微波修复在细胞凋亡原位检测中的应用

末端转移酶 (Td T)介导的 X-d UTP切口末端标记法(Td T-mediated X-d UTP nick end labeling TUNEL)是目前原位检测细胞凋亡最敏感、快速、特异的新方法 ,已广泛用于医学生物学领域 ,尤其对肿瘤的基础与临床研究。它的标记靶是凋亡细胞 DNA在核小体处断裂形成的 3′-OH,通过TDT将标记的脱氧核苷酸结合到 3′-OH末端上 ,通常所用核苷酸为 d UTP,再进行免疫组织化学方法观察。TUNEL法检测的成功与否和组织的修复有着十分密切的关系 ,一般石蜡包埋切片作 TUNEL检测 ,其修复的方法均采用蛋白酶 K处理。在实践操作中我们发现这…     

The thymic stromal cell line MTSC4 induced thymocyte apoptosis in a non-MHC-restricted manner

Mouse thymic stromal cell line 4 (MTSC4) is one of the stromal cell lines established in our laboratory. While losing the characteristics of epithelial cells, they express some surface markers shared with thymic dendritic cells (TDCs). To further study the biological functions of these cells, we compared the capability of MTSC4 with TDCs in the induction of thymocyte apoptosis, using thymic reaggregation culture system. Apoptosis of thymocytes induced by MTSC4 and TDCs was measured by Annexin V and PI staining and analyzed by flow cytometry. We found that MTSC4 selectively augmented the apoptosis of CD4^ 8^ (DP) thymocytes. This effect was Fas/FasL independent and could not be blocked by antibodies to MHC class I and class II molecules. In addition, MTSC4 enhanced the apoptosis of DP thymocytes from different strains of mice, which implies that MTSC4-induced thymocyte apoptosis is not mediated by the TCR recognition of self peptide/MHC molecules. In contrast to MTSC4, thymocyte apoptosis induced by TDCs was MHC-restricted. Thus, MHC-independent fashion of stromal-DP thymocyte interaction may be one of the ways to induce thymocyte apoptosis in thymus. Our study has also shown that the interaction of MTSC4 stromal cells and thymocytes is required for the induction of thymocyte apoptosis.     

拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

GnRH受体与TRAIL在前列腺肿瘤中表达的研究

目的研究促性腺激素释放激素受体(GnRH-R)和凋亡相关蛋白(TRAIL)在前列腺肿瘤中的表达及临床意义.方法采用SABC免疫组化法及原位定量法,对前列腺肿瘤和前列腺增生患者中GnRH-R和TRAIL的表达进行检测及半定量分析.结果在前列腺癌组织高分化的11例,中分化的4例以及低分化的5例中,各种不同分化的癌组织均显示不同程度的GnRH-R和TRAIL阳性免疫反应,原位定量显示GnRH-R与TRAIL的表达均分别随着组织分化程度增高而增高(P<0.05),并且TRAIL较GnRH-R阳性免疫反应强.结论 GnRH-R和TRAIL表达量可能与前列腺恶性肿瘤的发生与发展有关.     

肿瘤坏死因子家族新成员——TRAIL

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)或称凋亡素2配体(Apo2 ligand, Apo-2L), 是TNF家族的新成员.它是从表达序列标签库(expressed sequenced tag, EST)中寻找TNF的同源分子时发现的.TRAIL是一种分子质量为32.5 ku的Ⅱ型跨膜糖蛋白, 活性形式呈同源三聚体.TRAIL和可溶性的TRAIL强烈诱导肿瘤细胞株凋亡.新近发现的TRAIL受体DR4和DR5及TRID说明了TRAIL与TNF和Fas/Apo-1配体的作用途径是不同的.随着对TRAIL的受体及作用机理研究的深入, TRAIL很可能成为新一代抗肿瘤制剂.     

醋酸铅对体外培养大鼠肾小管上皮细胞的毒性损伤

探讨醋酸铅对体外培养的大鼠肾小管上皮细胞系HBZY-1的细胞毒性效应.用不同浓度的醋酸铅(30、40、50 μmol/L)作用HBZY-1细胞24 h、48 h和72 h,采用CellTiter-Glo(R)萤光细胞活性检测盒测定细胞存活情况;用流式细胞仪检测细胞凋亡率.结果显示经30、40、50μmol/L醋酸铅处理...     

