【作者】 陈金元；

【导师】 黄宗海；

【作者基本信息】 南方医科大学 ， 外科学， 2014， 博士

【摘要】 第一部分FLEP新辅助化疗对胃癌治疗效果的影响目的：给予胃癌患者FLEP新辅助化疗,通过比较化疗前后肿瘤组织病理学及影像学改变,研究该方案在胃癌治疗中的作用。方法：自2012年7月至2013年6月广东医学院附属医院胃肠外科收治的晚期胃癌36例,男29例,女7例,男女之比4.1：1,平均年龄51.6±14.8岁,病程1周～3年不等。将其随机分为两组(每组18例)：实验组(FLEP NACT+手术+术后辅助化疗组,即CSC组)及对照组(手术+术后辅助化疗组,即S组)。FLEP方案进行2-4周期NACT,2和4周期化疗结束后分别重新进行临床分期的评估,继而进行手术。术后行常规方案辅助化疗。结果：CSC组18例中,4例完全缓解(胃镜未见明显病灶,活检未发现癌细胞)；10例部分缓解(病灶明显缩小、浸润深度变浅)；其余4例病变无进展。有效率为77.8%(14/18)。CSC组18名患者大部分得到不同程度的临床降期,Ⅲ期以上患者所占比重由化疗前的66.7%降至11.1%。与S组相比,CSC组RO切除率显著增加,淋巴结转移数量显著下降,肿瘤的浸润范围明显缩小。CSC组所有患者均完成了NACT,2个周期的4例,8个周期的3例,4个周期的6例。按照新的药物毒副反应判定标准有2例后白细胞偏低,但计数都≥3×109/L；有1例粒细胞<1.5×109/L,所有患者未有Ⅲ级以上毒副反应发生。结论：FLEP方案NACT能显著降低胃癌的临床分期,减少淋巴结转移数量,提高胃癌根治性切除机会；同时不增加手术并发症的发生几率,NACT对胃癌手术操作和术后恢复无明显不良影响。第二部分FlEP新辅助化疗对胃癌肿瘤细胞生物学行为的影响目的：给予胃癌患者FLEP新辅助化疗,通过检测细胞凋亡指数(AI)、MVD及PCNA表达水平,探讨FLEP新辅助化疗对诱导细胞凋亡、抑制细胞增殖和肿瘤血管新生的作用,以助于评估治疗效果及判断其预后。方法：自2012年7月至2013年6月广东医学院附属医院胃肠外科收治的晚期胃癌36例,男29例,女7例,男女之比4.1：1,平均年龄51.6±14.8岁,病程1周～3年不等。将其随机分为两组(每组18例)：实验组(FLEP NACT+手术+术后辅助化疗组,即CSC组)及对照组(手术+术后辅助化疗组,即S组)。FLEP方案进行2-4周期NACT,2和4周期化疗结束后分别重新进行临床分期的评估,继而进行手术。术后行常规方案辅助化疗。结果：CSC组标本胃癌细胞AI最高为13.06%,最低为3.82%,平均为6.15士2.03%；S组标本胃癌细胞AI最高为8.64%,最低为2.37%,平均为4.44士1.68%,p值小于0.05,提示NACT后胃癌细胞凋亡指数比化疗前增高。CSC组胃癌细胞PCNA表达阳性率最高为85.94%,最低为41.18%,平均56.97士14.39%,S组胃癌细胞PCNA表达阳性率最高为59.07%,最低为19.23%,平均40.86士7.92%,差异有统计学意义,说明新辅助化疗后胃癌细胞PCNA表达较未化疗者明显降低。检测CSC组和S组MVD,结果显示两组MVD具有统计学差异,说明CSC组行新辅助化疗后MVD明显低于S组。结论：FLEP方案新辅助化疗可通过诱导进展期胃癌细胞的凋亡、抑制胃癌细胞增殖和肿瘤血管生成,以到达治疗效果。肿瘤微血管形成及细胞增殖可成为判断FLEP方案新辅助化疗用于恶性肿瘤转移治疗效果的重要指标之一。第三部分FLEP新辅助化疗对CD4+CD25+调节性T细胞的影响目的：给予胃癌患者FLEP新辅助化疗,通过分析外周血中CD4+CD25+调节性T (Treg)细胞比例,及其抑制T细胞增殖能力和免疫抑制细胞因子分泌,明确CD4+CD25+Treg细胞与胃癌临床分期关系,及其在胃癌FLEP新辅助化疗中的作用。方法：收集CSC组、S组患者及正常健康周外周血,分离外周血单个核细胞(PBMCs),流式细胞术检测外周血中CD4+CD25+Treg细胞比例,免疫磁珠分选法分离外周血中CD4+CD25+Treg细胞。流式细胞术检测分选所得CD4+CD25+Treg细胞纯度,台盼蓝染色检测其细胞活力,3H掺入法检测体外增殖能力。混合淋巴细胞反应分析CD4+CD25+Treg细胞抑制T细胞体外增殖能力。流式细胞术和ELISA检测CD4+CD25+Treg细胞的免疫抑制细胞因子的分泌。结果：与Ⅰ、Ⅱ期胃癌相比,Ⅲ、Ⅳ期胃癌患者外周血中CD4+CD25+Treg细胞占CD4+T细胞的比例显著增加。胃癌患者外周血中CD4+CD25+Treg细胞比例显著高于正常人；CSC组新辅助化疗后CD4+CD25+Treg细胞比例显著下降,而术后CD4+CD25+Treg细胞比例无显著变化；S组术后CD4+CD25+Treg细胞比例虽有所下降,却不具有统计学差异；CSC组与S组术后相比,CD4+CD25+Treg细胞比例明显下降,具有统计学差异。这说明手术对患者外周血中CD4+CD25+Treg细胞比例无影响,新辅助化疗可显著降低胃癌患者外周血中CD4+CD25+Treg细胞的比例。CD4+CD25+Treg细胞可显著抑制CD4+CD25-T细胞增殖,随着CD4+CD25+Treg细胞数的增加,这种抑制增殖的能力也相应增加；新辅助化疗后,CD4+CD25+Treg细胞对CD4+CD25-T细胞的抑制率显著下降；而手术并不明显改变CD4+CD25+Treg细胞对CD4+CD25-T细胞的抑制作用。胃癌患者CD4+CD25+Treg细胞分泌TGF-β、IL-4、IL-10水平要明显高于与健康人群；新辅助化疗后,CD4+CD25+Treg细胞分泌TGF-β、IL-4、IL-10水平显著降低；手术对上述细胞因子无影响。结论：胃癌分期越高,外周血中CD4+CD25+T细胞比例越高。新辅助化疗可显著降低胃癌患者外周血中CD4+CD25+T细胞的比例,抑制CD4+CD25+T细胞抑制CD4+CD25-T细胞的作用,影响免疫抑制因子工L-10和TGF-β等的分泌,是新辅助化疗通过调控机体免疫反应达到治疗作用的重要机制。更多还原

【Abstract】 Part Ⅰ The impact of neoadjuvant chemotherapy with FLEP regimen on the therapeutic effects of gastric carcinoma patientsObjective:Giving FLEP neoadjuvant chemotherapy to gastric cancer patients, and comparing the changes of tumor tissue pathology and imaging before and after chemotherapy, to evaluate the therapeutic effects of FLEP neoadjuvant chemotherapy for gastric cancer.Methods and materials:Collecting36cases of advanced gastric cancer admitted in general surgery fron Guangdong Medical college affiliated hospital(July2012to