光化学法脑缺血后细胞凋亡及其相关基因bcl-2表达的变化

采用ＴＵＮＥＬ染色及免疫组织化学技术对光化学法脑缺血后细胞凋亡及其相关基因ｂｃｌ－２表达的变化进行了研究。结果发现，缺血后１２ｈ，损伤侧皮层缺血区内凋亡细胞数及ｂｃｌ－２免疫反应阳性细胞数明显增加，一直持续至缺血后７２ｈ；并呈现下列时程变化：在缺血后３ｈ每张切片上几乎无凋亡细胞出现，以后逐渐增加，缺血后１２ｈ达到峰值，缺血后２４ｈ和缺血后７２ｈ逐渐减少，但仍高于假手术组水平。凋亡相关基因ｂｃｌ－２的表达在缺血后３ｈ以前不明显，缺血后１２ｈ逐渐增加，缺血后２４ｈ最多，以后逐渐下降。上述结果提示，缺血后凋亡细胞的时程变化可能与缺血后梗塞灶的发生和发展有关，而ｂｃｌ－２表达的变化可能与抑制细胞凋亡、发挥内源性细胞保护作用有关。     

MicroRNA‐181c targets Bcl‐2 and regulates mitochondrial morphology in myocardial cells

Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl‐2 family which includes pro‐ and anti‐apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA‐181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR‐181c could target the 3′ untranslated region of Bcl‐2, one of the anti‐apoptotic members of the Bcl‐2 family. Thus, we have suggested that miR‐181c was involved in regulation of Bcl‐2. In this study, we investigated this hypothesis using the Dual‐Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR‐181c. We found that the level of miR‐181c was inversely correlated with the Bcl‐2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR‐181c resulted in significant changes in the levels of caspases, Bcl‐2 and cytochrome C in these cells. The increased level of Bcl‐2 caused by the decrease in miR‐181c protected mitochondrial morphology from the tumour necrosis factor alpha‐induced apoptosis.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

阿尔茨海默病中mir-34a作用机制的探讨

目的探讨mir-34a在阿尔茨海默病发病机制中的作用。方法取3月龄和6月龄APPswe/PSΔE9小鼠脑组织,进行microRNA芯片的检测;利用real-time RT-PCR对芯片结果进行验证;采用western blot的方法检测APPswe/PSΔE9小鼠和对照小鼠脑组织中bcl2蛋白的表达情况;通过构建mir-34a稳定转染细胞系和mir-34aknockdown研究mir-34a与bcl2之间的关系;通过构建bcl23’UTR-荧光素酶报告载体,验证bcl23’UTR序列中包含mir-34a的结合位点。结果mir-34a在模型小鼠中表达水平明显升高,并且其表达水平与bcl2蛋白水平呈负相关;通过体外实验,我们发现mir-34a过表达可以明显降低bcl2蛋白水平,反之,当我们抑制mir-34a的表达以后则可以增加bcl2蛋白水平;荧光素酶报告载体实验表明bcl23’UTR序列中包含mir-34a的结合位点。结论bcl2可能是mir-34a重要的功能靶点,mir-34a的过表达可能通过下调bcl2的蛋白水平,从而参与AD的发生。     

Bcl-2在去甲二氢愈创木酸诱导恶性胶质瘤细胞凋亡中的变化

观察bcl-2在去甲二氢愈创木酸（NDGA）诱导恶性胶质瘤细胞系SHG-44细胞凋亡中的变化。利用光镜和电镜观察及TUNEL法检测培养细胞凋亡的情况。用免疫组织化学,原位杂交和图像分析等方法检测bcl-2基因mRNA和蛋白的表达水平。结果显示：（1）一定浓度的NDGA处理SHG-44细胞12-96h,均可诱导SHG-44细胞发生凋亡,且随着作用时间的延长,凋亡细胞数增加越明显;（2）免疫组织化学方法结果显示,NDGA处理细胞后,出现SHG-44细胞bcl-2蛋白表达水平降低,且随着作用时间的延长,这种趋势更加明显,与细胞凋亡的发生密切相关;（3）原位杂交结果显示,NDGA处理细胞不同时间后,出现SHG-44细胞bcl-2基因mRNA的表达水平降低,结果与免疫组织化学方法检测结果一致,上述结果表明,NDGA诱导SHG-44细胞凋亡过程中,bcl-2基因的表达降低,但其确切机制有待进一步深入研究。     

重组RA-538及反义c-myc腺病毒对人胃癌、食管癌和bcl-2高表达细胞系的作用及其分子机制的体内外研究

本课题研究RA53 8、反义c ymc重组腺病毒对人胃癌 (SGC790 1 )、食管癌(EC1 0 9、EC871 2 )、正常人胚肺 2BS ( 2BS)及bcl 2高表达细胞系的体内外生物学作用及其分子机制。结果显示Ad RA53 8及Ad ASc myc对SGC790 1细胞体内外均具有明显的生长抑制及凋亡诱导作用 ,并能抑制其c myc、bcl 2、cyclinD1基因的表达及刺激bax基因的表达。对EC1 0 9、EC871 2、2BS及bcl 2基因高表达细胞系均无明显的生长抑制及凋亡诱导作用 ,对这些细胞系中c myc、bcl 2基因的表达均无调节作用。结果表明Ad RA53 8、Ad ASc myc对胃癌细胞具有显著的体内外生长抑制及凋亡诱导作用 ,其作用是c myc、bcl 2、bax、cyclinD1等一系列基因表达变化及其相互作用的结果。对食管癌、2BS及bcl 2高表达细胞系则无类似作用。     

