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。     

不同保存条件对丙型肝炎病毒核酸稳定性的影响

目的探讨不同温度和时间条件下保存对血液中丙型肝炎病毒核酸(HCV RNA)稳定性的影响。方法采集12名丙型肝炎患者4 ml的外周血,分装、保存在不同温度及相同温度下的不同时间,利用实时荧光定量PCR检测其不同时间点血浆HCV RNA的水平(log IU/ml),并与该血液标本的起始HCV RNA水平做比较分析。结果当血液标本中的HCV RNA起始水平≥105IU/ml时,在25℃放置6 h后转至4℃保存18 h(a)、25℃放置12 h后转至4℃保存12 h(b)及4℃放置24 h(c)等3种保存条件,24 h时内检测到血液标本的HCV RNA水平105IU/ml组分别为(5.15±0.16)、(5.15±0.15)及(5.16±0.16)IU/ml(P>0.05),107 IU/ml组分别为(7.45±0.21)、(7.44±0.23)及(7.45±0.24)IU/ml(P>0.05);当血液标本中HCV RNA起始水平在临界范围[(3.38±0.14)IU/ml]时,在条件a、b保存24 h血液标本的HCV RNA含量分别为(2.28±1.52)IU/ml、0(均为P<0.01),而条件c保存24 h血液标本的HCV RNA含量为(3.28±0.20)IU/ml(P>0.05);当血液标本中HCV RNA起始水平为103 IU/ml时,在4℃保存48 h时HCV RNA含量为(2.31±1.54)IU/ml(P<0.01)。结论当血液中HCVRNA起始浓度较低时,HCV RNA的稳定性较差,短时常温保存都会造成病毒核酸的降解。     

Col1a1-GFP Transgene Expression in Developing Incisors

Previous studies have shown that terminal differentiation of odontoblasts is accompanied by dramatic increases in type I collagen synthesis. Recently transgenic mice in which green fluorescent protein (GFP) expression is under the control of the rat 3.6 (pOBCol3.6GFPtpz) and 2.3 (pOBCol2.3GFPemd) Col1a1 promoter fragments were generated. Our analysis of these GFP-expressing transgenic mice shows that the 2.3-kb promoter fragment directs strong expression of GFP only to bones and teeth, whereas the 3.6-kb fragment of promoter directs strong expression of GFP in bone and tooth, as well as in other type I collagen producing tissues. Our observations of incisors in these transgenic mice show high levels of GFP expression in functional odontoblasts and in differentiated osteoblasts. These observations show that expression of GFP reporter genes closely follow the patterns of expression of &#102 1(I) collagen in various tissues including odontoblasts.     

实时荧光定量PCR检测人宫颈癌细胞赖氨酰氧化酶样-4基因的表达

目的:利用实时荧光定量PCR检测赖氨酰氧化酶样-4(Lysyloxidase-like 4,LOXL4)基因在Hela、ME180、HCC94人宫颈癌细胞中的表达情况.方法:提取3株体外培养宫颈癌细胞株的总RNA,逆转录为cDNA,实时荧光定量PCR扩增,结合内参基因GAPDH及标准曲线比较LOXL4 mRNA在Hela、ME180和HCC94 3株体外培养的人宫颈癌细胞中的表达.结果:LOXL4基因在3株人宫颈癌细胞中均有表达,但表达水平存在统计学差异,以Hela的表达为参照(Ratio=LOXL4/GAPDH=1.00),ME180的表达水平为6.80E-3,HCC94细胞株表达丰度最低为5.62E-4.结论:建立了LOXL4基因在宫颈癌细胞表达的检测平台,为宫颈癌早期诊断及预测预后提供可借鉴的观察指标.     

