细胞凋亡的检测技术与方法

细胞凋亡在保证多细胞生物的健康生存过程中扮演着关键的角色。根据死亡细胞的形态学、生物化学和分子生物学特征,可以将凋亡细胞与坏死细胞区别开来。本文综述了常用的细胞凋亡检测技术与方法的基本原理,如形态学观察、荧光素染色、DNA阶梯、半胱氨酸天冬氨酸蛋白酶活性的检测等,并对各种方法的优点及局限性做一简要比较。用透射电镜进行超微结构观察仍是鉴定细胞凋亡的金标准。在选择细胞凋亡检测方法时,要充分了解各种方法的原理和应用范围,进行综合考虑。多种检测方法的联合应用常可获得准确的结果。     

Brn3b基因对视神经损伤条件下视网膜神经节细胞的保护作用

目的　探讨Brn3b过表达对视神经损伤条件下视网膜神经节细胞（retinal ganglion cells，RGCs）的保护作用。方法　选取雄性健康成年BALB/c小鼠60只，用微型视神经夹将小鼠右眼球后视神经夹持损伤，按视神经损伤后的天数依次分为第1、3、5、7、14天组，小鼠左侧正常眼作为空白组，明确视神经损伤天数条件。选取雄性健康成年BALB/c小鼠40只，用微量进样器以小鼠右眼玻璃体内注射的方法将腺相关病毒载体转染小鼠视网膜，建立Brn3b过表达模型并分组:Brn3b过表达组［转染Brn3b过表达腺相关病毒载体（Brn3b overexpressed adeno-associated viral vector，AAV-CMV-Brn3b）］和阴性对照组［转染空白腺相关病毒载体（blank adeno-associated viral vector，AAV-CMV-GFP）阴性对照物］，每组20只；之后，取Brn3b过表达组和阴性对照组各10只，用微型视神经夹将小鼠右眼球后视神经夹持损伤，构建模拟小鼠视神经损伤（controlled optic nerve crush，CONC）模型并分组:CONC-Brn3b过表达组和CONC-阴性对照组。利用视网膜铺片和切片免疫荧光标记相关蛋白表达量，检测Brn3b过表达对视神经损伤条件下RGCs、Brn3b和Caspase3蛋白表达的影响，并对其共定位情况做出统计分析。结果　与阴性对照组相比，Brn3b过表达组小鼠视网膜Brn3b的表达水平明显增加。在小鼠CONC模型制作后的第7天RGCs的总凋亡数量达到65%，第14天RGCs的凋亡数量未见进一步改变。免疫荧光标记的蛋白表达量及其共定位分析显示，在视神经损伤条件下，与CONC-阴性对照组相比，CONC-Brn3b过表达组小鼠RGCs的凋亡量以及凋亡因子Caspase3的表达量均明显减少，差异均有统计学意义（均为P＜0.001）。结论　Brn3b基因对视神经损伤条件下RGC具有明确的保护作用，Brn3b基因对凋亡因子Caspase3的表达可能具有一定的抑制作用。     

Discovery of Biaryl Amides as Potent,Orally Bioavailable, and CNS Penetrant RORγt Inhibitors

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CEACAM5 has different expression patterns in gastric non-neoplastic and neoplastic lesions and cytoplasmic staining is a marker for evaluation of tumor progression in gastric adenocarcinoma

Objective

The aim of this study was to investigate the expression patterns of CEACAM5 in non-neoplastic and neoplastic gastric lesions, as well as its application in the differential diagnosis and its relationship with tumor progression.

Methods

CEACAM5 expression was detected by immunohistochemical staining in the serial sections of the gastric neoplastic and non-neoplastic lesions. The impacts of CEACAM5 expression patterns on tumor progression were evaluated by statistics, the clinical and pathological data included sex, age, tumor extension, lymph node involvement and tumor staging.

