人乳腺癌MCF-7细胞系凋亡过程中bcl-2基因的结合蛋白

 Ｍ Ｃ Ｆ ７ 是 ｐ５３ 为野生型的人乳腺癌细胞系．为探讨该细胞系凋亡过程中 ｂｃｌ ２ 基因的下调机制,以 ５ 氟尿嘧啶（５ Ｆｕ）诱导凋亡,用 Ｐ Ｃ Ｒ 技术扩增出人 ｂｃｌ ２（ｈｂｃｌ ２）基因调控区长度为 ５５８ｂｐ 的片段,经亚克隆后的序列分析证实其准确性．用此 Ｐ Ｃ Ｒ 产物作为探针与 ５ Ｆｕ 刺激 １２ ｈ 后的 Ｍ Ｃ Ｆ ７ 核内蛋白质粗提物进行 Ｓｏｕｔｈｗ ｅｓｔｅｒｎ 印迹及凝胶阻滞（ Ｅ Ｍ Ｓ Ａ）分析．以未用 ５ Ｆｕ 刺激者为对照．结果显示,细胞核内一分子量为 ５３ ｋ Ｄ 的结合蛋白与 ｂｃｌ ２ 调控区的结合信号明显增强,而一种２０ ｋ Ｄ 的 Ｄ Ｎ Ａ 结合蛋白与该区的结合信号明显减弱．这些结果提示ｐ５３ 蛋白有可能是ｂｃｌ ２ 的负转录因子,２０ ｋ Ｄ 的 Ｄ Ｎ Ａ 结合蛋白有可能是ｂｃｌ ２ 的正转录因子．     

Indispensable role of Bcl2 in the development of the melanocyte stem cell

Bcl2 null mice display a characteristic loss of pigmentation demonstrating the importance of Bcl2 in the melanocyte (Mc) lineage. It was recently reported that this abnormal phenotype is due to the failure of melanocyte stem cell (MSC) maintenance and that Bcl2 is selectively important for the survival of MSCs. However, in our analysis of the same mouse, we observe a reduction in melanoblast (Mb) number in both epidermal and follicular populations. More importantly, there is a complete absence of MSCs. SCF downregulation in the epidermis is concomitant with the dramatic reduction in Mb numbers observed in the Bcl2 null, suggesting that Bcl2 is indispensable for the survival of Mbs in the absence of c-Kit signaling. Consistently, abrogation of c-Kit signaling in Bcl2 null mice depletes all Mbs and Mcs, whereas continuous expression of SCF in epidermal keratinocytes rescues the MSCs. Our results demonstrate that Bcl2 has a general role in Mb and Mc survival and is essential for the emergence of MSCs. Moreover, the results indicate that the first wave of Mcs that provide hair pigmentation is derived directly from epidermal Mbs bypassing MSCs. Furthermore, a Bcl2-independent mechanism of action of SCF in the Mc lineage is revealed as SCF c-Kit signaling is functional in the absence of Bcl2.     

Development of apoptosis-resistant dihydrofolate reductase-deficient Chinese hamster ovary cell line

Apoptosis-resistant dihydrofolate reductase-deficient CHO cell line (dhfr(-) CHO-bcl2) was developed by introduction of the bcl-2 gene into the dhfr(-) CHO cell line (DUKX-B11, ATCC CRL-9096) and subsequent selection of clones stably overexpressing Bcl-2 in the absence of selection pressure. When the dhfr(-) CHO-bcl2 cell line was used as a host cell line for development of a recombinant CHO (rCHO) cell line expressing a humanized antibody, it displayed stable expression of the bcl-2 gene during rCHO cell line development and no detrimental effect of Bcl-2 overexpression on specific antibody productivity. Taken together, the results obtained demonstrate that the use of an apoptosis-resistant dhfr(-) CHO cell line as the host cell line saves the effort of establishing an apoptosis-resistant rCHO cell line and expedites the development process of apoptosis-resistant rCHO cells producing therapeutic proteins.     

