Bochan M R, Goebel W S, Brahmi Z
Department of Microbiology, Indiana University School of Medicine, Indianapolis 46202-5200, USA.
Cell Immunol. 1995 Sep;164(2):234-9. doi: 10.1006/cimm.1995.1166.
In human NK cells and CTL it has been shown that release of lytic molecules is, at least in part, responsible for the lysis of target cells (TC). Of the various types of molecules thought to be involved in cell-mediated cytotoxicity (CMC), perforin and the serine proteases (granzymes A and B) are the best described. Using mammalian expression vectors (pRSV-neo and pSV2-neo), antisense constructs for perforin and granzyme B were independently electroporated into YT-INDY, a human non-MHC-restricted, IL-2-independent, cytotoxic lymphocyte. Transfected YT-INDY was then selected for expression of the plasmid by antibiotic G418 resistance. The presence of plasmid was confirmed by detection of the integrated plasmid G418 resistance gene using PCR. The presence of antisense perforin in YT-INDY (YT-xPFP) inhibited lytic ability by > 95% compared to YT-INDY transfected with plasmid alone or plasmid with unrelated antisense (YT-neo, YT-ctrl, respectively). Likewise, the presence of antisense GrB (YT-xGrB) inhibited the lytic ability of YT-INDY by > 95%. Western analysis revealed a 30% decrease in the level of perforin and a 55% decrease in granzyme B protein levels compared to YT-neo. Northern analysis using oligo probes complementary to perforin and granzyme B mRNA showed a decrease in their respective message levels. In conclusion, stably transfected antisense constructs for perforin and granzyme B essentially eliminated the lytic ability of YT-INDY. These results strongly indicate that both perforin and granzyme B are required by this human cytotoxic lymphocyte for effective TC lysis.
在人自然杀伤细胞和细胞毒性T淋巴细胞中，已表明溶细胞分子的释放至少部分负责靶细胞（TC）的裂解。在被认为参与细胞介导的细胞毒性（CMC）的各种类型分子中，穿孔素和丝氨酸蛋白酶（颗粒酶A和B）是描述得最为清楚的。使用哺乳动物表达载体（pRSV-neo和pSV2-neo），将针对穿孔素和颗粒酶B的反义构建体分别电穿孔导入YT-INDY，一种人非主要组织相容性复合体（MHC）限制的、白细胞介素-2非依赖性的细胞毒性淋巴细胞。然后通过抗生素G418抗性选择转染的YT-INDY以表达质粒。通过使用聚合酶链反应（PCR）检测整合的质粒G418抗性基因来确认质粒的存在。与单独转染质粒或转染无关反义质粒（分别为YT-neo、YT-ctrl）的YT-INDY相比，YT-INDY中反义穿孔素（YT-xPFP）的存在使裂解能力降低了95%以上。同样，反义颗粒酶B（YT-xGrB）的存在使YT-INDY的裂解能力降低了95%以上。蛋白质免疫印迹分析显示，与YT-neo相比，穿孔素水平降低了30%，颗粒酶B蛋白水平降低了55%。使用与穿孔素和颗粒酶B mRNA互补的寡核苷酸探针进行的Northern分析显示它们各自的信使水平下降。总之，稳定转染的针对穿孔素和颗粒酶B的反义构建体基本上消除了YT-INDY的裂解能力。这些结果强烈表明，这种人细胞毒性淋巴细胞有效裂解靶细胞需要穿孔素和颗粒酶B。
