發(fā)布時(shí)間：2018-04-28 13:54

  本文選題：雷帕霉素 + Notch1��； 參考：《北京中醫(yī)藥大學(xué)》2017年博士論文

【摘要】：惡性腫瘤是危害人類健康的主要慢性疾病之一,在祖國(guó)醫(yī)學(xué)中,屬"癥瘕""積聚""巖""瘤""噎膈"等范疇。林洪生教授致力于中西醫(yī)結(jié)合防治惡性腫瘤科研與臨床近40載,認(rèn)為腫瘤是整體屬虛,局部屬實(shí)的全身疾病的局部反應(yīng),"虛""毒""瘀"是腫瘤致病的主要病理因素。林洪生教授在全國(guó)中醫(yī)腫瘤工作者對(duì)中醫(yī)腫瘤病因病機(jī),理法方藥不斷研究的基礎(chǔ)上,繼承總結(jié)既往"扶正培本"理論,并進(jìn)一步凝練提出了更能針對(duì)腫瘤"虛" "毒" "瘀"致病特點(diǎn)的"固本清源"新理論。"固本"是顧護(hù)機(jī)體正氣以扶正,提高患者的防病抗病能力,糾正正氣不足的病理狀態(tài);"清源"是祛除腫瘤發(fā)生發(fā)展的致病因素,從源頭上控制腫瘤。林洪生教授通過(guò)循證醫(yī)學(xué)研究證實(shí)"固本清源"理論指導(dǎo)下的扶正活血解毒法可以延長(zhǎng)晚期非小細(xì)胞肺癌患者的生存期,提高患者的生活質(zhì)量,證實(shí)了該理論指導(dǎo)下的扶正活血解毒法治療腫瘤的臨床有效性。為了進(jìn)一步明確固本清源理論的生物學(xué)內(nèi)涵,以及探明扶正活血解毒中藥對(duì)腫瘤的作用機(jī)制,林洪生課題團(tuán)隊(duì)以腫瘤微環(huán)境和腫瘤干細(xì)胞為切入點(diǎn),對(duì)扶正活血解毒法指導(dǎo)下的中藥干預(yù)腫瘤千細(xì)胞的生物學(xué)行為做了深入研究。在既往師兄師姐博士課題中,從"固本"和"清源"兩方面證實(shí)扶正活血解毒中藥復(fù)方對(duì)腫瘤干細(xì)胞生物學(xué)行為的調(diào)控作用。為了深入研究不同治則中藥在"固本"和"清源"中醫(yī)腫瘤理論中所扮演的角色,以及他們對(duì)腫瘤干細(xì)胞生物學(xué)行為的干預(yù)作用機(jī)制,課題組針對(duì)單一治則中藥進(jìn)行研究,目前已經(jīng)初步證實(shí)活血中藥、解毒中藥對(duì)腫瘤干細(xì)胞在"清源"理論指導(dǎo)下的調(diào)控作用。但課題組仍未對(duì)扶正中藥干預(yù)腫瘤干細(xì)胞的生物學(xué)行為做以系統(tǒng)研究,所以為填補(bǔ)課題組的研究空白,畢業(yè)課題特選用扶正中藥人參的提取物人參皂苷Rh2(Ginenoside Rh2,GRh2)作為研究藥物,以前列腺癌DU145細(xì)胞系干細(xì)胞作為研究對(duì)象,探索扶正治則代表中藥人參的提取物GRh2對(duì)腫瘤干細(xì)胞生物學(xué)行為在"清源"理論方面的調(diào)控機(jī)制,以期進(jìn)一步明確扶正活血解毒法對(duì)腫瘤發(fā)生發(fā)展的干預(yù)作用,在豐富固本清源理論科學(xué)內(nèi)涵的同時(shí),也為中醫(yī)藥多通路多靶點(diǎn)調(diào)控腫瘤干細(xì)胞提供實(shí)驗(yàn)室依據(jù)。研究目的:1.明確GRh2通過(guò)下調(diào)Notch1信號(hào)通路調(diào)控腫瘤干細(xì)胞生物學(xué)行為,進(jìn)而抑制腫瘤增殖生長(zhǎng)的作用機(jī)制。2.探索GRh2聯(lián)合mTOR抑制劑提高腫瘤抑瘤效果的作用機(jī)制。研究方法:1.體內(nèi)實(shí)驗(yàn):采用前列腺癌DU145細(xì)胞系,通過(guò)懸浮球狀培養(yǎng)的方法分離富集、培養(yǎng)純化出腫瘤干細(xì)胞微球(Tumor Spheres)。通過(guò)流式細(xì)胞儀鑒定干細(xì)胞表型,并采用動(dòng)物成瘤實(shí)驗(yàn),將不同數(shù)量級(jí)的DU145和Tumor Spheres接種到NOD/SCID小鼠雙側(cè)腋下,觀察成瘤率和成瘤速度,證實(shí)Tumor Spheres的腫瘤干細(xì)胞特性。采用異種移植實(shí)驗(yàn)將Tumor Spheres接種到NOD/SCID小鼠腋下建立荷瘤小鼠模型,用不同劑量的GRh2尾靜脈注射干預(yù)荷瘤小鼠模型,監(jiān)測(cè)小鼠體重、瘤體體積,4周處死小鼠稱量瘤重,明確最佳藥物劑量。對(duì)瘤體的Notch1,HES1,Sox2,HIF-1α,jagged1等指標(biāo)進(jìn)行Western blot,免疫組化,Real-time PCR檢測(cè),明確不同藥物劑量下對(duì)相關(guān)蛋白和基因的調(diào)控作用。