中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
20期
9131-9137
,共7页
唐慧%董虹%高建梅%王金丽%王林坪%李丽%左荣霞%华映坤%严新民
唐慧%董虹%高建梅%王金麗%王林坪%李麗%左榮霞%華映坤%嚴新民
당혜%동홍%고건매%왕금려%왕림평%리려%좌영하%화영곤%엄신민
CIK细胞%血清微环境%干扰素Ⅱ型%白细胞介素13%CD4-CD8比值%Th1/Th2
CIK細胞%血清微環境%榦擾素Ⅱ型%白細胞介素13%CD4-CD8比值%Th1/Th2
CIK세포%혈청미배경%간우소Ⅱ형%백세포개소13%CD4-CD8비치%Th1/Th2
CIK cells%Serum microenvironment%Interferon type Ⅱ%Interleukin-13%CD4-CD8 ratio%Th1/Th2
目的：观察不同血清微环境中培养的细胞因子诱导的杀伤细胞（cytokine induced killer，CIK） CD4＋/CD8＋和Th1/Th2的动态改变，探讨最有利于恢复肿瘤患者CD4＋/CD8＋和Th1/Th2平衡的CIK细胞培养血清微环境和最适CIK细胞回输时机。方法将3例不同患者来源的外周血单个核细胞（peripheral blood mononuclear cell，PBMC）用含10%肿瘤患者自体抗凝血浆培养基（ZT）在体外诱导培养成CIK细胞，并设含10%胎牛血清培养基（FBS）和无血清培养基（GT）作为对照。连续记录CIK细胞在三组血清微环境中的增殖情况，并分别收集培养第0、7、14和21天的CIK细胞和细胞培养上清。MTT法检测CIK细胞毒活性。流式细胞术检测CIK细胞CD3＋、CD4＋和CD8＋免疫表型。ELISA法检测细胞培养上清中Th1和Th2优势细胞因子IFN-γ和IL-13浓度，二者浓度比即为Th1/Th2比值。结果（1）CIK细胞增殖情况：三个血清微环境组间无显著差异。（2）CIK细胞肿瘤杀伤活性：培养至第7、14和21天的CIK细胞对K562的杀伤活性在不同血清微环境组依次为：ZT：（79.95±12.23）%、（93.86±2.38）%、（66.34±7.54）%，FBS：（71.02±14.79）%、（83.72±5.63）%、（58.37±7.57）%，GT：（40.92±4.66）%、（52.61±8.05）%、（41.43±5.25）%，CIK细胞肿瘤杀伤活性在同一血清组不同时间点间比较结果为ZT和FBS组在第14天均显著高于第7天和第21天（P＜0.01）；GT组在不同时间点间无显著差异。在不同血清微环境组相同时间点间比较的结果发现在第7、14和21天三个时间点，ZT和FBS组均显著高于GT组（P＜0.01）。（3）CIK细胞中CD4＋/CD8＋的动态改变：同一血清组不同时间点间的比较结果显示在第0天，肿瘤患者来源的PMBC CD4＋/CD8＋倒置，ZT组CD4＋/CD8＋在第7天升高至2.25±1.67，显著高于第0天（P＜0.05），但随即在第14天和第21天又恢复倒置现象；FBS组CD4＋/CD8＋一直维持倒置现象，并在第21天降低至0.04±0.55，极显著低于第0天（P＜0.01）；GT组CD4＋/CD8＋亦一直维持倒置现象，且在4个时间点间无显著差异。在相同的时间点不同血清微环境组间比较的结果显示在第7天，CD4＋/CD8＋比例在ZT组显著高于FBS和GT组（P＜0.05）；在第14天和第21天，三组间无显著差异。（4）CIK细胞中Th1/Th2的动态改变：CIK细胞Th1/Th2在相同血清组不同时间点间比较发现ZT和GT组在4个时间点一直维持Th1/Th2倒置，且4个时间点间无显著差异；FBS组Th1/Th2在第7天和第14天均极显著高于第0天（P＜0.01），Th1/Th2倒置现象被纠正，但在第21天又再次出现Th1/Th2倒置。在不同血清微环境组相同时间点间比较发现在第7天和第14天，FBS组Th1/Th2极显著高于ZT和GT组（P＜0.01）。结论不同血清微环境培养的CIK细胞CD4＋/CD8＋和Th1/Th2均呈动态改变趋势。提示在临床进行CIK细胞治疗时，应综合考虑患者的自身免疫状态、所采用的细胞培养血清微环境及CIK细胞培养过程中CD4＋/CD8＋和Th1/Th2的动态改变，制定个性化的治疗方案。
