CAJ | 학술논문

目的：研究5-氟尿嘧啶（5-FU ）对原位肝癌小鼠组织中髓系抑制性细胞（ myeloid-derived suppressor cells ，MDSCs）的分布、比例变化及免疫功能的影响作用。方法采用H22细胞株原位移植法建立小鼠原位肝癌模型，造模后第7天将模型小鼠随机分为4组，分别给予0．4 ml PBS，10、30和50 mg/kg剂量的5-FU，连续给药10 d，同时设正常对照组，停药后采用免疫组织化学染色法测定小鼠肝脏中MDSCs的分布，用流式细胞术检测小鼠脾脏中MDSCs的比例，ELISA法检测小鼠血清中高表达精氨酸酶 I（Arginase -I，Arg-I）及IFN-γ的含量，化学比色法检测小鼠血清中一氧化氮合酶（ nitric oxide synthetase ，NOS）的活性，MTT法比较各组小鼠T细胞的增殖功能。结果模型小鼠随着给药剂量的升高，肿瘤体积缩小，肝脏与腹腔粘连程度减弱；模型组小鼠肝脏及脾脏中MDSCs比例均高于正常对照组（P＜0．05），其中PBS组比例最高，随着5-FU剂量的升高，MDSCs比例逐渐下调，除肝脏PBS组与10 mg/kg组外，其他组间差异有统计学意义（P＜0．05）；模型组Arg-I的含量及NOS的活性均高于正常对照组（P＜0．05），其中PBS组含量最高，随着5-FU剂量的升高，二者逐渐下调，组间差异有统计学意义（P＜0．05）；模型组血清IFN-γ的含量均低于正常对照组（P＜0．05），其中PBS 组含量最低，随着5-FU剂量的升高，IFN-γ含量逐渐上调，除PBS组与10 mg/kg组外，其他组间差异有统计学意义（P＜0．05）；T细胞增殖试验，脾细胞培养24 h时各组间增殖指数差异均无统计学意义，48 h时模型组均低于正常组，其中PBS组最低，随着5-FU剂量升高，增殖指数逐渐升高，除PBS组与10 mg/kg组外，其他组间差异有统计学意义（P ＜0．05）；肝、脾MDSCs比例与 Arg-I含量及NOS活性呈正相关，与血清IFN-γ含量及48 h T细胞增殖指数呈负相关。结论5-FU在一定程度上减少肝癌小鼠肝脏、脾脏中MDSCs的数量，缓解MDSCs对肝癌小鼠的免疫抑制作用，提高肝癌小鼠的免疫功能，抑制肿瘤的生长，并在该实验剂量范围内具有一定的量效依赖性。目的：研究5-氟尿嘧啶（5-FU ）對原位肝癌小鼠組織中髓繫抑製性細胞（ myeloid-derived suppressor cells ，MDSCs）的分佈、比例變化及免疫功能的影響作用。方法採用H22細胞株原位移植法建立小鼠原位肝癌模型，造模後第7天將模型小鼠隨機分為4組，分彆給予0．4 ml PBS，10、30和50 mg/kg劑量的5-FU，連續給藥10 d，同時設正常對照組，停藥後採用免疫組織化學染色法測定小鼠肝髒中MDSCs的分佈，用流式細胞術檢測小鼠脾髒中MDSCs的比例，ELISA法檢測小鼠血清中高錶達精氨痠酶 I（Arginase -I，Arg-I）及IFN-γ的含量，化學比色法檢測小鼠血清中一氧化氮閤酶（ nitric oxide synthetase ，NOS）的活性，MTT法比較各組小鼠T細胞的增殖功能。結果模型小鼠隨著給藥劑量的升高，腫瘤體積縮小，肝髒與腹腔粘連程度減弱；模型組小鼠肝髒及脾髒中MDSCs比例均高于正常對照組（P＜0．05），其中PBS組比例最高，隨著5-FU劑量的升高，MDSCs比例逐漸下調，除肝髒PBS組與10 mg/kg組外，其他組間差異有統計學意義（P＜0．05）；模型組Arg-I的含量及NOS的活性均高于正常對照組（P＜0．05），其中PBS組含量最高，隨著5-FU劑量的升高，二者逐漸下調，組間差異有統計學意義（P＜0．05）；模型組血清IFN-γ的含量均低于正常對照組（P＜0．05），其中PBS 組含量最低，隨著5-FU劑量的升高，IFN-γ含量逐漸上調，除PBS組與10 mg/kg組外，其他組間差異有統計學意義（P＜0．05）；T細胞增殖試驗，脾細胞培養24 h時各組間增殖指數差異均無統計學意義，48 h時模型組均低于正常組，其中PBS組最低，隨著5-FU劑量升高，增殖指數逐漸升高，除PBS組與10 mg/kg組外，其他組間差異有統計學意義（P ＜0．05）；肝、脾MDSCs比例與 Arg-I含量及NOS活性呈正相關，與血清IFN-γ含量及48 h T細胞增殖指數呈負相關。結論5-FU在一定程度上減少肝癌小鼠肝髒、脾髒中MDSCs的數量，緩解MDSCs對肝癌小鼠的免疫抑製作用，提高肝癌小鼠的免疫功能，抑製腫瘤的生長，併在該實驗劑量範圍內具有一定的量效依賴性。
목적：연구5-불뇨밀정（5-FU ）대원위간암소서조직중수계억제성세포（ myeloid-derived suppressor cells ，MDSCs）적분포、비례변화급면역공능적영향작용。방법채용H22세포주원위이식법건립소서원위간암모형，조모후제7천장모형소서수궤분위4조，분별급여0．4 ml PBS，10、30화50 mg/kg제량적5-FU，련속급약10 d，동시설정상대조조，정약후채용면역조직화학염색법측정소서간장중MDSCs적분포，용류식세포술검측소서비장중MDSCs적비례，ELISA법검측소서혈청중고표체정안산매 I（Arginase -I，Arg-I）급IFN-γ적함량，화학비색법검측소서혈청중일양화담합매（ nitric oxide synthetase ，NOS）적활성，MTT법비교각조소서T세포적증식공능。결과모형소서수착급약제량적승고，종류체적축소，간장여복강점련정도감약；모형조소서간장급비장중MDSCs비례균고우정상대조조（P＜0．05），기중PBS조비례최고，수착5-FU제량적승고，MDSCs비례축점하조，제간장PBS조여10 mg/kg조외，기타조간차이유통계학의의（P＜0．05）；모형조Arg-I적함량급NOS적활성균고우정상대조조（P＜0．05），기중PBS조함량최고，수착5-FU제량적승고，이자축점하조，조간차이유통계학의의（P＜0．05）；모형조혈청IFN-γ적함량균저우정상대조조（P＜0．05），기중PBS 조함량최저，수착5-FU제량적승고，IFN-γ함량축점상조，제PBS조여10 mg/kg조외，기타조간차이유통계학의의（P＜0．05）；T세포증식시험，비세포배양24 h시각조간증식지수차이균무통계학의의，48 h시모형조균저우정상조，기중PBS조최저，수착5-FU제량승고，증식지수축점승고，제PBS조여10 mg/kg조외，기타조간차이유통계학의의（P ＜0．05）；간、비MDSCs비례여 Arg-I함량급NOS활성정정상관，여혈청IFN-γ함량급48 h T세포증식지수정부상관。결론5-FU재일정정도상감소간암소서간장、비장중MDSCs적수량，완해MDSCs대간암소서적면역억제작용，제고간암소서적면역공능，억제종류적생장，병재해실험제량범위내구유일정적량효의뢰성。