BCL-2 promotes migration and invasiveness of human glioma cells

Malignant progression in gliomas is correlated with increased migratory capacity which involves metalloproteolytic activity. Here, we report that ectopic expression of BCL-2 in two malignant glioma sublines markedly promoted glioma cell migration from spheroids and invasion into Matrigel-coated membranes. Invasion of fetal rat-brain aggregates was enhanced by BCL-2. Zymography revealed activation of matrix metalloproteinase-2 (MMP-2) in BCL-2-expressing cells. BCL-2 expressing cells showed an increase in MMP-2/-3/-12 (LN-18), and MMP-9/-12 and cell surface urokinase-type plasminogen activator (u-PA) (LN-229) mRNA and a reduction in tissue inhibitors of metalloproteinases (TIMP)-2 mRNA (LN-229). Taken together, we propose a novel function for BCL-2 in the malignant phenotype of glioma cells, that is, to enhance migration and invasion by altering the expression of a set of metalloproteinases and their inhibitors.     

Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: the effect of culture mode and the anti-apoptotic gene, bcl-2

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.     

生物膜信号转导与细胞凋亡

胞外信号可经过相应的转导途径传至胞内,通过激活靶分子而产生细胞效应.细胞凋亡是受控于生物体精确调节的细胞主动消亡过程,具有独特而复杂的信号系统.特异性的胞膜蛋白及膜脂等皆可介导凋亡相关分子的级联激活,并通过活化凋亡关键调节分子Caspases蛋白酶家族,bcl-2基因家族及线粒体等而影响凋亡的进程.     

人乳腺癌细胞p53和bcl-2蛋白的表达状况及与激素受体的关系

为了进一步证明 p5 3和 bcl- 2癌基因蛋白的反向关系 ,我们用 SP法进行免疫细胞化学染色 ,观察了 p5 3、 bcl- 2、雌激素受体 (estrogen receptor,ER)、孕激素受体 (progesterone receptor,PR)在人乳腺癌细胞系 MCF7和 MDA- MB2 31中的表达情况 ,并应用图像分析系统对其阳性反应物进行定量。结果 :MCF7细胞表达 ER和 PR,而 MDA- MB2 31细胞不表达。二个细胞系均表达 p5 3,但 MCF7光密度 (OD)值为 0 .10 0 9± 0 .0 14,而 MDA- MB2 31细胞为 0 .16 78± 0 .0 42 ,后者明显高于前者 (P<0 .0 0 0 1)。二系细胞均可见到 bcl- 2阳性物质 ,但 MCF7表达的 OD值为 0 .10 45± 0 .0 2 0 8,而 MDA- MB2 31细胞仅为 0 .0 5 2 5± 0 .0 113(P<0 .0 0 0 1)。表明 bcl- 2的表达与 ER及 PR的存在有很强的相关性 ,并且与 p5 3的表达呈明显的相反关系     

B波段紫外线照射对ＮＨ３Ｔ３细胞bcl—2、n—myc、c—fos三种基因表达的影响

用免疫组织化学SPTM试剂盒标记,用Quantimet520型图像分析仪定量分析B波段紫外线照射对NIH3T3细胞bcl-2,n-myc,c-fos三种基因表达的影响,结果发现Bcl-2(bcl-2表达的蛋白质）定位于细胞质,N-myc,c-Fos(分别为n-myc,c-fos表达的蛋白质）在细胞核中高表达,在细胞质中低表达,三种基因蛋白在对照组（未受紫外线照射的正常NIH3T3细胞中）均有表达,NIH3T3细胞经B波段紫外线照射后,9小时前Bcl-2无显变化。9小时后急剧增高,超过最初值;N-myc于照射后1小时前无显变化。1小时后降低,9小时后显剧增高,并超过最初值;c-Fos于照射后即下降,0.5小时后回升,逐渐达到最初值。     

大鼠bcl-x_L cDNA的克隆和高滴度重组bcl-x_L腺相关病毒的制备

以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础     

bcl—2基因的转录调控

许多不同的外界刺激,包括生长因子和细胞因子等都能诱导ｂｃｌ－２基因的表达,而且很显然,这种爱诱导表达的Ｂｃｌ－２对于细胞的存活是至关重要的。因此,为了对ｂｃｌ－２的转录调控研究有了初步的了解,将从转录水平和转录后水平两个层次上谈谈目前在该领域中的最新进展。