纳米粒子介导反义mTOR基因转染抑制移植静脉内膜增生

目的:观察以纳米粒子为载体的外源性反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因，制备纳米级粒子混合物。建立自体静脉移植模型72只，随机分成转基因组(转染以纳米粒子为载体的反义mTOR基因)，空载体组(单纯转染纳米粒子包载的空载体)和对照组(不予特殊处理)。分别于术后3，7，14，28d取材。检测mTOR基因的mRNA及蛋白产物表达，以及血管平滑肌细胞(VSMC)凋亡的动态变化。 结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05)；转基因组内膜增生厚度于7，14，28d较其他组明显减少(P<0.01)；转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体。反义mTOR基因的表达能有效抑制自体移植静脉内膜的增生及促进VSMC凋亡。     

反义雷帕霉素靶蛋白基因局部转染对移植静脉内膜增生的影响

目的:观察以纳米粒子为载体的反义雷帕霉素靶蛋白(mTOR)基因局部转染对移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载mTOR基因,制备纳米级粒子混合物。检测其包埋率、体外释放情况及粒子大小。建立自体静脉移植模型,随机分成转基因组、空载体组、对照组。转基因组移植静脉转染以纳米粒子为载体的反义mTOR基因,空载体组单纯转染纳米粒子包载的空载体,对照组不予特殊处理。分别于术后3d、7d、14d、28d取材,常规HE、Verhoeff染色,RT-PCR、Westernblot检测mTOR基因的mRNA及蛋白的变化,TUNEL法观察血管平滑肌细胞(VSMC)凋亡的动态变化。结果:转基因组内膜中mTOR基因的mRNA及蛋白产物表达较其他两组明显减少(P<0.05);转基因组内膜增生厚度7d、14d、28d较其他组明显减少(P<0.01);转基因组凋亡细胞较其他组明显增高(P<0.05)。结论:纳米粒子可以作为转基因载体,反义mTOR基因的表达能够有效抑制自体移植静脉内膜的增生,促进VSMC的凋亡。     

RNA干扰沉默蛋白激酶B基因表达对移植静脉内膜增生的影响

摘要：目的:观察纳米粒子包载的靶向蛋白激酶B(PKB)基因的shRNA表达载体局部转染对大鼠移植静脉内膜增生的影响。方法:应用聚乳酸聚乙醇酸共聚物(PLGA)和聚乙烯醇(PVA)包载PKB的RNA干扰基因载体，制备纳米级粒子混合物。建立自体颈静脉-颈总动脉移植模型共72只，随机分成转基因组、空载体组和对照组。分别于术后3，7，14，28 d取材；常规HE及Verhoeff 染色,用Northern blot和Western blot检测PKB基因的mRNA及蛋白的变化，HE和Verhoeff 染色观察内膜厚度，TUNEL法观察血管平滑肌细胞(VSMC)凋亡的动态变化。结果:转基因组内膜中PKB基因的mRNA及蛋白产物表达较其他两组明显减少(P＜0.05)；术后7，14，28 d转基因组静脉内膜增生厚度较其他组明显减少(P＜0.01)；转基因组细胞凋亡率较其他组明显增高(P＜0.05)。结论:纳米粒子可以作为转基因载体；沉默PKB基因表达能有效地抑制自体移植静脉内膜的增生，促进VSMC的凋亡。     

p27kip1基因表达对大鼠移植静脉内膜平滑肌细胞增殖与凋亡的影响

目的观察以聚乳酸聚乙醇酸（PLGA）纳米粒子为载体的人p27^kip1基因局部转染自体移植静脉后，对大鼠静脉内膜平滑肌细胞增殖及凋亡的影响。方法Wistar大鼠120只建立自体静脉移植模型，随机分成3组。转基因组：移植静脉转染以PLGA纳米粒子为载体的p27^kip1基因；空白对照组：转染不含有p27^kip1基因的单纯PLGA纳米粒子；单纯静脉移植组：使用生理盐水。分别于术后3、7、14、28d取材，常规HE、Verhoeff弹力纤维染色，Western blot检测p27^kip1蛋白的表达，免疫组化（SABC）法检测增殖细胞核抗原（PCNA）的表达、TUNEL法观察内膜平滑肌细胞凋亡的动态变化。结果转基因组内膜中p27^kip1蛋白表达水平高于其他组；内膜平均厚度7、14、28d低于其他2组（P〈0．01）；转基因组内膜PCNA的表达7、14d明显受到抑制（P〈0．01），平滑肌细胞的凋亡细胞百分比于7、14d较对照组明显增加（P〈0．01），单纯静脉移植组与空白对照组之间各项监测指标差异无统计学意义。结论p27^kip1基因的过表达能够有效抑制自体静脉移植后的内膜增生（IH），促进平滑肌细胞的凋亡。     

