中国免疫学杂志
中國免疫學雜誌
중국면역학잡지
CHINESE JOURNAL OF IMMUNOLOGY
2014年
12期
1585-1590
,共6页
吴小丽%张婷%王昕婧%王凯%汪希鹏
吳小麗%張婷%王昕婧%王凱%汪希鵬
오소려%장정%왕흔청%왕개%왕희붕
上皮性卵巢癌%TAM%Treg/Th17%IL-10
上皮性卵巢癌%TAM%Treg/Th17%IL-10
상피성란소암%TAM%Treg/Th17%IL-10
Eoc%TAM%TregT/h 17%IL-10
目的：探讨M2型肿瘤相关巨噬细胞（TAM）是否为上皮性卵巢癌外周血和肿瘤微环境中Treg／Th17比例失衡的影响机制之一。方法：流式细胞术检测M2型细胞与CD4＋T细胞共培养后Treg／Th17比例变化，Western blot检测T 细胞中的转录因子 Foxp3及 RORrt 水平， ELISA 检测共培养上清中 IL-10的含量，结晶紫检测上清对肿瘤细胞增殖的影响， Transwell法检测上清对肿瘤细胞迁移能力的影响，分析TAM细胞对Treg／Th17失衡影响的机制。结果：①CD4＋T细胞与M2型巨噬细胞共培养后，Treg／Th17比例为0.76±0.33相比Control组0.41±0.25，M0组0.40±0.32，M1组0.31±0.16有显著增高（P＜0.05）；②共培养上清对Skov3细胞具有显著的促增殖能力，共培养1 d后，M2组为14942.43±434.19较Control 组：12445.57±179.34，CD3／28组12470.32±434.18明显上升（ P＜0.001）；共培养2 d后，M2组为30129.09±520.53较Control组25622.81±897.07，CD3／28组25721.62±1808.60显著提高（P＜0.05）；③共培养后的T细胞中，Treg特异性转录因子Foxp3上调（ P＝0.047），Th17特异性转录因子RORrt 出现下降趋势但无统计学差异（ P＝0.294）。④T细胞与M2细胞共培养3 d后上清中IL-10含量为（264.04±75.9）pg／ml，较CD3／28组（60.89±46.54）pg／ml，M0组（44.81±32.93）pg／ml，M1组（42.71±26.09）pg／ml均显著提高（P＝0.001）。结论：M2型TAM细胞促进Treg／T1h7比例增加，上调Treg特异性转录因子Foxp 3，下调Th17特异性转录因子RORrt。同时，TAM与T细胞共培养上清具有显著促进肿瘤细胞增殖和迁移的能力。而这一机制可能是与其促进IL-10分泌增多有关。
目的：探討M2型腫瘤相關巨噬細胞（TAM）是否為上皮性卵巢癌外週血和腫瘤微環境中Treg／Th17比例失衡的影響機製之一。方法：流式細胞術檢測M2型細胞與CD4＋T細胞共培養後Treg／Th17比例變化，Western blot檢測T 細胞中的轉錄因子 Foxp3及 RORrt 水平， ELISA 檢測共培養上清中 IL-10的含量，結晶紫檢測上清對腫瘤細胞增殖的影響， Transwell法檢測上清對腫瘤細胞遷移能力的影響，分析TAM細胞對Treg／Th17失衡影響的機製。結果：①CD4＋T細胞與M2型巨噬細胞共培養後，Treg／Th17比例為0.76±0.33相比Control組0.41±0.25，M0組0.40±0.32，M1組0.31±0.16有顯著增高（P＜0.05）；②共培養上清對Skov3細胞具有顯著的促增殖能力，共培養1 d後，M2組為14942.43±434.19較Control 組：12445.57±179.34，CD3／28組12470.32±434.18明顯上升（ P＜0.001）；共培養2 d後，M2組為30129.09±520.53較Control組25622.81±897.07，CD3／28組25721.62±1808.60顯著提高（P＜0.05）；③共培養後的T細胞中，Treg特異性轉錄因子Foxp3上調（ P＝0.047），Th17特異性轉錄因子RORrt 齣現下降趨勢但無統計學差異（ P＝0.294）。④T細胞與M2細胞共培養3 d後上清中IL-10含量為（264.04±75.9）pg／ml，較CD3／28組（60.89±46.54）pg／ml，M0組（44.81±32.93）pg／ml，M1組（42.71±26.09）pg／ml均顯著提高（P＝0.001）。結論：M2型TAM細胞促進Treg／T1h7比例增加，上調Treg特異性轉錄因子Foxp 3，下調Th17特異性轉錄因子RORrt。同時，TAM與T細胞共培養上清具有顯著促進腫瘤細胞增殖和遷移的能力。而這一機製可能是與其促進IL-10分泌增多有關。
목적：탐토M2형종류상관거서세포（TAM）시부위상피성란소암외주혈화종류미배경중Treg／Th17비례실형적영향궤제지일。방법：류식세포술검측M2형세포여CD4＋T세포공배양후Treg／Th17비례변화，Western blot검측T 세포중적전록인자 Foxp3급 RORrt 수평， ELISA 검측공배양상청중 IL-10적함량，결정자검측상청대종류세포증식적영향， Transwell법검측상청대종류세포천이능력적영향，분석TAM세포대Treg／Th17실형영향적궤제。결과：①CD4＋T세포여M2형거서세포공배양후，Treg／Th17비례위0.76±0.33상비Control조0.41±0.25，M0조0.40±0.32，M1조0.31±0.16유현저증고（P＜0.05）；②공배양상청대Skov3세포구유현저적촉증식능력，공배양1 d후，M2조위14942.43±434.19교Control 조：12445.57±179.34，CD3／28조12470.32±434.18명현상승（ P＜0.001）；공배양2 d후，M2조위30129.09±520.53교Control조25622.81±897.07，CD3／28조25721.62±1808.60현저제고（P＜0.05）；③공배양후적T세포중，Treg특이성전록인자Foxp3상조（ P＝0.047），Th17특이성전록인자RORrt 출현하강추세단무통계학차이（ P＝0.294）。④T세포여M2세포공배양3 d후상청중IL-10함량위（264.04±75.9）pg／ml，교CD3／28조（60.89±46.54）pg／ml，M0조（44.81±32.93）pg／ml，M1조（42.71±26.09）pg／ml균현저제고（P＝0.001）。결론：M2형TAM세포촉진Treg／T1h7비례증가，상조Treg특이성전록인자Foxp 3，하조Th17특이성전록인자RORrt。동시，TAM여T세포공배양상청구유현저촉진종류세포증식화천이적능력。이저일궤제가능시여기촉진IL-10분비증다유관。
