硅纳米载体携带RNA干扰质粒逆转人结肠癌细胞多药耐药的实验研究

目的 探讨硅纳米颗粒作为基因转染载体携带干扰质粒MRP2siRNA逆转人结肠癌耐药细胞株HT29/L-OHP的多药耐药,为硅纳米作为靶向基因治疗和化疗药物的有效性打下基础. 方法 制备硅纳米载体,用硅纳米载体和脂质体分别将多药耐药基因MRP2的干扰重组质粒pGensil-MRP2siRNA,转染人结肠癌耐药细胞株HT29/L-OHP,观察两种载体转染人结肠癌耐药细胞株HT29/L-OHP后绿色荧光的表达,利用RT-PCR和Western-blot检测MRP2的表达,CCK-8实验测定转染干扰质粒后顺铂对人结肠癌耐药细胞株HT29/L-OHP细胞增殖抑制作用,并比较两种载体的逆转效率. 结果 在人结肠癌耐药细胞株HT29/L-OHP中,两种载体携带的siRNA都明显抑制了MRP2 mRNA及蛋白的表达,两种载体逆转效果差异无统计学意义（P＞0.05）.在相同浓度化疗药物的作用下,两种载体的RNA干扰组细胞凋亡比例显著高于对照组（P＜0.01）,表明细胞耐药性显著下降,两种载体细胞凋亡差异无统计学意义（P＞0.05）. 结论 硅纳米能有效的携带pGensil-MRP2siRNA质粒转染人结肠癌细胞人结肠癌耐药细胞株HT29/L-OHP,并且对结肠癌耐药细胞的MRP2的表达有明显抑制作用,取得良好的逆转耐药效果.     

LYRM1基因绿色荧光蛋白融合载体的构建及表达

[目的]构建LYRM1基因绿色荧光蛋白融合载体,进而观察LYRM1基因编码蛋白在细胞内的定位情况.[方法]运用RT-PCR技术从人网膜脂肪组织中分离LYRM1基因的完整编码框,将其亚克隆到绿色荧光蛋白表达载体pEGFP-N2,脂质体转染3T3-L1前体脂肪细胞,激光共聚焦显微镜下观察融合蛋白的表达情况.[结果]PCR、酶切鉴定及测序结果表明重组质粒构建正确.激光共聚焦显微镜下观察到pEGFP-N2转染的细胞中绿色荧光均匀分布于整个细胞,而pEGFP-LYRM1转染的细胞中绿色荧光主要集中在细胞核区域.[结论]成功构建了LYRM1绿色荧光蛋白融合载体,LYRM1编码蛋白在细胞中定位于胞核中.     

hPARP-1高表达HaCaT细胞株的建立

目的建立hPARP-1过表达的HaCaT细胞株,观察过表达该基因后所引起的生物学效应。方法常规提取人皮肤角质细胞HaCaT的总RNA,RT-PCR扩增PARP-1全长cDNA基因片段,构建含PARP-1全长cDNA基因片段的真核表达载体pEGFP-PARP-1,通过BglⅡ、xhoⅠ双酶切及测序鉴定其正确性。利用脂质体将pEGFP-N2和pEGFP-PARP-1分别转染人皮肤角质细胞HaCaT,通过G418加压筛选,建立稳定高表达hPARP-1的HaCaT-PARP-1细胞株,并采用Realtime Q-PCR和Western blotting检测其mRNA和蛋白的表达水平。结果 RT-PCR扩增产物经BglⅡ、xhoⅠ双酶切后,与pEGFP-N2连接构建的重组体经酶切和测序,结果与GeneBank报道序列一致。Realtime Q-PCR及Western blotting结果显示,转染pEGFP-PARP-1质粒的细胞其PARP-1在mRNA及蛋白的表达水平均显著高于对照组。结论 PARP-1高表达细胞株成功建立。     

家兔pEGFP-N1/rCNP质粒的构建及其在人脐静脉内皮细胞中的表达

[目的]构建真核表达载体pEGFP-N1/rCNP并转染体外培养的人脐静脉内皮细胞,观察CNP基因在其中的表达。[方法]利用RT-PCR方法从家兔腹主动脉组织扩增获得CNP基因全场全长编码区,克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体pEGFP-N1上,构建重组载体pEGFP-N1/rCNP。利用LipofectaminTM 2000脂质体将重组质粒转染人脐静脉内皮细胞。[结果]重组质粒pEGFP-N1/rCNP构建成功;将其转染人脐静脉内皮细胞,观察到目的基因CNP表达。[结论]成功构建了家兔腹主动脉CNP基因的真核表达载体pEGFP-N1/rCNP,并可转染人脐静脉内皮细胞。     