反义寡核苷酸抑制Survivin基因表达对移植静脉内膜增生的影响

目的研究反义寡脱氧核苷酸（ASODN）抑制Survivin基因表达对移植静脉内膜增生的抑制作用。方法Wistar大鼠60只，建立自体静脉移植模型，术后随机分为：对照组、Survivin ASODN 50、200μg组、正义对照组、Lipofectin＋pluronic等五个组，施加不同的处理因素，在移植后1、2周取材。组织形态学方法比较内膜增生程度，逆转录．聚合酶链反应（RT-PCR）检测Survivin基因的mRNA表达，Westem blot检测Survivin基因的蛋白产物表达，免疫组织化学方法检测Survivin及增殖细胞核抗原（PCNA）的表达，脱氧核苷酸转移酶末端标记法（TUNEL）检测血管平滑肌细胞（VSMC）凋亡的变化。结果移植后1、2周内膜增生明显，局部转染50μg Survivin ASODN组内膜增生明显受抑制（P〈0．05），200μg组受抑制程度更为明显（P〈0．05）；与对照组相比，Survivln ASODN组Survivin的mRNA及蛋白产物表达显著减少（P〈0．05），PCNA阳性表达同时减少，而TUNEL阳性细胞却明显增加。结论Survivin ASODN可显著抑制移植静脉的内膜增生，其作用可能是通过抑制Survivin基因及其蛋白产物表达，从而抑制VSMC增殖、促进其凋亡而实现的。     

转染survivin反义寡脱氧核苷酸对移植血管内膜增生的影响

目的研究survivin基因的反义寡脱氧核苷酸（ASODN）对移植血管内膜增生的影响。方法Wistar大鼠60只，建立自体静脉移植模型，术后随机分为5组：对照组，survivin ASODN 50μg组，200μg组，正义对照组，lipoectin＋pluronic组。分别施加不同的处理因素，在移植后1，2周取材。用组织形态学方法比较内膜增生程度；用半定量RT-PCR检测survivin基因的mRNA表达；Western blot检测survivin基因的蛋白产物表达；免疫组化方法检测survivin及增殖细胞核抗原（PCNA）的表达，TUNEL法检测血管平滑肌细胞（VSMC）凋亡。结果静脉移植1～2周内膜增生明显，局部转染50μg survivin ASODN后明显抑制内膜增生（P〈0．05），200μg组受抑制程度较50μg组更为显著（P〈0．05）。静脉移植后，对照组survivin的mRNA及蛋白产物表达显著增加，而survivin ASODN组却显著减少（P〈0．05），VSMC中PCNA表达也同时减少，而TUNEL阳性细胞明显增加。结论survivin ASODNs可显著抑制移植静脉的内膜增生；其作用可能是通过抑制survivin的基因及蛋白产物表达，促进VSMC凋亡而实现的。     

纳米粒子载体携带反义转化生长因子-β1基因的局部定位转染对大鼠自体移植静脉内膜增生的影响

目的探讨纳米粒子携带反义转化生长因子(TGF-)β1局部定位转染对大鼠自体移植静脉内膜增生的影响。方法45只大鼠制成自体静脉移植模型后,分成纳米粒子DNA组、纳米粒子空载体组和生理盐水对照组,进行局部定位转染,2周后观察移植静脉内膜的变化,应用逆转录聚合酶链反应(RTPCR)、Westernblot以及免疫组织化学等方法检测反义基因对内源性TG-Fβ1表达的影响。结果基因转染后两周,内膜厚度为(20.5±4.7)μm,明显小于两个对照组(P<0.01)。RTPCR和Westernblot结果表明,基因转染组的TGF-β1mRNA和蛋白的表达明显低于空载体组及生理盐水对照组。结论纳米粒子可有效地作为基因的转移载体;纳米粒子携带反义TGF-β1可以减少内源性TGF-β1基因的表达,抑制细胞外基质(ECM)的合成,从而达到抑制内膜增生的目的。     

