奥沙利铂对结直肠癌肿瘤微环境中MDSCs分化调控的影响及机制初探
发布时间：2018-11-07 19:41
【摘要】：研究背景及目的结直肠癌(colorectal cancer,CRC)是全球第三大高发肿瘤,发病机制复杂。基于奥沙利铂(Oxaliplatin,OXP)的全身化疗联合手术切除是目前CRC治疗的主要方案。但CRC对OXP的耐药性限制了其临床疗效,而其中潜在的机制尚不清楚。越来越多的研究证实,化疗药物在治疗结直肠癌等实体肿瘤时不仅可以攻击肿瘤本身,还能影响肿瘤微环境(tumormicroenvironment,TME),对抗瘤效果和预后具有重要的作用。其中MDSCs作为肿瘤微环境中的一种重要的免疫调节细胞,在肿瘤微环境下可因受不同因素影响向M1/M2型巨噬细胞分化,从而起到抑瘤或促瘤的作用,这可能介导肿瘤化疗药物耐药,而其中的机制尚不明确。NLRP3/Caspasel信号通路是炎性小体作用的重要途径,抑制炎性小体活性后MDSCs中IRF5表达明显升高,而IRF5与巨噬细胞分化密切相关,是M1型巨噬细胞的特异性标志之一。通过生物信息学分析,课题组发现IRF5启动子区域存在着caspase-1的酶切识别序列,这可能是调控MDSCs分化的靶点之一。本研究旨在探讨奥沙利铂在结直肠癌治疗中对肿瘤微环境内MDSCs分化调控的影响,并对其中的机制进行初探,为结直肠癌奥沙利铂耐药治疗找寻新的思路。方法1、分别构建Balb/c小鼠的DSS诱导的慢性肠炎模型和CT26皮下移植瘤模型,分为空白对照组(PBS)、低剂量OXP组(OXP 1mg/kg)、高剂量OXP组(OXP1Omg/kg)处理小鼠,采用肠道组织HE染色验证肠炎模型,采用皮下瘤组织HE染色和Ki67染色验证皮下瘤模型,应用流式细胞技术对外周血,脾脏、骨髓、皮下瘤等组织中MDSCs(CD11b+Gr-1+)及其MCH-II+的表达比例。2、取Balb/c小鼠骨髓细胞体外诱导生成MDSCs。取CT26培养上清模拟肿瘤微环境和非肿瘤微环境,对比MDSCs在不同浓度OXP处理组中MDSCs(CD11b+Gr-1 +)及其MCH-II+的表达比例。3、构建鼠IRF5、IRF5 mutation质粒,293t细胞分组转染质粒48h后提蛋白,Western blot分析IRF5、Caspase1蛋白表达情况。4、统计学分析:实验数据采用SPSS13.0软件进行统计学分析,两组间的比较采用两独立样本t检验进行,多组间的比较采用单向方差分析one way ANOVA。方差齐,采用LSD法,如方差不齐,采用Dunnett T3法,将P0.05定义为具有显著性的统计学差异。结果1、在DSS诱导的慢性肠炎小鼠模型中,随着OXP剂量的增高,MDSCs的比例在骨髓、外周血、脾脏中均明显下降;脾脏中CD11b+Gr-1high亚群比例明显下降,而其MHC-II+比例明显升高。2、在CT26荷瘤小鼠中,随着OXP剂量的增高,MDSCs的比例在骨髓、外周血、脾脏、肿瘤中均明显升高;皮下瘤中MDSCs中表达MHC-II+的比例明显下降。3、在体外模拟无OXP作用的肿瘤微环境中,同非肿瘤微环境相比,MDSCs细胞群中,Group1细胞群比例降低,Group2细胞群比例升高。其中Group1群中仅CD11b+Gr-1low细胞群的比例下降,而其表达MHC-II+比例升高;Group2组中仅CD11b+Gr-1high细胞群的比例升高,而其表达MHC-II+的比例下降。低浓度的OXP对于体外模拟的肿瘤微环境和非肿瘤微环境中MDSCs的分化均无明显影响。4、Caspase1可与IRF5结合,作用于DY酶切位点分解IRF5,进而下调IRF5的表达。结论1、OXP在肿瘤微环境中对于MDSCs的分化调控具有两面性。一方面,OXP药物本身可对MDSCs起到杀伤的作用,并促进MDSCs向M1型巨噬细胞分化。另一方面,在肿瘤微环境中,OXP在杀伤肿瘤细胞的过程中,肿瘤细胞可释放出相关因子,大量募集MDSCs,并在肿瘤局部抑制MDSCs向M1型巨噬细胞分化,从而形成有利于肿瘤生存的肿瘤微环境。2、在体外培养MDSCs时,在无OXP作用的情况下,肿瘤细胞自身分泌的相关因子可抑制MDSCs向M1型巨噬细胞分化,从而形成有利于肿瘤生长的肿瘤微环境。低浓度的OXP对于体外肿瘤微环境和非肿瘤微环境中MDSCs的分化均无明显影响。3、Caspase1可与IRF5结合,作用于DY酶切位点分解IRF5,进而下调IRF5的表达。
[Abstract]:The background of the study and the purpose of colorectal cancer (CRC) are the third most high-incidence tumors in the world, and the pathogenesis is complicated. Combined surgical resection of systemic chemotherapy based on oxaliplatin (OXP) is the main protocol of CRC. However, the resistance of CRC to OXP has limited its clinical efficacy, and the underlying mechanism is not clear. More and more studies have confirmed that chemotherapy drugs can not only attack the tumor itself but also influence the tumor microenvironment (TME) in the treatment of solid tumors such as colorectal cancer, and play an important role in the treatment of tumor effect and prognosis. MDSCs, as an important immunomodulatory cell in tumor microenvironment, can differentiate into M1/ M2-type macrophages by different factors in a tumor microenvironment, which can play a role in tumor-inhibiting or tumor-stimulating, which may mediate the drug resistance of tumor chemotherapy, and the mechanism is not clear. NLRP3/ Casasel signal pathway is an important approach to the action of inflammatory small body, and the expression of IRR5 in MDSCs is significantly increased after the inhibition of inflammatory cell activity, while IRR5 is closely related to the differentiation of macrophages, and is one of the specific markers of M1-type macrophages. Through the bioinformatics analysis, the research group found that the IRF5 promoter region has an enzyme-cut recognition sequence of caspase-1, which may be one of the targets for regulating the differentiation of MDSCs. The purpose of this study was to study the effect of oxaliplatin on the differentiation and control of MDSCs in tumor microenvironment during the treatment of colorectal cancer, and to explore the mechanism of oxaliplatin in the treatment of colorectal cancer. Method 1, DSS-induced chronic enteritis model and CT26 subcutaneous transplantation tumor model of Balb/ c mice were respectively constructed. The model was divided into blank control group (PBS), low-dose OXP group (OXP 1mg/ kg) and high-dose OXP group (OXP1Omg/ kg). The expression of MDSCs (CD11b + Gr-1 +) and MCH-II + in peripheral blood, spleen, bone marrow and subcutaneous tumor and the expression of