CAJ | 학술논문

目的：探讨类风湿关节炎（RA）患者滑液（SF）自然杀伤（NK）细胞NK-22促成纤维样滑膜细胞（FLS）增殖作用及相关信号通路。方法采用流式细胞术分选6例RA患者SF中NK-22细胞（2×104），体外悬浮培养2周，佛波酯（20 ng/ml）和离子霉素（0.5μmol/L）刺激后ELISA检测NK-22细胞培养上清IL-22浓度。组织块培养法原代培养RAFLS，NK-22细胞培养上清分别作用于RAFLS24、48、72 h，MTT法检测FLS增殖；同时加入IL-22抗体检测FLS增殖情况。RT-PCR检测rhIL-22及AG490作用后RAFLS Stat3 mRNA的表达，Western blot检测RAFLS p-Stat3蛋白含量。结果①流式分选2×104 NK-22细胞，细胞纯度>90%；①PMA和ionomycin刺激后NK-22细胞培养上清中IL-22浓度为1273.42±254.48 pg/ml；①PMA和ionomycin刺激后NK-22细胞培养上清作用于RAFLS 24 h、48 h、72 h，可明显刺激RAFLS增殖（P<0.05）。IL-22抗体能明显抑制NKp44+NK细胞上清诱导的RA FLS增殖（P<0.01）；①rhIL-22（1 ng/ml）能诱导RAFLS p-Stat3表达（P<0.05）；①AG490能抑制rhIL-22诱导的RAFLS p-Stat3表达（P<0.05）。结论 RA患者SFNK-22细胞体外可分泌高浓度的IL-22，通过激活STAT3信号通路进而刺激RAFLS增殖。
목적：탐토류풍습관절염（RA）환자활액（SF）자연살상（NK）세포NK-22촉성섬유양활막세포（FLS）증식작용급상관신호통로。방법채용류식세포술분선6례RA환자SF중NK-22세포（2×104），체외현부배양2주，불파지（20 ng/ml）화리자매소（0.5μmol/L）자격후ELISA검측NK-22세포배양상청IL-22농도。조직괴배양법원대배양RAFLS，NK-22세포배양상청분별작용우RAFLS24、48、72 h，MTT법검측FLS증식；동시가입IL-22항체검측FLS증식정황。RT-PCR검측rhIL-22급AG490작용후RAFLS Stat3 mRNA적표체，Western blot검측RAFLS p-Stat3단백함량。결과①류식분선2×104 NK-22세포，세포순도>90%；①PMA화ionomycin자격후NK-22세포배양상청중IL-22농도위1273.42±254.48 pg/ml；①PMA화ionomycin자격후NK-22세포배양상청작용우RAFLS 24 h、48 h、72 h，가명현자격RAFLS증식（P<0.05）。IL-22항체능명현억제NKp44+NK세포상청유도적RA FLS증식（P<0.01）；①rhIL-22（1 ng/ml）능유도RAFLS p-Stat3표체（P<0.05）；①AG490능억제rhIL-22유도적RAFLS p-Stat3표체（P<0.05）。결론 RA환자SFNK-22세포체외가분비고농도적IL-22，통과격활STAT3신호통로진이자격RAFLS증식。
Objective To investigate the role of natural killer-22 (NK-22) cells in the synovial fluid in the proliferation of fibroblast-like synoviocytes (FLS) in patients with rheumatoid arthritis (RA) and explore the possible signal pathway involved. Methods NK-22 cells in the SF of RA patients were sorted by flow cytometry. NK-22 cells were cultured for two weeks and the purity was detected by flow cytometry before stimulation with 20 ng/ml phorbol 12-myristate 13-acetate and 0.5 μmol/L ionomycin for 4 h. The level of interleukin-22 (IL-22) in the culture medium supernatant was then measured with ELISA. The proliferation of FLS in the presence of the culture supernatant of NK-22 cells was assessed with MTT assay at 24, 48 and 72 h, and the effect of IL-22 antibody on FLS proliferation was also observed. Real-time PCR and Western blotting were used to detect Stat3 mRNA and p-Stat3 protein levels, respectively, in the FLS exposed to rhIL-22 and AG490. Results NK-22 cells were successfully sorted by flow cytometry with a purity exceeding 90%. The levels of IL-22 in the supernatant of NKp44+NK cell culture averaged 1273.42±254.48 pg/ml. The FLS proliferated rapidly 24, 48, and 72 h after the addition of culture supernatant of NK-22 cells (P<0.05). IL-22 antibody obviously inhibited the proliferation of FLS induced by NK-22 cell culture supernatant (P<0.05). Exposure of the FLS to rhIL-22 obviously increased cellular Stat3 expression levels, which were significantly lowered by the addition of AG490 (P<0.05). Conclusion NK-22 cells in the SF of RA patients can produce high concentrations of IL-22 to promote the proliferation of FLS through the STAT3 signal pathway.