POR基因敲除、回补及过表达LO2细胞株的构建及作为AFB_(1)染毒模型的初步应用

为了构建POR基因稳定敲除(POR^(KO))、回补(PORCO)、过表达(POR^(OE))的LO2细胞株,以便为后续深入开展POR基因在AFB_(1)致毒性机理中的功能研究提供细胞模型。本研究首先利用CRISPR/Cas9技术设计2条靶向POR基因的sgRNA并克隆至lentiCRISPR v2载体,进行慢病毒包装与感染,筛选出LO2 POR^(KO)单克隆细胞后进行测序及Western blot鉴定。其次用同源重组方法构建pcDNA3.1(+)/myc-His B-POR重组质粒,经对POR^(KO)细胞和野生型细胞转染分别构建LO2 PORCO及LO2 POR^(OE)细胞株,以G-418筛选后进行Western blotting鉴定。再用4μmol·L^(-1)及8μmol·L^(-1)AFB_(1)分别对野生型、LO2 POR^(KO)、LO2 PORCO、LO2 POR^(OE)细胞株染毒48 h,观察细胞形态并测定细胞存活率。结果表明,靶向exon5的sgRNA-2可成功敲除POR基因,得到LO2 POR^(KO)细胞株;成功构建表达POR重组蛋白的LO2 PORCO及LO2 POR^(OE)细胞株。对AFB_(1)处理后的各细胞存活率测定结果表明,相较于野生型LO2细胞43.66%±0.58%和27.90%±0.46%的存活率,LO2 POR^(KO)细胞存活率具有极显著性差异,均为100%(P<0.0001);LO2 PORCO细胞存活率仍有显著性差异,分别为57.07%±0.74%和42.54%±0.68%(P<0.05);LO2 POR^(OE)细胞存活率则显著降低,分别为24.58%±0.92%和17.99%±0.81%(P<0.01)。综上所述,本研究成功构建了LO2 POR^(KO)、LO2 PORCO、LO2 POR^(OE)细胞株,并以AFB_(1)染毒处理分别研究了各细胞株的存活率和细胞形态学变化,发现POR基因对AFB_(1)所致细胞毒性的影响巨大。研究结果为后续深入开展POR基因在AFB_(1)致毒性机理中的功能研究奠定了基础。

稳定表达人细胞色素P450氧化还原酶(POR)的Flp-In^(TM) CHO细胞系的建立

目的建立稳定表达人细胞色素P450氧化还原酶(POR)的Flp-In^(TM) CHO细胞系,为进一步建立稳定双表达人POR与人细胞色素P450(CYP)的细胞系奠定基础。方法构建POR重组慢病毒并感染Flp-In^(TM) CHO细胞,荧光显微镜观察绿色荧光蛋白表达,挑选转染细胞进行嘌呤霉素筛选和单克隆培养,以获得稳定转染细胞株。利用丝裂霉素C(MMC)细胞毒实验、实时荧光定量PCR和Western blot法检测细胞POR的表达,获得稳定表达POR的Flp-In^(TM) CHO-POR细胞株。构建稳定双表达POR和CYP2C19的Flp-In^(TM) CHO-POR细胞(Flp-In^(TM) CHO-POR-2C19)和单表达CYP2C19的Flp-In^(TM) CHO细胞(Flp-In^(TM) CHO-2C19),并用环磷酰胺(CPA)测定CYP2C19酶活性。结果与感染阴性对照病毒的Flp-In^(TM) CHO细胞相比,感染POR重组慢病毒的Flp-In^(TM) CHO细胞MMC代谢活性升高,POR mRNA和蛋白的表达水平增加,说明获得了可稳定表达POR的Flp-In^(TM) CHO-POR细胞。与Flp-In^(TM) CHO细胞相比,Flp-In^(TM) CHO-2C19的CPA代谢活性无显著差异,而Flp-In^(TM) CHO-POR-2C19的CPA代谢活性增加且明显高于Flp-In^(TM) CHO-2C19细胞。结论成功建立了稳定表达POR且可进一步用于CYP转基因细胞构建的Flp-In^(TM) CHO-POR细胞系。