Objective To investigate the effects of 5-fluorouracil (5-FU) on the distribution, pro-portion and immunosuppressive activities of myeloid-derived suppressor cells (MDSCs) in a mouse model of liver cancer.Methods Orthotopic transplantation of H22 cell line was performed to establish the mouse model of orthotopic liver cancer.Forty mice were randomly divided into four groups (n=10) on the 7th day after transplantation.Three different doses of 5-FU (10 mg/kg, 30 mg/kg and 50mg /kg) and 0.4 ml of PBS were respectively given to the mice in each group for 10 consecutive days .A control group without canc-er cell transplantation was set up correspondingly .The distribution of MDSCs in mice liver tissues was detec-ted by immunohistochemistry .The proportions of MDSCs in spleen tissues were tested by flow cytometry . The levels of IFN-γand Arginase-I ( Arg-I) in serum samples were analyzed by enzyme-linked immunosor-bent assay.The activities of nitric oxide synthetase (NOS) in serum samples were tested by chemical colori-metry.The proliferation of T lymphocytes was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltet-razolium bromide (MTT) test.Results 5-FU reduced the tumor size and alleviated the adhesion between liver tissues and abdominal cavity in mice with liver cancer in a dose-dependent manner .The proportions of MDSCs in liver and spleen tissues from mice in four model groups were significantly higher than those in con - trol group (P<0.05).The highest proportions of MDSCs were observed in PBS treated group , followed by 10 mg/kg of 5-FU treated group , 30 mg/kg of 5-FU treated group and 50 mg/kg of 5-FU treated group .Ex-cept for comparing between PBS treated group and 10 mg/kg of 5-FU treated group, there were significant differences with the proportions of MDSCs in liver tissues among other groups (P<0.05).The levels of Arg-I and the activities of NOS in mice with liver cancer were higher than those in control group (P<0.05).The mice in PBS treated group showed the highest level of Arg-I and the strongest activities of NOS that were de-creased as concentrations of 5-FU increased( P<0.05).The levels of IFN-γin serum samples from mice with liver cancer were lower than those in control group , and the lowest level was detected in PBS treated group.IFN-γlevels were increased as concentrations of 5-FU elevated.Except for comparing between PBS treated group and 10 mg/kg of 5-FU treated group , significant differences with the levels of IFN-γin serum samples (P<0.05) were observed among other groups .No significant differences with the proliferation of T cells were observed among all groups after 24 hours of culture (P>0.05),but that were decreased in model groups than those in control group after 48 hours of culture (P<0.05).T cells from mice in PBS treated group showed the lowest level of proliferation .T cell proliferation was enhanced upon the treatment of 5-FU in a dose-dependent manner .Except for comparing between PBS treated group and 10 mg/kg of 5-FU treated group, significant differences with T cell proliferation were observed among other groups (P<0.05).The proportions of MDSCs in liver and spleen tissues were positively correlated with the levels of Arg -I and the activities of NOS, but negatively correlated with IFN-γlevel and T cell proliferation at the time point of 48 h.Conclusion To a certain extent , 5-FU could reduce the numbers of MDSCs in liver and spleen tis-sues from mice with liver cancer , inhibit immunosuppressive activities of MDSCs and restrain the growth of the tumor in a dose-dependent manner .