耐药的

The loss or diminishment of OprD2 was the main reason for the imipenem resistance of P.aeruginosa in our hospital, but its effect on meropenem resistance deserved further study.

在对亚胺培南耐药的菌株中，合并有其他β-内酰胺类抗生素耐药的菌株达到62.34%(48/77)。32株Pa外膜蛋白电泳结果显示OprD2相对含量在耐药组为0～3.5%(其中有一株为8.70%)，敏感组为6.20%～10.20%。

In order to explore the clinical prospect of Peimine, we select acute leukemia patients as objects, and investigate the followings:(1) Relationship between P170′s expression and its clinical effect in AL;(2) Mechanism of Thurberg Fritillary Bulb and Peimine in reversing MDR in AL;(3) Combined with chemotherapeutic drugs, the ability of Thurberg Fritillary Bulb to decrease P170's high expression and its clinical efficacy in AL management;(4) Relationship between Traditional Chinese syndrome and P170′s expression or clinical efficacy.

为了探讨浙贝母的临床应用前景，考虑到取材的方便性，我们选择了急性白血病为研究对象，做了以下几方面的研究工作：(1)P170在急性白血病中的表达及其与临床疗效的关系；(2)浙贝母散剂和贝母甲素逆转急性白血病细胞多药耐药的机制探讨；(3)浙贝母散剂临床合用化疗时对急性白血病细胞多药耐药蛋白P170的逆转作用及其临床疗效；(4)中医证型与P170表达及临床耐药关系的研究。

Advance basic research has confirmed that the alkaloids extracted from thunberg fritillary bulb which is widely used in clinical medicine in vitro and in animal experiments have reversed MDR of leukemia. It will increase resistance of acute leukemia cells anticancer drug concentration and lower resistance the expression of Pgp. Pre-Clinical Experimental Research also shows that the conventional chemotherapy combined with the powder of thunberg fritillary bulb for the treatment of acute leukemia. Clinical complete response rate was higher, especially for patients with refractory or relapsed leukemia patients better clinical remission rate. In addition, Caladium particles Slices with chemotherapy in the treatment of RAL Clinical studies also show the Chinese refractory leukemia improve clinical efficacy safe and reliable.

先期基础研究已证实：临床常用的化痰散结中药浙贝母提取的生物碱体外及动物实验均有逆转白血病多药耐药的生物活性，能增加耐药的急性白血病细胞内抗癌药物浓度，降低耐药蛋白Pgp的表达；临床预实验研究也表明，常规化疗方案配合浙贝母粉用于急性白血病临床治疗，其临床完全缓解率明显高于对照组，尤其对难治及复发白血病患者临床缓解率更佳；此外，单药浙贝母颗粒配合化疗治疗RAL的临床研究亦显示中药在提高难治性白血病临床疗效方面安全可靠。

Objective To investigate the reversal effect of curcumin on HL 60 /ADR cell line.

目的 研究姜黄素对白血病耐药细胞HL 60 /ADR多药耐药的逆转作用。

The stable clones are further identified by RT-PCR and Western blot; 6 MTT assay is used to investigate the effect of ZNRD1 on the cell growth of cells (AGS, SGC7901, MKN28, NIH3T3, GES-1); 7 Soft agar assay is used to investigate the effect of ZNRD1 on the clonality of cells (AGS, MKN28); 8 Nude mice assay is used to investigate the effect of ZNRD1 on the cell growth of gastric cancer cells (AGS, MKN28); 9 Flow cytometry is used to investigate the effect of ZNRD1 on the cell cycle distribution of cells (AGS, MKN28, NIH3T3, GES-1); 10 Flow cytometry is used to investigate the effect of ZNRD1 on the cell apoptosis of cells (AGS, MKN28, NIH3T3); 11 MTT assay is used to investigate the effect of ZNRD1 on the drug sensitivity of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR) in vitro; 12 SRCA is used to investigate the effect of ZNRD1 on the drug sensitivity of gastric cancer cells (SGC7901, SGC7901/VCR) in vivo; 13 Flow cytometry is used to investigate the effect of ZNRD1 on adriamycin accumulation of cancer cells (SGC7901, SGC7901/VCR, HL-60, HL-60/VCR); 14 Transmission electron microscope is used to investigate the effect of ZNRD1 on the sensitivity of SGC7901 cells towards drug-induced apoptosis; 15 Flow cytometry and DNA ladder assay are used to investigate the effect of ZNRD1 on the sensitivity of cells (SGC7901, SGC7901/VCR, HL-60/VCR) towards drug-induced apoptosis; 16 Microarray is used to investigate the profiling of ZNRD1-responsive genes in gastric cancer cells (AGS, MKN28, SGC7901, SGC7901/VCR); 17 RT-PCR and Western blot are used to identify the results of microarray; 18 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of cyclin D1; 19 Reporter gene assay is used to investigate the effect of ZNRD1 on the transcriptional activity of MDR1; 20 Kinase assay is used to investigate the effect of ZNRD1 on the activity of cyclin E-CDK2 kinase; 21 The antisensenucleic acids of p21 is used to inhibit the expression of p21, and flow cytometry is used to investigate the effect of p21 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 22 The antisensenucleic acids of p27 is used to inhibit the expression of p27, and flow cytometry is used to investigate the effect of p27 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 23 Liposome is used to up-regulate the expression of Skp2, and flow cytometry is used to investigate the effect of Skp2 on ZNRD1-induced cell cycle arrest in gastric cancer cells; 24 Western blot is used to investigate the effect of ZNRD1 on the stability of Skp2 and p27 in gastric cancer cells; 25 MVD assay is used to investigate the effect of ZNRD1 on the angiopoietic activity of gastric cancer cells; 26 ELISA is used to investigate the effect of ZNRD1 on the expression of VEGF165 in gastric cancer cells; 27 The roles of DARPP-32 in MDR of gastric cancer cells are investigated using gene transfection, MTT assay, SRCA, flow cytometry and DNA ladder assay.