目的：觀察不同血清微環境中培養的細胞因子誘導的殺傷細胞（cytokine induced killer，CIK） CD4＋/CD8＋和Th1/Th2的動態改變，探討最有利于恢複腫瘤患者CD4＋/CD8＋和Th1/Th2平衡的CIK細胞培養血清微環境和最適CIK細胞迴輸時機。方法將3例不同患者來源的外週血單箇覈細胞（peripheral blood mononuclear cell，PBMC）用含10%腫瘤患者自體抗凝血漿培養基（ZT）在體外誘導培養成CIK細胞，併設含10%胎牛血清培養基（FBS）和無血清培養基（GT）作為對照。連續記錄CIK細胞在三組血清微環境中的增殖情況，併分彆收集培養第0、7、14和21天的CIK細胞和細胞培養上清。MTT法檢測CIK細胞毒活性。流式細胞術檢測CIK細胞CD3＋、CD4＋和CD8＋免疫錶型。ELISA法檢測細胞培養上清中Th1和Th2優勢細胞因子IFN-γ和IL-13濃度，二者濃度比即為Th1/Th2比值。結果（1）CIK細胞增殖情況：三箇血清微環境組間無顯著差異。（2）CIK細胞腫瘤殺傷活性：培養至第7、14和21天的CIK細胞對K562的殺傷活性在不同血清微環境組依次為：ZT：（79.95±12.23）%、（93.86±2.38）%、（66.34±7.54）%，FBS：（71.02±14.79）%、（83.72±5.63）%、（58.37±7.57）%，GT：（40.92±4.66）%、（52.61±8.05）%、（41.43±5.25）%，CIK細胞腫瘤殺傷活性在同一血清組不同時間點間比較結果為ZT和FBS組在第14天均顯著高于第7天和第21天（P＜0.01）；GT組在不同時間點間無顯著差異。在不同血清微環境組相同時間點間比較的結果髮現在第7、14和21天三箇時間點，ZT和FBS組均顯著高于GT組（P＜0.01）。（3）CIK細胞中CD4＋/CD8＋的動態改變：同一血清組不同時間點間的比較結果顯示在第0天，腫瘤患者來源的PMBC CD4＋/CD8＋倒置，ZT組CD4＋/CD8＋在第7天升高至2.25±1.67，顯著高于第0天（P＜0.05），但隨即在第14天和第21天又恢複倒置現象；FBS組CD4＋/CD8＋一直維持倒置現象，併在第21天降低至0.04±0.55，極顯著低于第0天（P＜0.01）；GT組CD4＋/CD8＋亦一直維持倒置現象，且在4箇時間點間無顯著差異。在相同的時間點不同血清微環境組間比較的結果顯示在第7天，CD4＋/CD8＋比例在ZT組顯著高于FBS和GT組（P＜0.05）；在第14天和第21天，三組間無顯著差異。（4）CIK細胞中Th1/Th2的動態改變：CIK細胞Th1/Th2在相同血清組不同時間點間比較髮現ZT和GT組在4箇時間點一直維持Th1/Th2倒置，且4箇時間點間無顯著差異；FBS組Th1/Th2在第7天和第14天均極顯著高于第0天（P＜0.01），Th1/Th2倒置現象被糾正，但在第21天又再次齣現Th1/Th2倒置。在不同血清微環境組相同時間點間比較髮現在第7天和第14天，FBS組Th1/Th2極顯著高于ZT和GT組（P＜0.01）。結論不同血清微環境培養的CIK細胞CD4＋/CD8＋和Th1/Th2均呈動態改變趨勢。提示在臨床進行CIK細胞治療時，應綜閤攷慮患者的自身免疫狀態、所採用的細胞培養血清微環境及CIK細胞培養過程中CD4＋/CD8＋和Th1/Th2的動態改變，製定箇性化的治療方案。
목적：관찰불동혈청미배경중배양적세포인자유도적살상세포（cytokine induced killer，CIK） CD4＋/CD8＋화Th1/Th2적동태개변，탐토최유리우회복종류환자CD4＋/CD8＋화Th1/Th2평형적CIK세포배양혈청미배경화최괄CIK세포회수시궤。방법장3례불동환자래원적외주혈단개핵세포（peripheral blood mononuclear cell，PBMC）용함10%종류환자자체항응혈장배양기（ZT）재체외유도배양성CIK세포，병설함10%태우혈청배양기（FBS）화무혈청배양기（GT）작위대조。련속기록CIK세포재삼조혈청미배경중적증식정황，병분별수집배양제0、7、14화21천적CIK세포화세포배양상청。MTT법검측CIK세포독활성。류식세포술검측CIK세포CD3＋、CD4＋화CD8＋면역표형。ELISA법검측세포배양상청중Th1화Th2우세세포인자IFN-γ화IL-13농도，이자농도비즉위Th1/Th2비치。결과（1）CIK세포증식정황：삼개혈청미배경조간무현저차이。（2）CIK세포종류살상활성：배양지제7、14화21천적CIK세포대K562적살상활성재불동혈청미배경조의차위：ZT：（79.95±12.23）%、（93.86±2.38）%、（66.34±7.54）%，FBS：（71.02±14.79）%、（83.72±5.63）%、（58.37±7.57）%，GT：（40.92±4.66）%、（52.61±8.05）%、（41.43±5.25）%，CIK세포종류살상활성재동일혈청조불동시간점간비교결과위ZT화FBS조재제14천균현저고우제7천화제21천（P＜0.01）；GT조재불동시간점간무현저차이。재불동혈청미배경조상동시간점간비교적결과발현재제7、14화21천삼개시간점，ZT화FBS조균현저고우GT조（P＜0.01）。（3）CIK세포중CD4＋/CD8＋적동태개변：동일혈청조불동시간점간적비교결과현시재제0천，종류환자래원적PMBC CD4＋/CD8＋도치，ZT조CD4＋/CD8＋재제7천승고지2.25±1.67，현저고우제0천（P＜0.05），단수즉재제14천화제21천우회복도치현상；FBS조CD4＋/CD8＋일직유지도치현상，병재제21천강저지0.04±0.55，겁현저저우제0천（P＜0.01）；GT조CD4＋/CD8＋역일직유지도치현상，차재4개시간점간무현저차이。