反义寡脱氧核苷酸抑制survivin基因表达对移植静脉内膜增生的影响

目的探讨生存素（survivin）的反义寡脱氧核苷酸（ASODNs）抑制survivin基因表达对移植静脉内膜增生的抑制作用。方法Wistar大鼠60只，建立自体静脉移植模型，然后随机均分为对照组、survivin ASODNS 50μg和200μg组、正义寡脱氧核苷酸组（ODNs组）及Lipofectin＋pluronic组共5组，施加不同的处理因素，分别在静脉移植术后7和14d取材。组织形态学方法比较移植静脉内膜增生程度，半定量RT—PCR法检测survivin基因mRNA的表达，Western blot检测survivin基因的蛋白产物表达，免疫组化法检测survivin及增殖细胞核抗原（PCNA）的表达，TUNEL检测血管平滑肌细胞（VSMC）凋亡的变化。结果对照组、ODNs组和Lipofectin＋pluronic组移植术后7d、14d移植静脉内膜增生明显，但3组间差异无统计学意义（P〉0．05），局部转染50μg survivin ASODNs能明显抑制其内膜增生（P〈0．05），200μg组受抑制程度更明显（P〈0．05）。对照组survivin mRNA及蛋白产物表达显著增加，而survivin ASODNs组却显著减少（P〈0．05），VSMC中PCNA表达也同时减少（P〈0．05），而VSMC凋亡明显增加（P〈0．05）。结论survivin ASODNs可显著抑制移植静脉内膜增生，其作用可能是通过抑制survivin的基因及蛋白产物表达，从而减轻VSMC增殖，促进其凋亡而实现的。     

野生型p53基因转染和低能量激光照射防治移植静脉再狭窄的实验研究

目的：探索转基因疗法和激光疗法防治移植静脉远期再狭窄的可行性及作用机制。方法：建立兔颈外静脉颈总动脉移植模型，分为（1）对照组，（2）绿色荧光蛋白（GFP）基因转染组，（3）p53基因转染组，（4）低能量激光照射组，（5）p53基因转染并低能量激光照射组。术后4周，免疫组织化学方法检测外源p53基因的表达及增殖细胞核抗原（PCNA），应用DNA片段末端标记法（TUNEL）标记凋亡细胞。HE、Masson及维多利亚兰染色后，应用计算机图像分析系统检测移植静脉内膜、中膜增生情况。结果：术后4周，与对照组相比，GFP基因转染组移植静脉血管平滑肌细胞（VSMC）增殖率、凋亡率差异无显著性，移植静脉内膜和中膜厚度无明显变化；p53基因转染组VSMC增殖率降低61％，凋亡率增加25％，移植静脉内膜和中膜厚度分别减少60％、33％，内膜厚度/中膜厚度比值(I/M)减少37％；应用低能量激光照射组VSMC增殖率降低41.5％，细胞凋亡率增加40.9％，移植静脉内膜和中膜厚度分别减少了58.5％、18.0％，I/M比值减少47.2％；转染p53基因同时应用低能量激光照射组VSMC增殖率较对照组降低61.7％，细胞凋亡率增高47.0％，移植静脉内膜和中膜厚度分别减少69.7％、44.4％，I/M比值减少44.5％。结论：转染野生型p53基因和低能量激光血管外照射可以抑制静脉VSMC增殖，促进移植静脉VSMC凋亡，使移植静脉内膜和中膜的增生减轻，具有防治移植静脉远期再狭窄的作用。     

