双参颗粒调控髓源性抑制细胞构筑肺转移前微环境的机制研究

发布时间：2018-07-07 11:13

本文选题：转移前微环境 + MDSC　；参考：《中国中医科学院》2017年博士论文

【摘要】：研究背景:转移前微环境是防治肿瘤转移的新靶点,被称为可能引起肿瘤治疗模式变革的重要研究方向。转移前微环境的概念首先由Kaplan在Nature杂志中提出,2016年在Cancer Cell杂志上给予明确定义:一种具有支持性和接纳性的组织微环境,通过一系列分子和细胞改变形成“指定”的转移位点,或为肿瘤细胞(“种子”)提供“肥沃”的“土壤”以利于其种植,从而为肿瘤定植于远端器官提供条件,促进肿瘤转移。转移前微环境的干预成为不能及时或完全切除原位肿瘤的患者预防或减少远端器官转移的重要手段,通过干预转移前微环境以抑制肿瘤细胞转移逐渐成为肿瘤领域研究的热点,被誉为变革肿瘤肿瘤模式的新方向。S1pr1-stat3 激活的髓源性抑制细胞(Myeloid derived suppressor cells,MDSCs)在转移前微环境构筑及肺转移过程中起到重要的支持作用。肿瘤细胞从原位溢出渗入血管、到达靶器官种植转移是一十分复杂的过程,其中任何一个环节的失败都会阻止肿瘤细胞的转移,研究显示MDSCs在肺转移前微环境中聚集是转移前微环境构建的关键,MDSCs与肿瘤细胞之间的交互作用是循环肿瘤细胞种植转移的关键限速环节。而肿瘤细胞s1pr1-stat3信号通路激活引起的MDSCs细胞相应信号通路活化及转移前微环境中stat3的广泛激活是MDSCs细胞得以在转移前微环境中持续募集及生存的保证。通过干预MDSCs细胞S1pr1-stat3系统而抑制肺转移前微环境形成是抑制肺转移的可靠手段。选择抗肿瘤转移有效的中药方剂,发挥多靶点的优势,干预转移前微环境的形成可能为中药治疗肿瘤提供新的研究方向。中医治疗肿瘤重在从整体上扶助人体正气,预防肿瘤细胞的转移。益气活血法是在临床实践中摸索的抗肿瘤转移的有效方法,在该法指导下结合气机升降理论由花宝金教授创立的双参颗粒(西洋参、冬虫夏草及三七)具有抑制肺转移的临床疗效。且前期的初步实验证实,双参颗粒可显著减少肺中MDSCs的比例。双参颗粒是否通过抑制MDSCs细胞中s1pr1-stat3信号通路的激活起到抑制肺转移前微环境形成的作用是本研究的主要内容。揭示双参颗粒干预肺转移前微环境抗肿瘤转移的机制对进一步配伍提高抗肿瘤转移效率的研究有重要意义。且从理论上为中医治未病,“先安未受邪之地”的理论提供了实验基础及科学依据,对中医治疗肿瘤模式的变革具有现实意义。研究目的:理论研究:以气机升降理论为指导的益气活血方剂在“先安未受邪之地”思想指导下抗肿瘤转移的作用机制研究。实验研究:分析气机升降理论与益气活血法结合创立的双参颗粒,探讨其在干预转移前微环境防治肿瘤转移中的作用,开展如下实验:(1)体内实验:①观察双参颗粒通过干预转移前微环境对荷瘤小鼠原位肿瘤及肺转移的影响,②研究双参颗粒对肺转移前微环境中MDSCs以及其s1pr1/stat3信号通路的影响③研究双参颗粒对肺转移前微环境特异性肿瘤源性细胞因子(tumor-derived secreted factors,TDSFs)的作用;④双参颗粒对转移前微环境生物标记物(Fibronectin,Lox,Versican,MMP9)表达的影响。(2)体外实验:①探讨双参颗粒对肿瘤细胞分泌肺特异性TDSFs的作用,②在明确肿瘤细胞s1pr1-stat3信号通路激活对MDSCs分化影响之后,探讨双参颗粒对该过程的干预作用,③体外建立共培养模型,模拟转移前微环境,探讨双参颗粒对转移前微环境特异性生物标记物表达的作用。总体而言,探讨气机升降与益气活血结合指导的双参颗粒干预肺转移前微环境抗肺转移的效果及其相关机制。研究方法:(1)构建Lewis肺癌C57BL/6小鼠肺转移模型,采用小动物活体成像和定期取材(肺、瘤体)两种方法,观察双参颗粒对原位肿瘤及肺转移的影响。(2)利用荧光显微镜、免疫组化、酶标仪荧光定量3种方法评估转移前微环境动物模型的构建。(3)利用ELISA方法,检测肺转移前微环境与转移微环境中肺特异性TDSFs(VEGF、TGF-β、GM-CSF 及 TNF-α)的表达水平。(4)利用流式细胞检测仪定量分析肺转移前微环境中MDSCs的比例及表型变化。(5)Western blot、免疫组化染色方法评估肺转移前微环境中转移前微环境特异性生物标记物的水平。(6)利用共培养技术构建转移前微环境体外模型,观察双参颗粒醇提剂对模型中TDSFs、转移前微环境特异性生物标记物的影响。(7)在共培养模型中,明确s1pr1-stat3信号通路激活的Lewis细胞对髓系细胞向MDSCs分化的作用,及双参颗粒的干预效果。研究结果:(1)双参颗粒抑瘤效果:小动物活体成像结果显示:双参颗粒组及5-FU组均可明显抑制小鼠瘤体p/sec/cm2/sr(P0.05);14d天双参颗粒有较明显的抑制原位肿瘤生长的作用p/sec/cm2/sr(P0.05),28d与对照组比较,原位肿瘤质量相似(P0.05);5-Fu组在28d时仍有较显著的抑制瘤体作用(P0.05)。(2)抑瘤率:在接种Lewis细胞后第14d、28d分别处死小鼠,剥离瘤体称重计算抑瘤率:在14d、28d时双参组和5-Fu的抑瘤率分别为38.2%、4.2%;41.8%、20.1%。(3)肺转移:接种 Lewis 细胞后第 14d、17d、20d、23d、26d、29d、32d、35d分别处死小鼠,采用布氏液处理后肉眼观察转移灶、冰冻切片荧光显微镜观察、组织研碎酶标仪荧光定量分析、石蜡包埋切片HE染色观察4种方法评估肺转移情况,26d前双参颗粒组和5-Fu组均较对照组转移减少(P0.05),29d后双参颗粒组和对照组肺转移相似(P0.05)。(4)肺中MDSCs比例:和对照组相比,双参颗粒组小鼠肺中MDSCs比例显著降低(33.9%vs.9.9%,P0.O5)。(5)血中肿瘤源性分泌性细胞因子:采用ELISA法,定量分析接瘤后14d、28d小鼠血中VEGF、TGF-β、GM-CSF及TNF-α的表达水平,结果和对照组相比,14d时SSG只对GM-CSF和TNF-α有促进恢复正常的作用(P0.05),对VEGF、TGF-β无显著作用;28d时SSG可显著降低血中TGF-β、GM-CSF的表达水平(P0.05),对VEGF及TNF-α无显著影响。(6)肺转移前微环境中特异性生物标记物:Versican、Fibronectin、Lox、MMP9被认为是和肺转移前微环境相关的生物标记物。我们采用免疫组化染色方法、Western blot定性和定量评估各指标的表达情况,两种方法的结果基本一致,双参颗粒治疗组较对照组均有显著的下降(PO.05)。5-Fu与双参组无显著优势(P0.05)。(7)双参颗粒对体外转移前微环境模型中TDSFs的影响:在共培养模型中分别观察12h、24h共培养上清液中TDSFs的变化,发现共培养12h时各组TDSFs较对照组无显著变化(P0.05)。24小时后TGF-β及GM-CSF在双参颗粒组有显著降低(P0.05),VEGF和TNF-α无显著变化。5-Fu对各种TDSFs均无显著抑制作用。(8)双参颗粒对s1pr1-stat3信号通路的作用。我们用实时定量荧光PCR方法检测s1pr1、stat3基因在各相关组织(肺、骨髓、肿瘤)细胞的表达情况,同时用western blot方法进行验证。我们发现双参颗粒对肿瘤组织、骨髓组织及肺脏组织中两种基因及蛋白的表达均有抑制作用(P0.05)。5-Fu只对肿瘤组织中两种基因及蛋白的表达有抑制作用(P0.05),对骨髓及肺中两种基因及蛋白的表达作用不明显(P0.05)。