慢病毒载体介导Mdr1基因转染人骨髓间充质干细胞的实验研究

本研究探讨多药耐药（mdr1）基因体外转染人骨髓间充质干细胞（BMMSC）以应用于基因治疗的可行性、安全性。体外分离、培养、鉴定MSC;采用具有高效转染非分裂期细胞的慢病毒载体（1entiviralvector,LV）系统将mdr1基因导入BMMSC中;采用RT—PCR和GFP荧光技术检测目的基因的表达,台盼蓝染色及MTT法检测转染后细胞的增殖能力。结果表明：慢病毒感染MSC的感染复数（multiplicityofinfection,MOI）为10,最佳感染率可达80％;Msc表面低表达CD34、HLA—DR、CD31、CD45,高表达CD44、CDl05、CD90、CDl3;GFP荧光表达自72小时开始出现,以后逐渐增强;转染细胞中显示目的基因mRNA的表达;转染对MSC存活及增殖几乎无影响（P〉0．05）。结论：慢病毒载体可成功转染人骨髓间充质干细胞并使其mdrl表达增高,转染对MSC存活及增殖基本无影响。     

多药耐药基因在基因治疗中的应用

人类多药耐药基因(mdr1)转染造血干细胞已在实验研究获得成功,现已进入Ⅰ期临床试验,用于肿瘤化疗中保护骨髓造血干细胞。mdr1与其他基因共同构建的双顺反子逆转录病毒载体转染可同时表达mdr1和其他基因,如为mdr1和其他耐药基因,则可用于联合化疗中保护骨髓造血干细胞;如为mdr1和其他治疗基因,mdr1则作为其他治疗基因的选择标志。     

多药耐药基因在人骨髓CD34+细胞中的表达及对化疗药物的耐受性

目的　探讨人多药耐药 (MDR1)基因过表达能否提高骨髓造血干 /祖细胞对化疗药物的耐受性。方法　应用免疫磁性分选系统 (miniMACS)体外分离、纯化骨髓CD34 细胞并进行扩增 ;采用脂质体介导的基因转移方法 ,将人MDR1基因转染骨髓CD34 细胞 ,并运用流式细胞术检测基因转导前后造血干 /祖细胞中MDR1基因的编码产物 Pl70糖蛋白表达和功能变化。MTT法检测基因转导前后造血干 /祖细胞对化疗药物耐受性的改变。结果　MDR1基因转导后 4 8h骨髓造血干 /祖细胞Pl70抗原表达为 (2 3 6± 2 34) % ,明显高于转导前 (11 2± 2 2 ) % (P <0 0 1)。P170的功能活性被Rh 12 3的摄取和排除试验证实。转基因后细胞表现为典型的多药耐药表型 ,对P170谱的多种化疗药的耐受性提高了约 2～ 8倍 ,对非P170谱的顺铂、氨甲喋呤耐受性没有改变。结论　人多药耐药基因能提高骨髓造血干 /祖细胞对多种化疗药物的耐受性 ,表现为典型的多药耐药表型。     

多药耐药基因转染的脐血细胞对白血病小鼠化疗的骨髓保护作用

目的探讨转染多药耐药(mdr1)基因的脐血单个核细胞(MNC)对急性髓系白血病小鼠的骨髓保护作用及疗效。方法通过逆转录病毒介导的方法将含有人全长cDNAmdr1基因导入脐血MNC,即逆转录病毒上清液与脐血MNC体外共培养;将接种人髓系白血病细胞系(HL60细胞)的SCID小鼠分成3组A组(观察组)经鼠尾静脉注射转染mdr1的脐血MNC2×106/只,共2次;B、C两对照组小鼠以同样剂量、方法分别注射未转染mdr1的脐血MNC和等容积的生理盐水。3组白血病SCID小鼠在每周递增高三尖杉酯碱剂量化疗下,通过检测小鼠外周血白细胞数、瘤细胞阳性率、组织病理和HL60细胞表面抗原(CD33)等观察转基因小鼠和对照小鼠对抗癌药物的耐受性及抗肿瘤疗效。同时分别采用PCR技术、免疫组化方法和柔红霉素排出试验检测mdr1基因在小鼠体内的表达和功能。结果①体外成功地将mdr1基因导入脐血MNC,转染率达30%左右;②用HL60细胞2×106接种于经亚致死量照射的SCID小鼠可成功制成白血病动物模型;③采用程序性移植转基因细胞方法成功建立了mdr1转基因脐血细胞移植小鼠模型,并可作为白血病临床前期体内评价mdr1基因保护骨髓作用;④转基因脐血细胞移植小鼠对高三尖杉酯碱耐受性高于正常剂量的5~6倍,外周血白细胞维持在3.0×109/L左右,外周血涂片瘤细胞降至5%以     