在最佳藥物劑量的給藥基礎(chǔ)上,進(jìn)一步探索GRh2聯(lián)合化療藥物環(huán)磷酰胺,以及聯(lián)合mTOR抑制劑雷帕霉素對(duì)抑瘤效果的增強(qiáng)作用。2.體外實(shí)驗(yàn):對(duì)分離富集、培養(yǎng)純化的Tumor Spheres進(jìn)行誘導(dǎo)分化、增殖水平、活性氧簇、細(xì)胞周期以及特異性蛋白和基因等檢測(cè)實(shí)驗(yàn),進(jìn)一步明確Tumor Spheres的腫瘤干細(xì)胞特性。用CCK8法檢測(cè)GRh2在不同時(shí)間點(diǎn),不同藥物濃度對(duì)DU145或腫瘤干細(xì)胞的增殖影響。用相應(yīng)的試劑盒檢測(cè)GRh2對(duì)腫瘤干細(xì)胞的周期影響、凋亡誘導(dǎo)作用、活性氧簇調(diào)控作用以及表面標(biāo)志物的影響。為了進(jìn)一步探索GRh2調(diào)控腫瘤干細(xì)胞生物學(xué)行為機(jī)制,并對(duì)動(dòng)物實(shí)驗(yàn)結(jié)果加以驗(yàn)證,收集藥物干預(yù)的不同濃度、不同時(shí)間點(diǎn)的腫瘤干細(xì)胞,采用 Western blot 和 Real-time PCR 技術(shù)對(duì) Notch1,HES1,Sox2,HIF-1α,jagged1等指標(biāo)進(jìn)行檢測(cè)。采用CCK8法觀察GRh2聯(lián)合mTOR抑制劑或PI3K抑制劑對(duì)腫瘤干細(xì)胞增殖的影響。并于細(xì)胞層面,進(jìn)一步探索GRh2聯(lián)合mTOR抑制劑對(duì)腫瘤干細(xì)胞的抑制作用機(jī)制。對(duì)動(dòng)物實(shí)驗(yàn)結(jié)果進(jìn)行驗(yàn)證。研究結(jié)果1.采用懸浮球狀培養(yǎng)的方法在無(wú)血清培養(yǎng)基中可以使對(duì)數(shù)生長(zhǎng)期DU145細(xì)胞分離富集形成Tumor Spheres,培養(yǎng)3代可達(dá)到純化的目的,流式細(xì)胞技術(shù)鑒定其高表達(dá)前列腺癌干細(xì)胞表型 CD44(+)CD24(-/low)(p0.05)。2.動(dòng)物成瘤實(shí)驗(yàn),接種Tumor Spheres側(cè)腫瘤成瘤速度和成瘤率均高于接種DU145細(xì)胞側(cè)(p0.05)。3.不同劑量的GRh2干預(yù)下的荷瘤小鼠,體重?zé)o統(tǒng)計(jì)學(xué)差異(p0.05),動(dòng)物瘤體體積測(cè)量和取材剝瘤的瘤重稱量提示0.5mg/kg小鼠體重的尾靜脈給藥劑量,對(duì)腫瘤的抑瘤率最高(p0.05)。4.Western blot和免疫組化結(jié)果顯示,0.5mg/kg小鼠體重的給藥劑量,可下調(diào)JAG1,HES1,Sox2,HIF-1 α,NICD,NTM,p-mTOR 蛋白表達(dá)水平。5.Real-timePCR結(jié)果顯示,0.5mg/kg小鼠體重的給藥劑量,可顯著下調(diào)Notch1,HES1,jagged1的mRNA表達(dá)水平,對(duì)Sox2,HIF-1 α的mRNA表達(dá)水平具有上調(diào)作用。6.GRh2與化療藥環(huán)磷酰胺聯(lián)用,可提高抑瘤率。7.GRh2與mTOR抑制劑聯(lián)用,可提高抑瘤率,Western blot結(jié)果提示兩者聯(lián)用可提高p-mTOR,NTM,NICD的抑制作用,免疫組化結(jié)果提示兩者聯(lián)用可提高HES1,HIF-1α的抑制作用。8.腫瘤干細(xì)胞具有更強(qiáng)的自我更新能力和分化潛能,細(xì)胞周期檢測(cè)發(fā)現(xiàn)腫瘤干細(xì)胞大多數(shù)處于細(xì)胞周期的GO/G1期�；钚匝醮厮綑z測(cè)發(fā)現(xiàn)腫瘤干細(xì)胞具有更低水平的活性氧簇水平。9.腫瘤干細(xì)胞高表達(dá)HES1,p-mTOR蛋白,高表達(dá)Notch1,HES1,HIF-1α,Sox2,jagged 1 的 mRNA。10.CCK8結(jié)果顯示,24h、48h、72h的GRh2干預(yù)DU145細(xì)胞的IC50分別為62.833uM,54.746uM,51.796uM。24h,48h 的 GRh2 干預(yù)腫瘤干細(xì)胞單細(xì)胞的 IC50 分別為71.834uM,70.579uM。24h,48h的GRh2干預(yù)腫瘤干細(xì)胞球的IC50分別為83.969uM,73.562uM。11.細(xì)胞周期結(jié)果提示,50uM的GRh2將DU145細(xì)胞周期阻滯在S期,75uM的GRh2將DU145細(xì)胞周期阻滯在G1期。50uM和75uM可促使腫瘤干細(xì)胞由靜止?fàn)顟B(tài)G1期向S期和G2期發(fā)展,100uM將腫瘤干細(xì)胞周期阻滯在G2/M期。