(9)双参颗粒对髓系细胞向MDSCs分化的作用:首先,我们通过sew2871(s1pr1激活剂)激活Lewis细胞s1pr1-stat3信号通路。激活的Lewis细胞和髓系细胞共培养,流式细胞仪结果显示MDSCs占髓系细胞的比例较对照组明显增加(43%vs.5%,P0.05)。其次,我们观察了双参颗粒对MDSCs分化的影响,发现野生Lewis细胞对MDSCs分化作用不明显,双参颗粒作用也不显著;而双参颗粒醇提剂可通过抑制Lewis细胞s1pr1的表达,从而抑制髓系细胞向MDSCs细胞的分化(43%vs.0.7%)。5-Fu有相同的效果,两组和对照组比较均有统计学差异(P0.05)。(9)双参颗粒对体外转移前微环境模型中肺转移前微环境特异性生物标记物的作用:通过对模型中肺细胞和MDSCs细胞混合物做western blot检测,我们发现,和单纯的肺细胞或MDSCs对照组相比,双参颗粒对转移前微环境模型中 Version、Fibronectin、Lox、MMP9 均有抑制作用(P0.05)。研究结论:(1)气机升降理论指导的益气活血方剂双参颗粒不仅对原位肿瘤有一定的抑制作用,还可通过调控转移前微环境防治肿瘤细胞在远端靶器官种植转移。(2)双参颗粒可有效减少肺转移前微环境中MDSCs的含量及降低肿瘤细胞分泌的部分TDSFs。(3)Lewis肺癌细胞s1pr1-stat3信号通路的激活与骨髓细胞向MDSCs的转化、肺脏s1pr1-stat3激活有关。双参颗粒通过降低肺转移前微环境中s1pr1-stat3信号通路的激活程度及MDSCs在肺脏转移前微环境中的聚集,减少转移前微环境标记物Version、Fibronectin、Lox、MMP9的表达水平,逆转肺转移前微环境,减少肿瘤细胞肺转移。
[Abstract]:Background: the pre transfer microenvironment is a new target for the prevention and treatment of tumor metastasis. It is known as an important research direction for the change of tumor therapy patterns. The concept of pre transfer microenvironment was first proposed by Kaplan in Nature magazine. In 2016, the Cancer Cell magazine gave a clear definition: a supportive and receptive organizational microenvironment. A "designated" transfer site is formed through a series of molecular and cell changes, or a "fertile" "soil" for tumor cells ("seeds") to help its planting, thus providing conditions for tumor colonization of distal organs and promoting tumor metastasis. The important means to prevent or reduce the metastasis of distal organs is the hot spot in the field of cancer by interfering with the transfer of the microenvironment to inhibit the metastasis of tumor cells. It is known as the new direction of the tumor tumor tumor model.S1pr1-stat3 activated myeloid suppressor cells (Myeloid derived suppressor cells, MDSCs) in the pre transfer microring It is a very complicated process that tumor cells infiltrate into the blood vessels from the site and reach the target organ, and the failure of any one of them will prevent the metastasis of the tumor cells. The study shows that the aggregation of MDSCs in the microenvironment before the lung metastasis is the construction of the microenvironment before metastasis. The key point is that the interaction between MDSCs and tumor cells is the key speed limit for the implantation and metastasis of circulating tumor cells, and the extensive activation of the STAT3 in the microenvironment before the activation of the MDSCs cell signaling pathway activation by the s1pr1-stat3 signaling pathway of the tumor cells and the extensive activation of the MDSCs cells in the microenvironment before the metastasis can be continuously raised in the microenvironment before the metastasis and in the microenvironment. The guarantee of survival. By interfering with the S1pr1-stat3 system of MDSCs cells, inhibiting the formation of the microenvironment before the lung metastasis is a reliable means of inhibiting the lung metastasis. The emphasis is on helping the body's positive gas and preventing the metastasis of tumor cells. Yiqi Huoxue method is an effective method for anti tumor metastasis in clinical practice. Under the guidance of this method, the clinical effect of double ginseng granules (Panax quinquefolium, Cordyceps sinensis and 37) founded by Professor Hua Baojin, combined