POR相关基因多态性对华法林维持剂量的影响

目的:探讨POR相关基因多态性对华法林维持剂量个体差异的影响。方法:2017年1月至2019年3月,在中山市人民医院、中山大学附属第一医院进行复查的188名机械瓣膜置换术患者被纳入研究,国际化标准比值及华法林维持剂量由患者的电子病历获得。POR A503V基因型检测采用直接测序法,POR 831-35C>T的基因型检测采用PCR-RFLP法。统计分析POR A503V、POR 831-35C>T各基因型对华法林剂维持剂量的影响。结果:POR A503V各基因型的平均华法林维持剂量分别为CC型(2.75±1.42)mg/d、CT型(2.78±1.41)mg/d、TT型(3.10±1.46)mg/d,组间差异无统计学意义;排除混杂因素后,组间差异仍无统计学意义。POR 831-35C>T各基因型的平均剂量为CC型(2.55±1.36)mg/d、CT型(2.90±1.40)mg/d、TT型(3.05±1.55)mg/d,CT型、TT型的剂量均显著高于CC型(P <0.05);排除混杂因素后,CT型、TT型组的剂量分别比CC型组高10.7%、16.0%(P<0.05)。结论:POR 831-35C>T突变后与华法林维持剂量升高相关,患者在制定华法林个体化用药方案时应参考POR 831-35C>T基因型。

CYP3A5、ABCB1和POR基因多态性对肾移植受者他克莫司浓度、剂量及肾功能的长期影响

目的:研究CYP3A5、ABCB1和POR基因多态性对术后3个月以上肾移植受者他克莫司(FK506)剂量、浓度以及肾功能的影响。方法:选取某院行同种异体肾移植手术的320例患者为研究对象,采用聚合酶链式反应连接的限制性片段长度多态性(PCR-RFLP)分析法和测序法检测患者CYP3A5、ABCB1和POR基因型;由于CYP3A5*3基因突变导致CYP3A5酶失活,因此将CYP3A5*3*3定义为CYP3A5非表达者(n=163),将CYP3A5*1*1和CYP3A5*1*3定义为CYP3A5表达者(n=157),比较CYP3A5表达者和非表达者间FK506血药浓度、剂量、浓度剂量比及肾小球滤过率(eGFR)的差异。结果:在移植后1、4年,CYP3A5表达的FK506血药浓度低于非表达者(P=0.0067,P=0.0004),其剂量在移植后长达8年高于非表达者(P=0.0004),而浓度剂量比则低于非表达者(P=0.0060),差异均有统计学意义。ABCB13435、ABCB11236和POR基因多态性对FK506血药浓度、剂量及浓度剂量比均无影响,差异无统计学意义(P=0.3040,P=0.1190,P=0.3450)。在移植后3、6个月和2年后,CYP3A5非表达者的eGFR高于CYP3A5表达者,差异有统计学意义(P=0.0240,P=0.0187,P=0.0309),提示CYP3A5基因多态性对移植肾功能有长期影响。结论:CYP3A5基因多态性对肾移植受者FK506的浓度、剂量、浓度剂量比及移植后的肾功能有长期影响。

肺移植患者术后使用他克莫司1年CYP3A5、CYP3A4、ABCB1、POR~*28基因多态性与他克莫司个体化用药的关系研究

目的:研究肺移植患者术后使用他克莫司1年CYP3A5、CYP3A4、ABCB1、POR*28基因多态性与他克莫司给药剂量(D)和稳态血药浓度/给药剂量比值(c0/D)的关系。方法:采用回顾性分析方法,选取2017年5月-2018年5月期间在中日友好医院接受肺移植术的46例受试者为研究对象,统计受试者术后使用他克莫司1年后他克莫司的c0和D,并计算c0/D。收集受试者CYP3A5(rs776746)、CYP3A4(rs2242480、rs28371759)、ABCB1(rs1045642、rs2032582、rs1128503)和POR*28(rs1057868)位点的基因型,对基因多态性与D、c0/D的关系进行统计学分析。结果:本研究中涉及位点的基因型频率均符合Hardy-Weinberg平衡(P>0.05)。维持他克莫司c0在治疗窗范围内的条件下,受试者的CYP3A5(rs776746)和CYP3A4(rs2242480)基因型多态性对他克莫司的D、c0/D有显著影响(P<0.05);其他位点各基因型之间的D、c0/D差异均无统计学意义(P>0.05)。联合CYP3A5(rs776746)和CYP3A4(rs2242480)两个位点分析受试者CYP3A代谢型发现,同时携带CYP3A5(rs776746)*1和CYP3A4(rs2242480)*1G等位基因的快代谢型受试者与只携带CYP3A5(rs776746)*1或CYP3A4(rs2242480)*1G等位基因的正常代谢型受试者和不携带CYP3A5(rs776746)*1和CYP3A4(rs2242480)*1G等位基因的慢代谢受试者比较,D、c0/D差异具有统计学意义(P<0.05),其中快代谢型受试者的他克莫司D最高,慢代谢型受试者的他克莫司D最低。结论:检测CYP3A5(rs776746)和CYP3A4(rs2242480)基因多态性对肺移植患者术后使用他克莫司1年后他克莫司的个体化给药具有指导意义。