MCH-II + in peripheral blood, spleen, bone marrow and subcutaneous tumor were studied by flow cytometry. The expression of MDSCs (CD11b + Gr-1 +) and MCH-II + in different concentrations of OXP treatment group was compared with MDSCs, and the expression of IRF5 and Caspas1 protein was analyzed by Western blot. Statistical analysis: The SPSS 13.0 software was used for statistical analysis. The comparison between the two groups was carried out by two independent samples, one way ANOVA was used for the comparison between the two groups. The variance is the same as that of the LSD method, such as the variance of the variance, and the Dunnett's T3 method is used to define the difference between the statistical significance and the difference. Results 1. In the model of DSS-induced chronic enteritis, with the increase of OXP, the proportion of MDSCs decreased significantly in the bone marrow, peripheral blood and spleen, and the proportion of CD11b + Gr-1high in the spleen decreased significantly, while the proportion of MHC-II + increased significantly. As the dose of OXP increased, the proportion of MDSCs increased significantly in the bone marrow, peripheral blood, spleen and tumor; the ratio of the expression of MHC-II + in the MDSCs in the subcutaneous tumor was significantly decreased. The proportion of group 1 cells decreased and the proportion of group 2 cells increased. Only the proportion of the CD11b + Gr-1low cell population in the Group1 group was decreased, and the expression of MHC-II + was increased; only the proportion of the CD11b + Gr-1high cell group in the Group 2 group was increased, and the proportion of the expression of MHC-II + decreased. The low concentration of OXP has no significant effect on the differentiation of MDSCs in the in vitro simulated tumor microenvironment and the non-tumor microenvironment. Conclusion 1. OXP has two-sidedness in the differentiation and control of MDSCs in the tumor microenvironment. On the one hand, the OXP drug itself can kill MDSCs and promote the differentiation of MDSCs to M1-type macrophages. on the other hand, in the tumor microenvironment, in the course of killing the tumor cells, the OXP can release the relevant factors, raise the MDSCs in a large amount, and locally inhibit the MDSCs to differentiate into the M1-type macrophages, thereby forming a tumor microenvironment conducive to the survival of the tumor. When MDSCs are cultured in vitro, in the absence of OXP, the relevant factors secreted by the tumor cells can inhibit the differentiation of MDSCs to the M1-type macrophages, thereby forming a tumor microenvironment which is beneficial to the growth of the tumor. The low concentration of OXP has no significant effect on the differentiation of MDSCs in the in vitro tumor microenvironment and the non-tumor microenvironment.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2317379
[Abstract]:The background of the study and the purpose of colorectal cancer (CRC) are the third most high-incidence tumors in the world, and the pathogenesis is complicated. Combined surgical resection of systemic chemotherapy based on oxaliplatin (OXP) is the main protocol of CRC. However, the resistance of CRC to OXP has limited its clinical efficacy, and the underlying mechanism is not clear. More and more studies have confirmed that chemotherapy drugs can not only attack the tumor itself but also influence the tumor microenvironment (TME) in the treatment of solid tumors such as colorectal cancer, and play an important role in the treatment of tumor effect and prognosis. MDSCs, as an important immunomodulatory cell in tumor microenvironment, can differentiate into M1/ M2-type macrophages by different factors in a tumor microenvironment, which can play a role in tumor-inhibiting or tumor-stimulating, which may mediate the drug resistance of tumor chemotherapy, and the mechanism is not clear. NLRP3/ Casasel signal pathway is an important approach to the action of inflammatory small body, and the expression of IRR5 in MDSCs is significantly increased after the inhibition of inflammatory cell activity, while IRR5 