影响胚胎培养液pH及CO2平衡的因素探讨

目的探讨胚胎培养液在CO2平衡及使用过程中pH的动态变化特性。方法应用血气分析仪检测人输卵管液（HTF）培养液在CO2平衡过程中不同时间、CO2浓度、液量、加油厚度以及失去CO2支持后的二氧化碳分压（PCO2）和pH的变化。结果胚胎培养液添加10％合成血清代用品（SSS）后,[HCO3^-]降低2．19mmol／L,pH降低0．022.1．0ml培养液CO2完全平衡时间为5h,加油后延长至15h,培养液经5．0％CO2完全平衡的最终pH为7．270-7．280。培养液液面越高平衡效果越差,≥6．0ml培养液CO2平衡15h后pH尚未稳定。CO2浓度由5．0％提高到6．0％,培养液pH降低0．060。培养液加油越厚其平衡效果越差,矿物油预先经过CO2平衡能明显提高平衡效果。失去CO：支持的无油培养液在静止状况下pH稳定时间为23min,而加油培养液的pH稳定时间超过60min。结论胚胎培养液CO2平衡宜采用适量多管和≥15h的过夜平衡方法;失去CO2支持的无油平衡培养液的操作时间应尽量短,而加油培养液可稍延长;应尽量减少培养液的加油厚度,使用预先CO2平衡的矿物油能明显提高平衡效果;应实测培养液pH来客观评价CO2平衡效果或CO2气体质量,选择适宜的CO2气体浓度、平衡方式和平衡时间。     

胚胎培养液中添加白蛋白对pH的影响

目的了解胚胎培养液添加白蛋白对pH的影响。方法在HTF受精液、CM卵裂液、G1卵裂液和CSC全程培养液中添加不同浓度的白蛋白制剂,在经过5.0%CO_2过夜平衡后用血气分析仪检测其HCO_3浓度和pH。结果白蛋白制剂的HCO_3~-浓度明显低于培养液;培养液中添加白蛋白会降低HCO_3~-浓度和pH,白蛋白浓度与pH呈高度负相关(P0.01);不同品牌培养液的pH,模型存在较明显差异,HTF和CM的pH明显低于G1和CSC(P0.01)。结论胚胎培养液中添加白蛋白会降低pH且与培养液的品牌相关,应通过实测胚胎培养液的pH来确定CO_2浓度,尤其是搭配使用不同品牌产品的时候,仅仅依靠经验或厂家提供的参数是不可靠的。     

褪黑素对人卵裂期胚胎发育的影响及机制研究

目的探讨褪黑素对人第3天(D3)卵裂期胚胎发育成囊胚的影响及其作用机制。方法收集2012年10月至2014年10月在本中心行IVF-ET助孕治疗患者捐赠的D3玻璃化冷冻胚胎,复苏后存活率100%且未发生卵裂球溶解的胚胎纳入研究(n=143),随机分为褪黑素组(培养液中加入1×10-7 mol/L褪黑素,n=71)和对照组(不添加褪黑素,n=72)。两组胚胎均放于37℃、5%CO2培养箱中培养72h,统计囊胚及优质囊胚形成率;采用鲁米诺化学发光法检测培养液中总活性氧(ROS)水平;实时荧光定量PCR法分析抗氧化酶类基因CAT、GPx、SOD1、SOD2及凋亡相关基因Bax、Bcl-2的表达。结果褪黑素组的囊胚形成率(42.25%)显著高于对照组(26.38%)(P0.05),优质囊胚形成率(30.00%)略高于对照组(26.32%),但无显著性差异(P0.05)。培养72h后两组胚胎培养液中的ROS水平比较(513.33vs.556.67RLUs)无显著性差异(P0.05)。褪黑素处理后,胚胎内过氧化氢酶基因CAT的相对表达量(1.68±0.43)较对照组(1.00±0.03)显著上调(P0.05),其他基因包括Bax、Bcl-2、SOD1、SOD2、GPx的表达则没有显著性差异(P0.05)。结论褪黑素能够促进人卵裂期胚胎继续发育形成囊胚,其机制可能与上调胚胎中过氧化氢酶基因CAT的表达有关。     

