【作者】 李伟；

【导师】 王国斌； 陶凯雄；

【作者基本信息】 华中科技大学 ， 外科学， 2014， 博士

【摘要】 目的：结直肠癌已成为第三大癌症发生和死亡原因,手术切除仍然是可能治愈结直肠癌的唯一治疗方法,然而结直肠癌的术后复发还是临床亟待解决的问题。手术在治疗肿瘤的同时也常常带来创伤,手术创伤可释放多种损伤相关分子模式分子包括HMGB1,同时也通过多种机制引起免疫抑制,而免疫抑制如MDSC与肿瘤的进展密切相关。结直肠癌手术创伤对肿瘤腹腔种植复发的影响还不清楚。本研究旨在通过动物肿瘤手术模型探索手术创伤在结直肠肿瘤腹腔局部复发中作用。方法：通过腹部手术模拟结直肠肿瘤的手术创伤,收集术后24小时腹腔渗液,用HMGB1的ELISA试剂盒检测HMGB1水平；给于外源性HMGB1注射小鼠腹腔三次,每天一次,收集腹腔灌洗液,流式检测MDSC在CD45中的比例和计数MDSC在灌洗液中的数量；体外培养结肠癌细胞系CT26,皮下种植CT26细胞,制造小鼠负瘤模型；两周后麻醉小鼠,剥除肿瘤并制造腹腔内手术损伤,模拟腹腔肿瘤切除手术。用HMGB1Box-A阻断HMGB1,用Gemcitabine清除MDSC,收集小鼠腹腔灌洗液,流式检测腹腔灌洗液中MDSC的比例和数量；给HMGB1阻断组、Gemcitabine去除组、单纯手术组和对照组腹腔创伤小鼠种植CT26细胞,两周后对肿瘤种植负荷评分。收集HMGB1阻断、Gemcitabine去除组、单纯手术组和对照组腹腔创伤术后腹腔中MDSC,分别用DAF-FM DA荧光探针和DCFH-DA荧光探针标记MDSC,流式检测NOS活性和ROS水平。应用NOS抑制剂(L-NAME)和ROS清除剂(Tiron)注射创伤术后腹腔,同时腹腔种植CT26,两周后进行腹腔肿瘤种植评分,评估L-NAME和Tiron对CT26腹腔种植的影响。结果：正常小鼠手术后腹腔渗液的HMGB1浓度平均为4237.67ng/ml,负瘤小鼠手术后腹腔渗液的HMGB1浓度平均为3300.67ng/ml,负瘤小鼠无手术时,腹腔液中HMGB1浓度平均为53.97ng/ml。正常小鼠腹腔中只有很少量的MDSC存在,只占CD45阳性细胞的2%左右,而腹腔中只有数量有限的MDSC,约为2.4×10^3个MDSC细胞；100ng/ml HMGB1处理组小鼠腹腔中MDSC比例占CD45阳性细胞的10%,lml腹腔灌洗液含有MDSC数量为3.9×10^4；1000ng/ml HMGB1处理组小鼠腹腔中MDSC比例占CD45阳性细胞的30%,lml腹腔灌洗液中有1×10^5左右个MDSC细胞,与对照组相比此差异具有统计学意义(p<0.05)。HMGB1拮抗剂HMGB1Box-A处理后,小鼠腹腔灌洗液中MDSC占CD45阳性细胞比例为12%,lml小鼠腹腔灌洗液中MDSC数量为0.75×10^6；单纯手术组小鼠腹腔灌洗液中MDSC比例为73%,1ml小鼠腹腔灌洗液中MDSC可达6.0×10^6,另外术前给以Gemcitabine去除小鼠体内的MDSC后,趋化至小鼠腹腔中MDSC比例约为28%,lml小鼠腹腔灌洗液中MDSC数量减少为1.3×10^6；负瘤小鼠无手术组,小鼠腹腔灌洗液中MDSC比例为2.3%,lml小鼠腹腔灌洗液中MDSC为2.0×10^3。小鼠腹膜肿瘤种植评分结果显示非手术组、HGMB1Box-A处理组、Gemcitabine组和手术组小鼠壁层腹膜肿瘤种植总评分分别为：2.0±1.0,5.0±1.0,7.3±3.0和10.4±3.5,组内比较差异具有统计学意义(p=0.008)；而总的腹腔种植转移评分分别为：6.7±1.5,11.0±1.0,13.5±3.8和18.8±3.5,组内比较差异有统计学意义(p=0.001)。同时,腹腔种植肿瘤病灶的最大直径分别为：0.3±0.1cm、0.3±0.1cm,0.5±0cm和0.8±0.3cm,组内比较差异具有统计学意义(p=0.013)。NOS和ROS活性检测结果显示手术创伤募集的MDSC中DAF-FMDA探针荧光强度较无手术组明显增加,而Gecitabine和]HMGB1Box-A组DAF-FMDA探针荧光较手术组减弱；手术组与Gemcitabine处理组和无手术组小鼠腹腔灌洗液的MDSC中DCFH-DA探针荧光强度差异不大,而手术组与HMGB1Box-A处理组小鼠腹腔灌洗液的MDSC中DCFH-DA探针荧光强度有显差异。然而,HMGB1Box-A处理组较无手术组小鼠腹腔灌洗液的MDSC中DCFH-DA探针荧光强度也明显减弱。用NOS和ROS抑制剂干预后,评估肿瘤腹腔种植转移负荷结果显示手术组、Tiron处理组和L-NAME组小鼠壁层腹膜肿瘤种植总评分分别为：8.5±1.3,6.3±3.7和4.8±1.3,组内比较差异不明显(p=0.134)；而总的腹腔种植转移评分分别为：16.3±1.0,12.3±3.7和9.7±1.5,组内比较差异有统计学意义(p=0.011)。同时,腹腔种植肿瘤病灶的最大直径分别为：0.7±0.1cm、0.5±0.1cm和0.5±0.1cm,组内比较差异具有统计学意义(p=0.044)结论：我们通过动物肿瘤手术模型研究表明手术创伤导致损伤组织释放损伤相关分子HMGB1,HMGB1可以募集大量的髓系来源免疫抑制性细胞MDSC趋化至腹腔,阻断HMGB1可抑制MDSC的趋化,减轻结肠癌细胞CT26腹腔种植,腹腔术后趋化的MDSC通过产生NOS促进结肠癌细胞种植灶的形成和生长,通过产生ROS促进CT26肿瘤种植灶的增大。更多还原