12.75uM的GRh2可誘導(dǎo)腫瘤干細(xì)胞凋亡。13.GRh2可上調(diào)DU145細(xì)胞和腫瘤干細(xì)胞的活性氧簇水平。14.GRh2可下調(diào)腫瘤干細(xì)胞表面標(biāo)志物CD44(+)CD24(-/low)的水平,且劑量越高,下調(diào)越明顯。15.Western blot結(jié)果提示,50uM和75uM的GRh2分別干預(yù)腫瘤干細(xì)胞24h和48h,均可下調(diào)蛋白NTM,HES1,Sox2。16.Real-time PCR 結(jié)果提示,50uM 和 75uM 的 GRh2 干預(yù) 24h,對(duì) Notch1,HES1,HIF-1α,Sox2,JAG1的mRNA均有下調(diào)作用。50uM的GRh2干預(yù)48h,對(duì)Sox2和JAG1的mRNA均有上調(diào)作用,而對(duì)Notch1,HES1,HIF-1α無(wú)顯著作用。75uM的GRh2干預(yù)48h,對(duì)Notch1,HES1,HIF-1α,Sox2,JAG1的mRNA均有上調(diào)作用。17.CCK8結(jié)果顯示,24h和48h組內(nèi)比較,LY的抑制率均高于RAPA(p0.01),RAPA+GRh2的抑制率均高于RAPA(p0.01),LY+GRh2的抑制率均高于LY(p0.01);24h和48h組間比較,24h的RAPA和LY抑制率均低于48h(p0.01),而RAPA+GRh2組和LY+GRh2組在24h和48h的抑制率比較無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)18.Western blot實(shí)驗(yàn)對(duì)HES1蛋白進(jìn)行檢測(cè),發(fā)現(xiàn)GRh2組,聯(lián)合組,RAPA組對(duì)HES1蛋白表達(dá)量均有下調(diào)作用,均具有統(tǒng)計(jì)學(xué)差異(p0.01)。且聯(lián)合組抑制效果最明顯。19.Western blot實(shí)驗(yàn)對(duì)p-mTOR蛋白進(jìn)行檢測(cè),發(fā)現(xiàn)GRh2組,聯(lián)合組,RAPA組對(duì)p-mTOR蛋白表達(dá)量均有下調(diào)作用,均具有統(tǒng)計(jì)學(xué)差異(p0.01)。且聯(lián)合組抑制效果最明顯。結(jié)論:1.動(dòng)物成瘤實(shí)驗(yàn)和腫瘤干細(xì)胞生物學(xué)功能檢測(cè)均證實(shí)Tumor Spheres具有腫瘤干細(xì)胞自我更新,分化增殖等生物學(xué)特性。2.體內(nèi)實(shí)驗(yàn)中,GRh2尾靜脈注射,每周給藥2次的用藥方式,0.5mg/kg小鼠體重的給藥劑量對(duì)腫瘤干細(xì)胞自我更新能力抑制作用最強(qiáng)。3.體內(nèi)研究證實(shí)GRh2可以通過(guò)下調(diào)Notch1/HES1信號(hào)通路調(diào)控腫瘤干細(xì)胞的生物學(xué)行為,進(jìn)而抑制腫瘤的增殖生長(zhǎng)。4.體內(nèi)實(shí)驗(yàn)證實(shí)GRh2聯(lián)合化療藥物環(huán)磷酰胺可以提高抑瘤效果,增加化療療效。5.體內(nèi)實(shí)驗(yàn)證實(shí)GRh2聯(lián)合mTOR抑制劑可以通過(guò)進(jìn)一步下調(diào)Notch1信號(hào)通路和mTOR信號(hào)通路,提高抑瘤效果。6.體外實(shí)驗(yàn)中,GRh2可以抑制腫瘤干細(xì)胞的增殖;GRh2可促使腫瘤干細(xì)胞由細(xì)胞周期的靜止?fàn)顟B(tài)G1期向S期和G2期發(fā)展;GRh2可誘導(dǎo)腫瘤干細(xì)胞凋亡;GRh2可上調(diào)細(xì)胞內(nèi)活性氧簇水平;GRh2可抑制具有腫瘤干細(xì)胞表面標(biāo)志物表達(dá)的細(xì)胞增殖。7.體外實(shí)驗(yàn)證實(shí)GRh2可以通過(guò)下調(diào)Notch1/HES1信號(hào)通路調(diào)控腫瘤干細(xì)胞的生物性行為,驗(yàn)證動(dòng)物實(shí)驗(yàn)結(jié)論。8.體外實(shí)驗(yàn)證實(shí)GRh2聯(lián)合mTOR抑制劑可以通過(guò)進(jìn)一步下調(diào)HES1和p-mTOR蛋白,提高對(duì)腫瘤干細(xì)胞的抑制作用。