June2013). Randomly dividing them into two groups (18cases in each group):the experimental group (FLEP NACT+surgery+postoperative adjuvant chemotherapy group, the CSC group) and control group (surgery+postoperative adjuvant chemotherapy group, the group S). FLEP-based NACT for2～4cycles, and evaluating the clinical stage after2or4cycles of NACT. Routine postoperative adjuvant chemotherapy after surgery.Results:In18cases from group CSC, four people were complete remission,10 people partially alleviated and four people had no progress. The effective rate was77.8%(14/18). The clinical stage of gastric cancer reduced in group CSC. Compared with S group, the RO resection rate increased significantly, lymph node metastasis reduced and tumor infiltrating significantly narrowed down in group CSC. According to the grading evaluation criterion of NCI-CTC adverse reactions, the numbers of leucocytes were on the low side in2patients after chemotherapy, skin pigmentation occured in1case, belonging to the adverse reaction grade Ⅰ. No patient had Ⅲ magnitude adverse reactions.Conclusions:FLEP-based neoadjuvant chemotherapy can significantly reduce the clinical stage of gastric cancer, reduce lymph node metastasis, and improve the radical resection opportunities of gastric cancer. At the same time, FLEP-based neoadjuvant chemotherapy do not increase the risk of surgical complications, the difficulty of gastric cancer surgery and has no obvious effects on the recovery of postoperative patients.Part Ⅱ The impact of neoadjuvant chemotherapy FLEP on biological behavior of gastric cancer tumor cellsObjective:Giving FLEP neoadjuvant chemotherapy to gastric cancer patients, and detecting tumor cell apoptosis index (AI), MVD and PCNA expression level, to discusses the effects of FLEP neoadjuvant chemotherapy on apoptosis induction, cell proliferation inhibition and tumor angiogenesis and help evaluate and judging treatment effects and prognosis.Methods and materials:Collecting36cases of advanced gastric cancer admitted in general surgery fron Guangdong Medical college affiliated hospital(July2012to June2013). Randomly dividing them into two groups (18cases in each group):the experimental group (FLEP NACT+surgery+postoperative adjuvant chemotherapy group, the CSC group) and control group (surgery+postoperative adjuvant chemotherapy group, the group S). FLEP-based NACT for2-4cycles, and evaluating the clinical stage after2or4cycles of NACT. Routine postoperative adjuvant chemotherapy after surgery.Results:There are significant differences between AI of gastric cancer tumor cells in group CSC and S, indicating higher AI of gastric cancer tumor cells after neoadjuvant chemotherapy. There are also significant differences between the positive rate of PCNA expression of gastric cancer tumor cells in group CSC and S, indicating lower PCNA expression of gastric cancer tumor cells after neoadjuvant chemotherapy.Detecting MVD of group CSC and S showed significant differences, suggesting lower MVD after neoadjuvant chemotherapy.Conclusions:FLEP neoadjuvant chemotherapy can induce apoptosis of advanced gastric cancer tumor