is closely related to the differentiation of macrophages, and is one of the specific markers of M1-type macrophages. Through the bioinformatics analysis, the research group found that the IRF5 promoter region has an enzyme-cut recognition sequence of caspase-1, which may be one of the targets for regulating the differentiation of MDSCs. The purpose of this study was to study the effect of oxaliplatin on the differentiation and control of MDSCs in tumor microenvironment during the treatment of colorectal cancer, and to explore the mechanism of oxaliplatin in the treatment of colorectal cancer. Method 1, DSS-induced chronic enteritis model and CT26 subcutaneous transplantation tumor model of Balb/ c mice were respectively constructed. The model was divided into blank control group (PBS), low-dose OXP group (OXP 1mg/ kg) and high-dose OXP group (OXP1Omg/ kg). The expression of MDSCs (CD11b + Gr-1 +) and MCH-II + in peripheral blood, spleen, bone marrow and subcutaneous tumor and the expression of MCH-II + in peripheral blood, spleen, bone marrow and subcutaneous tumor were studied by flow cytometry. The expression of MDSCs (CD11b + Gr-1 +) and MCH-II + in different concentrations of OXP treatment group was compared with MDSCs, and the expression of IRF5 and Caspas1 protein was analyzed by Western blot. Statistical analysis: The SPSS 13.0 software was used for statistical analysis. The comparison between the two groups was carried out by two independent samples, one way ANOVA was used for the comparison between the two groups. The variance is the same as that of the LSD method, such as the variance of the variance, and the Dunnett's T3 method is used to define the difference between the statistical significance and the difference. Results 1. In the model of DSS-induced chronic enteritis, with the increase of OXP, the proportion of MDSCs decreased significantly in the bone marrow, peripheral blood and spleen, and the proportion of CD11b + Gr-1high in the spleen decreased significantly, while the proportion of MHC-II + increased significantly. As the dose of OXP increased, the proportion of MDSCs increased significantly in the bone marrow, peripheral blood, spleen and tumor; the ratio of the expression of MHC-II + in the MDSCs in the subcutaneous tumor was significantly decreased. The proportion of group 1 cells decreased and the proportion of group 2 cells increased. Only the proportion of the CD11b + Gr-1low cell population in the Group1 group was decreased, and the expression of MHC-II + was increased; only the proportion of the CD11b + Gr-1high cell group in the Group 2 group was increased, and the proportion of the expression of MHC-II + decreased. The low concentration of OXP has no significant effect on the differentiation of MDSCs in the in vitro simulated tumor microenvironment and the non-tumor microenvironment. Conclusion 1. OXP has two-sidedness in the differentiation and control of MDSCs in the tumor microenvironment. On the one hand, the OXP drug itself can kill MDSCs and promote the differentiation of MDSCs to M1-type macrophages. on the other hand, in the tumor microenvironment, in the course of killing the tumor cells, the OXP can release the relevant factors, raise the MDSCs in a large amount, and locally inhibit the MDSCs to differentiate into the M1-type macrophages, thereby forming a tumor microenvironment conducive to the survival of the tumor. When MDSCs are cultured in vitro, in the absence of OXP, the relevant factors secreted by the tumor cells can inhibit the differentiation of MDSCs to the M1-type macrophages, thereby forming a tumor microenvironment which is beneficial to the growth of the tumor. The low concentration of OXP has no significant effect on the differentiation of MDSCs in the in vitro tumor microenvironment and the non-tumor microenvironment.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前1条
1 Peter Laszlo Lakatos;Laszlo Lakatos;;Risk for colorectal cancer in ulcerative colitis:Changes,causes and management strategies[J];World Journal of Gastroenterology;2008年25期
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