[Abstract]:Malignant tumor is one of the main chronic diseases that harm human health. In the Chinese medicine, it belongs to "sticking to" "accumulated" "rock", "tumor", "choking" and "choking", and so on. Professor Lin Hongsheng is committed to the research and clinical study of the combination of traditional Chinese and Western medicine for the prevention and treatment of malignant tumor for nearly 40 years. Professor Lin Hongsheng, on the basis of the continuous research on the etiology and pathogenesis of traditional Chinese medicine and the traditional Chinese medicine, has inherited the theory of "Fu Zheng Ben" and put forward a new theory of "consolidating the source", which is more capable of "deficiency", "poison" and "stasis". To protect the positive gas of the body, improve the patient's ability to prevent disease and disease, and correct the pathological condition of insufficient positive gas; "Qingyuan" is the pathogenic factor of removing the development of tumor and controlling the tumor from the source. Through the evidence-based medicine research, Professor Lin Hongsheng confirmed that the method of "fixing the source of blood" can prolong the late non small cells. The survival period of the patients with lung cancer and the improvement of the quality of life of the patients confirmed the clinical effectiveness of the treatment of tumor by the theory of Fuzheng Huoxue detoxification. In order to further clarify the biological connotation of the solid source theory, and to explore the mechanism of the action of Fuzheng Huoxue and detoxifying Chinese medicine on the tumor, the Lin Hongsheng project team was based on the microenvironment and swelling of the tumor. The tumor stem cell is the breakthrough point, and the biological behavior of the traditional Chinese medicine under the guidance of Fuzheng Huoxue detoxification is studied. In the past, two aspects of "Gu Ben" and "Qingyuan" were used to verify the regulation effect of Fuzheng Huoxue detoxifying Chinese medicine compound on the biological behavior of cancer stem cells. Tongzhi is the role of traditional Chinese medicine in the theory of "Gu Ben" and "Qing Yuan", as well as the mechanism of their intervention on the biological behavior