巨细胞病毒pp65抗原检测的新方法

目的传统的检测CMVpp65抗原的方法是间接免疫荧光,操作麻烦,步骤较多,整个实验需要6个多小时,而且只能定性。需要寻找一种简便的可以定量的方法来检测CMVpp65抗原阳性细胞。因此我们设计了流式的方法来检测CMVpp65抗原阳性细胞。方法我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行了流式标记移植病人外周血中的WBC,操作步骤明显简便了,只需不到2 h就可完成检测,而且可以定量检测CMVpp65抗原阳性细胞。同时我们用荧光定量PCR检测病人尿中的CMV基因的拷贝数。结果流式检测阳性率高的标本,用间接免疫荧光方法检测也为阳性,同时流式检测强阳性的病人尿用荧光定量PCR检测到CMV基因,表明病人处于CMV病毒血症。结论我们用间接免疫荧光试剂盒中的一抗和购自invitrogen公司的二抗进行流式标记,成功检测了移植病人的CMVpp65抗原阳性细胞的百分比,新方法可以定量,与荧光定量PCR方法检测病人尿中的CMV基因的结果符合率较高,可以推广应用。     

食品中非法添加和滥用化学染料的分析研究

建立了食品中碱性黄、碱性嫩黄、碱性橙II、酸性橙II、罗丹明B、对位红、苏丹类1-4号11种有机染料的高效液相色谱分析方法。在0.2-10μg/mL的浓度范围内线性相关系数良好,相关系数范围为0.9996-0.9999,检测限0.05μg/mL。该方法涉及的化学试剂少,操作简便,结果准确。对阳性样品进一步采用液质联用技术确证。     

Rhodamine B increases hypothalamic cell apoptosis and disrupts hormonal balance in rats

ObjectiveTo investigate whether orally exposure to rhodamine B could be changes the expression of Bax, Bcl-2 of the hypothalamic, and also levels of Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) in female rats.MethodsTwenty eight virgin female Wistar rats were divided into four groups, including control group, group exposed to dose of 4.5, 9 and 18 milligram/200 gram body weight (mg/200 g BW) of rhodamine B daily for 36 days. The hypothalamic expressions of Bax and Bcl-2 were examined immunohistochemically. The levels of serum FSH and LH were determined by the enzyme-linked immunosorbent assay (ELISA) technique.ResultsThe level of Bax was significantly higher in the rhodamine B treatment group compred to control group (P<0.05). Out of the 4.5, 9, and 18 mg/200 g BW doses of rhodamine B treatment, only the two highest doses significantly decreased the Bcl-2 levels compared to the control group (P<0.05). The serum FSH and LH levels were significantly lower in all dose's rhodamine B treatment groups compared with the control (P<0.05).ConclusionIn conclusion, rhodamine B increases hypothalamic cell apoptosis and disrupts hormonal balance in rats.     

Combining different assays and chemical analysis to characterize the genotoxicity of waters impacted by textile discharges

Waters receiving textile discharges can exhibit genotoxic and mutagenic activity, which has been related to the presence of dyes and aromatic amines as synthesis precursors or byproducts. The aim of this study was to identify dyes and aromatic amines in water samples impacted by textile discharges, and to evaluate the genotoxic responses of these samples using the Salmonella/microsome assay in strains TA98 and YG1041, and the Fpg‐modified comet assay in the RTL‐W1 fish cell line. The genotoxicity of river samples downstream of the discharge was greater than the upstream samples in both of the Ames tests. The Fpg‐modified comet assay detected similar levels of DNA damage in the upstream and downstream samples. Mutagenicity was not detected with TA98, except for the Quilombo River samples, but when YG1041 was used as the tester strain mutagenicity was detected for all sites with a very different profile in upstream sites relative to the other sites. The mutagenic response strongly indicated that aromatic amines or dyes were contributing to the mutagenic activity downstream. The impact of textile discharges was also confirmed by chemical analysis, because the highest concentrations of azo dyes and aromatic amines were detected in the river downstream. This study shows the value of combining assays measuring complementary endpoints to better characterize the mutagenicity of environmental samples, with the advantage that this approach provides an indication of what classes of compounds are responsible for the effect. Environ. Mol. Mutagen. 57:559–571, 2016. © 2016 Wiley Periodicals, Inc.