with the theory of Qi and Qi, has the clinical effect of inhibiting lung metastasis. Preliminary experiments confirmed that double ginseng granules can significantly reduce the proportion of MDSCs in the lung. The main content of this study is whether double ginseng granules can inhibit the formation of microenvironment before lung metastasis by inhibiting the activation of s1pr1-stat3 signaling pathway in MDSCs cells. The mechanism of double ginseng granules to intervene in the microenvironment before lung metastasis is revealed. It is of great significance to further study the efficiency of anti tumor metastasis. In theory, the theory provides the experimental basis and scientific basis for the theory of the treatment of non disease in traditional Chinese medicine. It is of practical significance to the reform of the mode of cancer treatment in traditional Chinese medicine. Study on the mechanism of anti tumor metastasis of blood prescription under the guidance of "Xian an unaffected land". Experimental study: to analyze the role of double ginseng granules established by the combination of Qi lifting theory and Yiqi Huoxue Method in the prevention and control of tumor metastasis before the intervention and transfer of microenvironment. The following experiments were carried out: (1) in vivo experiments: (1) observation of double ginseng granules The effect of pre transferred microenvironment on the tumor and lung metastasis of tumor bearing mice was studied. (2) the effect of double ginseng Granule on MDSCs and its s1pr1/stat3 signaling pathway in the microenvironment before lung metastasis was studied. The effect of double ginseng Granule on tumor-derived secreted factors (TDSFs) before lung metastasis was studied. The effect of Shuang Shen Granule on the expression of biomarkers (Fibronectin, Lox, Versican, MMP9) in microenvironment before transfer. (2) in vitro experiment: (1) to explore the effect of double ginseng granules on the secretion of lung specific TDSFs by tumor cells. 2. After the effect of s1pr1-stat3 signaling pathway activation on the differentiation of MDSCs in tumor cells, the effect of double ginseng Granule on this process was discussed. A co culture model was established in vitro to simulate the microenvironment before transfer, and to explore the effect of double ginseng granules on the expression of microenvironment specific biomarkers before transfer. 1) the model of lung metastasis of Lewis lung cancer C57BL/6 mice was constructed. The effects of double ginseng granules on the in situ tumor and lung metastasis were observed with two methods of living body imaging and regular sampling (lung, tumor body). (2) using fluorescence microscopy, immunohistochemistry, and fluorescence quantitative enzyme labeling method to evaluate the construction of animal models of microenvironment before transfer. (3) use of ELISA Methods to detect the expression level of lung specific TDSFs (VEGF, TGF- beta, GM-CSF and TNF- alpha) in the microenvironment before lung metastasis. (4) the quantitative analysis of the proportion and phenotypic changes of MDSCs in the microenvironment before lung metastasis by flow cytometry. (5) Western blot, immunohistochemical staining method to evaluate the pre metastasis microenvironment