中华皮肤科杂志
中華皮膚科雜誌
중화피부과잡지
Chinese Journal of Dermatology
2011年
1期
18-22
,共5页
ABO血型系统%分子模拟%细胞毒性试验,免疫%黑素细胞
ABO血型繫統%分子模擬%細胞毒性試驗,免疫%黑素細胞
ABO혈형계통%분자모의%세포독성시험,면역%흑소세포
ABO blood-group system%Molecular mimicry%Cytotoxicity tests,immunologic%Melanocytes
目的 建立稳定表达跨膜型血型A抗原模拟肽疫苗的恶性黑素瘤细胞株B16,并检测体外细胞毒活性.方法 采用脂质体转染法将前期构建的模拟肽疫苗稳定转染B16细胞进行表达,RT-PCR及Western印迹法检测模拟肽/Fas融合基因及巨噬细胞炎症蛋白3β(Mip3β)的mRNA及蛋白表达.细胞计数试剂盒-8(CCK-8)法检测疫苗介导的补体依赖性细胞毒效应(CDC)及抗体依赖的细胞介导细胞毒效应(ADCC).结果 RT-PCR在预期位置检测到特异性条带.Western印迹表明,表达产物具有特异的结合抗血型A抗体活性.析因分析提示不同分组之间CDC效应总体差异有统计学意义(F=244.522,P＜0.01),ADCC效应总体差异也有统计学意义(F=71.593,P＜0.01).组间比较示M-pIRES组CDC效应与空质粒pIRES组无统计学差异,两组均明显低于P/F-M-pIRES组和P/F-pIRES组;P/F-M-pIRES组、P/F-pIRES组ADCC效应明显强于M-pIRES组与pIRES组,P/F-M-pIRES组ADCC效应明显强于P/F-pIRES组(F=15.42,P＜0.05).结论 跨膜型血型A抗原模拟肽疫苗可在B16细胞膜稳定表达,且可通过介导CDC和ADCC发挥对黑素瘤细胞B16的杀伤作用.
目的 建立穩定錶達跨膜型血型A抗原模擬肽疫苗的噁性黑素瘤細胞株B16,併檢測體外細胞毒活性.方法 採用脂質體轉染法將前期構建的模擬肽疫苗穩定轉染B16細胞進行錶達,RT-PCR及Western印跡法檢測模擬肽/Fas融閤基因及巨噬細胞炎癥蛋白3β(Mip3β)的mRNA及蛋白錶達.細胞計數試劑盒-8(CCK-8)法檢測疫苗介導的補體依賴性細胞毒效應(CDC)及抗體依賴的細胞介導細胞毒效應(ADCC).結果 RT-PCR在預期位置檢測到特異性條帶.Western印跡錶明,錶達產物具有特異的結閤抗血型A抗體活性.析因分析提示不同分組之間CDC效應總體差異有統計學意義(F=244.522,P＜0.01),ADCC效應總體差異也有統計學意義(F=71.593,P＜0.01).組間比較示M-pIRES組CDC效應與空質粒pIRES組無統計學差異,兩組均明顯低于P/F-M-pIRES組和P/F-pIRES組;P/F-M-pIRES組、P/F-pIRES組ADCC效應明顯彊于M-pIRES組與pIRES組,P/F-M-pIRES組ADCC效應明顯彊于P/F-pIRES組(F=15.42,P＜0.05).結論 跨膜型血型A抗原模擬肽疫苗可在B16細胞膜穩定錶達,且可通過介導CDC和ADCC髮揮對黑素瘤細胞B16的殺傷作用.
목적 건립은정표체과막형혈형A항원모의태역묘적악성흑소류세포주B16,병검측체외세포독활성.방법 채용지질체전염법장전기구건적모의태역묘은정전염B16세포진행표체,RT-PCR급Western인적법검측모의태/Fas융합기인급거서세포염증단백3β(Mip3β)적mRNA급단백표체.세포계수시제합-8(CCK-8)법검측역묘개도적보체의뢰성세포독효응(CDC)급항체의뢰적세포개도세포독효응(ADCC).결과 RT-PCR재예기위치검측도특이성조대.Western인적표명,표체산물구유특이적결합항혈형A항체활성.석인분석제시불동분조지간CDC효응총체차이유통계학의의(F=244.522,P＜0.01),ADCC효응총체차이야유통계학의의(F=71.593,P＜0.01).조간비교시M-pIRES조CDC효응여공질립pIRES조무통계학차이,량조균명현저우P/F-M-pIRES조화P/F-pIRES조;P/F-M-pIRES조、P/F-pIRES조ADCC효응명현강우M-pIRES조여pIRES조,P/F-M-pIRES조ADCC효응명현강우P/F-pIRES조(F=15.42,P＜0.05).결론 과막형혈형A항원모의태역묘가재B16세포막은정표체,차가통과개도CDC화ADCC발휘대흑소류세포B16적살상작용.
Objective To establish a stable cell line expressing transmembrane form of human blood group A antigen mimotope vaccine by transfecting malignant melanoma cell line B16, and to detect the cytotoxicity of the vaccine against melanoma cells. Methods Cultured B16 cells were classified into 4 groups, i.e.,P/F-M-pIRES group [transfected with the recombinant plasmid mimotope peptide/Fas-macrophage inflammatory protein (Mip)-pIRES], P/F-pIRES group (transfected with the recombinant plasmid mimotope peptide/FaspIRES), M-pIRES group (transfected with the recombinant plasmid Mip-pIRES), and pIRES group (transfected with the empty plasmid pIRES). B 16 cells were transfected through Lipofectamine 2000. Subsequently, RT-PCR and Western blotting were performed to detect the mRNA and protein expressions of the mimotope peptide/Fas fusion gene and Mip3β in transfected B16 cells. Cell counting kit-8 (CCK-8) was used to evaluate the vaccinemediated complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC)against B16 cells. Results RT-PCR yielded specific DNA fragments with expected size. Western blotting revealed the anti-A antibody-binding activity of the recombinant mimotope peptide/Fas fusion protein. Factor analysis indicated significant differences in CDC (F = 244.522, P ＜ 0.01 ) and ADCC (F = 71.593, P ＜ 0.01 )against B16 cells between the 4 groups. Group comparisons demonstrated more intense CDC and ADCC in P/FM-pIRES and P/F-pIRES groups compared with M-plRES and pIRES groups, stronger ADCC in P/F-M-pIRES group in comparison with P/F-pIRES group (F = 15.42, P ＜ 0.05), but no significant difference in CDC was observed between M-pIRES and pIRES group. Conclusions The transmembrane form of human blood group A antigen mimotope vaccine could be stably expressed in B16 cells, and mediate ADCC and CDC against B16 cells in vitro.
