Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa’s staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa’s staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase. The transduction did not affect the expression of Fas molecule on cells.     

Cloning and characterization of a cDNA encoding human differentiation antigen 5D4

A 1 846 bp cDNA is isolated from a human tonsil cell λgt 11 cDNA library (ATCC No. 37546) with mAb 5D4 reactive strongly with human B cell line 3D5, but weakly with human B cell line Daudi and human T cell line Jurkat as a probe. RT-PCR also shows a strong reaction in 3D5 cell and a weak reaction in Daudi and Jurkat cell for 5D4 mRNA. There is an open reading frame from 88 to 1 209 bp in 5D4 cDNA encoding a 374 AA protein. Both the Northern blot analysis and the two consecutive stop codens before start coden demonstrate that the cDNA is a full-length cDNA. Secondary structure prediction suggests that there are a region from 295 to 334 AA in the protein with strong hydrophobicity and a transmembrane helix region with high score from 313 to 334 AA with an orientation from the inside to the outside of the cell.     

人Man2c1转基因小鼠外周血中性粒细胞和CD8+T细胞的调节

目的以转hMan2c1基因小鼠为模型,分析hMan2c1基因在转基因小鼠脾脏的蛋白表达和活性,研究hMan2c1基因对于机体免疫系统的影响。方法Western blot方法检测hMan2c1基因在脾脏的蛋白表达并检测小鼠脾脏α-甘露糖苷酶活性;血常规分析外周血中各种血细胞的比例;以BSA作为抗原观察机体的免疫应答;流式细胞技术观察外周血中CD4 、CD8 、B、NK细胞的数量。结果Western blot结果显示,与野生型小鼠比较,hMan2c1基因在转基因小鼠脾脏组织有明显的表达,α-甘露糖苷酶活性明显增高,中性粒细胞明显升高。BSA刺激后,转基因小鼠外周血免疫细胞中的CD8 T淋巴细胞明显高于野生型小鼠。结论hMan2c1基因在小鼠脾脏表达引起α-甘露糖苷酶活性显著升高,并进一步影响淋巴细胞的生成,增加中性粒细胞和CD8 T淋巴细胞对免疫原的应答。     

615纯系与昆明杂种新生小鼠去胸腺衰弱性疾病发生的比较

近二十年来,大量事实证明,胸腺是一个与免疫有关的重要器官。动物切除胸腺后,特别是新生动物,其淋巴系统发育受阻,淋巴结、脾脏以及周围血中小淋巴细胞丢失显著。在部分动物,还发生所谓衰弱性疾病(wasting disease)(Miller 1961,Parrott 1962)。切去胸腺的动物对化学致癌物及肿瘤病毒的敏感性也发生变化。但是这些变化因动物种属和     

SKW6细胞在转导6A8 a-甘露糖苷酶反义cDNA后对抗Fas抗体诱导的凋亡

鉴于蛋白质糖基化的重要生物学意义, 以腺相关病毒为载体, 把编码6A8 a-甘露糖苷酶的cDNA 3′端1358 bp的反向片段转导入EB病毒转化的B细胞株SKW6, 探讨6A8 a-甘露糖苷酶表达抑制对抗Fas抗体诱导凋亡的影响. 反义6A8转导成功及表达用Northern杂交及RT-PCR检测, 6A8 a-甘露糖苷酶表达用Con A结合试验检测. Giemsa染色, AnnexinⅤ染色及梯形DNA电泳显示, Fas抗体能诱导SKW6细胞凋亡, 但6A8 a-甘露糖苷酶表达抑制的细胞对Fas抗体诱导的凋亡发生抵抗, 而转导正义6A8或空载载体则无影响. 6A8 a-甘露糖苷酶表达状况对SKW6细胞表面Fas分子表达没有影响.     

SKW6细胞在转导6A8 α-甘露糖苷酶反义cDNA后对抗Fas抗体诱导的凋亡

鉴于蛋白质糖基化的重要生物学意义,以腺相关病毒为载体,把编码6A8 α-甘露糖苷酶的cDNA 3′端1358 bp的反向片段转导入EB病毒转化的B细胞株SKW6,探讨6A8 α-甘露糖苷酶表达抑制对抗Fas抗体诱导凋亡的影响.反义6A8转导成功及表达用Northern杂交及RT-PCR检测,6A8 α-甘露糖苷酶表达用Con A结合试验检测.Giemsa染色,AnnexinⅤ染色及梯形DNA电泳显示,Fas抗体能诱导SKW6细胞凋亡,但6A8 α-甘露糖苷酶表达抑制的细胞对Fas抗体诱导的凋亡发生抵抗,而转导正义6A8或空载载体则无影响.6A8 α-甘露糖苷酶表达状况对SKW6细胞表面Fas分子表达没有影响.     

编码人分化抗原5D4的全长cDNA克隆及其编码蛋白质的二级结构分析

用 3D5细胞染色强阳性 ,而Daudi与Jurkat细胞染色弱阳性的单克隆抗体5D4,从一个人扁桃体细胞λgt1 1cDNA文库 (ATCCNo .375 46 )筛选到一个长 1 846bp的cDNA克隆 .RT_PCR显示 3D5细胞、Daudi细胞和Jurkat细胞 5D4mRNA均阳性 ,但 3D5细胞明显强于后两者 . 5D4cDNA中 88～ 1 2 0 9bp为一个开放阅读框架 ,编码374个氨基酸的蛋白质 .Northernblot分析结果和起始密码子前 5′端非编码区有 2个连续的终止密码子表明 ,这是一个全长cDNA .从其开放阅读框架推断的蛋白质的氨基酸序列中 2 95～ 334aa为一强疏水区 ,预测 31 3～ 334aa为一分值极高的跨膜螺旋区 ,其方向最可能是由膜内到膜外 .     

Resistance of SKW6 cell to apoptosis induction with anti-Fas antibody upon transduction of a reverse fragment to a cDNA encoding human 6A8 α-mannosidase

The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 α-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa's staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-