재상동적시간점불동혈청미배경조간비교적결과현시재제7천，CD4＋/CD8＋비례재ZT조현저고우FBS화GT조（P＜0.05）；재제14천화제21천，삼조간무현저차이。（4）CIK세포중Th1/Th2적동태개변：CIK세포Th1/Th2재상동혈청조불동시간점간비교발현ZT화GT조재4개시간점일직유지Th1/Th2도치，차4개시간점간무현저차이；FBS조Th1/Th2재제7천화제14천균겁현저고우제0천（P＜0.01），Th1/Th2도치현상피규정，단재제21천우재차출현Th1/Th2도치。재불동혈청미배경조상동시간점간비교발현재제7천화제14천，FBS조Th1/Th2겁현저고우ZT화GT조（P＜0.01）。결론불동혈청미배경배양적CIK세포CD4＋/CD8＋화Th1/Th2균정동태개변추세。제시재림상진행CIK세포치료시，응종합고필환자적자신면역상태、소채용적세포배양혈청미배경급CIK세포배양과정중CD4＋/CD8＋화Th1/Th2적동태개변，제정개성화적치료방안。
Objective Dynamic changes of CD4+/CD8+ and Th1/Th2 in cytokine induced killer (CIK) cells cultured in vitro in different serum microenvironments were observed, both the optimal cell culture serum microenvironment and the optimum transfusion timepoint of CIK cells will beneficial to reconstruct the balance of CD4+/CD8+ and Th1/Th2 in tumor patients were discussed. Methods Peripheral blood mononuclear cells (PBMCs) derived from 3 tumor patients were induced into CIK cells in vitro, CIK cells were cultured in medium containing 10%autologous blood plasma derived from tumor patients (ZT), medium containing 10%fetal bovine serum (FBS) and serum free medium (GT) were set as control groups. Proliferation of CIK cells cultured in 3 different serum microenvironments were recorded continuously, CIK cells and cell culture supernatants were collected respectively on D0, D7, D14 and D21. Cytotoxicity of CIK cells were determined by MTT, immune phenotypes of CD3+, CD4+ and CD8+ were detected by flow cytometry. Protein levles of IFN-γand IL-13 in cell culture supernatants were determined by ELISA, Th1/Th2 was calculated as the concentration ratio of IFN-γand IL-13. Results (1) CIK cells proliferation: there were no significant differences in 3 serum microenvironment groups. (2) Cytotoxicity of CIK cells: K562 killing activity of CIK cells on D7, D14 and D21 in 3 serum microenvironments were: ZT: (79.95±12.23)%, (93.86±2.38)% and (66.34±7.54)%, FBS: (71.02±14.79)%, (83.72±5.63)% and (58.37±7.57)%, GT: (40.92±4.66)%, (52.61±8.05)% and (41.43±5.25)% respectively. Cytotoxicity of CIK cells were significantly