本文選題：轉移前微環(huán)境 + MDSC　； 參考：《中國中醫(yī)科學院》2017年博士論文

【摘要】：研究背景:轉移前微環(huán)境是防治腫瘤轉移的新靶點,被稱為可能引起腫瘤治療模式變革的重要研究方向。轉移前微環(huán)境的概念首先由Kaplan在Nature雜志中提出,2016年在Cancer Cell雜志上給予明確定義:一種具有支持性和接納性的組織微環(huán)境,通過一系列分子和細胞改變形成“指定”的轉移位點,或為腫瘤細胞(“種子”)提供“肥沃”的“土壤”以利于其種植,從而為腫瘤定植于遠端器官提供條件,促進腫瘤轉移。轉移前微環(huán)境的干預成為不能及時或完全切除原位腫瘤的患者預防或減少遠端器官轉移的重要手段,通過干預轉移前微環(huán)境以抑制腫瘤細胞轉移逐漸成為腫瘤領域研究的熱點,被譽為變革腫瘤腫瘤模式的新方向。S1pr1-stat3 激活的髓源性抑制細胞(Myeloid derived suppressor cells,MDSCs)在轉移前微環(huán)境構筑及肺轉移過程中起到重要的支持作用。腫瘤細胞從原位溢出滲入血管、到達靶器官種植轉移是一十分復雜的過程,其中任何一個環(huán)節(jié)的失敗都會阻止腫瘤細胞的轉移,研究顯示MDSCs在肺轉移前微環(huán)境中聚集是轉移前微環(huán)境構建的關鍵,MDSCs與腫瘤細胞之間的交互作用是循環(huán)腫瘤細胞種植轉移的關鍵限速環(huán)節(jié)。而腫瘤細胞s1pr1-stat3信號通路激活引起的MDSCs細胞相應信號通路活化及轉移前微環(huán)境中stat3的廣泛激活是MDSCs細胞得以在轉移前微環(huán)境中持續(xù)募集及生存的保證。通過干預MDSCs細胞S1pr1-stat3系統(tǒng)而抑制肺轉移前微環(huán)境形成是抑制肺轉移的可靠手段。選擇抗腫瘤轉移有效的中藥方劑,發(fā)揮多靶點的優(yōu)勢,干預轉移前微環(huán)境的形成可能為中藥治療腫瘤提供新的研究方向。中醫(yī)治療腫瘤重在從整體上扶助人體正氣,預防腫瘤細胞的轉移。益氣活血法是在臨床實踐中摸索的抗腫瘤轉移的有效方法,在該法指導下結合氣機升降理論由花寶金教授創(chuàng)立的雙參顆粒(西洋參、冬蟲夏草及三七)具有抑制肺轉移的臨床療效。且前期的初步實驗證實,雙參顆�？娠@著減少肺中MDSCs的比例。雙參顆粒是否通過抑制MDSCs細胞中s1pr1-stat3信號通路的激活起到抑制肺轉移前微環(huán)境形成的作用是本研究的主要內容。揭示雙參顆粒干預肺轉移前微環(huán)境抗腫瘤轉移的機制對進一步配伍提高抗腫瘤轉移效率的研究有重要意義。且從理論上為中醫(yī)治未病,“先安未受邪之地”的理論提供了實驗基礎及科學依據(jù),對中醫(yī)治療腫瘤模式的變革具有現(xiàn)實意義。研究目的:理論研究:以氣機升降理論為指導的益氣活血方劑在“先安未受邪之地”思想指導下抗腫瘤轉移的作用機制研究。實驗研究:分析氣機升降理論與益氣活血法結合創(chuàng)立的雙參顆粒,探討其在干預轉移前微環(huán)境防治腫瘤轉移中的作用,開展如下實驗:(1)體內實驗:①觀察雙參顆粒通過干預轉移前微環(huán)境對荷瘤小鼠原位腫瘤及肺轉移的影響,②研究雙參顆粒對肺轉移前微環(huán)境中MDSCs以及其s1pr1/stat3信號通路的影響③研究雙參顆粒對肺轉移前微環(huán)境特異性腫瘤源性細胞因子(tumor-derived secreted factors,TDSFs)的作用;④雙參顆粒對轉移前微環(huán)境生物標記物(Fibronectin,Lox,Versican,MMP9)表達的影響。