中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2010年
11期
982-986
,共5页
HLA-G%异构体%转染%细胞毒性
HLA-G%異構體%轉染%細胞毒性
HLA-G%이구체%전염%세포독성
HLA-G%Isoforms%Transfection%Cytotoxicity
目的 探讨膜结合型HLA-G1～G4异构体的表达及对NK细胞杀伤功能的影响.方法 通过基因克隆及转染,分别建立稳定表达HLA-G1～G4抗原的人绒癌JAR细胞株.采用RTPCR、流式细胞术、Western blot及免疫细胞化学法分析、鉴定转染细胞中HLA-G的mRNA及蛋白表达.通过加载HLA-G高亲和性KIPAQFYIL抗原肽,观察对HLA-G表达的影响.LDH释放法检测HLA-G1～G4表达对NK细胞杀伤活性的影响.结果 RT-PCR、Western blot及免疫细胞化学结果显示,HLA-G1～G4/pVITRO2-mcs重组质粒成功转染HLA-G表达阴性的人绒癌JAR细胞株.FACS分析显示HLA-G1抗原能在JAR-HLA-G1细胞株表面表达,HLA-G2～G4抗原不能有效到达细胞表面.体外杀伤试验发现表达HLA-G1～G4抗原的细胞均能抑制NK细胞的杀伤活性(P＜0.05);加载HLA-G高亲和性KIPAQFYIL抗原肽对HLA-G表达无明显影响,对NK细胞杀伤抑制程度也未见明显改变.结论 HLA-G1～G4能够明显抑制NK细胞的杀伤活性,提示不同膜结合型HLA-G异构体分子均能作为免疫耐受分子,具备免疫调节功能.
目的 探討膜結閤型HLA-G1～G4異構體的錶達及對NK細胞殺傷功能的影響.方法 通過基因剋隆及轉染,分彆建立穩定錶達HLA-G1～G4抗原的人絨癌JAR細胞株.採用RTPCR、流式細胞術、Western blot及免疫細胞化學法分析、鑒定轉染細胞中HLA-G的mRNA及蛋白錶達.通過加載HLA-G高親和性KIPAQFYIL抗原肽,觀察對HLA-G錶達的影響.LDH釋放法檢測HLA-G1～G4錶達對NK細胞殺傷活性的影響.結果 RT-PCR、Western blot及免疫細胞化學結果顯示,HLA-G1～G4/pVITRO2-mcs重組質粒成功轉染HLA-G錶達陰性的人絨癌JAR細胞株.FACS分析顯示HLA-G1抗原能在JAR-HLA-G1細胞株錶麵錶達,HLA-G2～G4抗原不能有效到達細胞錶麵.體外殺傷試驗髮現錶達HLA-G1～G4抗原的細胞均能抑製NK細胞的殺傷活性(P＜0.05);加載HLA-G高親和性KIPAQFYIL抗原肽對HLA-G錶達無明顯影響,對NK細胞殺傷抑製程度也未見明顯改變.結論 HLA-G1～G4能夠明顯抑製NK細胞的殺傷活性,提示不同膜結閤型HLA-G異構體分子均能作為免疫耐受分子,具備免疫調節功能.
목적 탐토막결합형HLA-G1～G4이구체적표체급대NK세포살상공능적영향.방법 통과기인극륭급전염,분별건립은정표체HLA-G1～G4항원적인융암JAR세포주.채용RTPCR、류식세포술、Western blot급면역세포화학법분석、감정전염세포중HLA-G적mRNA급단백표체.통과가재HLA-G고친화성KIPAQFYIL항원태,관찰대HLA-G표체적영향.LDH석방법검측HLA-G1～G4표체대NK세포살상활성적영향.결과 RT-PCR、Western blot급면역세포화학결과현시,HLA-G1～G4/pVITRO2-mcs중조질립성공전염HLA-G표체음성적인융암JAR세포주.FACS분석현시HLA-G1항원능재JAR-HLA-G1세포주표면표체,HLA-G2～G4항원불능유효도체세포표면.체외살상시험발현표체HLA-G1～G4항원적세포균능억제NK세포적살상활성(P＜0.05);가재HLA-G고친화성KIPAQFYIL항원태대HLA-G표체무명현영향,대NK세포살상억제정도야미견명현개변.결론 HLA-G1～G4능구명현억제NK세포적살상활성,제시불동막결합형HLA-G이구체분자균능작위면역내수분자,구비면역조절공능.
Objective To establish the expression of membrane-bound HLA-G1-G4 isoforms in choriocarcinoma cell line JAR and to investigate its roles in NK cytotoxicity in vitro. Methods Stable expression of HLA-G1, -G2, -G3 and -G4 in JAR cells was established by gene cloning and transfection.HLA-Gtranscripts and protein expression in the transfected JAR cells was tested by RT-PCR, flow cytometry, Western blot and immunocytochemistry, respectively. High-affinity peptide KIPAQFYIL pulsing was performed to evaluate its effects on HLA-G expression. Effects of HLA-G1-G4 isoforms on NK cytotoxicity was performed with lactic dehydrogenase (LDH) releasing method. Results RT-PCR, Western blot and immunocytochemistry results showed that exogenous HLA-G1-G4 gene were successfully transfected and proteins were stably expressed in the HLA-G negative JAR cells; Flow cytometry data showed that only HLAG1, but not HLA-G2-G4 isoform was detectable in those transfected JAR cells and the peptide pulsing did not affect their expression status. However, all HLA-G1-G4 isoform expressed JAR cells could significantly decreased the NK cell cytotoxicity (P＜0.05). Conclusion HLA-G1-G4 isoform expression could dramatically inhibit NK-92 cell lysis, indicating that membrane-bound HLA-G isoforms are importantly immunotolerant and may play immune regulation roles in various physio-pathological situations.