淋球菌por,tbpB基因序列变异和系统进化探讨

目的分析遵义地区淋球菌por,tbp B基因序列变异及系统进化关系。方法对淋球菌por,tbp B基因进行测序鉴定,利用NG-MAST分型法对por,tbp B基因进行比对编号。用MEGA5.22软件中的邻接法构建系统发育树,并以Kimura 2-parameter模型计算序列间的遗传距离;Felsenstein法计算同义替换率(dS)和非同义替换率(dN)。以RDP3软件对por,tbp B基因中的基因重组事件进行分析。结果 65株淋球菌共鉴定出19种por基因型和22种tbp B基因型。19种por基因型被划分为2个进化簇群,基因间平均遗传距离0.09。22种tbp B基因型被划分为3个进化簇群,基因间平均遗传距离0.13。por,tbp B基因的dN<dS。在tbp B基因中检测出8个基因重组事件,而por基因仅有1个。结论本地区淋球菌por基因与tbp B基因的进化速度与方式存在一定差异,其中tbp B基因主要以基因重组的方式参与进化,por基因则可能以突变或缺失的方式参与进化。纯选择作用是维持本地区por,tbp B基因多态性的重要机制。

欧美杨POR组培再生系统研究

以欧美杨POR(Populus×euramericana)幼嫩茎段、叶片2种外植体为材料,研究诱导不定芽、茎伸长和生根的合适培养条件。结果表明,TDZ在芽诱导过程中有显著的促进作用,不定芽诱导最适培养基为MS+TDZ 0.05mg·L-1+6-BA 0.05 mg·L-1+NAA 0.05 mg·L-1+蔗糖30 g·L-1,茎段和叶片分化率分别为97.3%和88.6%;丛生芽的茎伸长诱导最适培养基为MS+KT 0.5 mg·L-1+GA31 mg·L-1+果糖30 g·L-1,茎段和叶片来源的丛生芽的茎伸长率分别为95.5%和90%,茎伸长培养20 d,每个外植体超过3 cm的芽有3个以上。抽茎芽可直接转入生根培养基,在培养基1/2 MS+IBA 0.05 mg·L-1+NAA 0.05 mg·L-1+蔗糖30 g·L-1中生根率可达92.2%,生根数量多,苗体健康。

HepG2细胞中CYP3A、SLCO1B1和POR基因型检测

目的:检测Hep G2细胞中CYP3A、SLCO1B1、POR的单核苷酸多态性(SNP)位点CYP3A4*1G、CYP3A4*22、CYP3AP1*3、CYP3A5*3、SLCO1B1 T521C、SLCO1B1 A388G、POR*28的基因型。方法:从Hep G2细胞中提取基因组DNA,PCR扩增目的片段,对PCR扩增产物测序后分析上述7个多态性位点的基因型。结果与结论:CYP3A4*1G、CYP3A4*22、CYP3AP1*3、CYP3A5*3、SLCO1B1 T521C、SLCO1B1 A388G、POR*28的基因型分别为GA、CC、GA、AG、TC、AG、CC。CYP3A4*1G下游596 bp处基因型为CT,SLCO1B1 T521C下游50 bp和76 bp处基因型分别为TC和CT,POR*28下游281 bp处基因型为GA。

Transjugular intrahepatic portosystemic shunts and portal hypertension-related complications