多药耐药基因在骨髓移植模型中的表达

目的 探讨多药耐药基因（mdrl)在骨髓移植小鼠模型中表达情况，及转染mdrl的骨髓细胞对化疗中骨髓保护的可行性。方法 以小鼠骨髓细胞为靶细胞，通过逆转录病毒介导，将mdrl转入骨髓细胞，通过将转染基因的骨髓细胞回输同种小鼠体内的骨髓移植动物模型，观察不同时期移植小鼠骨髓细胞中mdrl的表达及功能，结果 （1）成功地将mdrl导入小鼠骨髓细胞中，转染率达到35%；（2）采用程序性移植方法成功建立了mdrl转基因鼠骨髓移植模型；（3）mdrl在移植小鼠细胞内稳定，有效地表达，观察1-5个月mdrl转染的细胞在受体鼠骨髓单个核细胞中所占比例分别为8.0%，8.0%,7.5%,4.0%,3.0%;(4)mdrl转染的骨髓细胞在化疗中有明显的骨髓保护作用。结论 从一个侧面证实了通过mdrl转染骨髓细胞在化疗中进行骨髓保护是一种长期，稳定，有效的方法。     

mdr1基因shRNA表达载体逆转白血病耐药细胞系K562/ADM多药耐药的实验研究

目的 探讨mdr1短发夹RNA（mdr1 shRNA）对人红白血病耐阿霉素细胞系K562／ADM的耐药逆转作用。方法 编码设计合成靶位mdr1基因mRNA具有19个碱基发夹结构互补的寡核苷酸模板，构建2个shRNA表达载体pSilencer^Tm3．1-H1 neo mdr1—A和mdr1—B，将其稳定转染K562／ADM细胞。用RT—PCR法检测转染后K562／ADM细胞mdr1 mRNA表达，Western blot检测P-糖蛋白（P—gP）表达，流式细胞术和MTT法分别检测K562／ADM细胞凋亡和对阿霉素的敏感性，用激光共聚焦荧光显微镜观察并测定细胞内柔红霉素的积累。结果 在pSilencer^TM3．1-H1 neomdr1—A和mdr1—B shRNA表达载体稳定转染的K562／ADM细胞，mdr1mRNA表达分别减少到转染前的35．9％（P〈0．05）和27．5％（P〈0．01）；同时Western blot结果显示P—gP表达被明显而特异地抑制，对阿霉素的耐药性由79倍分别减低到38倍和30倍；并且，细胞内荧光强度与对照组相比显著增加（P〈0．05），与阿霉素联合应用凋亡细胞百分率分别增加至18．1％（P〈0．05）和54．4％（P〈0．01）。结论 靶向mdr1基因shRNA表达载体可有效逆转耐药，使耐药的肿瘤细胞恢复对化疗药物的敏感性。     