人卵泡抑素基因真核表达载体构建和体外表达

目的:体外构建携带人卵泡抑素基因(FS)cDNA的真核表达载体,并观察其在幼年叙利亚地鼠肾细胞(BHK21)中的表达。方法:从新鲜人卵泡抽提液细胞中提取RNA,应用RT-PCR方法扩增人FScDNA序列,克隆到pMD-18T载体中,构成pMD-FS重组克隆质粒。经测序鉴定后,双酶切得到人FS基因cDNA序列,插入到真核表达载体pEGFP-N1中,构成pEGFP-FS重组表达质粒。pEGFP-FS重组表达质粒经酶切和PCR鉴定,通过脂质体2000介导瞬时转染BHK21细胞,并检测绿色荧光蛋白(GFP)的荧光表达。结果:成功构建pMD-FS重组克隆质粒,FScDNA序列测序结果与GenBank中公布的序列(Genbank NM_013409.1)同源性为100%;成功构建pEGFP-FS重组表达质粒,将其转染BHK21细胞后,观察到绿色荧光蛋白的表达。结论:本实验成功构建了人FS的pMD-FS重组克隆质粒和pEGFP-FS重组表达质粒,为进一步研究FS对哺乳动物卵泡发育的影响奠定了基础。     

利用流感病毒反向遗传学操作系统表达绿色荧光蛋白(EGFP)

目的 构建基于RNP复合体的流感病毒反向遗传学操作系统,并利用该系统表达绿色荧光蛋白.方法 将人源pol Ⅰ启动子分别插入真核表达载体pcDNA3和原核质粒pUC18,获得pol Ⅰ/pol Ⅱ双启动子表达载体pCHBW和pol Ⅰ单启动子表达载体pPol IR;来源于禽流感病毒H5N1湖北分离株A/Chicken/Hubei/489/2004的基因被克隆到pCHBW上,获得pCHBW-PB2、pCHBW-PB1、pCHBW-PA、pCHBW-NP;将荧光蛋白基因置换NA基因的编码区进而克隆到pPol IR上获得pPol IR-NA (EGFP);这些质粒共转染293T细胞并观察荧光.结果 所构建的载体均经酶切和测序验证正确.pCHBW-PB2、pCHBW-PB1、pCHBW-PA、pCHBW-NP和pPol IR-NA (EGFP)共转染293T细胞后能观察到绿色荧光,Western-blot实验证实被转染的293T细胞表达绿色荧光蛋白(EGFP).结论 基于RNP复合体的反向遗传操作系统构建成功,并表达绿色荧光蛋白.     

全长型人Smac基因真核表达载体构建及表达

目的 构建全长型(full length,FL)人Smac基因(hSmac)真核表达载体pcDNA3.1 -FL hSmac,并在肝癌细胞中表达.方法 应用逆转录聚合酶链式反应(RT-PCR)技术从人宫颈癌细胞株Hela中扩增到Smac CDs基因片段,构建重组真核表达载体peDNA3.1 -FL hSmae.PCR、酶切鉴定重组质粒正确后,通过脂质体介导将其和作为阴性对照的空载体pcDNA3.1 分别转染人肝癌细胞株(SMMC-7721).同时转染含EGFP的质粒pcDNA3.1 -EGFP.利用荧光显微镜和流式细胞术检测转染效率.采用RT-PCR、western blot法检测外源基因Smac的表达.结果 PCR扩增片段与预期片段大小相符,插入片段测序结果与GenBank公布的一致,表明人Smac基因克隆成功.且鉴定证实真核表达载体pcDNA3.1 -FL hSmac构建成功.流式细胞术检测到转染效率高达48%,荧光显微镜下也可见非常明亮的绿色荧光.无论是在mRNA水平还是蛋白水平上,转染后的细胞中外源基因Smac表达均明显增加.结论 成功构建重组真核表达载体pcDNA3.1 -FL hSmac,并在肝癌细胞中明显表达,为研究Smac基因在细胞凋亡过程中的调控作用.以及探讨以Smac基因为基础的肿瘤基因治疗策略提代基础依据.     