【Abstract】 Aims:Colorectal cancer has been the third incidence of cancer and the third cause of tumor death. Surgery remains the only treatment that has the possibility to completely cure colorectal tumor. However, the postoperative recurrence of colorectal tumor is a urgent issue needing to be addressed by clinical researchers. When surgery is used to treat cancer, itself is a trauma to body. Surgical trauma induces immune suppression through various mechanisms and immunosuppression is closely associated with tumor progression. The effect of surgical trauma in colorectal cancer therapy on the local recurrence of colorectal tumor in peritoneal cavity is remained to be known. This study was aimed at uncovering the contribution of surgical trauma to the local implantation in peritoneal cavity by a model of animal tumor surgery.Methods:Abdominal operation was made as the model of surgery trauma in colorectal tumor ectomy. Abdominal exudation was collected at24h after surgery, and detected by ELISA kit for HMGB1level. Exogenous HMGB1was injected into peritoneal cavity of mice one time per day for three days and then peritoneal lavage was collected. The ratio of MDSC to CD45positive cells and the number of MDSC in lavage were measure by flow cytometer. CT26cell was cultured and injected subcutaneously in mice which worked as tumor loading model. Two weeks later, implanted tumors were excised and surgical trauma in peritoneal cavity was made which mimiced operation for the abdominal tumor. HMGB1was blocked with HMGB1Box-A and MDSC was deleted with gemcitabine. The peritoneal lavage was collected and detected by flow cytometer for the percent of MDSC in CD45positive cells and the quantity of MDSC in the lavage. CT26cell was injected into different group mice including HMGB1blocking group, gemcitabine deleting group, mere surgery group and control group. Two weeks later, the tumor metastatic score was evaluated. Cells in the peritoneal lavage from HMGB1blocking group, gemcitabine deleting group, mere surgery group and control group were collected and detected by flow cytometer using DAF-FM and DCFH-DA probe for the NOS activity and ROS level respectively. CT26cell was injected into peritoneal cavity after surgery trauma followed by NOS inhibitor (L-NAME) and ROS scavenger (Tiron) injection at one time per day for three times. Two weeks later, the peritoneal metastatic score was evaluated.Results:The level of HMGB1in exudate from normal mice with surgery is4237.67ng/ml, and the concentration of HMGB1for tumor bearing mice with surgery is3300.67ng/ml. For the mice without surgery, the concentration is53.97ng/ml. Control mice had small percent of MDSC in CD45positive cells and small quantity of MDSC in lml lavage, corresponding to2%and2.4×10^3.100ng/ml HMGB1treated mice had10%MDSC in CD45positive cells