粒细胞集落刺激因子受体及其阳性免疫细胞在结直肠癌发生发展中的表达变化

Expression of granulocyte colony-stimulating factor receptor in colitis-associated colonic carcinogenesis

目的探讨粒细胞集落刺激因子受体（G-CSFR）在结肠炎相关结直肠癌（CAC）发生发展过程中的表达水平，并分析其阳性表达的免疫细胞亚群。方法联合应用氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）诱导C57BL/6小鼠，建立CAC动物模型，模拟结直肠癌疾病发展中的3个阶段，即炎症恢复期（AD1）、轻度不典型增生期（AD2）和原位癌形成期（AD3）。于AD1、AD2和AD3结束后，分别取结直肠组织制备单细胞悬液，应用流式细胞仪和荧光定量PCR法检测粒细胞集落刺激因子（G-CSF）和粒细胞集落刺激因子受体（G-CSFR）的表达，并分析G-CSFR在T细胞、巨噬细胞、中性粒细胞等免疫细胞中的表达情况。结果小鼠结直肠炎-癌转化模型成功建立，可以有效模拟结直肠炎-癌的发生和发展过程。实时荧光定量PCR检测结果显示，AD1组、AD2组和AD3组小鼠结直肠组织中G-CSF mRNA的表达水平分别为对照组的1.2倍、7.3倍和18.0倍，其中AD2组与对照组、AD3组与对照组比较，差异均有统计学意义（均P〈0.05）。AD1组、AD2组和AD3组小鼠结直肠组织中G-CSFR mRNA的表达水平分别为对照组的1.5倍、2.2倍和4.5倍，其中AD3组与对照组比较，差异有统计学意义（P〈0.05）。流式细胞仪检测结果显示，对照组、AD1组、AD2组和AD3组小鼠结直肠组织中CD45＋G-CSFR＋细胞的比例分别为（21.84±1.77）%、（41.48±4.15）%、（44.84±8.54）%和（57.76±1.95）%，AD2组和AD3组均明显高于对照组（均P〈0.05）。对照组、AD1组、AD2组和AD3组小鼠结直肠组织中CD45＋G-CSFR＋巨噬细胞的比例分别为（21.54±5.88）%、（47.14±5.25）%、（42.49±7.80）%和（29.25±8.24）%；CD45＋G-CSFR＋T细胞的比例分别为（30.04±6.87）%、（29.65±8.08）%、（33.75±7.37）%和（33.32±9.85）%；CD45＋G-CSFR＋中性粒细胞的比例分别为（2.39±2.10）%、（4.05±1.56）%、（3.62±2.67）%和（2.26±0.85）%。各组小鼠结直肠组织中巨噬细胞亚群和T细胞亚群占CD45＋G-CSFR＋细胞群的比例均明显高于粒细胞亚群所占的比例（均P〈0.05）。对照组与AD1组、对照组与AD2组、AD1组与AD3组、AD2组与AD3组小鼠结直肠组织中G-CSFR＋巨噬细胞含量的比较，差异均有统计学意义（均P〈0.05）。结论CAC发生发展过程中，G-CSFR的表达明显上调，其阳性表达的巨噬细胞随疾病进展而富集于结肠组织，提示其参与CAC的发生发展。

Objective To investigate the expression of granulocyte-colony stimulating factor receptor （G-CSFR） in a mouse model of colitis-assoclated cancer （CAC） , and the roles of G-CSFR positive immune cells in the development of CAC. Methods The C57BL/6 mouse model of CAC was established by azoxymethane and dextran sulphate sodium. Three different stages in the development of CAC, including inflammation （ AD1 ） , mild dysplasia （AD2） and adenocareinoma （AD3） were simulated. Colon tissue was digested into single cell suspension and the expressions of G-CSF and G-CSFR were analyzed by real-time PCR and fluorescence activated cell sorter （FACS）.The expressions of G-CSFR on T cell, macrophage and neutrophil were analyzed by FACS. Results The establishment of mouse model can effectively simulate the disease progression of CAC. The results of real-time PCR detection showed that the expression level of G-CSF mRNA in ADI, AD2 and AD3 groups were 1.2, 7.3 and 18.0-fold changes of the control group, respectively. The differences between AD2, AD3 and control groups were statistically significant （P〈0.05）. G-CSFR mRNA levels in ADI, AD2 and AD3 groups were 1.5, 2.2 and 4.5-fold changes of the control group, respectively. The difference between AD3 and control groups was statistically significant （P〈0.05）. FACS showed that the percentages of CD45±G-CSFR± cells in eolorectal tissues of the control group, AD1, AD2 and AD3 groups were （21.84±1.77）%, （41.48±4.15）%, （44.84±8.54）% and （57.76±1.95）%, respectively.The percentages of CD45^＋G-CSFR^＋ cells in AD2 and AD3 groups were significantly higher than that of control group （P〈0.05）. The percentages of CD45^＋-CSFR^＋macrophage in the colorectal tissues of the control group, ADI, AD2 and AD3 groups were （21.54±5.88）%, （47.14±5.25）%, （42.49±7.80）% and （ 29.25± 8.24） %, respectively. The percentages of CD45^＋G-CSFR^＋T cells in these groups were （ 30.04± 6.87） %, （ 29.65 ± 8.08 ） %, （ 33.75 ± 7.37 ） % and （ 33.32 ± 9.85 ） %, respectively. The percentages of CD4^＋-CSFR^＋granulocyte were （2.39±2.10）%, （4.05±1.56）%, （3.62±2.67）% and （2.26±0.85）%, respectively （P〈0.05）. The percentages of G-CSFR± macrophage and G-CSFR^＋T cells were significantly higher than that of G-CSFR^＋granulocyte （P〈0.05）.The differences between ADI and control group, AD2 and control group, AD1 and AD2 group, AD2 and AD3 group were statistically significant （P〈0.05）. Conclusions The expression of G-CSFR is significantly up-regulated in the development of CAC. The enrichment of G-CSFR^＋macrophages in the colon tissue suggests G-CSFR^＋macrophages participate in the development of CAC.