促凋亡因子Smac过表达增强胰腺癌Panc-1细胞化疗敏感性研究

目的探讨胞浆过表达促凋亡因子Sm ac对胰腺癌细胞Panc-1化疗敏感性的影响。方法应用RT-PCR获得Sm accDNA(不含线粒体定位序列),定向克隆入pEGFP-N1质粒载体中,经限制性酶切、PCR及测序鉴定。利用脂质体将pEGFP-N1/Sm ac重组体转染胰腺癌细胞株Panc-1,应用荧光显微镜检测Sm ac-EGFP绿色荧光融合蛋白表达;流式细胞仪测定顺铂、5-FU诱导的转染细胞凋亡率;四甲基偶氮唑蓝(MTT)法检测细胞生长抑制率。结果荧光显微镜下可见转染pEGFP-N1/Sm ac的细胞胞浆自发绿色荧光;在顺铂、5-FU作用下,N1/Sm ac转染组细胞凋亡率分别为(36.8±5.5)%、(47.6±6.9)%,明显高于未转染组(12.3±3.2)%、(19.2±4.7)%及空载体转染组(16.9±3.7)%、(22.5±5.8)%,其差异有统计学意义(P<0.01)。N1/Sm ac转染组细胞的生长抑制率亦明显高于未转染组及空载体转染组(P<0.01)。结论胞浆过表达Sm ac活性分子可明显促进顺铂、5-FU诱导的Panc-1细胞凋亡,增强其对化疗药物的敏感性,为探索基于凋亡的胰腺癌治疗新策略提供实验依据。     

载体介导的人支气管上皮细胞DNA聚合酶β基因的靶向RNA干扰

目的运用载体介导的RNA干扰技术靶向抑制人DNA聚合酶β基因在支气管上皮细胞中的表达,为研究DNA聚合酶β在环境化学污染物所致DNA损伤修复中的功能和机制作准备。方法利用分子克隆技术构建含pU6启动子的人DNA聚合酶β基因RNA干扰特异性绿色荧光蛋白C1载体重组子“pEGFP-C1-U6-dsRNA”;以脂质体法将载体重组子转染人支气管上皮细胞,同时以空白细胞和空载体转染细胞作对照;在经G418筛选后,以荧光显微成像技术观察细胞的转染效果,以蛋白印迹法分析转染后细胞中的DNA聚合酶β表达情况。结果在转染重组子的细胞中,DNA聚合酶β的表达明显下调,仅相当于正常细胞的17·3%。结论人支气管上皮细胞DNA聚合酶β基因的靶向抑制成功。     

HBsAg重组腺病毒载体的构建及在树突状细胞的表达

目的构建能高效转导HBsAg基因的重组腺病毒Ad-S,证实其能在小鼠树突状细胞内表达,以用于基因治疗的实验研究。方法应用AdEasyTMXL腺病毒载体系统,首先将HBV S基因克隆到p-shuttle-CMV形成重组穿梭载体,用PmeⅠ酶切使之线性化,电转化到BJ5183-AD-1细胞中形成重组腺病毒Ad-S质粒,后者以PacⅠ酶切线性化,转染到AD293细胞包装为重组腺病毒Ad-S。Ad-GFP或Ad-S以MOI为100转染经GM-CSF、IL-4和TNF-α等细胞因子诱导培养的小鼠骨髓来源的树突状细胞,用流式细胞仪分析细胞荧光表达,Western blot、RIA和ELISA法检测HBsAg的表达。结果PCR、序列测定和酶切鉴定证明HBsAg重组腺病毒Ad-S构建正确,扩增到的病毒颗粒滴度为108/ml;流式细胞仪分析细胞荧光表达率为(92.93±2.35)%,荧光强度达236.67±45.72;Western blot、RIA和ELISA法均检测到Ad-S转染的树突状细胞能表达HBsAg。结论HBsAg重组腺病毒Ad-S构建正确并能高效转染树突状细胞和表达HBsAg,为进一步研究腺病毒介导HBsAg基因修饰的DC疫苗的免疫治疗作用奠定了基础。     

The human drama of genetics: 'hard' and 'soft' media representations of inherited breast cancer

This paper examines media coverage of 'breast cancer genetics', and explores its implications for public understanding. We present a content analysis of coverage in British newspapers and look at a variety of popular forms, including women's magazines, television soap opera and radio drama. Genetic/inherited risk receives a great deal of coverage across a wide range of media formats and outlets. Much of this attention has focused on individuals from 'high risk families' and dilemmas around prophylactic mastectomies. Through examining media coverage, combined with interviews with media personnel and their sources, we show why this story proved so attractive to the media and highlight the different production values which influence coverage. Finally, we introduce preliminary findings from focus group discussions to demonstrate how such 'human interest' framing has engaged audience attention and influenced public understandings. The paper concludes by highlighting the implications for analysing, predicting, and engaging with, media representations of science.     