and3.9×10^4MDSC in lml lavage and1000ng/ml HMGB1treated mice had30%MDSC in CD45positive cells and1×10^5MDSC in lml lavage. Both of them were higher than control group (p＜0.05). HMGB1Box-A treated mice had12%MDSC in CD45positive cells and0.75×10^6MDSC in lml lavage, while mere surgery mice had73%MDSC in CD45positive cells and6.0×10^6/ml lavage. Gemcitabine treated mice had28%MDSC in CD45positive cells and1.3×10^6/ml lavage. Tumor bearing mice without surgery had2.3%MDSC in CD45positive cells and2.0×10^3/ml lavage. The metastasis score showed that the total parietal peritoneal score for control group, HMGB1blocking group, gemcitabine deleting group and mere surgery group were2.0±1.0,5.0±1.0,7.3±3.0and10.4±3.5respectively, the difference in groups is significant (p=0.008). Total peritoneal score control group, HMGB1blocking group, gemcitabine deleting group and mere surgery group were6.7±1.5,11.0±1.0,13.5±3.8and18.8±3.5respectively. The difference in groups is significant (p=0.013). The result for NOS activity showed that MDSC in mere surgery group had higher fluorescence signal of DAF-FM DA compared with control group while both of gemcitabine and HMGB1Box-A groups had lower fluorescence signal compared with surgery group. For the ROS level, MDSC in surgery group, gemcitabine group and control group had similar fluorescence signal of DCFH-DA, while MDSC in HMGB1group had significant lower fluorescence signal than that in surgery group. However, MDSC in HMGB1group also had significant lower fluorescence signal than that in control group. When treated with NOS inhibitor and ROS scavenger, mice in surgery group, Tiron group and L-NAME group had total parietal peritoneal metastasis score corresponding to8.5±1.3,6.3±3.7and4.8±1.3respectively, and there was no significant difference in the groups (p=0.134). But, total peritoneal metastasis score for the three groups were16.3±1.0,12.3±3.7and9.7±1.5respectively, and there was significant difference in the groups (p=0.011). Meanwhile, the maximum diameter for the implanted tumor in three groups were0.7±0.1cm,0.5±0.1cm and0.5±0.1cm respectively, and there was significant difference in the groups (p=0.044).Conclusion:We used the model of animal tumor surgery to show that abdominal surgical trauma induce HMGB1which recruits large number of MDSC into peritoneal cavity. Blocking HMGB1could inhibit recruitment of MDSC and alleviate peritoneal implantation of colorectal cancer cell. The recruited MDSC produce NO to promote the form and growth of CT26implantation and ROS to favor the growth of CT26implantation.更多还原