(2)體外實驗:①探討雙參顆粒對腫瘤細胞分泌肺特異性TDSFs的作用,②在明確腫瘤細胞s1pr1-stat3信號通路激活對MDSCs分化影響之后,探討雙參顆粒對該過程的干預作用,③體外建立共培養(yǎng)模型,模擬轉移前微環(huán)境,探討雙參顆粒對轉移前微環(huán)境特異性生物標記物表達的作用�？傮w而言,探討氣機升降與益氣活血結合指導的雙參顆粒干預肺轉移前微環(huán)境抗肺轉移的效果及其相關機制。研究方法:(1)構建Lewis肺癌C57BL/6小鼠肺轉移模型,采用小動物活體成像和定期取材(肺、瘤體)兩種方法,觀察雙參顆粒對原位腫瘤及肺轉移的影響。(2)利用熒光顯微鏡、免疫組化、酶標儀熒光定量3種方法評估轉移前微環(huán)境動物模型的構建。(3)利用ELISA方法,檢測肺轉移前微環(huán)境與轉移微環(huán)境中肺特異性TDSFs(VEGF、TGF-β、GM-CSF 及 TNF-α)的表達水平。(4)利用流式細胞檢測儀定量分析肺轉移前微環(huán)境中MDSCs的比例及表型變化。(5)Western blot、免疫組化染色方法評估肺轉移前微環(huán)境中轉移前微環(huán)境特異性生物標記物的水平。(6)利用共培養(yǎng)技術構建轉移前微環(huán)境體外模型,觀察雙參顆粒醇提劑對模型中TDSFs、轉移前微環(huán)境特異性生物標記物的影響。(7)在共培養(yǎng)模型中,明確s1pr1-stat3信號通路激活的Lewis細胞對髓系細胞向MDSCs分化的作用,及雙參顆粒的干預效果。研究結果:(1)雙參顆粒抑瘤效果:小動物活體成像結果顯示:雙參顆粒組及5-FU組均可明顯抑制小鼠瘤體p/sec/cm2/sr(P0.05);14d天雙參顆粒有較明顯的抑制原位腫瘤生長的作用p/sec/cm2/sr(P0.05),28d與對照組比較,原位腫瘤質量相似(P0.05);5-Fu組在28d時仍有較顯著的抑制瘤體作用(P0.05)。(2)抑瘤率:在接種Lewis細胞后第14d、28d分別處死小鼠,剝離瘤體稱重計算抑瘤率:在14d、28d時雙參組和5-Fu的抑瘤率分別為38.2%、4.2%;41.8%、20.1%。(3)肺轉移:接種 Lewis 細胞后第 14d、17d、20d、23d、26d、29d、32d、35d分別處死小鼠,采用布氏液處理后肉眼觀察轉移灶、冰凍切片熒光顯微鏡觀察、組織研碎酶標儀熒光定量分析、石蠟包埋切片HE染色觀察4種方法評估肺轉移情況,26d前雙參顆粒組和5-Fu組均較對照組轉移減少(P0.05),29d后雙參顆粒組和對照組肺轉移相似(P0.05)。(4)肺中MDSCs比例:和對照組相比,雙參顆粒組小鼠肺中MDSCs比例顯著降低(33.9%vs.9.9%,P0.O5)。(5)血中腫瘤源性分泌性細胞因子: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的表達水平,結果和對照組相比,14d時SSG只對GM-CSF和TNF-α有促進恢復正常的作用(P0.05),對VEGF、TGF-β無顯著作用;28d時SSG可顯著降低血中TGF-β、GM-CSF的表達水平(P0.05),對VEGF及TNF-α無顯著影響。(6)肺轉移前微環(huán)境中特異性生物標記物:Versican、Fibronectin、Lox、MMP9被認為是和肺轉移前微環(huán)境相關的生物標記物。