奧沙利鉑對結(jié)直腸癌腫瘤微環(huán)境中MDSCs分化調(diào)控的影響及機(jī)制初探
發(fā)布時(shí)間：2018-11-07 19:41
【摘要】：研究背景及目的結(jié)直腸癌(colorectal cancer,CRC)是全球第三大高發(fā)腫瘤,發(fā)病機(jī)制復(fù)雜�；趭W沙利鉑(Oxaliplatin,OXP)的全身化療聯(lián)合手術(shù)切除是目前CRC治療的主要方案。但CRC對OXP的耐藥性限制了其臨床療效,而其中潛在的機(jī)制尚不清楚。越來越多的研究證實(shí),化療藥物在治療結(jié)直腸癌等實(shí)體腫瘤時(shí)不僅可以攻擊腫瘤本身,還能影響腫瘤微環(huán)境(tumormicroenvironment,TME),對抗瘤效果和預(yù)后具有重要的作用。其中MDSCs作為腫瘤微環(huán)境中的一種重要的免疫調(diào)節(jié)細(xì)胞,在腫瘤微環(huán)境下可因受不同因素影響向M1/M2型巨噬細(xì)胞分化,從而起到抑瘤或促瘤的作用,這可能介導(dǎo)腫瘤化療藥物耐藥,而其中的機(jī)制尚不明確。NLRP3/Caspasel信號(hào)通路是炎性小體作用的重要途徑,抑制炎性小體活性后MDSCs中IRF5表達(dá)明顯升高,而IRF5與巨噬細(xì)胞分化密切相關(guān),是M1型巨噬細(xì)胞的特異性標(biāo)志之一。通過生物信息學(xué)分析,課題組發(fā)現(xiàn)IRF5啟動(dòng)子區(qū)域存在著caspase-1的酶切識(shí)別序列,這可能是調(diào)控MDSCs分化的靶點(diǎn)之一。本研究旨在探討奧沙利鉑在結(jié)直腸癌治療中對腫瘤微環(huán)境內(nèi)MDSCs分化調(diào)控的影響,并對其中的機(jī)制進(jìn)行初探,為結(jié)直腸癌奧沙利鉑耐藥治療找尋新的思路。方法1、分別構(gòu)建Balb/c小鼠的DSS誘導(dǎo)的慢性腸炎模型和CT26皮下移植瘤模型,分為空白對照組(PBS)、低劑量OXP組(OXP 1mg/kg)、高劑量OXP組(OXP1Omg/kg)處理小鼠,采用腸道組織HE染色驗(yàn)證腸炎模型,采用皮下瘤組織HE染色和Ki67染色驗(yàn)證皮下瘤模型,應(yīng)用流式細(xì)胞技術(shù)對外周血,脾臟、骨髓、皮下瘤等組織中MDSCs(CD11b+Gr-1+)及其MCH-II+的表達(dá)比例。2、取Balb/c小鼠骨髓細(xì)胞體外誘導(dǎo)生成MDSCs。取CT26培養(yǎng)上清模擬腫瘤微環(huán)境和非腫瘤微環(huán)境,對比MDSCs在不同濃度OXP處理組中MDSCs(CD11b+Gr-1 +)及其MCH-II+的表達(dá)比例。3、構(gòu)建鼠IRF5、IRF5 mutation質(zhì)粒,293t細(xì)胞分組轉(zhuǎn)染質(zhì)粒48h后提蛋白,Western blot分析IRF5、Caspase1蛋白表達(dá)情況。4、統(tǒng)計(jì)學(xué)分析:實(shí)驗(yàn)數(shù)據(jù)采用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,兩組間的比較采用兩獨(dú)立樣本t檢驗(yàn)進(jìn)行,多組間的比較采用單向方差分析one way ANOVA。方差齊,采用LSD法,如方差不齊,采用Dunnett T3法,將P0.05定義為具有顯著性的統(tǒng)計(jì)學(xué)差異。結(jié)果1、在DSS誘導(dǎo)的慢性腸炎小鼠模型中,隨著OXP劑量的增高,MDSCs的比例在骨髓、外周血、脾臟中均明顯下降;脾臟中CD11b+Gr-1high亞群比例明顯下降,而其MHC-II+比例明顯升高。2、在CT26荷瘤小鼠中,隨著OXP劑量的增高,MDSCs的比例在骨髓、外周血、脾臟、腫瘤中均明顯升高;皮下瘤中MDSCs中表達(dá)MHC-II+的比例明顯下降。3、在體外模擬無OXP作用的腫瘤微環(huán)境中,同非腫瘤微環(huán)境相比,MDSCs細(xì)胞群中,Group1細(xì)胞群比例降低,Group2細(xì)胞群比例升高。其中Group1群中僅CD11b+Gr-1low細(xì)胞群的比例下降,而其表達(dá)MHC-II+比例升高;Group2組中僅CD11b+Gr-1high細(xì)胞群的比例升高,而其表達(dá)MHC-II+的比例下降。低濃度的OXP對于體外模擬的腫瘤微環(huán)境和非腫瘤微環(huán)境中MDSCs的分化均無明顯影響。4、Caspase1可與IRF5結(jié)合,作用于DY酶切位點(diǎn)分解IRF5,進(jìn)而下調(diào)IRF5的表達(dá)。結(jié)論1、OXP在腫瘤微環(huán)境中對于MDSCs的分化調(diào)控具有兩面性。一方面,OXP藥物本身可對MDSCs起到殺傷的作用,并促進(jìn)MDSCs向M1型巨噬細(xì)胞分化。另一方面,在腫瘤微環(huán)境中,OXP在殺傷腫瘤細(xì)胞的過程中,腫瘤細(xì)胞可釋放出相關(guān)因子,大量募集MDSCs,并在腫瘤局部抑制MDSCs向M1型巨噬細(xì)胞分化,從而形成有利于腫瘤生存的腫瘤微環(huán)境。2、在體外培養(yǎng)MDSCs時(shí),在無OXP作用的情況下,腫瘤細(xì)胞自身分泌的相關(guān)因子可抑制MDSCs向M1型巨噬細(xì)胞分化,從而形成有利于腫瘤生長的腫瘤微環(huán)境。低濃度的OXP對于體外腫瘤微環(huán)境和非腫瘤微環(huán)境中MDSCs的分化均無明顯影響。3、Caspase1可與IRF5結(jié)合,作用于DY酶切位點(diǎn)分解IRF5,進(jìn)而下調(diào)IRF5的表達(dá)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.34
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
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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