox.F)草酰辅酶A脱羧酶基因(oxc)的分离、克隆及其在293细胞中的表达.方法 提取中国人肠道Ox.F的基因组DNA,PCR扩增oxc基因片段并克隆入真核表达载体pEGFP-C1,通过限制性内切酶酶切电泳和测序鉴定基因片段.将重组质粒脂质体转染至293细胞,利用RT-PCR和Western blot分别从mRNA和蛋白水平检测oxc基因在真核细胞中的表达.结果 中国人肠道产甲酸草酸杆菌oxc基因全长1707 bp,与Gene Bank中的序列比较,碱基序列的同源性为93.61%,氨基酸残基序列的同源性为97.18%.重组质粒转染293细胞后24～48 h,可观察到明亮的绿色荧光,从mRNA和蛋白水平上可以检测到oxc基因在真核细胞中的表达.结论 中国人肠道产甲酸草酸杆菌中可以分离出oxc基因;中国人肠道产甲酸草酸杆菌oxc基因存在一定的变异;oxc基因可在真核细胞293细胞中表达.     

产甲酸草酸杆菌甲酰辅酶A转移酶基因和草酰辅酶A脱羧酶基因慢病毒表达载体的构建及鉴定

目的 构建国人肠道来源产甲酸草酸杆菌(OxCF)甲酰辅酶A转移酶(FCoAT)基因(FRC)和草酰辅酶A脱羧酶(OCoAD)基因(OXC)的重组慢病毒pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED,为高草酸尿症的基因治疗奠定基础.方法 采用Taq高保真DNA聚合酶从OxCF基因组中扩增FRC基因和OXC基因片段,用DNA凝胶回收试剂盒切胶回收,用DNA连接试剂盒将回收产物分别与pMD18-T Simple载体连接获得FRC和OXC的克隆载体,并转化大肠杆菌DH5α感受态细胞,挑取阳性克隆并测序,筛选携带完整目的基因质粒pMD18T simple-FRC和pMD18T simple-OXC,用BamH Ⅰ酶切获得目的基因,连接FRC和携带绿色荧光蛋白的pLenti6.3/v5DEST-IRES-EGFP,连接OXC与表达红色荧光蛋白的pLenti6.3/v5 DEST-IRES-DsRED,转化DH5α后挑取阳性克隆并测序.构建的慢病毒过表达载体pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED转染HEK293T细胞,包装并测滴度.结果 聚合酶链反应(PCR)从OxCF基因组中扩增分获得1.3kb和1.7kb的DNA片段,与Genbank中FRC( U82167)间碱基序列匹配率为95.88％,存在53个碱基的变异;与Genbank中OXC( M77128)间碱基序列匹配率为93.61％,存在109个碱基的变异;测序证实pLenti6.3-FRC-IRES-EGFP和pLenti6.3-OXC-IRES-DsRED分别含有大小正确的正向FRC cDNA和OXC cDNA,转染HEK293T细胞实验24h后,在荧光显微镜下相应可见大量绿色荧光和红色荧光,两种慢病毒滴度分别为1.15×108 TU/ml和9.75×107 TU/ml.结论 成功构建表达OxCF中草酸分解关键基因FRC和OXC的重组慢病毒载体,并通过转染293细胞制备了高滴度慢病毒.     

乙肝病毒X 蛋白对正常肝细胞生物钟基因表达的影响

目的：探讨乙肝病毒X蛋白（HBx）对人正常肝细胞中生物钟基因表达的影响。 方法：分别将HBx表达质粒（pcDNA3.1-HBx）与空载体质粒（pcDNA3.1）转染入人正常肝细胞系L02后,用RT-PCR与Western blot法检测细胞HBx mRNA与蛋白的表达;real-time PCR检测细胞中生物钟基因CLOCK、BMAL1、Per1、Per2、Per3、Cry1、Cry2、CKIε的表达。 结果：RT-PCR与Western blot结果显示,L02细胞转染HBx表达质粒后有明显的HBx mRNA与蛋白表达,而转染空载体质粒的L02细胞无HBx mRNA与蛋白表达。与转染空载体质粒的L02细胞比较,转染HBx表达质粒的L02细胞CLOCK与Cry1的mRNA表达明显升高,而BMAL1、Per1、Per2、Per3、Cry2、CKIε的mRNA表达明显降低,差异均有统计学意义（均P<0.05）。 结论：HBx能使正常肝细胞中的生物钟基因表达发生改变,破坏肝细胞正常的生物节律,这可能为其导致肝癌形成的机制之一。     