逆病毒介导EGFP基因转染人骨髓间充质干细胞的实验研究

目的探讨逆转录病毒介导增强型绿色荧光蛋白（EGFP）转染人骨髓间充质干细胞（hBM-MSC）的可行性。方法携带EGFP的重组逆转录病毒载体（EGFP-RV）转染体外培养扩增的hBM-MSC,嘌呤霉素筛选,获得表达EGFP的hBM-MSC／EGFP,以未转染的hBM-MSC为对照组,观察两组细胞的形态、生长特性、表面分子表达及向脂肪细胞、成骨细胞分化的潜能。结果EGFP-RV转染hBM-MSC后,获得稳定表达EGFP的hBM-MSC／EGFP;hBM．MSC／EGFP的细胞形态、生长特性、表面分子表达及向脂肪细胞、成骨细胞分化的潜能与未转染的hBM-MSC无差别。结论逆转录病毒介导的EGFP可作为人骨髓间充质干细胞的良好示踪剂。     

多药耐药基因MDR1转染胎盘源间充质干细胞的研究

目的 将全长外源多药耐药基因转入间充质干细胞使其外源基因得以表达。方法 从胎盘组织中分离培养间充质干细胞，构建携带全长外源多药耐药基因的逆转录病毒，将此逆转录病毒转染间充质干细胞，绿色荧光显微镜检测外源基因的表达，westem blot检测MDR1基因表达产物。结果 绿色荧光显微镜可观测到转染后间充质干细胞有绿色荧光蛋白的表达，westem blot检测到全长多药耐药基因编码的p-糖蛋白的表达。结论 mdr1逆转录病毒载体可有效转染人胎盘源MSC，转染的外源基因可充分表达。     

mdr-1和DHFR双基因共转染人CD34+细胞拓宽造血细胞耐药谱的体外研究

目的　探讨将多药耐药基因 (mdr 1)和二氢叶酸还原酶基因 (DHFR)同时导入人CD3 4 细胞 ,以拓宽造血细胞耐药谱 ,改善骨髓耐受联合化疗的可行性。方法　将以造血细胞中高表达的逆转录病毒载体FMCF为基本结构骨架 ,通过引入IRES序列构建获得含mdr 1和DHFR(L2 2Y)双耐药基因的逆转录病毒载体pSF DIM ,通过脂质体介导包装 ,单嗜性和双嗜性包装细胞上清交叉感染提高病毒滴度。低温离心病毒上清转染人脐血CD3 4 细胞 ,用流式细胞仪检测P gp的表达 ,基因组PCR检测外源性耐药基因的整合 ,CFU GM培养观察耐药性变化。结果　逆转录病毒载体pSF DIM转导人脐血CD3 4 细胞后 ,P gp的表达较未转基因组增加了 10 .98% ;基因组PCR同时检测到两种外源性耐药基因的整合 ;与未转基因组比较 ,在 48nmol/L甲氨蝶呤和 10ng/ml及 12ng/ml紫杉醇 (商品名Taxol)浓度水平 ,CFU GM集落形成显著增加 (P <0 .0 5 )。结论　重组双耐药基因逆转录病毒载体pSF DIM可以有效介导mdr 1和DHFR双耐药基因进入人脐血CD3 4 细胞并且获得共表达 ,拓宽了造血细胞耐药谱     

增强型绿色荧光蛋白转染猪骨髓间充质干细胞特性观察

目的：借助质粒将增强型绿色荧光蛋白（EGFP）基因转染猪骨髓干细胞（MSC）,观察转染后EGPP在（MSC）内表达情况及MSC生长特性。方法：1月龄小型香猪为MSC供体,EGFP基因转染MSC,计算转染效率,流式细胞仪鉴定G418筛选后EGFP基因稳定表达率和转染后细胞增殖周期改变。结果：转染率为（36．9±3．4）％;G418筛选后EGFP表达率75．6％,EGFP转染MSCG0／G1期MSC比例较对照组明显增加（P〈0．01）,S＋G2／M期MSC比例减少（P〈0．01）。结论：猪MSC经基因转染和G418筛选,获得EGFP稳定表达;转染和G418筛选后MSC增殖能力降低,G0／G1期MSC比例增加,S＋G2／M期MSC比例下降。     