Portal hypertension(PH)plays an important role in the natural history of cirrhosis,and is associated with several clinical consequences.The introduction of transjugular intrahepatic portosystemic shunts(TIPS)in the 1980s has been regarded as a major technical advance in the management of the PH-related complications.At present,polytetrafluoroethylene-covered stents are the preferred option over traditional bare metal stents.TIPS is currently indicated as a salvage therapy in patients with bleeding esophageal varices who fail standard treatment.Recently,applying TIPS early(within 72 h after admission)has been shown to be an effective and life-saving treatment in those with high-risk variceal bleeding.In addition,TIPS is recommended as the second-line treatment for secondary prophylaxis.For bleeding gastric varices,applying TIPS was able to achieve hemostasis in more than 90%of patients.More trials are needed to clarify the efficacy of TIPS compared with other treatment modalities,including cyanoacrylate injection and balloon retrograde transvenous obliteration of gastric varices.TIPS should also be considered in bleeding ectopic varices and refractory portal hypertensive gastropathy.In patients with refractory ascites,there is growing evidence that TIPS not only results in better control of ascites,but also improves long-term survivalin appropriately selected candidates.In addition,TIPS is a promising treatment for refractory hepatic hydrothorax.However,the role of TIPS in the treatment of hepatorenal and hepatopulmonary syndrome is not well defined.The advantage of TIPS is offset by a risk of developing hepatic encephalopathy,the most relevant postprocedural complication.Emerging data are addressing the determination the optimal time and patient selection for TIPS placement aiming at improving long-term treatment outcome.This review is aimed at summarizing the published data regarding the application of TIPS in the management of complications related to PH.

基于Por/E的电机线控制动器结构设计

在Pro/E环境下,利用其内部开发程序,针对电机线控制动器的盘式制动器结构进行参数化设计,包括把圆周运动转化为直线运动的机构、增力机构、圆柱齿轮减速机构等.通过设计制动钳支架、摇臂杆、圆柱齿轮、制动盘、制动块总成、传动件等几何参数以及建立它们之间的基本参数关系,利用Pro/E中的拉伸、旋转功能将草绘图转换成空间轮廓图,并进行着色形成实体,在组件环境下将各零件组装为一体,完成电机线控盘式制动器的结构设计,有效地缩短了产品设计周期.

POR与SANDA程序的测井算法与应用比较

POR程序和SANDA程序都是分析泥质砂岩的常用测井解释程序,它们之间既是一脉相承,又存在许多不同之处。本文主要从泥质含量与孔隙度计算原理及在实际解释中的应用等方面将这两种程序进行比较研究。

基于POR/E的印涂铝盖拉深模具工作零件参数化建模技术研究

以四川隆鑫科技有限公司的1617铝套冲压拉深模具的工作零件参数化建模过程为载体,介绍了在POR/E环境条件下拉深凸模与凹模的建模方法,凸模与凹模的装配方法,阐述了应用POR/E软件对冲压拉深模具参数化建模的实用技巧和经验,以期对冲压模具的参数化建模有一定的借鉴意义.

POR基因多态性与华法林维持剂量关系的研究

目的探讨POR基因多态性与华法林维持剂量的相关性。方法共纳入185例中国汉族人工心脏机械瓣膜置换术患者,采用Sequenom MassARRAYSystem检测VKORC1及POR相关SNPs,采用PCR-RFLP法检测CYP2C9*3基因型。采用ANOVA或t检验考察目的 SNPs与患者华法林维持剂量的关系。结果在CYP2C9*1*1携带者中,POR rs17685 T等位基因携带者(TT型和CT型)华法林维持剂量明显高于CC型携带者(3.50±1.07)mg·d-1vs(3.14±0.94)mg·d-1,P=0.03。在CYP2C9*1*1及VKORC1 rs9934438 GA/GG携带者中,POR rs17685 T等位基因携带者(TT型和CT型)的华法林维持剂量明显高于CC型携带者(4.76±0.90)mg·d-1vs(4.08±1.03)mg·d-1,P=0.04。未发现POR rs2868177与华法林维持剂量存在相关性。结论在中国汉族人工心脏机械瓣膜置换术患者中,POR rs17685 T突变与华法林剂量上调相关,该基因型检测将有助于指导华法林的临床合理应用。