cells, inhibit gastric cancer tumor cell proliferation and tumor angiogenesis, reaching effective treatment. Tumor microvascular formation and cell proliferation can be important indexes for judging therapeutic effects prognosis of FLEP-based neoadjuvant chemotherapy for malignant tumor.Part Ⅲ The impact of neoadjuvant chemotherapy FLEP on CD4+CD25+regulatory T cells in the peripheral blood of patients with gastric cancerObjective:Giving FLEP neoadjuvant chemotherapy to gastric cancer patients, and analyzing the ratio of CD4+CD25+regulatory T (Treg) cells peripheral blood, the ability of CD4+CD25+Treg cells suppressing T cell proliferation and immune suppression secreted factors of CD4+CD25+Treg cells, to clear the association between CD4+CD25+Treg cells and clinical stage of gastric cancer, and its role in FLEP neoadjuvant chemotherapy for gastric cancer.Methods and materials:Collecting peripheral blood of healthy people and patients in group CSC and S for separation of peripheral blood mononuclear cells (PBMCs). Then testing proportions of CD4+CD25+Treg cells in peripheral blood by flow cytometry and separating CD4+CD25+Treg cells from peripheral blood by MACs. Detecting the purity of sorted CD4+CD25+Treg cells, the cell vitality by trypan blue staining, and proliferation in vitro by H mixed method. Analyzingthe ability of CD4+CD25+Treg cells suppressing T cell proliferation in vitro by mixed lymphocyte reaction. Detecting immune suppression secreted factors of CD4+CD25+Treg cells by flow cytometry and ELISA.Results:Compared with Ⅰ, Ⅱ stage gastric cancer, the proportion of CD4+CD25+Treg cells in peripheral blood from Ⅲand IVgastric cancer patients increased significantly, the proportion of CD4+CD25+Treg cells in peripheral blood from gastric cancer patients is higher than healthy people; the proportion in group CSC after neoadjuvant chemotherapy decreased; compared with the group S, the proportion in group CSC also decreased, suggesting that neoadjuvant chemotherapy can significantly reduce the proportion of CD4+CD25+Treg cells in peripheral blood from gastric cancer patients. CD4+CD25+Treg cells can significantly inhibit the CD4+CD25"T cells proliferation, and the ability increased accordingly with the increasing number of CD4+CD25+Treg cells; after neoadjuvant chemotherapy, the ability of CD4+CD25+Treg cells suppressing T cell proliferation dropped significantly. CD4+CD25+Treg cells from gastric cancer could secrete higher TGF-P, IL-4and IL-10level than healthy people; after neoadjuvant chemotherapy, immune suppression secreted factors of CD4+CD25+Treg cellsdecreased.Conclusions:The higher the staging of gastric cancer is associated with higher proportion of CD4+CD25+Treg cells in peripheral blood. Neoadjuvant chemotherapy can significantly lower the ratio of CD4+CD25+Treg cells in gastric cancer patients, inhibit the ability of CD4+CD25+Treg cells suppressing T cell proliferation, and impair immune suppression secreted factors of CD4+CD25+Treg cells, indicating an important mechanism for neoadjuvant chemotherapy to achieve therapeutic effects by modulating the immune responses.更多还原