新加良附方对移植性人胃癌细胞Survivin与Caspase-3蛋白表达影响

目的:观察新加良附方对移植性人胃癌细胞(BGC-823)Survivin与Caspase-3蛋白表达的影响.方法:建立移植性人胃癌动物模型,并将动物模型随机分为模型对照、5-Fu组及新加良附方高、中、低剂量5组.新加良附大、中、小剂量组给药量分别为10g/kg、5g/kg和2.5g/kg;5-Fu组给药剂量为17mg/kg;模型组给予等量无菌生理盐水.连续给药、给水15天处死模型小鼠分离肿瘤,瘤块称重,石蜡包埋,并将肿瘤组织切片,免疫组化法检测Survivin与Caspase-3蛋白表达.结果:新加良附方高、中剂量组能上调肿瘤组织Caspase蛋白表达,与模型对照组比较,有统计学意义(P＜0.05);新加良附方高、低剂量组均能下调Survivin蛋白表达,与模型对照组比较,有统计学意义(P＜0.05).结论:新加良附方能通过上调肿瘤组织Caspase蛋白表达,下调Survivin蛋白表达,从而诱导胃癌细胞发生凋亡.预示新加良附方在抗肿瘤方面具有进一步深入研究价值.     

Binding activity of H-Ras is necessary for in vivo inhibition of ASK1 activity

H-Ras is well known as one of the essential components of Ras/Raf/MEK/ERK cascade, which is a critical prosurvival signaling mechanism in most eukaryotic cells. Ras targets Raf/MEK/ERK cascade by integrating and transmitting extracellular signals from growth factor receptors to Raf, leading to the propagation of signals to modulate a serious of cellular survival events. Apoptosis signal-regulating kinasel (ASK1) serves as a general mediator of cell death because it is responsive to a variety of death signals. In this study, we found that H-Ras interacted with ASK1 to cause the inhibition of both ASK1 activity and ASKl-induced apoptosis in vivo, which was reversed only partially by addition of RafS621 A, an antagonist of Raf, whereas MEK inhibitor, PD98059, and PI3K inhibitor, LY294002, did not disturb the inhibitory effect of H-Ras on ASK-1-induced apoptosis. Furthermore, by means of immunoprecipitate and kinase assays, we demonstrated that the interaction between H-Ras and ASK1 as well as the inhibition of ASKI activity were dependent on the binding activity of H-Ras. These results suggest that a novel mechanism may be involved in H-Rasmediated cell survival in addition to the well established MEK/ERK and PI3K/Akt kinase-dependent enhancement of cell survival.     

肠道疾病中肠上皮细胞的死亡

目前,由肠上皮细胞死亡引发的肠损伤仍然是危险且难以预测的严重疾病之一。肠上皮细胞、免疫系统和微生物组之间相互关联以维持正常肠道稳态和肠屏障的完整性。在微生物入侵时,肠上皮细胞进入死亡程序以维持肠上皮功能,并且保持其持续更新能力,维护组织稳态。但是脱离稳态的肠上皮细胞死亡会导致肠通透性增加和肠屏障功能障碍,导致多种急性和慢性肠道疾病,例如肠缺血/再灌注、败血症、炎症性肠病(inflammatory bowel diseases, IBD),坏死性小肠结肠炎(necrotizing enterocolitis, NEC)等。在病理生理状态下,过量的肠上皮细胞凋亡性死亡导致慢性炎症状态,而后转向坏死性凋亡细胞死亡机制,其诱导的病理特征比细胞凋亡的情况下更多,此外还可能诱导其它溶细胞性死亡机制,例如细胞焦亡和铁凋亡,从而增加肠道疾病的病理指征。但是,目前关于慢性肠道疾病中肠上皮细胞死亡机制的研究仍然存在空白。目前亟需对慢性肠道疾病(包括败血症,IBD,NEC和肠缺血/再灌注)中特定的细胞死亡机制进行深入了解,以开发针对此类疾病的新型的有效治疗策略。本文旨在综述肠上皮细胞不同死亡机制(例如细胞凋亡,细胞坏死,坏死性凋亡,细胞焦亡和铁凋亡)在急、慢性肠道疾病中的研究进展。