before metastasis in lung metastasis The level of environmental specific biomarkers. (6) using co culture technique to construct an in vitro model of microenvironment before transfer, and observe the effect of double ginseng granule alcohol extract on TDSFs and microenvironment specific biomarkers in the model. (7) in co culture model, the Lewis cells activated by s1pr1-stat3 signaling pathway to myeloid cells to MDSCs The results of the study were as follows: (1) the effect of double ginseng Granule on tumor suppression: the imaging results of small animals in vivo showed that the p/sec/cm2/sr (P0.05) of the mice could be obviously inhibited by the double ginseng granule group and the 5-FU group; the effect of double ginseng Granule on the growth of the primary tumor was obviously p/sec/cm2/sr (P0.05), and the ratio of 28d to the control group was compared with that of the control group. The mass of tumor in situ was similar (P0.05), while group 5-Fu still had a significant inhibition of tumor body action (P0.05) at 28d. (2) the tumor suppressor rate was killed in Lewis cells after 14d, 28d was killed respectively, and the tumor suppressor rate of the stripped tumor weight calculation was 38.2%, 4.2%, 41.8%, 20.1%. (3) lung metastasis: inoculation of Lewis thin. The mice were killed at 14d, 17D, 20d, 23d, 26D, 29d, 32D, and 35d respectively. The metastases were observed by the broth solution with the naked eye, the frozen section fluorescence microscope was observed, the fluorescence quantitative analysis of the tissue fragment enzyme scale was observed, and the paraffin embedded slice HE staining was used to observe the pulmonary transfer in 4 methods. Both the double ginseng granule group and the 5-Fu group before 26D were reduced to the control group. Less (P0.05), 29d after double ginseng granule group and control group similar (P0.05). (4) the proportion of MDSCs in the lung: compared with the control group, the proportion of MDSCs in the lung of the double ginseng granule group was significantly lower (33.9%vs.9.9%, P0.O5). (5) the tumor derived secretory cytokines in the blood were collected with ELISA, and the quantitative analysis of VEGF, TGF- beta, TGF- beta in 28d mice The expression level of F- alpha, compared with the control group, SSG only promoted the recovery of GM-CSF and TNF- alpha at 14d (P0.05), and had no significant effect on VEGF, TGF- beta. SSG could significantly reduce TGF- beta in blood, and no significant effect on the expression level of GM-CSF. (6) specific biomarkers in the microenvironment before lung metastasis: An, Fibronectin, Lox, MMP9 were considered as biomarkers related to the microenvironment before lung metastasis. We used immunohistochemical staining method, Western blot qualitative and quantitative evaluation of the expression of each index, the results of the two methods were basically the same. The double ginseng granule treatment group had a significant decrease (PO.05).5-Fu and double ginseng group no significant difference compared with the control group. (P0.05). (7) the effect of double ginseng granules on TDSFs in the microenvironment model before the transfer in vitro: the changes of TDSFs in the co culture supernatant of 12h and 24h were observed in the co culture model, and