Results

There was no CEACAM5 expression in normal gastric epithelial cells. In hyperplastic polyps, CEACAM5 was expressed with apical membranous staining in the hyperplastic and prolonged gastric pit adjacent to the surface. Intestinal metaplasia (IM) expressed CEACAM5 mainly with membranous pattern, and some cases showed membranous staining mixed with cytoplasmic staining. GIN expressed CEACAM5 mainly with membranous staining, but the mixed staining of cytoplasmic and membranous patterns increased, and especially in the high grade GIN, cytoplasmic staining of CEACAM5 began to occur. Compared with IM and GIN, CEACAM5 expression patterns of hyperplastic polyp showed a significant difference (P = 0.000). IM, low grade GIN and the whole GIN showed no significant difference in CEACAM5 expression patterns (P = 0.355), but IM and high grade GIN showed a significant difference (P = 0.027). There was a significant difference between low and high grade GIN (P = 0.002). GIN and well-differentiated carcinomas showed no significant difference (P = 0.070), but low grade GIN and well differentiated carcinomas showed a significant difference (P = 0.006). In gastric adenocarcinomas, CEACAM5 expression patterns showed a significant difference in tumor grading (P = 0.010) and Laurén classification (P = 0.001). In histological grading, well differentiated carcinomas showed more membranous staining than moderately and poorly differentiated, and more cytoplasmic CEACAM5 staining was detected in moderately and poorly differentiated carcinomas. Similar to that, in Laurén classification, intestinal carcinomas showed more membranous staining, and diffuse carcinomas showed more cytoplasmic staining. Moreover, CEACAM5 expression patterns showed a significant difference in tumor extension (P = 0.012), lymph node involvement (P = 0.015) and tumor staging (P = 0.002), suggesting that CEACAM5 should be involved in tumor progression. In advanced carcinomas, CEACAM5 was expressed with more cytoplasmic staining regardless of the histological classification.

Conclusion

CEACAM5 had different expression patterns in gastric non-neoplastic and neoplastic lesions. The CEACAM5 expression patterns were associated with tumor progression. Membranous staining of CEACAM5 might be a marker of premalignancy in gastric lesions, and cytoplasmic CEACAM5 might enhance tumor invasion and migration and be an evaluated marker for progressive and advanced gastric cancer. Also, it might be useful for the differential diagnosis of gastric premalignant lesions.     

Investigation and influencing factors about well-being level of elderly chronic patients during COVID-19 postpandemic period in Beijing

The Corona Virus Disease 2019 (COVID-19) pandemic has huge impacts on the world, including human health and economic decline. The COVID-19 has severe infectivity, especially the elderly with chronic diseases will cause various complications after infection and accelerate the disease process. In addition, COVID-19 will also affect their mental health. Therefore, the mental health of elderly patients with chronic diseases cannot be ignored. The aim of this study was to investigate the well-being level of elderly people with chronic disease during COVID-19 postpandemic period in Beijing and analysis related influencing factors, so as to provide a basis for improving the well-being level of elderly chronic patients during the postpandemic period.Elderly patients with chronic diseases who met the inclusion criteria in 5 different administrative regions in Beijing were selected to carry out a questionnaire survey. The contents of the questionnaire included general data, the Memorial University of Newfoundland Happiness scale and the awareness situation of the COVID-19 pandemic. A total of 500 questionnaires were distributed by WeChat and 486 valid questionnaires were collected. The t test and one-way analysis of variance were used to compare Memorial University of Newfoundland Happiness scores between 2 or more groups, multiple linear regression analysis was used to conduct multiple factor analysis to explore the related factors about well-being level of elderly chronic patients.A total of 109 cases (22.43%) were evaluated high well-being level, 319 cases (65.64%) were evaluated moderate well-being level and 58 cases (11.93%) were evaluated low well-being according to the Memorial University of Newfoundland Happiness (MUNSH) scores rating. The multiple linear regression indicated that the education level, number of chronic diseases, medical expenses, frequency of children''s visits, taking care of grandchildren or not, and group activity frequency significantly affected the well-being of patients with chronic diseases during COVID-19 postpandemic period in Beijing (P < .05).Most elderly patients with chronic diseases had moderate or above sense of well-being during postpandemic period, but we should still pay attention to the mental health of those elderly chronic patients with low education level, much comorbidity, more medical expenses, less visits by children, not take care of grandchildren and never participate in group activities.     