Objective:To investigate the impact and mechanisms of TAM to the imbalance of Treg /Th17 in the Eoc microenvi-ronment.Mte hods:Build the in vitro M2 macrophage model ,which was like TAMs .Use flow cytometry to detect the difference of the Treg/Th17 before and after the co-culture of M2 macrophage and CD 4+T cells.Use Western bolt to detect the change of T cell transcription factor and ELISA to detect the IL-10 levels in the supernatant after co-culture.Use crystal violet methods to detect the influence to the ovarian tumor cell proliferation between the different co-culture supernatants and the Transwell to detect the influence to the ovarian tumor cell migration.Thus to analysis the how TAMs influence the imbalance of Treg/Th17 in Eoc microenvironments.R esults:①After coc-ultured with M2 macrophage ,the ratio of Treg/Th17 was( 0.76 ±0.33 ) significant increased compared with control (0.41±0.25) ,M0( 0.40±0.32) and M1(0.31±0.16) (P<0.05).②After co-cultured,the supernatant of M2 group has a significant ability to promote the proliferation of Skov-3 cells.After co-cultured for 1 day, the Skov-3 cell number of M2 group was 14 942.43 ±434.19 , which was significantly higher than the control group ( 12 445.57 ±179.34 ) and CD3/28 group (12 470.32±434.18)(P<0.001).After co-cultured for 2 days,the Skov-3 cell number of M2 group was 30 129.09±520.53 ,which was significantly higher than the control group (25 622.81±897.07) and CD3/28 group(25 721.62±1 808.60) (P<0.05).③After co -cultured with M2 macrophage , the Treg-specific transcription factor Foxp 3 increased ( P=0.047 ) compared with control and M 1 group .④After co-cultured with M2 macrophage for 3 days,the concentration of IL1-0 in the supernatant was(264.04±75.9)pg/ml, which was significantly higher than CD 3/28 group ( 60.89 ±46.54 ) pg/ml,M 0 group ( 44.81 ±32.93 ) pg/ml, M1 group ( 42.71 ± 26.09)pg/ml(P=0.001).Conclusion: M2 macrophage induces the increase of the radio of Treg /Th17 as well as the increase of Treg-specific transcription factor Foxp 3 and the decrease of Th17 -specific transcription factor ROR-γt.Meanwhile , the co-culture supernatant of M2 macrophage and CD4+T cell have the ability to promote the proliferation and migration of ovarian cancer cell ,the mechanism which ,may related to the IL -10 in the supernatant .