应用杂交瘤技术制备ZNRD1的首个单克隆抗体；2）利用RT-PCR、Western blot和免疫组化检测ZNRD1在胃癌组织、胃炎组织、正常胃上皮组织、胃癌细胞和正常胃组织上皮细胞中的表达；3）构建ZNRD1的小干扰RNA载体，并测序鉴定；4）利用脂质体将ZNRD1的真核表达载体及其空载体转染胃癌细胞（AGS、SGC7901、MKN28）和小鼠成纤维细胞（NIH3T3），G418筛选后进行鉴定；5）利用脂质体将ZNRD1的小干扰RNA载体及其空载体转染药敏胃癌细胞（SGC7901）、正常胃组织上皮细胞（GES-1）、对长春新碱耐药的胃癌细胞（SGC7901/VCR）、药敏白血病细胞（HL-60）、对长春新碱耐药的白血病细胞（HL-60/VCR），G418筛选后进行鉴定；6）利用MTT实验检测ZNRD1高/低表达对细胞（AGS、SGC7901、MKN28、NIH3T3、GES-1）生长的影响；7）通过软琼脂克隆形成实验检测上调ZNRD1对AGS、MKN28细胞克隆形成能力的影响；8）通过裸鼠成瘤实验检测上调ZNRD1对AGS、MKN28细胞体内成瘤性的影响；9）通过流式细胞仪分析ZNRD1高/低表达对细胞（AGS、MKN28、NIH3T3、GES-1）的细胞周期的影响；10）通过流式细胞仪分析上调ZNRD1对细胞（AGS、MKN28、NIH3T3）的凋亡的影响；11）通过MTT实验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）体外药物敏感性的影响；12）通过肾包膜下移植法检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR）体内药物敏感性的影响；13）通过流式细胞仪分析ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60、HL-60/VCR）内阿霉素蓄积和泵出的影响；14）通过透射电镜检测上调ZNRD1对SGC7901细胞凋亡敏感性的影响；15）通过流式细胞仪和DNA梯度试验检测ZNRD1高/低表达对细胞（SGC7901、SGC7901/VCR、HL-60）凋亡敏感性的影响；16）通过基因芯片检测ZNRD1高/低表达对胃癌细胞内基因表达谱的影响；17）利用RT-PCR、Western blot对基因芯片的结果进行鉴定；18）利用报告基因实验检测ZNRD1对cyclin D1的启动子活性的调节作用；19）利用报告基因实验检测ZNRD1高/低表达对MDR1的启动子活性的调节作用；20）利用激酶试验检测ZNRD1对cyclin E-CDK2 激酶活力的影响；21）利用反义核酸技术抑制p21的表达；通过流式细胞仪检测抑制p21对ZNRD1介导的细胞周期阻滞的影响；22）利用反义核酸技术抑制p27的表达；通过流式细胞仪检测抑制p27对ZNRD1介导的细胞周期阻滞的影响；23）利用脂质体转染法上调Skp2的表达；通过流式细胞仪检测上调Skp2对ZNRD1介导的细胞周期阻滞的影响；24）利用Western blot检测ZNRD1对p27和Skp2的蛋白稳定性的影响；25）利用微血管密度实验检测ZNRD1对AGS、MKN28细胞裸鼠移植瘤微血管形成的影响；26）利用ELISA检测ZNRD1对AGS、MKN28细胞培养上清和移植瘤匀浆中VEGF165含量的影响；27）利用脂质体转染法、MTT实验、肾包膜下移植法、流式细胞仪和DNA梯度试验检测新耐药相关分子DARPP-32对细胞（SGC7901、SGC7901/VCR、对阿霉素耐药的胃癌细胞SGC7901/ADR）多药耐药表型的影响；利用脂质体转染法和MTT实验检测下调ZNRD1对DARPP-32介导的胃癌多药耐药的调控作用。

We selected mecA gene and femA factor asβ-lactam antibiotic-resistant genes, ermA, ermC, msrAgene as macrolides-lincosamids antibiotic-resistant genes, norA, grlA, gyrA, gene as Quinolonesantibiotic-resistant genes, and tetM gene as Tetracycline antibiotic-resistant genes on the basis of the latestresearch. We designed primers with GenBank enunciable gene sequence, cloned the drug resistantgenes, and sequenced these genes.