细菌内同源重组快速构建和制备表达hSDF-1α的重组腺病毒

目的:利用细菌内同源重组快速构建携带hSDF-1α cDNA重组腺病毒质粒和制备表达hSDF-1α重组腺病毒. 方法: 制备感受态BJ5183,将pAdEasy-1转化BJ5183,制备含pAdEasy-1的BJ5183感受态,将线性化的pShuttle-EGFP-hSDF-1α转化含pAdEasy-1的BJ5183感受态菌. 采用细菌中同源重组法构建重组腺病毒质粒pAd-EGFP-hSDF-1α. pAd-EGFP-hSDF-1α用Pac Ⅰ线性化后,再用LipoVec介导其转染至AD293细胞内包装扩增出重组腺病毒颗粒,采用CsCl密度梯度离心法纯化重组腺病毒Ad-EGFP-hSDF-1α. 采用荧光显微镜下观察LipoVec介导的重组腺病毒质粒的转染效果及病毒包装情况. 结果:pShuttle-EGFP-hSDF-1α成功地转化了含pAdEasy-1的BJ5183,并在其内发生了同源重组. 荧光显微镜下观察证实了pAd-EGFP-hSDF-1α转染AD293后,产生了重组腺病毒Ad-EGFP-hSDF-1α. 病毒滴度为4.1×1015 pfu/L. 结论:用细菌内同源重组法可快速、高效制备表达hSDF-1α的高滴度重组腺病毒. 为研究hSDF-1α在动员骨髓源干细胞迁移到组织损伤部位修复组织损伤的研究奠定了基础.     

细胞穿透肽PEP-1介导增强型绿色荧光蛋白转导人结直肠癌SW480细胞

背景与目的:细胞穿透肽是近年来发现的一类能介导大分子物质跨膜转导的小分子肽段,本实验研究细胞穿透肽PEP-1携带大分子蛋白穿透人结直肠癌细胞SW480的能力.方法:用基因工程的方法制备并纯化EGFP蛋白和PEP-1-EGFP融合蛋白,将纯化后的两种蛋白与体外培养的人结直肠癌细胞共同孵育,在荧光显微镜下直接观察PEP-1介导目的蛋白EGFP转导入SW480细胞的能力.结果:EGFP蛋白不能进入细胞内,PEP-1-EGFP融合蛋白和SW480细胞共同孵育1h后可见有明亮绿色荧光分布于胞浆和胞核.结论:PEP-1能有效携带目的蛋白进入人结直肠癌细胞.     

Upright position mechanical ventilation: An alternative strategy for ALI/ARDS patients?

Use of body positioning to improve oxygenation in mechanically ventilated patients with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) has been well documented. However, neither prone position ventilation nor side lying ventilation has been reported to improve the survival. Whether there is a body position superior to routine supine position or other positions as therapeutic adjunct for ventilated patients with ALI and ARDS? We propose the hypothesis that upright position ventilation may be helpful to improve oxygenation and benefit patients with ALI/ARDS. According to the existing physiologic and pathophysiologic data of upright position investigation, we suppose that improvement of V/Q matching, increased functional residual capacity, alveolar recruitment, accelerated diaphragm recovery, early gastric emptying and enteric feeding may be a potential protect mechanism of upright position ventilation. Whether this can be translated into improvement in patient outcome should be further tested in clinical trial.     

PCR产物靶向克隆法构建原核表达载体pET15b-CAT

目的:将人过氧化氢酶(Catalase,CAT)cDNA的PCR扩增产物定向克隆到pET15b载体上以构建重组质粒pET15b-CAT.方法:扩增目的基因的PCR引物的5′端加上与线性化载体同源的碱基序列,以使PCR的扩增产物两端分别带上15个和线性化载体两端同源的碱基序列.以pZeoSV2( )-CAT为模板进行靶向克隆所需的人CAT cDNA扩增.将纯化的人CAT cDNA PCR产物与经XhoⅠ和BamHⅠ酶切的线性化载体pET15b以6:1摩尔数之比混合于含重组酶的反应液中,25℃反应30 min,将PCR产物定向克隆于目标载体pET15b上,获得重组质粒pET15b-CAT.重组质粒经PCR,XhoⅠ酶切和DNA测序鉴定.结果:人CAT cDNA的PCR扩增产物与经Xho Ⅰ和BamHⅠ酶切的线性化载体pET15b发生了同源重组反应,经鉴定采用PCR产物靶向克隆法成功构建了pET15b-CAT.结论:PCR产物靶向克隆法是一种简便、高效地将PCR产物定向克隆于目标载体的方法,而无需对PCR产物进行酶切、末端抹平、载体去磷酸化及连接反应,适用于任何载体、任何插入片段和任意酶切位点.