higher on D14 compared to D7 and D21 both in ZT and FBS groups (P<0.01). While cytotoxicity of CIK cells had significant differences when compared between 3 different serum microenvironments at the same timepoints (including D7, D14 and D21), ZT and FBS group were significantly higher than GT (P<0.01). (3) Dynamic changes of CD4+/CD8+ in cultured CIK cells: Comparison results between different timepoints in the same serogroup displayed CD4+/CD8+ inversion on D0, then increased to 2.25±1.67 on D7 in ZT group, which was significantly higher than D0 (P<0.05), while recovered to CD4+/CD8+inversion status on D14 and D21; In FBS group, CD4+/CD8+ maintain inversion status from D0 to D14, then decreased to 0.04±0.55 on D21, which was significantly lower than D0 (P<0.01);In GT group, CD4+/CD8+also maintain inversion status from D0 to D21, and there was no significant difference between four timepoints. Comparison results between different serum microenvironments at same timepoint showed that CD4+/CD8+ in ZT group was significantly higher than both FBS and GT group on D7 (P<0.05); While there was no significant difference between 3 serogroups both on D14 and D21. (4) Dynamic changes of Th1/Th2 in cultured CIK cells:Comparison results of Th1/Th2 between different timepoints in the same serogroup displayed that Th1/Th2 ratio presented as inversion status on all timepoints, and there was no significant difference between four timepoints;In FBS group, Th1/Th2 ratio were significantly higher on D7 and D14 than D0 (P<0.01), while it recovered to inversion status on D21. Comparison results between different serum microenvironments at same timepoint showed that on D7 and D14, Th1/Th2 ratio in FBS group was significantly higher than ZT and GT group (P<0.01). Conclusions CD4+/CD8+ and Th1/Th2 of CIK cells cultured in different serum microenvironments presented dynamic changing trends. Personalized CIK cells treatment solution should established based on immune status of individual patient, serum microenvironment used for cell culture, and dynamic changes of CD4+/CD8+and Th1/Th2 during CIK cells culture process.