Aspirin resistance

Treatment failures occur with any drug and aspirin is no exception. Evidence is growing to indicate that there are subpopulations that do not respond to antithrombotic action of aspirin. The term 'aspirin resistance' has been used to describe a number of different phenomena, including inability of aspirin to: (i) protect against cardiovascular events despite its regular intake; (ii) to affect various laboratory tests, reflecting platelet activity. Research on aspirin resistance yielded interesting results in clinical pharmacology and pharmacogenetics. Future studies will show whether genotyping for polymorphisms might be of value in everyday clinical use of aspirin. Present data indicate that in survivors of recent myocardial infarction or unstable angina, patients receiving coronary artery bypass grafts, as well as in subjects with hypercholesterolemia, aspirin resistance has to be considered when implementing antithrombotic therapy. However, in individual patients the available laboratory tests are of no particular use to predict reliably the clinical outcome or to guide in making therapeutic decision. Prospective clinical trials seem necessary to reach such conclusions.     

Gefitinib reverses breast cancer resistance protein-mediated drug resistance

Breast cancer resistance protein (BCRP) is an ATP binding cassette transporter that confers resistance to a series of anticancer agents such as 7-ethyl-10-hydroxycamptothecin (SN-38), topotecan, and mitoxantrone. In this study, we evaluated the possible interaction of gefitinib, a selective epidermal growth factor receptor tyrosine kinase inhibitor, with BCRP. BCRP-transduced human epidermoid carcinoma A431 (A431/BCRP) cells acquired cellular resistance to gefitinib, suggesting that BCRP could be one of the determinants of gefitinib sensitivity in a certain sort of cells. Next, the effect of gefitinib on BCRP-mediated drug resistance was examined. Gefitinib reversed SN-38 resistance in BCRP-transduced human myelogenous leukemia K562 (K562/BCRP) or BCRP-transduced murine lymphocytic leukemia P388 (P388/BCRP) cells but not in these parental cells. In addition, gefitinib sensitized human colon cancer HT-29 cells, which endogenously express BCRP, to SN-38. Gefitinib increased intracellular accumulation of topotecan in K562/BCRP cells and suppressed ATP-dependent transport of estrone 3-sulfate, a substrate of BCRP, in membrane vesicles from K562/BCRP cells. These results suggest that gefitinib may overcome BCRP-mediated drug resistance by inhibiting the pump function of BCRP. Furthermore, P388/BCRP-transplanted mice treated with combination of irinotecan and gefitinib survived significantly longer than those treated with irinotecan alone or gefitinib alone. In conclusion, gefitinib is shown to interact with BCRP. BCRP expression in a certain sort of cells is supposed to be one of the determinants of gefitinib sensitivity. Gefitinib inhibits the transporter function of BCRP and reverses BCRP-mediated drug resistance both in vitro and in vivo.     

Gentamicin resistance in Pseudomonas aeruginosa: R-factor-mediated resistance

By disk diffusion antimicrobial susceptibility testing, 11% of 313 consecutive strains of Pseudomonas aeruginosa, examined during July to October 1973, were resistant to gentamicin (minimal inhibitory concentration 12.5 to >100 mug/ml), and a further 31% were moderately resistant (6.25 to 12.5 mug/ml) to gentamicin at the University of Alberta Hospital in Edmonton, Canada. Of 45 gentamicin-resistant strains from that hospital, none possessed R-factors or gentamicin-inactivating enzymes. Eight of 13 strains obtained from three American sources, which contained gentamicin-acetylating (12 strains) or -adenylating (1 strain) activity, conjugally transferred both gentamicin resistance and antibiotic-inactivating activity. P. aeruginosa recipients were much more effective for detection of transferable gentamicin resistance than Escherichia coli recipients, although not all P. aeruginosa were equally as effective as recipients. One strain, POW 151, transferred resistance to both carbenicillin and gentamicin as well as to several other antibiotics. R-factors detected belonged to P-2 and P-3 (Com 6, C) incompatibility groups. Expression of gentamicin resistance due to acetylation of gentamicin was subject to marked phenotypic lag, especially in recipient strain P. aeruginosa 280. This was shown to result in the failure to detect gentamicin resistance transfer if the concentration of gentamicin in selection media was too high (>2.5 mug/ml for strain 280). Some but not all recipients were changed in pyocine type upon acquisition of R-factors.     