根据国内外最新文献报道，筛选出对β-内酰胺类抗生素耐药的mecA基因及辅助因子femA；对大环内酯类及林可霉素类抗生素耐药的ermA、ermC、msrA基因，对喹诺酮类抗菌药物耐药的norA、grlA和gyrA基因及对四环素类抗生素耐药的tetM基因作为拟选的主要耐药基因，分别设计PCR扩增的特异性引物，进行克隆。

Drug sensitive test and three-dimensional test220 strains of Pa were isolated from hospitalized patients between 2003 and 2007. K-B method was used to tested the susceptibility of 10 different antibiotics. IRPa was screened by testing the minimal inhibitory concentration of imipemem by using agar diluiion method.The susceptibility of these IRPa to the antibiotics was analysised. Three-dimensional test was used to identify the different kinds of beta lactamases from 220 strains of Pa.2.Carbarpenems hydrolytic enzyme genes and oprD2 gene were detectedamong the selected IRPa strains, PCR method was performed to detect carbapenemase genes which included GES、KPC、SPM、VIM、IMP、GIM gene and the oprD2 gene;Multiplex PCR were used to detect OXA genes and plasmid-mediated AmpC beta lactamase genes; The expression of the chromosomal AmpC beta lactamases and oprD2 genes in IRPa strains were analyzed by Real-time PCR.3.Identification and characterization of integronsIntegrase gene was detected by PCR, and the classification of integrons was performed by using restriction fragment length polymorphism.PCR was performed to detect the qacE△1-sull gene,and the gene cassetes which are located at variable region of integrons in the strains were detected to be positive.

方法1、药敏实验和三维实验收集2003～2007年临床分离的220株Pa，对这些菌株采用K-B法测定10种临床常用抗生素的药敏情况，同时采用琼脂稀释法检测亚胺培南的最低抑菌浓度(Minimal inhibitory concentration，MIC)，筛选出对亚胺培南耐药的铜绿假单胞菌，并分析其对其它抗生素的药物敏感率；采用三维实验的方法分析220株Pa产β内酰胺酶的类型。2、碳青霉烯类水解酶和oprD2蛋白的检测针对鉴定的IRPa菌株，采用普通PCR方法检测具有碳青霉烯水解作用的β内酰胺酶耐药基因(GES、KPC、SPM、VIM、IMP、GIM基因)和oprD2基因，采用多重PCR的方法检测OXA型基因和质粒携带的AmpC酶基因，用荧光定量RT-PCR方法检测oprD2蛋白基因表达情况；同时对产AmpC酶的Pa(25株，含IMP耐药和敏感株)用RT-PCR方法检测AmpC酶基因的表达量情况。3。

It showed that drug-resistant genes are important mechanism in which MRSA showed drug resistance of macrocyclic lactones antibacterials;The Panton-Valentine Leukocidin gene in Multiple drug-resistance MRSA were higher than.

多重耐药的MRSA中红霉素耐药基因ermA/B/C检出率较高，是MRSA对大环内酯类抗生素耐药的主要机制；杀白细胞毒素基因PVL检出率较高，是MRSA的重要致病因子。

Presently, the studies on multidrug resistance mechanism, reversor and associated exterior experiments in vivo and in vitro from different points of view and levels show that multidrug resistance is the result of common action in many factors. In recent years nonrepresentative mechanism of multidrug resistance becomes more and more significant.

肿瘤细胞产生耐药性成为许多肿瘤治疗成败的关键，也是血液肿瘤化疗失败的重要原因，目前人们从不同的角度和水平对耐药的发生机制、逆转耐药的药物研究及相应的体内外实验等进行研究，指出多药耐药是多种因素共同作用的结果。

Objective:To explore the mechanism of reversal of mutidrug resistance of GBC-SD cell lines by grape seed polyphenols.

目的： 研究葡萄籽多酚逆转先天性耐药细胞株GBC-SD耐药的机制，寻找高效低毒的耐药逆转剂。

It has been put forward that there exists single Ball point and double Ball points on the symmetrical connecting-rod curves of equilateral mechanisms.

从鲍尔点的形成原理出发，分析对称连杆曲线上鲍尔点的产生条件，提出等边机构的对称连杆曲线上有单鲍尔点和双鲍尔点。

The factory affiliated to the Group primarily manufactures multiple-purpose pincers, baking kits, knives, scissors, kitchenware, gardening tools and beauty care kits as well as other hardware tools, the annual production value of which reaches US$ 30 million dollars.

集团所属工厂主要生产多用钳、烤具、刀具、剪刀、厨具、花园工具、美容套等五金产品，年生产总值3000万美元，产品价廉物美、选料上乘、质量保证，深受国内外客户的青睐