我們采用免疫組化染色方法、Western blot定性和定量評估各指標的表達情況,兩種方法的結果基本一致,雙參顆粒治療組較對照組均有顯著的下降(PO.05)。5-Fu與雙參組無顯著優(yōu)勢(P0.05)。(7)雙參顆粒對體外轉移前微環(huán)境模型中TDSFs的影響:在共培養(yǎng)模型中分別觀察12h、24h共培養(yǎng)上清液中TDSFs的變化,發(fā)現(xiàn)共培養(yǎng)12h時各組TDSFs較對照組無顯著變化(P0.05)。24小時后TGF-β及GM-CSF在雙參顆粒組有顯著降低(P0.05),VEGF和TNF-α無顯著變化。5-Fu對各種TDSFs均無顯著抑制作用。(8)雙參顆粒對s1pr1-stat3信號通路的作用。我們用實時定量熒光PCR方法檢測s1pr1、stat3基因在各相關組織(肺、骨髓、腫瘤)細胞的表達情況,同時用western blot方法進行驗證。我們發(fā)現(xiàn)雙參顆粒對腫瘤組織、骨髓組織及肺臟組織中兩種基因及蛋白的表達均有抑制作用(P0.05)。5-Fu只對腫瘤組織中兩種基因及蛋白的表達有抑制作用(P0.05),對骨髓及肺中兩種基因及蛋白的表達作用不明顯(P0.05)。(9)雙參顆粒對髓系細胞向MDSCs分化的作用:首先,我們通過sew2871(s1pr1激活劑)激活Lewis細胞s1pr1-stat3信號通路。激活的Lewis細胞和髓系細胞共培養(yǎng),流式細胞儀結果顯示MDSCs占髓系細胞的比例較對照組明顯增加(43%vs.5%,P0.05)。其次,我們觀察了雙參顆粒對MDSCs分化的影響,發(fā)現(xiàn)野生Lewis細胞對MDSCs分化作用不明顯,雙參顆粒作用也不顯著;而雙參顆粒醇提劑可通過抑制Lewis細胞s1pr1的表達,從而抑制髓系細胞向MDSCs細胞的分化(43%vs.0.7%)。5-Fu有相同的效果,兩組和對照組比較均有統(tǒng)計學差異(P0.05)。(9)雙參顆粒對體外轉移前微環(huán)境模型中肺轉移前微環(huán)境特異性生物標記物的作用:通過對模型中肺細胞和MDSCs細胞混合物做western blot檢測,我們發(fā)現(xiàn),和單純的肺細胞或MDSCs對照組相比,雙參顆粒對轉移前微環(huán)境模型中 Version、Fibronectin、Lox、MMP9 均有抑制作用(P0.05)。研究結論:(1)氣機升降理論指導的益氣活血方劑雙參顆粒不僅對原位腫瘤有一定的抑制作用,還可通過調控轉移前微環(huán)境防治腫瘤細胞在遠端靶器官種植轉移。(2)雙參顆�？捎行p少肺轉移前微環(huán)境中MDSCs的含量及降低腫瘤細胞分泌的部分TDSFs。(3)Lewis肺癌細胞s1pr1-stat3信號通路的激活與骨髓細胞向MDSCs的轉化、肺臟s1pr1-stat3激活有關。雙參顆粒通過降低肺轉移前微環(huán)境中s1pr1-stat3信號通路的激活程度及MDSCs在肺臟轉移前微環(huán)境中的聚集,減少轉移前微環(huán)境標記物Version、Fibronectin、Lox、MMP9的表達水平,逆轉肺轉移前微環(huán)境,減少腫瘤細胞肺轉移。
[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.
【學位授予單位】：中國中醫(yī)科學院
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R273

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