草酸铂、5-氟尿嘧啶联合多药耐药基因反义RNA对耐药直肠癌细胞杀伤作用的研究

目的应用基因克隆技术,将多药耐药基因（multidrug resistance gene,MDR1）反义RNA转染入对氟尿嘧啶（5-FU）耐药直肠癌细胞（8348R）中,封闭正义MDR1的转录和表达,联合应用草酸铂及5-FU共同作用于8348R,观察其联合杀伤作用.方法构建含MDR1反义RNA的真核表达质粒PC-MDR1;以绿色荧光蛋白（greenfluorescence protein,GFP）基因作为报告基因,观察在草酸铂作用下GFP基因对8348R细胞的转染效率,用克隆形成试验观察草酸铂对PC-MDR1质粒转染8348R细胞的影响;用MTT试验检测草酸铂联合5-FU及MDR1反义RNA对8348R细胞的杀伤效率.结果转染PC-MDR1质粒后,8348R细胞在5-FU作用下较转染前活性明显下降,转染后细胞出现明显的S期和G2/M期阻滞,细胞凋亡比例显著升高;草酸铂将PC-MDR1质粒对直肠癌细胞的转染效率提高18倍,IC50剂量草酸铂、5-FU联合MDR1反义RNA可大大提高对8348R细胞的杀伤效率,总体杀伤效率可达到75%.结论应用草酸铂、5-FU联合MDR1反义RNA对直肠癌耐药细胞的协同杀伤作用,可望成为治疗对5-FU耐药的直肠癌的有效方法.     

活体转染肝细胞生长因子基因对小鼠急性肝功能衰竭的治疗作用

目的 研究肝细胞生长因子（HGF）基因治疗对小鼠急性肝衰模型的治疗作用。方法 构建人肝细胞生长因子（hHGF）表达载体，利用脂质体介导法在活体内转染肝细胞生长因子基因，利用荧光显微镜检测肝组织内绿色荧光蛋白（GFP）表达了解目的基因的表达，用酶联免疫吸附试验（ELISA）法检测血清中人肝细胞生长因子的含量，通过观察小鼠急性肝衰模型的生存率、肝功能变化、肝组织病理改变来检测肝细胞生长因子基因对急性肝衰的治疗作用，通过检测肝组织中PC—NA指数的变化了解肝脏的增殖能力的变化。结果 荧光显微镜下可见肝组织内有绿色荧光蛋白的表达，转染人肝细胞生长因子基因后在血清中可检测到hHGF的表达，而且可持续1周以上，与对照组相比。转染hHGF基因组的生存率明显提高（40．0％vs11．5％，P〈0．05），血清ALT、TBi明显降低，肝组织中PCNA指数也明显升高。结论 活体转染肝细胞生长因子基因可获得表达，而且肝细胞生长因子基因对急性肝衰小鼠有治疗作用。     

表达甲酰辅酶A转移酶的大肠杆菌对大鼠尿草酸浓度的影响

目的探讨喂饲表达甲酰辅酶A转移酶（FCoAT）的大肠杆菌Nissle1917（EcN-Frc）对大鼠尿草酸浓度的影响。 方法将30只雄性SD大鼠随机分为六组（编号A-F）,每组5只。除A组外,其余五组在食物中每克添加1%草酸。B组每次喂食前30 min,喂食1 ml EcN-Frc培养液,C、D、E、F组喂食1 ml EcN-Frc悬液（活细菌数10³）。C、D、E、F组喂饲细菌的方式分别为每日1次,每3 d一次,每周一次和观察期内首日喂养一次。实验周期为2周,分别于喂养当日,喂养后3、6、9、14 d分别记录大鼠健康情况和24 h尿草酸排泄量。 结果口服ECN-Frc能有效减低高草酸饮食大鼠的24 h尿草酸排泄量。含10³活菌数的EcN-Frc在喂养后3 d即可发挥作用。1次喂服10³个活菌数的ECN-Frc,2周后仍能有效降低大鼠尿中草酸浓度。所有大鼠观察期内均体健,未见明显异常。 结论低剂量口服EcN-Frc能有效降低高草酸饮食大鼠的24 h尿草酸排泄量,且安全,耐受性好。     

转肝细胞生长因子基因肝细胞模型的建立

目的：利用脂质体介导法在体外建立转肝细胞生长因子（HGF）基因的人肝细胞模型。方法：建立HGF真核细胞表达载体，利用脂质体介导法在体外将HGF基因转染入人肝细胞，利用荧光显微镜观察、免疫组织化学、原位杂交方法检测HGF真核细胞表达载体的转录和表达情况。结果：以阳离子脂质体LipofectAMINE为载体将HGF基因转染人肝细胞后，经400mg/L的G418筛选后可形成抗性克隆；Neo基因原位杂交结果显示转染基因的细胞有阳性表达；荧光显微镜下观察到有绿色荧光；免疫组织化学证实转染HGF基因的肝细胞有HGF蛋白的表达。结论：HGF基因可被成功转染入人肝细胞并能有效表达，这可能为肝病的基因治疗提供一种新途径。     