一款带POR的可校正低功耗电压基准源

为了解决低功耗、深亚微米工艺条件下基准源的精度问题,本文通过引入工作在亚阈值区MOS管和数字微调技术,提出一种带POR的可校正超低功耗电压基准源,并采用TSMC 90nm COMS工艺对其进行仿真验证。仿真结果表明:该基准源总电流仅为0.876μA,温度系数为24.4 10-6/℃,微调电压步进为25mV。本文提出的基准源与传统基准源比,具有低温漂、可校正、低功耗等优点,适用于便携式低功耗电子产品。

过氧钒烟酸络合物(POR)逆转Zucker肥胖大鼠胰岛素抵抗的作用

目的 探讨过氧钒烟酸络合物（ＰＯＲ）对胰岛素抵抗的逆转作用，ＰＯＲ促进葡萄糖摄取与胰岛素促进作用在机制上的异同。方法方法 以皮下渗透式输注ＰＯＲ，观察ＰＯＲ对Ｚｕｃｋｅｒ大鼠胰岛素抵抗的缓解作用，检测ＰＯＲ对具有胰岛素严重抵抗的Ｚｕｃｋｅｒ肥胖大鼠睾丸脂肪垫脂肪细胞对葡萄糖摄取的促进作用，以及对ＰＬ－３Ｋ Ｐ１１０亚单位酪氨酸磷酸化的影响，比较ＰＯＲ和胰岛素促进ＧＬＵＴ４的转位对ＰＩ－３Ｋ的依赖性。结果 ＰＯＲ可以使肥胖伴胰岛素抵抗大鼠的空腹血糖明显降低，对糖负荷的处理能力明显提高；ＧＴＴ曲线下胰岛素的水平明显降低，提示对胰岛素的敏感性提高。葡萄糖载体的转位水平与ＰＩ－３Ｋ活性并不平行，胰岛素促进的葡萄糖摄取作用对ＰＩ－３Ｋ有依赖性，过氧钒络合物（ＰＯＲ）对葡萄糖载体转位的促进作用不依赖于ＰＩ－３Ｋ，表现为ＰＯＲ促进的葡萄糖摄取下被ＰＩ－３Ｋ抑制剂（Ｗｏｒｔｍａｎｎｉｎ）抑制。结论 ＰＯＲ在１／１０钒酸盐剂量下能逆转Ｚｕｃｋｅｒ大鼠的胰岛素抵抗，胰岛素对葡萄糖载体的激活作用是ＰＩ－３Ｋ依赖性的，而过氧钒（ＰＯＲ）则可通过其他途径激活葡萄糖载体，具体机制可能与载体的直接激活有关。

POR*28基因多态性与中国肾移植患者他克莫司稳定剂量的相关性研究

目的:探讨POR*28单核苷酸多态性(SNP)与中国肾移植患者他克莫司稳定剂量的相关性,为制定该人群个体化剂量调整方案提供参考。方法:共纳入350例中国肾移植患者,CYP3A5*3和POR*28基因检测由生工生物工程(上海)股份有限公司采用Mass ARRAY Analyzer Compac质谱检测。酶放大免疫测定法(EMIT)测定他克莫司血药谷浓度。分析上述基因型与他克莫司稳定剂量的相关性。结果:CYP3A5*3、POR*28等位基因频率均符合Hardy-Weinberg遗传平衡。CYP3A5*3 GG基因型肾移植患者稳定剂量比AG和AA基因型患者显著降低。基于CYP3A5分层分析,在CYP3A5表达与不表达组中,POR不同基因型均与他克莫司稳定剂量有显著相关性。至少携带一个T等位基因的POR*28患者的他克莫司的平均稳定剂量与携带CC野生型的患者之间差异均具有统计学意义(P=0.0037和P=0.0031)。多重线性回归分析显示,CYP3A5*3与POR*28基因分别可以解释36.6%与1.7%他克莫司稳定剂量个体化差异。结论:中国肾移植受者CYP3A5*3和POR*28基因型与他克莫司稳定剂量显著相关,移植前进行检测将有助于他克莫司的临床个体化用药。

Rejuvenation of Ovary and Thin Endometrium by Autologous PRP Injection in POR and Recurrent Implantation Failure