受精液平衡方式对新建胚胎实验室鼠胚实验的影响

目的探讨授精当日配皿与前日配皿对昆明小白鼠卵母细胞体外受精及胚胎发育的影响,为人类辅助生殖技术(ART)胚胎实验室选择最佳配皿方案提供参考。方法选择SPF级昆明小白鼠进行实验,对同一批次雌鼠统一超促排卵后按照随机原则分为当日配皿组(当日配制授精皿)与前日配皿组(前日配制授精皿)进行鼠胚实验。授精前1日配制含10%SPS的Quinn’s 1020液,置于37℃、5%CO_2、5%O_2的饱和湿度培养箱中过夜平衡后当日配皿为当日配皿组;于授精前1日配制含10%SPS的Quinn’s 1020液,并直接配皿后置于37℃、5%CO_2、5%O_2的饱和湿度培养箱过夜平衡为前日配皿组。观察并比较两组不同受精液平衡方式中小鼠卵母细胞的受精及胚胎发育情况。结果当日配皿组完成124个小鼠促排卵周期,总获卵4 731枚,受精率为68.2%,卵裂率97.7%,囊胚形成率76.7%;前日配皿组完成80例促排卵周期,总获卵2 683枚,受精率为84.5%,卵裂率99.6%,囊胚形成率88.8%。前日配皿组的受精率、卵裂率和囊胚形成率均显著高于当日配皿组(P0.01)。结论受精培养液的平衡方式对昆明小白鼠卵母细胞的受精及胚胎发育可产生显著影响,前日配皿较当日配皿更有利于小鼠卵母细胞的受精及后期的胚胎发育。     

胰岛素对小鼠早期胚胎体外发育的影响

目的　探讨胰岛素对小鼠体内外来源早期胚胎体外发育的影响。　方法　用添加不同浓度胰岛素和胰岛素+葡萄糖的CZB培养液对小鼠体内外来源胚胎进行体外培养 ,观察囊胚发育率和囊胚细胞数的变化。　结果　小鼠 2 细胞胚胎体外培养 10 2h后 ,0 .0 5 μg/ml胰岛素添加组扩张囊胚率、孵化囊胚率和囊胚细胞数均显著高于其他各浓度添加组。葡萄糖加入显著提高了这一效应。添加 0 .0 5 μg/ml胰岛素和葡萄糖的CZB培养液同样显著提高体外受精 (IVF)胚胎的孵化囊胚率和囊胚细胞数。　结论　添加胰岛素和葡萄糖培养液对体内或IVF小鼠胚胎发育具有相同的促进作用。     

培养液成份变化对胚胎发育的影响

培养液成份对胚胎发育影响重大。培养液的设计理念是尽可能接近生理条件下的培养环境。序贯培养液分为卵裂期培养液和囊胚期培养液,设计依据是胚胎在不同发育阶段对培养条件有不同的成份需要。单一培养液不同于序贯培养液,将同一混合成份的培养液覆盖了从卵裂期到囊胚期的胚胎体外培养过程,其设计思路是"让胚胎选择"自身需要的营养物质。研究培养液对胚胎发育的影响,需要深入了解能量底物及缓冲系统在支持细胞内稳定性方面的作用。卵裂期胚胎以丙酮酸、乳酸代谢为主,当胚胎发育到桑椹胚期以后,葡萄糖的消耗量急剧增加。而囊胚培养液中存在的过量乳酸盐,增加了囊胚非整倍体率的发生。培养液中使用缓冲液,可以稳定胞内pH,维持胚胎持续发育。培养液对人类胚胎发育的影响非常复杂,选择合适而必要的成份,可减少体外操作对胚胎的应激。     