重组腺病毒介导MDA-7/IL-24对Hep3B选择性杀伤和抑制增殖的研究

目的观察黑色素瘤分化相关基因7（MDA-7／IL-24）基因对人肝癌细胞Hep3B和正常的肝细胞L02的作用，并且探讨其该作用机制。方法将携带人MDA-7／IL-24基因的腺病毒Ad.mda-7感染人正常肝细胞L02和肝癌细胞Hep3S，逆转录-聚合酶链式反应（RT—PCR）和ELISA方法观察MDA-7／IL-24基因的表达，噻唑蓝染色法（MTT）观察MDA-7／IL-24对肝癌细胞的生长抑制，Hoechst染色和Annexin-V和PI双染后流式细胞仪检二种细胞的凋亡，利用PI染色后流式细胞仪检测细胞周期，RT-PCR方法检测bcl-2的表达变化。结果Ad．mda-7能介导外源基因MDA-7／IL-24在肝癌细胞株Hep3B和正常细胞L02中高效表达，细胞培养上清液中MDA-7／IL-24蛋白的表达（Hep3B：L02分别为790：810ng／L）。MDA-7／IL-24能明显抑制肝癌细胞的生长（抑制率分别是83％和1．2％），能促进肝癌细胞的凋亡（58％：2．2％），阻滞肝癌细胞在G2／M期（48．29％：7．95％）。而对正常的肝细胞没有促凋亡和增殖阻滞作用；能明显的抑制Hep3B的凋亡抑制基因bcl-2的表达。结论Ad．mda-7能介导MDA-7／IL-24基因在人肝癌细胞中高效表达，选择性的杀伤肝癌细胞Hep3B，促进细胞增殖阻滞，其机制是通过抑制bcl-2的表达诱导肿瘤细胞凋亡。     

The construction of an oxalate-degrading intestinal stem cell population in mice: a potential new treatment option for patients with calcium oxalate calculus

About 80% of all urological stones are calcium oxalate, mainly caused by idiopathic hyperoxaluria (IH). The increased absorption of oxalate from the intestine is the major factor underlying IH. The continuous self-renewal of the intestinal epithelium is due to the vigorous proliferation and differentiation of intestinal stem cells. If the intestinal stem cell population can acquire the ability to metabolize calcium oxalate by means of oxc and frc transgenes, this will prove a promising new therapy option for IH. In our research, the oxalate-degrading genes of Oxalobacter formigenes (Oxf)—the frc gene and oxc gene—were cloned and transfected into a cultured mouse-derived intestinal SC population to give the latter an oxalate-degrading function. Oxf was isolated and cultivated and the oxalate-degrading genes—frc and oxc—were cloned. The dicistronic eukaryotic expression vector pIRES-oxc-frc was constructed and transferred into the mouse stem cell population. After selection with G418, the expression of the genes was identified. The oxalate-degrading function of transfected cells was determined by transfection into the intestinal stem cell population of the mouse. The change in oxalate concentration was determined with an ion chromatograph. The recombinant plasmid containing oxc and frc genes was transfected into the stem cell population of the mouse and the expression of the genes found normal. The cell population had acquired an oxalate-degrading function. The oxc and frc genes could be transfected into the intestinal stem cell population of the mouse and the cells acquired an oxalate-degrading function.     

克隆与表达产甲酸草酸杆菌代谢基因Frc及临床意义

目的研究产甲酸草酸杆菌草酸代谢基因Frc转化大肠杆菌BL21后稳定表达代谢草酸相关性酶--甲酰辅酶A转移酶(FCoAT)对草酸的降解效能。方法成功培养产甲酸草酸杆菌后,采用PCR方法从产甲酸草酸基因组中获得Frc基因,克隆到pMDTM19-T载体上进行测序,得到正确的基因片段。经双酶切将目的基因片段插入到原核表达载体PGEX-4T-2上,测序正确后,转化大肠杆菌BL-21,IPTG诱导表达GST-FCoAT融合蛋白,表达产物行western-blot鉴定分析。结果重组克隆载体pMDTM19-Frc经测序鉴定序列正确。成功构建融合原核表达质粒PGEX-4T-2-Frc,大肠杆菌BL21成为载体,并稳定表达可溶性融合蛋白GST-FCoAT的同时获得代谢草酸潜能。结论克隆产甲酸草酸杆菌Frc基因,成功构建PGEX-4T-2-Frc载体,并转化大肠杆菌BL21,能稳定表达可溶性融合蛋白GST-FCoAT,具有代谢草酸潜能,为肠道细菌获得代谢草酸潜能,减少胃肠道内草酸的吸收,降低尿液中草酸含量和临床防治草酸结石的研究奠定理论基础。     