there was no significant change of TDSFs in each group of 12h (P0.05).24 hours after co culture (P0.05), TGF- beta and GM-CSF in the double ginseng granule group decreased significantly (P0.05). No significant changes in.5-Fu and TNF- alpha have no significant inhibitory effects on various TDSFs. (8) the effect of double ginseng granules on the s1pr1-stat3 signaling pathway. We detected the expression of s1pr1, STAT3 gene in the related tissues (lung, bone marrow, tumor) cells by real-time quantitative fluorescent PCR, and verified by western blot method. We found double ginseng The expression of two genes and proteins in tumor tissue, bone marrow tissue and lung tissue were inhibited (P0.05).5-Fu only inhibited the expression of two genes and proteins in tumor tissues (P0.05), and the expression of two genes and proteins in bone marrow and lung was unknown (P0.05). (9) double ginseng granules differentiated myeloid cells to MDSCs First, we activated the Lewis cell s1pr1-stat3 signaling pathway through the sew2871 (s1pr1 activator). The activated Lewis cells and the myeloid cells were co cultured. The flow cytometry showed that the proportion of MDSCs in the medullary cells was significantly increased (43%vs.5%, P0.05). The effect of wild Lewis cells on MDSCs differentiation was not obvious, and the effect of double ginseng granules was not significant. The double ginseng granule alcohol extract could inhibit the expression of s1pr1 in Lewis cells and inhibit the differentiation of myeloid cells to MDSCs cells (43%vs.0.7%).5-Fu. The two groups were significantly different from the control group (P0.05). (9) double ginseng granules were used. The role of microenvironment specific biomarkers before lung metastasis in the microenvironment model in vitro was detected by Western blot detection of the mixture of lung cells and MDSCs cells in the model. We found that, compared with the simple lung cells or the MDSCs control group, the double ginseng granule had the inhibition of Version, Fibronectin, Lox, and MMP9 in the pre transferred microenvironment model. P0.05. Conclusions: (1) Yiqi Huoxue Prescription double ginseng granule not only has a certain inhibitory effect on the tumor in situ, but also can control the metastasis of tumor cells in the distal target organ by regulating the microenvironment before the transfer. (2) double ginseng granules can effectively reduce the content and decrease of MDSCs in the microenvironment before the lung metastasis. The activation of the s1pr1-stat3 signaling pathway of partial TDSFs. (3) Lewis lung cancer cells secreted by tumor cells is related to the transformation of bone marrow cells to MDSCs and the activation of s1pr1-stat3 in the lung. By reducing the activation of s1pr1-stat3 signaling pathway in the microenvironment before lung metastasis and the accumulation of MDSCs in the microenvironment before the lung metastasis, the double ginseng granule reduces the metastasis before metastasis. The expression level of Version, Fibronectin, Lox and MMP9 in microenvironment reverses the microenvironment before lung metastasis and reduces lung metastasis of tumor cells.
【学位授予单位】：中国中医科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R273

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