脑死亡供体鼠吸入一氧化碳对受体鼠移植肺损伤的影响

目的 评价脑死亡供体鼠吸入一氧化碳(CO)对受体鼠移植肺损伤的影响.方法 雄性Wistar大鼠24只,体重250～300 g,随机分为3组(n=8),接受非脑死亡供体肺组(NBD组)供体鼠颅内置入Fogarty导管,但不诱导脑死亡,观察2.5 h;接受吸入氧气的脑死亡供体肺组(BDO2组)供体鼠确认脑死亡后吸入40%氧气2 h;接受吸入CO的脑死亡供体肺组(BDCO组)供体鼠确认脑死亡后吸入40%氧气和0.025%CO混合气2 h.处理结束后取供体左肺,进行原位异体肺移植,受体鼠每30 min进行一次动脉血气分析.肺移植成功后2 h处死受体鼠,采集右股动脉血样,采用嘌呤氧化酶法测定血浆超氧化物歧化酶(SOD)活性;采用硫代巴比妥酸法测定丙二醛(MDA)浓度;采用ELISA法测定血浆白细胞介素(IL)-6、肿瘤坏死因子(TNF)-α和IL-10浓度.计算移植肺组织湿/干重比(W/D);测定移植肺组织髓过氧化物酶(MPO)活性,并进行移植肺组织损伤评分.结果 与C组比较,BDO2组和BDCO组PaO2/FiO2、BE、pH值和血浆SOD活性、IL-10浓度降低,移植肺组织W/D、MPO活性、损伤评分和血浆MDA、IL-6、TNF-α浓度升高(P<0.05);与BD02组比较,BDCO组PaO2/FiO2、BE、pH值和血浆SOD活性、IL-10浓度升高,移植肺组织W/D、MPO活性、损伤评分和血浆MDA、IL-6、TNF-α浓度降低(P<0.05).结论 脑死亡供体鼠吸入CO 2 h可减轻受体鼠移植肺损伤,其机制可能是吸入CO提高了移植肺的抗氧化能力,减轻了移植后局部和全身炎性反应.     

氯化锶激活对小鼠体细胞核移植胚胎卵裂率和囊胚率的影响

目的:建立氯化锶(SrCl2)诱导小鼠体细胞核移植胚胎激活的最佳方法。方法:首先构建及鉴定小鼠核移植胚胎,在注核完成后,使用以下实验激活核移植胚胎:实验1,采用5 mmol/L和10 mmol/L SrCl2处理核移植胚胎1～8 h,观察时间对卵裂率和囊胚率的影响;实验2,在4、6 h采用1～20 mmol/L多种浓度SrCl2处理核移植胚胎,观察不同浓度对卵裂率和囊胚率的影响;实验3,研究10 mmol/L SrCl2配合不同培养液激活核移植胚胎的效果;实验4,观察10 mmol/L SrCl2联合蛋白激酶抑制剂6-DMAP、细胞松驰素(CB)对体外培育核移植胚胎的影响。结果:研究发现当浓度一致时(5 mmol/L),6 h组卵裂率(38.9%)与1 h组(6.7%)、2 h组(22.8%)、3 h组(22.8%)、4 h组(25.6%)比较差异均有显著性(P<0.05),但与5 h组(28.9%)、7 h组(34.4%)、8 h组(28.9%)均无显著差异(P>0.05);当时间固定时(6 h),SrCl2浓度为10 mmol/L获得的卵裂率和囊胚率(68.9%和7.2%)较高,较1 mmol/L组(28.3%和0%)、2.5 mmol/L组(35.6%和0%)、5 mmol/L组(37.8%和1.1%)、7.5 mmol/L组(60.6%和2.2%)、15 mmol/L组(51.7%和1.1%)和20 mmol/L组(41.7%和1.1%)均高(P<0.05);不同成分培养液实验显示,含Ca2+和Mg2+KSOM组的胚胎卵裂率较低,仅为27.8%,与不含Ca2+组(69.4%)、不含Ca2+/Mg2+组(66.1%)和添加EDTA组(68.3%)比较差异均显著(P<0.05);虽然SrCl2联合各培养液对核移植胚胎体外发育影响不大,但SrCl2联合CB组囊胚细胞计数(45.40±2.23)较未联合组(30.15±1.12)、联合6-DMAP组(34.95±1.38)、联合6-DMAP+CB组(37.45±1.43)均显著增多(P<0.05)。结论:10 mmol/L SrCl2单独处理6 h或联合CB均能较好地激活小鼠体细胞核移植胚胎。     