Presence of Oxalobacter formigenes in the intestinal tract is associated with the absence of calcium oxalate urolith formation in dogs

The incidence of calcium oxalate (CaOx) urolithiasis in dogs has increased steadily over the last two decades. A potential mechanism to minimize CaOx urolithiasis is to reduce enteric absorption of dietary oxalate by oxalate-metabolizing enteric bacteria. Enteric colonization of Oxalobacter formigenes, an anaerobe which exclusively relies on oxalate metabolism for energy, is correlated with absence of hyperoxaluria or CaOx urolithiasis or both in humans and laboratory animals. We thus hypothesized that decreased enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis in dogs. Fecal samples from dogs with CaOx uroliths, clinically healthy, age-, breed- and gender-matched dogs, and healthy non-stone forming breed dogs were screened for the presence of O. formigenes by quantitative PCR to detect the oxalyl CoA decarboxylase (oxc) gene, and by oxalate degrading biochemical activity in fecal cultures. Prevalence of O. formigenes in dogs with CaOx uroliths was 25%, compared to 50% in clinically healthy, age-, breed- and gender-matched dogs, and 75% in healthy non-stone forming breeds. The presence of oxc genes of O. formigenes was significantly higher in healthy non-stone forming breed dogs than in the dogs with CaOx stones. Further, dogs with calcium oxalate stones and the stone-forming breed-matched controls showed comparable levels of biochemical oxalate degrading activity. We conclude that the absence of enteric colonization of O. formigenes is a risk factor for CaOx urolithiasis.     

Detection and identification of oxalate-degrading bacteria in human feces.

BACKGROUND: Oxalate is detoxified (catabolized) via the action of two enzymatic proteins, formyl coenzyme A transferase (encoded by the frc gene) and oxalyl coenzyme A decarboxylase (encoded by the oxc gene), contained in the cytosol of Oxalobacter formigenes that colonizes the human intestinal tract. It is speculated that oxalate-degrading bacteria decrease oxalate absorption from the intestines and their absence in the gastrointestinal tract correlates with the formation of calcium-oxalate urolithiasis. METHODS: Two methods of detection and identification of this bacterial strain were studied in human fecal samples collected from Japanese subjects. Genomic DNA was isolated from bacterial culture, and specific 16S rDNA was amplified by polymerase chain reaction (PCR) followed by sequencing. The oxc gene was amplified directly from human feces by PCR using the specific primers. RESULTS: Oxalate-degrading bacteria were identified by comparing the sequences of 16S rDNA. The oxc gene was directly detected from human feces by PCR. It was ascertained that a combined PCR detection method using both 16S rDNA and the oxc gene allows for identification of O. formigenes in human fecal samples. CONCLUSION: This detection and identification method of oxalate-degrading bacteria using 16S rDNA and oxc gene should be applied in examination of clinical samples.     

Detection of Oxalobacter formigenes in human feces and study of related genes in a new oxalate-degrading bacterium

The first objective of the present study was to examine the presence of Oxalobacter formigenes (an oxalate-degrading bacterium in the human intestine) according to sex in a large number of Japanese. The second objective was to study the presence of three related genes in Bifidobacterium breve, which is considered to be a new oxalate-degrading bacterim. Fecal samples were collected from 55 male and 37 female healthy volunteers. O. formigenes was detected by a polymerase chain reaction (PCR) and a culture-based method. DNA was amplified by the PCR method including the site of important base sequences of each gene in order to detect oxc, frc, and OxlT. O. formigenes was present in 80% of 54 male and 62% of 34 female subjects in the PCR-based assay, while it was present in 62% of 40 male and 50% of 24 female subjects in the culture-based assay. The partial base sequences of the three related genes in B. breve were determined. The RNA polymerase-binding site in promoters and the rho-independent termination sequence were preserved in oxc and frc. In conclusion, the rate of the presence of O. formigenes was the same as in previous reports. Female subjects showed a 15% lower rate than males. B. breve is considered to be an oxalate-degrading bacterium since it was found to have oxalic acid-degrading ability and three genes involved in oxalate degradation.     