Worker education in the primary prevention of occupational dermatoses

This paper reports the evaluation of a skin care education programmeconducted on a fine chemicals manufacturing site where over1,000 employees are located. Approximately 60% are involvedin chemical manufacture. Over a 12 month period production staffreceived training in prevention of occupational dermatoses linkedto a site-wide poster initiative. The incidence of new casesof occupational dermatoses fell from 0.055 (70 cases in 1,277employees) to 0.021 (27 cases in 1,277 employees) before andafter the intervention respectively (p<0.0001). After otherfactors such as chemicals handled, observer bias and changesin reporting related to socioeconomic climate were taken intoaccount it is concluded that this study demonstrates the importanceof worker education as a tool for primary prevention of disease.Training materials such as video and poster presentations maybe effectively used in the chemical manufacturing industry asan adjunct to prevention and control of exposure to substanceshazardous to the skin. Such methods may also be used in otherindustries where there are significant risks of dermatoses.     

Regional Variation in Travel-related Illness acquired in Africa,March 1997–May 2011

To understand geographic variation in travel-related illness acquired in distinct African regions, we used the GeoSentinel Surveillance Network database to analyze records for 16,893 ill travelers returning from Africa over a 14-year period. Travelers to northern Africa most commonly reported gastrointestinal illnesses and dog bites. Febrile illnesses were more common in travelers returning from sub-Saharan countries. Eleven travelers died, 9 of malaria; these deaths occurred mainly among male business travelers to sub-Saharan Africa. The profile of illness varied substantially by region: malaria predominated in travelers returning from Central and Western Africa; schistosomiasis, strongyloidiasis, and dengue from Eastern and Western Africa; and loaisis from Central Africa. There were few reports of vaccine-preventable infections, HIV infection, and tuberculosis. Geographic profiling of illness acquired during travel to Africa guides targeted pretravel advice, expedites diagnosis in ill returning travelers, and may influence destination choices in tourism.     

Self-reported health problems among Swedish miners one year after unemployment

Unemployment is considered to be a public health concern sincedeterioration in the health of the unemployed is often anticipated.However, for some groups, such as miners, unemployment mightimprove health due to a cessation of potentially harmful occupationalexposures. This study evaluates the health of 79 miners in oneSwedish iron-ore mine, and 226 age-matched controls from thegeneral population, during one year after the closure of themine. The participants received a questionnaire regarding medicalhistory and subjective symptoms at the beginning of the studyperiod, and after one year. Statistically significant negativeeffects on self-reported health attributable to unemploymentwere not found, although neuropsychiatric symptoms were morecommon among the unemployed miners. The miners reported a statisticallysignificant improvement in grip force (p=0.031). They had asignificantly higher prevalence of symptoms associated withmining related exposures when compared with the population controls;pain in the upper extremities [relative risk (RR)=2.27, 95%confidence interval (Cl)=1.44–3.59), back pain (RR=1.84;Cl=1.237–2.75), vasospastic disease of the fingers (RR=2.05;Cl=1.18–3.57) and obstructive respiratory symptoms (attacksof dyspnea and wheezing: RR=3.67; Cl=1.167–11.6).     

Excès de cas humains et animaux de tularémie en France au cours de l’hiver 2007–2008 : émergence ou phénomène isolé ?

Context

Tularemia is a zoonosis affecting humans and hares in France. We describe the results of surveillance in both species, in 2007 and 2008.

Methods

Human tularemia cases are mandatorily notifiable in France since 2003. In hares, surveillance relies on volunteer hunter associations in all districts of the country. Data from mandatory reports and volunteer surveillance in 2007/2008 were analyzed and compared with previous results.

Results

In 2007/2008, 144 cases were reported in humans and 117 cases in hares. This was a 100% increase compared to previous years. Human cases differed from those of previous years only by the frequency of contact with breeding animals. Human cases without any documented risk exposure were also more frequent.

Conclusion

An increase of tularemia cases occurred in 2007/2008 in both species. Complementary studies are needed to identify the species reservoir in France to understand the causes of this peak of cases.     