Insulin resistance

Insulin resistance is closely associated with fat accumulation in liver. Thus, it has been suggested that insulin resistance is one of the important factor in development of non-alcoholic steatohepatitis(NASH). For example, insulin resistance in adipocyte results in increased lipolysis and delivery of free fatty acids(FFAs) to the liver, which induce fatty liver. If there is insulin resistance in skeletal muscle, hyperinsulinemia and/or hyperglycemia might increase fat accumulation in liver, through, at least in part, increased sterol-regulatory element binding protein-1c(SREBP-1c) activation. However, hepatic insulin resistance might prevent fat accumulation in liver, because insulin strongly induces lipogenesis. Thus, the tissue specific insulin resistance should be considered in the pathogenesis of NASH.     

Aspirin resistance

OBJECTIVE: To review the literature addressing the problem of aspirin resistance in patients with vascular disease. DATA SOURCES: A MEDLINE search (1966-February 2002) was performed. Key search terms included aspirin, resistance, resistant, failure, tolerance, and nonresponder. English-language studies were identified as well as pertinent references from these articles. DATA SYNTHESIS: Aspirin resistance has been reported in patients with cardiovascular, cerebrovascular, and peripheral vascular disease. Because of differences in the definition of resistance, variations in detection methods, and a lack of controlled trials, the true significance of the problem remains unknown. Multiple mechanisms for resistance have been proposed, including increased reactivity to platelet aggregating factors, genetic polymorphism, and alternate pathways for thromboxane synthesis. The studies to date have failed to demonstrate consistent relationships between aspirin's platelet-inhibiting effects, the impact of dosage escalation, and clinical outcomes. CONCLUSIONS: For many patients, aspirin is an effective antithrombotic agent. However, patients taking aspirin may demonstrate highly variable responses to in vitro tests for platelet aggregation and may experience breakthrough thromboembolic events. Although this phenomenon has been termed aspirin resistance, the lack of a uniform definition or agreement on diagnostic criteria precludes definitive recommendations at this time. In addition, strategies are needed to identify patients at risk for aspirin resistance who might benefit from alternative or combined antiplatelet therapy.     

Antibiotic resistance

Resistance to antibiotics is economically and physiologically costly. Control of antibiotic resistance will require aggressive implementation of numerous strategies. Ongoing surveillance is needed to monitor known antibiotic types and to be able to identify the development of other potential types. Early intervention is needed to combat the rising rate of resistance. Persistent use of hygiene measures and controlled use of antibiotics will limit the spread of antibiotic resistance. Health care providers need to monitor adherence to control measures. Hand and environmental control measures remain a critical component of staff education activities. Active management of infections with non-pharmacologic treatments should be promoted. Motivational campaigns will reinforce positive infection control behaviors. Consistent surveillance of antibiotic use will help fulfill the CDC directive to combat antibiotic resistance and keep the population healthy.     

Insulin resistance

There are several mechanisms about high blood pressure induced by insulin resistance in type 2 diabetes. Hyperinsulinemia is rare in Japanese type 2 diabetic patients. Therefore, hyperinsulinemia is not a major cause of high blood pressure in Japanese type 2 diabetic patients. Diabetic nephropathy was associated with high blood pressure. There was a relation between diabetic nephropathy and insulin resistance. Coexistence of essential hypertension with type 2 diabetes. Both essential hypertension and type 2 diabetes were related with insulin resistance. Vascular endothelial dysfunction was associated with insulin resistance. High blood pressure was partially caused by the endothelial dysfunction. The degree of insulin induced vasodilation was reduced in the type 2 diabetic patients with insulin resistance.