In India, the problem of infertility is growing and in the last 5 years, it has gone up to 20% - 30%. This ongoing prospective clinical study brings forth a novel, innovative, effective, simple, affordable, easily performed outpatient procedure (OP) and a promising therapeutic method in rejuvenating the Ageing Ovaries and Thin Endometrium, with autologous Platelet Rich Plasma (PRP). This clinical study proves to give a better result in rejuvenating Ovary and treating the Thin Endometrium. This pilot study included five women (28 - 44 years) with Poor Ovarian Response (POR), Premature Ovarian Insufficiency (POI) and Perimenopause and Thirty-nine women (22 - 43 years.) with recurrent implantation failure due to Thin Endometrium were subjected to autologous PRP instillation under Ultrasound Guidance, and Hysteroscopic guided PRP. After PRP, a significant output was obtained, with improved Anti Mullerian Hormone (AMH) and Antral Follicle Count (AFC) and out of five women three women conceived by Intra Cytoplasmic Sperm Injection (ICSI). PRP injected in women with Poor Ovarian Response found successful ovarian rejuvenation within 1 - 3 months and had a 60% of pregnancy rate, PRP into the endometrium had 53.8% successful pregnancies. We have not encountered any complications.

Por una Cabeza与电影音乐

在电影产业高速发展又高度发达的今天,电影从单一的视觉艺术的过去进入到了一个视听均衡的新阶段。电影中的音乐对于推动剧情发展、烘托影片主题,渲染画面情绪,调节气氛等都有着重要作用。音乐Por una Cabeza在影片《闻香识女人》,《辛德勒名单》以及《真实的谎言》中均有出现,与不同影片结合的同一原声,在电影作品中又有着不同的表现。

Photoluminescence of Por-Si with High-ordered Mosaic Structure Received at Long Anodic Etching p-Si (100) in the Electrolyte with an Internal Current Source

Photoluminescence spectra and nature of light-emitting centers of a porous silicon (por-Si) samples are given. The por- Si samples had high-ordered mosaic structure, which was received under long anodic etching p-Si(100) in electrolyte with an internal current source. The photoluminescence spectra were monitored at room temperature before and after annealing in air and vacuum. Comparative analysis of photoluminescence spectra of the por-Si samples annealed at different temperatures in air and vacuum shows that the thermal annealing conditions has significant effect on the intensity and spectral content of the photoluminescence spectra. The nature of the luminescence emission centers at different temperatures and annealing conditions was discussed.

Effects of heme precursors on CYP1A2 and POR expression in the baculovirus/Spodoptera frugiperda system

Objective：CYP1A2 and NADPH-CYP450 oxidoreductase（POR）were expressed in the baculovirus/ Spodoptera frugiperda（sf9）system.The aim of this study was to investigate the effects of heme precursors on the expression of CYP1A2 and POR.Methods：The heme precursors[δ-Aminolaevulinic Acid（5-ALA）,Fe^3＋and hemin]were introduced into the system to evaluate their effects on the expression of CYP1A2,POR and their co-expression.All the proteins were identified using immunoblotting,CO-difference spectroscopy,or cytochrome c assay.Results：In the present study,functional CYP1A2 and POR were successfully expressed in the baculovirus/sf9 system,and both of them showed high activities.Co-addition of 5-ALA and Fe^3＋significantly improved expression of CYP1A2 by about 50%compared with the addition of 5-ALA,Fe^3＋or hemin alone.Either co-addition of 5-ALA and Fe^3＋or addition of 5-ALA or Fe^3＋alone improved the POR expression level 2 fold and its activity 7-10 fold compared with control（no addition）.However,unlike CYP1A2,there was no difference between the co-addition and addition of these heme precursors alone.Different ratios of BvCYP1A2 to BvPOR also affected the co-expression of CYP1A2 and POR,with a 3：1 ratio of BvCYP1A2/BvPOR significantly increasing their co-expression.Surprisingly,the addition of 0.1 mM 5-ALA or Fe^3＋alone,but not their co- addition,could significantly improve the CYP1A2 and POR co-expression（P〈0.05）.Conclusion：5-ALA and Fe^3＋increased the expression of CYP1A2 and POR in a baculovirus/sf9 system,but the pattern of their expression was different between their expression alone and co-expression.