摘要
目的 采用形态学和分子生物学方法 ,分析转导 6A8α 甘露糖苷酶的反义DNA导致的B细胞株BJAB死亡的性质。方法 用Giemsa染色、annexin Ⅴ荧光染色、梯形DNA电泳以及透射电子显微镜等方法 ,分析细胞死亡的性质 ;应用ConA结合试验 ,检测转基因细胞 6A8α 甘露糖苷酶活性的改变 ;应用Fluo 3探针测定细胞胞浆 [Ca2 +]i。结果 用重组腺相关病毒 (rAAV)颗粒成功地将反义 6A8DNA转导入了EB病毒转化的B细胞株BJAB。在克隆中 ,转导反义 6A8DNA的BJAB细胞在生长到 3～ 1 0个细胞时死亡 ,而野生型、转导空载或转导正义 6A8DNA的BJAB细胞生长良好。转导反义 6A8DNA的BJAB细胞呈聚集状生长 ,且有大量细胞肿胀 ,最终死亡。Giemsa染色光镜观察、an nexin Ⅴ荧光染色、梯形DNA电泳及透射电镜观察结果表明 ,这种死亡是一种oncosis(肿胀死 )样死亡。ConA结合试验表明 ,BJAB细胞在转导反义 6A8DNA后的结合率升高 ,而转导正义 6A8DNA后的结合率则下降。另外 ,转导反义 6A8DNA的细胞胞浆 [Ca2 +]i升高。结论 转导反义 6A8DNA引起BJAB细胞发生oncosis样死亡 ,这种死亡可能与 6A8α 甘露糖苷酶表达被抑制有关。
Objective To characterize the oncosis like death of B lymphoma cell line BJAB with transduction of an antisense DNA to a cDNA encoding 6A8 α mannosidase. Methods Giemsa′s staining, annexin Ⅴ Fluos staining, DNA ladder electrophoresis and transmission EM microscopy were carried out to characterize the features of the BJAB cell death. Con A binding assay was applied to detect the expression of α mannosidase. Fluo 3 probe was used to detect the intracellular [Ca 2+ ]i. Results After transduction with antisense 6A8 DNA mediated by the rAAV, BJAB cells eventually undergo death in cloning, preceded by cell aggregation, cell swelling and the appearance of membrane blebbing. Giemsa's staining examination, annexin Ⅴ Fluo staining, DNA ladder tests and EM microscopy revealed that the cell death of BJAB caused by the antisense 6A8 DNA transduction might be an oncosis like cell death. Con A binding assay to the cells transduced with the antisense 6A8 DNA was enhanced, but reduced in the cells transduced with the sense 6A8 DNA, indicating a hypo and a hyper expression of 6A8 α mannosidase in the former and the later cells. The intracellular [Ca 2+ ]i was increased in the rAAV antisense 6A8 DNA transduced cells. Conclusion Transduction with antisense 6A8 DNA causes an oncosis like cell death of BJAB cells, which might be related to the aberrant glycosylation of proteins resulted from a blocking of 6A8 α mannosidase expression by the transcripts of the antisense 6A8 DNA.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2001年第5期480-485,共6页
Chinese Journal of Microbiology and Immunology
基金
国家自然科学基金重点项目 ( 3960 30 0 )
中国医学科学院创新资助项目