雷帕霉素靶蛋白反义RNA真核表达载体的构建及其在血管平滑肌细胞中的表达

目的构建雷帕霉素靶蛋白（mTOR）的反义RNA真核表达载体，观察其对血管平滑肌细胞（VSMC）功能的影响。方法提取人VSMC总RNA，逆转录．聚合酶链反应（RT-PCR）扩增mTOR基因cDNA序列，经pGEM-T载体克隆后双酶切，将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1，转染VSMC，Westernblot法检测反义表达载体对mTOR蛋白表达的影响，流式细胞仪检测细胞周期的变化。结果经RT-PCR获得664bp产物，T载体克隆后，酶切确定该片段为mTOR基因cDNA，进而构建反义RNA真核表达载体pEGFP-C1-mTOR，测序证明序列正确后转染VSMC，证实其能够显著抑制mTOR蛋白产物表达，转染72h的mTOR蛋白产物表达抑制率达82％，S期细胞由15％降低为7％，凋亡细胞增至9％。VSMC增殖过程在Gx／Go期→S期受阻。结论成功构建mTOR基因的反义RNA真核表达载体。     

2015年乳腺癌辅助化疗进展

【摘要】〓乳腺癌是危害我国女性健康的头号杀手,尽管近年来辅助化疗的研究进展突飞猛进,但临床中仍有不少问题未能明确,如辅助化疗的合适人群、化疗的开始时间、蒽环及紫杉类的地位和用法、强化维持治疗的作用、疗效及预后的生物标志物等。本文结合乳腺癌辅助化疗在临床上的常见问题和2015年各大乳腺癌会议阐述乳腺癌辅助化疗的最新进展。     

Alterations in T-Cell Subset Frequency in Peripheral Blood in Obesity

Background: Obesity affects the regulation of immune and inflammatory responses. This study characterizes differences in peripheral blood lymphocyte phenotype in obese humans. Methods: Frequencies of lymphocyte subsets among peripheral blood mononuclear cells were compared between 10 obese (BMI ≥35) and 10 lean subjects, as determined by antibodies directed against cluster differentiation (CD) markers. Results: Obese patients demonstrated an increased frequency of CD3+CD4+ T-cells (mean difference 12%, P=0.004), a decreased frequency of CD3+CD8+ T-cells (mean difference 9.4%, P=0.016) and an increased frequency of CD3+CD8+CD95+ T-cells (mean difference 13.3%, P=0.032). No other differences among T-cell or monocyte subsets were noted. Conclusions: Obesity is associated with alterations in frequencies of peripheral CD4+ and CD8+ T-cells and aberrations in the expression of CD95 among CD8+ T-cells. These data suggest both CD4+ and CD8+ T-cell compartments, as well as the regulation of CD95 expression on CD8+ T-cells, as targets for further study into obesity's effects on the immune system.     

西宁地区烧伤病人动脉血气分析观察

对高海拔地区的27例烧伤病人动脉血气变化进行了分析和观察。结果证明:无论是存活病人还是死亡病人伤后均存在有低氧血症问题。并且在死亡病人和烧伤合并吸入性损伤病人其低氧血症的发生早于单纯烧伤病人。提示:吸入性损伤病人应立即行气管切开术以保障氧气供给,单纯烧伤病人可常规吸氧以维持正常血 PaO_2,ARDS 均发生在合并吸入性损伤的病人,高频喷射通气技术对纠正低氧血症有一定效果。     

Controversies in Fistula in Ano

Managing a complex fistula in ano can be a daunting task for most surgeons; largely due to the two major dreaded complications—recurrence & fecal incontinence. It is important to understand the anatomy of the anal sphincters & the aetiopathological process of the disease to provide better patient care. There are quite a few controversies associated with fistula in ano & its management, which compound the difficulty in treating fistula in ano. This article attempts to clear some of those major controversies.     