The design of hazard risk assessment matrices for ranking occupational health risks and their application in mining and minerals processing

Two hazard risk assessment matrices for the ranking of occupational health risks are described. The qualitative matrix uses qualitative measures of probability and consequence to determine risk assessment codes for hazard-disease combinations. A walk-through survey of an underground metalliferous mine and concentrator is used to demonstrate how the qualitative matrix can be applied to determine priorities for the control of occupational health hazards. The semi-quantitative matrix uses attributable risk as a quantitative measure of probability and uses qualitative measures of consequence. A practical application of this matrix is the determination of occupational health priorities using existing epidemiological studies. Calculated attributable risks from epidemiological studies of hazard-disease combinations in mining and minerals processing are used as examples. These historic response data do not reflect the risks associated with current exposures. A method using current exposure data, known exposure-response relationships and the semi-quantitative matrix is proposed for more accurate and current risk rankings.     

Occupational health hazards in mining: an overview

This review article outlines the physical, chemical, biological, ergonomic and psychosocial occupational health hazards of mining and associated metallurgical processes. Mining remains an important industrial sector in many parts of the world and although substantial progress has been made in the control of occupational health hazards, there remains room for further risk reduction. This applies particularly to traumatic injury hazards, ergonomic hazards and noise. Vigilance is also required to ensure exposures to coal dust and crystalline silica remain effectively controlled.     

Manufacture and use of nanomaterials: current status in the UK and global trends

This paper provides an overview of the production and use of nanomaterials (NMs), particularly in the UK. Currently, relatively few companies in the UK are identifiable as NM manufacturers, the main emphasis being the bulk markets in metals and metal oxides, and some niche markets such as carbon nanotubes and quantum dots. NM manufacturing in the UK does not reflect the global emphasis on fullerenes, nanotubes and fibres. Some assumptions have been made about the types of NM that are likely to be imported into the UK, which currently include fullerenes, modified fullerenes and other carbon-based NMs including nanotubes. Many university departments, spin-offs and private companies have developed processes for the manufacture of NMs but may only be producing small quantities for research and development (R&D) purposes. However, some have the potential to scale up to produce large quantities. The nanotechnology industry in the UK has strong R&D backup from universities and related institutions. This review has covered R&D trends at such institutions, and appropriate information has been added to a searchable database. While several companies are including NMs in their products, only a few (e.g. manufacturers of paints, coatings, cosmetics, catalysts, polymer composites) are using nanoparticles (NPs) in any significant quantities. However, this situation is likely to change rapidly. There is a need to collect more information about exposure to NPs in both manufacturing and user scenarios. As the market grows, and as manufacturers switch from the micro- to the nanoscale, the potential for exposure will increase. More research is required to quantify any risks to workers and consumers.     

The rise and fall in incidence of malignant mesothelioma from a British Naval Dockyard, 1979-1999

BACKGROUND: The incidence of malignant mesothelioma in Britain is predicted to rise over the next 15-25 years because of past failure to protect the workforce against inhalation of asbestos. In British Naval dockyards, alternative insulation materials and respiratory protection were introduced from the mid-1960s. Aims This study was carried out to investigate the effects of these control measures on mesothelioma deaths in dockyard workers. METHODS: Cases of mesothelioma of the pleura and peritoneum between 1979 and 1999 in workers from the Devonport Naval Dockyard, south-west England, were sought from coroners' and medico-legal records. RESULTS: Three hundred and one cases were identified, 7% peritoneal. The peak incidence occurred in 1991 with 25 cases per annum (quadratic model fit R(2) = 74.2%, P < 0.001) and we predict that by 2003 the incidence will fall to fewer than five cases per annum. The mean time between first exposure and presentation was 48.5 years [95% confidence interval (CI) = 47.3-49.8], but this was significantly shorter in the more heavily exposed trades, when compared with the less heavily exposed (42 years, 95% CI = 39.0-45.0, versus 49.5 years, 95% CI = 48.2-50.9). Those with higher exposure were also at significantly greater risk of peritoneal disease (P < 0.023, Fisher's exact test). CONCLUSION: The reduction in incidence of mesothelioma is greater than can be accounted for by reduction in numbers of dockyard workers over the last 50 years. Changes in insulation materials and improved industrial hygiene measures introduced into the Devonport Dockyard from the mid-1960s have resulted in an earlier decline in the incidence of malignant mesothelioma than that predicted for the British workforce as a whole.