细胞凋亡过程中bcl-2基因的甲基化

为探讨凋亡过程中,ｂｃｌ－２基因下调与该基因甲基化状态的关系,用５－氟尿嘧啶（５－Ｆｕ）诱导小鼠成纤维细胞ＮＣ３Ｈ１０,ＴＣ３Ｈ１０及人乳腺癌细胞ＭＣＦ－７的凋亡,分别检测了这三种细胞凋亡过程中ｂｃｌ－２的表达变化,与其调控区及编码区的甲基化状况．我们曾观察到５－Ｆｕ作用２４～４８ｈ出现细胞存活率下降,ＤＮＡ梯状断裂及细胞周期凋亡峰显现等典型凋亡现象．Ｎｏｒｔｈｅｒｎ杂交显示,在５－Ｆｕ作用１２ｈ时ｂｃｌ－２ｍＲＮＡ水平已明显降低．由此,我们用小鼠ｂｃｌ－２（ｍｂｃｌ－２）及人ｂｃｌ－２（ｈｂｃｌ－２）基因调控区ＰＣＲ扩增片段及ｂｃｌ－２编码区（ｃＤＮＡ）片段作为探针,与５－Ｆｕ作用１２ｈ的细胞ＤＮＡ的ＭｓｐⅠ／ＨｐａⅡ酶切产物进行Ｓｏｕｔｈｅｒｎ杂交,以未作用的细胞ＤＮＡ同样酶切杂交为对照．通过杂交带谱的变化,分析ｂｃｌ－２基因的甲基化状况．结果显示：ｍｂｃｌ－２及ｈｂｃｌ－２在５－Ｆｕ作用１２ｈ后调控区甲基化水平增高,但其编码区甲基化状态皆未出现可检出的变化．上述结果提示：ｂｃｌ－２基因调控区甲基化水平升高可能与该基因下调有关     

小鼠成纤维细胞凋亡过程中P53与bcl-2表达的时序性

为探讨细胞凋亡过程中,ｂｃｌ－２与Ｐ５３,这两种凋亡关键性基因的相互关系,用５－氟尿嘧啶（５－Ｆｕ）诱导小鼠正常与恶性转化的成纤维细胞的凋亡,观察了这两种基因在凋亡过程中表达变化的时序．经流式细胞计检测,这两种细胞在５－Ｆｕ作用２４ｈ均出现了凋亡峰,细胞存活率随之下降,ＤＮＡ电泳显现梯状断裂．Ｎｏｒｔｈｅｒｎ杂交结果显示,在５－Ｆｕ作用６ｈ后两种细胞Ｐ５３ｍＲＮＡ水平已明显增高,而ｂｃｌ－２ｍＲＮＡ水平则在作用１２ｈ方明显降低．Ｐ５３上调与ｂｃｌ－２下调的明显时序性,说明Ｐ５３具备作为ｂｃｌ－２基因负调控转录因子的条件．由此,为进一步了解凋亡过程的ｂｃｌ－２基因下调机理提供了线索     

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, Bcl-2 and NF-kappaB pathways

During rat hepatocarcinogenesis preneoplastic lesions (PNL) emerge which may persist (pPNL) and be sites of progress to cancer or suffer remodeling (rPNL) tending to disappear. Cellular and molecular mechanisms involved in both phenotypes are not sufficiently elucidated. pPNL and rPNL cellular proliferation and apoptosis were evaluated in rats submitted to the resistant hepatocyte (RH) model, and an adjusted growth index (AGI) was established. p53, Bcl-2, and NF-kappaB p65 subunit expression was evaluated by immunohistochemistry in pPNL and rPNL. p65 expression and NF-kappaB activation was evaluated by Western blot assays in whole livers. A lower number of BrdU-stained hepatocyte nuclei/mm(2) and higher number of apoptotic bodies (AB) per mm(2) were observed in remodeling compared to pPNL. Cytoplasmic p53 accumulation is related to increased hepatocarcinoma malignancy. We observed that 71.3% pPNL and 25.4% rPNL (P < 0.05) presented p53 staining in the cytoplasm. Similarly, 67.7% pPNL and 23.1 % rPNL (P < 0.05) presented increased Bcl-2 staining. Thirty-two percent pPNL and 15.6% rPNL (P < 0.05) presented p65 staining. Compared to normal rats, increase (P < 0.05) of hepatic p65 expression and NF-kappaB activation in rats submitted to the RH model was observed. In agreement to previous studies hepatic pPNL and rPNL differ regarding cell proliferation and apoptosis. Moreover, persistence and remodeling involve differences in p53, Bcl-2, and NF-kappaB pathways. These data point to molecular pathways that may direct preneoplastic lesions to spontaneously regress or to progress to cancer. J. Cell. Biochem. 103: 538-546, 2008. (c) 2007 Wiley-Liss, Inc.