β-半乳糖苷酶在体内外成骨细胞中的表达

目的 研究β—半乳糖苷酶(β—gal)在成骨细胞中的表达状况，为阐明MorquioB综合征的发病机制提供依据。方法 裸鼠各器官和骨组织标本行X-gal染色检测。抽取羊和人骨髓行骨髓基质细胞(BMSCs)培养，分为4组：I：Adv-hBMP-2转染组；Ⅱ：Adv—β—gal转染组；Ⅲ：未转染组；Ⅳ：地塞米松诱导组。分别行X-gal染色和RT-PCR检测β—gal的表达。结果 裸鼠骺板两侧、骨膜内面及松质骨的成骨细胞和破骨细胞可见多量β—gal的表达。未转染BMSCs组有少量β—gal的表达，其他3组细胞的β—gal表达增高。结论成骨细胞和破骨细胞可表达多量β—gal，该两种细胞的β—gal缺乏可能是MorquioB综合征骨骼异常的直接原因。     

Fluid-phase transcytosis in the primate epididymis in vitro and in vivo

Ligated tubules from the corpus epididymidis of men and monkeys were incubated in medium containing horseradish peroxidase (HRP) as a marker for fluid-phase endocytosis. HRP was localized by light and electron microscopy after 0, 15, 30 and 60 min of incubation. Movement between the cells was prevented by tight junctions, but bypass of this barrier was apparently achieved by an intracellular vesicular mechanism leading to a time-dependent appearance of HRP in the lumen. Uptake of HRP into basal cells and capture by the lysosomal apparatus of principal cells were also observed. HRP-filled vesicles also appeared in the basal, mid and apical cytoplasm of epithelial cells in the caput 1 h after injection of the tracer into the epididymal circulation of the monkey, suggesting that this pathway also operates in vivo.     

Surgical management of ductal carcinoma in situ in Australia in 1995

Background: In the present paper we describe the presentation and management of ductal carcinoma in situ (DCIS) of the breast in women in Australia in 1995. This representative, national data set provides a historical comparator for studies examining DCIS management that follow. Methods: Surgeons identified by population‐based cancer registries as having treated a new diagnosis of DCIS between 1 April and 30 September 1995 completed a questionnaire on the presentation and management of each case. Results: Two hundred and five surgeons supplied treatment details on 418 DCIS tumours in 415 women . Half of all tumours were detected at BreastScreen clinics and a further 25% were detected at other mammography centres. Twenty‐six percent of tumours were palpable at presentation, 33% were multifocal and 55% were high grade (including comedocarcinoma). Breast conserving therapy (BCT) rather than mastectomy was utilized in 260 (62%) of cases. Tumours that were of low grade, small in size and not multifocal were more likely to be treated by BCT. Surgeons seeing six or more DCIS cases in the 6‐month period were more likely to utilize BCT. Of the conservatively treated cases, 22% were referred for a radiation oncology consultation. The most common reasons for treating DCIS with mastectomy were that the tumour was too extensive or multifocal (63%), it extended to margins of the specimen (42%), or patient concerns about recurrence (34%). Conclusions: In 1995 the majority of DCIS was treated with breast conserving surgery alone. Surgeons treating more DCIS cases were more likely to perform conservative surgery than surgeons treating only one DCIS case in the study period.     

Smoking-Attributable mortality in Spain in 2016

IntroductionSmoking-attributable mortality (SAM) is a valuable indicator that can be used to characterize the course and health burden of the smoking epidemic. The aim of this paper was to estimate SAM in Spain in 2016 in the population aged 35 and over, using the best available evidence.MethodsA smoking prevalence-dependent analysis based on the estimation of population-attributable fractions was performed. Smoking prevalence (never, former, and current smokers) was calculated from a combination of the Spanish Health Survey (2016) and the European Health Survey (2014); the relative risk of death among current and former smokers was taken from the follow-up of various cohorts; and mortality rates were obtained from National Center for Statistics data. SAM estimates are presented globally, and by sex, age groups, and major disease categories: cancer, cardiometabolic diseases and respiratory diseases.ResultsIn 2016, 56,124 deaths were attributed to tobacco consumption, 84% in men (47,000), and 50% in the population aged over 74 (27,795). Overall, 50% of SAM was due to cancer (28,281), 65% of which was lung cancer. One in 4 attributable deaths (13,849) occurred before the age of 65.ConclusionsOne in 7 deaths in Spain in 2016 were attributable to smoking. This estimation of SAM clearly highlights the great impact of smoking on mortality in Spain, mainly due to lung cancer and chronic obstructive pulmonary disease.