罗比卡因和利多卡因对小鼠体外受精和早期胚胎发育的影响

目的 :应用昆明小鼠胚胎体外培养模式和小鼠体外受精模型探讨罗比卡因与利多卡因对体外受精和早期胚胎发育的影响。方法 :( 1 ) 2 -细胞期胚胎培养 :取 4～ 6周昆明雌鼠 ,下午 4～ 6时腹腔注射孕马血清 ( PMSG) 5IU,48h后腹腔注射人绒毛膜促性腺激素 ( h CG) 5IU超排卵 ,随即雌雄合笼。次日早上 9时检查阴栓 ,阳性者于注射 h CG42h后取 2 -细胞期胚胎 ,分别放入含 1、1 0及 1 0 0μg/ ml不同浓度的罗比卡因和利多卡因的M1 6培养液中培养 ,培养液中加入输卵管上皮 ,72 h后观察桑椹胚或囊胚形成率。 ( 2 )体外受精 :从 F1代 ( C57× CBA)杂交雄鼠附睾和输精管取精子 ,从昆明雌鼠取卵母细胞 ,卵母细胞体外受精前暴露于不同浓度的利多卡因和罗比卡因 3 0 min,统计体外受精率。结果 :罗比卡因只在高浓度 1 0 0 μg/ ml时对 2 -细胞至囊胚期的胚胎发育产生明显抑制影响。利多卡因则在 1μg/ ml的浓度即对 2 -细胞期胚胎发育产生明显抑制。而 1 0和 1 0 0μg/ ml的利多卡因和罗比卡因均未对体外受精率有明显影响。结论 :提示罗比卡因可能用于体外受精技术中有关配子和合子取出或植入时的麻醉。     

不同压力CO_2气腹对人胃癌MKN-45细胞生长的影响

目的研究体外模拟不同压力CO2气腹环境对人胃癌MKN-45细胞生长增殖能力的影响。方法实验组于体外建立模拟CO2气腹环境,全自动气腹机维持气腹压力分别为9、12、15mm Hg,作用时间均为4h,对照组直接放入37℃、5%CO2孵箱中培养。处理后第0、0.5、1、1.5、2、2.5、3h,用pH计检测细胞培养液pH值变化;第1、2、3、4、5、6、7天,MTT法检测细胞增殖情况。结果15mmHgCO2气腹处理后胃癌细胞增殖较对照组下降显著(P<0.05),但9、12mmHg组与对照组差异无统计学意义(P>0.05)。CO2气腹处理后细胞培养液pH值较对照组下降非常显著(P<0.01),但在恢复正常培养后最长3h即恢复正常。胃癌细胞增殖在第1天与不同压力CO2气腹处理后细胞培养液的即时pH值有显著相关性(P<0.05),而第2、3、4、5、6、7天均无相关性(P>0.05)。结论模拟9、12mmHgCO2气腹对胃癌MKN-45细胞增殖没有显著影响,模拟15mmHgCO气腹对胃癌MKN-45细胞增殖有显著的抑制作用。     

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。     

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.     

MicroRNAs in hepatic pathophysiology in diabetes

MicroRNAs(miRNAs or miRs) are small approximately 22 nucleotide RNA species that are believed to regulate diverse metabolic and physiological processes.In the recent past,several reports have surfaced that demonstrate the role of miRNAs in various biological processes and numerous disease states.For a disease as complex as diabetes,the emergence of miRNAs as key regulators leading to the disease phenotype has added a novel dimension to the area of diabetes research.On the other hand,the liver,a metabolic hub,contributes in a major way towards maintaining normal glucose levels in the body as it can both stimulate and inhibit hepatic glucose output.This equilibrium is frequently disturbed in diabetes and hence,the liver assumes special significance considering the correlation between altered hepatic physiology and diabetes.While the understanding of the mechanisms behind this altered hepatic behavior is not yet completely understood,recent reports on the status and role of miRNAs in the diabetic liver have further added to the complexities of the knowledge of hepatic pathophysiology in diabetes.Here,we bring together the various miRNAs that play a role in the altered hepatic behavior during diabetes.     

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.