Molecular identification of Oxalobacter formigenes with the polymerase chain reaction in fresh or frozen fecal samples.

OBJECTIVE: To develop a simple and rapid polymerase chain reaction (PCR) method for detecting Oxalobacter formigenes (which degrades oxalate in the gut) in fecal specimens from healthy volunteers and patients with urolithiasis, and to determine whether O. formigenes can be detected in frozen or fresh fecal samples. MATERIALS AND METHODS: Whole bacterial DNA was isolated directly from fresh and frozen fecal samples obtained from 30 healthy volunteers free from urolithiasis and from fresh fecal samples obtained from 38 patients with urolithiasis. Genus-specific oligonucleotide sequences were designed, corresponding to homologous regions residing in the oxc gene that encodes for oxalyl-coenzyme A decarboxylase. A PCR-based assay was used on both fresh and frozen fecal samples, and the nucleotide sequences analysed to confirm oxc. RESULTS: A PCR product of 416 bp encoding the oxc gene was detected in 23 (77%) of 30 healthy volunteers free from urolithiasis and in 14 (37%) of 38 patients with urolithiasis. In healthy volunteers, the results of PCR for the fresh and the frozen samples were identical in each subject. The nucleotide sequence analysis showed that the sequence of the amplified product was compatible with that of oxc. CONCLUSION: O. formigenes can be identified easily and efficiently using this PCR-based detection system. The colonization rate of O. formigenes in patients with urolithiasis was significantly lower than that in healthy volunteers known to be free from urolithiasis. Furthermore, as the PCR-based assay results in the frozen fecal samples were identical to those from fresh samples in each subject, immediate processing of fecal samples may not be necessary to detect O. formigenes in the clinical setting.     

Molecular epidemiology of fecal Oxalobacter formigenes in healthy adults living in Seoul,Korea

BACKGROUND AND PURPOSE: Oxalobacter formigenes is a member of the intestinal flora that degrades oxalate. This bacterium maintains an important symbiotic relation with its hosts by regulating oxalic acid absorption in the intestine as well as oxalic acid concentrations in plasma. We tried to define the prevalence of fecal O. formigenes positivity in healthy adults. MATERIALS AND METHODS: Whole-bacterial DNA was isolated directly from fresh stool samples obtained from 233 healthy adults known to be free of urolithiasis. Genus-specific oligonucleotide sequences corresponding to homologous regions residing in the oxc gene that encodes oxalyl-coenzyme A decarboxylase were designed. A PCR-based assay was done on the stool samples. RESULTS: A PCR product of 416 bp encoding the oxc gene was detected in 197 of the 233 stool samples (76.8%). Adjusted to the Seoul population census 1995, the calibrated fecal O. formigenes-positive rate was estimated to be 76.7%: 79.2% in men and 74.2% in women, with no significance difference according to age or sex. CONCLUSION: These results suggest that O. formigenes inhabits the intestine of three fourths of the normal Korean populations. These data provide a base for further studies to uncover the relation between O. formigenes and urolithiasis.     

Quantitative analysis of colonization with real-time PCR to identify the role of Oxalobacter formigenes in calcium oxalate urolithiasis

The objective of the study was to quantitatively measure the number of Oxalobacter formigenes (O. formigenes) colonizations in the gastrointestinal tract in calcium oxalate-forming patients with real-time polymerase chain reaction (PCR). Calcium oxalate-forming patients (n: 27) were included in the study. Serum calcium, sodium, potassium, urea and creatinine levels, as well as 24?h urine levels of calcium and oxalate were measured. The numbers of O. formigenes colonies in stool samples were detected by real-time PCR. One or two metabolic abnormalities were detected in 15 of 27 patients. The O. formigenes levels in patients with metabolic disturbance were significantly decreased when compared to the patients with no metabolic abnormalities (p: 0.038). The undetectable levels of O. formigenes were encountered in one of five patients with hypercalciuria, in three of four patients with hyperoxaluria and in four of six patients with both hypercalciuria and hyperoxaluria. In nine patients with a history of stone recurrence, O. formigenes colonization was significantly lower than the patients with the first stone attack (p: 0.001). O. formigenes formation ceased or significantly diminished in patients with calcium oxalate stones with a coexistence of both hyperoxaluria and hypercalciuria. The measurement of O. formigenes colonies by real-time PCR seemed to be an inconvenient and expensive method. For this reason, the real-time PCR measurements can be spared for the patients with stone recurrences and with metabolic abnormalities like hypercalciuria and hyperoxaluria. The exact measurement of O. formigenes may also help more accurate programming of O. formigenes-based treatments.