of tumor stem cells. The subject group studies the traditional Chinese medicine. At present, it has preliminarily confirmed the regulation effect of Chinese traditional Chinese medicine on the theory of "clearing the source" of cancer stem cells in the theory of "clearing the source" of cancer stem cells. However, the subject group still did not systematically study the biological behavior of the intervention of traditional Chinese medicine on tumor stem cells. So in order to fill the blank of the research group, the subject of graduation is to use the extract of ginsenoside Rh2 (Ginenoside Rh2, GRh2) as the research drug. The former adenocarcinoma DU145 cell line stem cells are used as the research object. Suofu Zheng Zhi, which represents the regulation mechanism of the biological behavior of the tumor stem cells on the "Qingyuan" theory of the biological behavior of the cancer stem cells, is to further clarify the intervention effect of the method of activating blood activating blood and detoxification to the development of tumor. In order to enrich the scientific connotation of the theory of the GRh2, it also regulates the tumor stem for the multi channel and multi target points of traditional Chinese medicine. Cells provide laboratory basis. Objective: 1. to clarify the mechanism of GRh2 to regulate the biological behavior of tumor stem cells by down regulation of Notch1 signaling pathway and to inhibit the proliferation and growth of tumor,.2. explore the mechanism of GRh2 combined with mTOR inhibitor to improve tumor suppressor effect. Research methods: 1. in vivo experiment: the use of prostate cancer DU145 cell line, The tumor stem cell microspheres (Tumor Spheres) were isolated and enriched by the method of suspension spheroidal culture. The phenotype of stem cells was identified by flow cytometry, and the animal tumor formation experiment was used to inoculate the DU145 and Tumor Spheres of different quantities to the bilateral axillary of NOD/SCID mice. The rate of tumor formation and the rate of tumor formation were observed and the Tumor Spheres was confirmed. Tumor Spheres was inoculated to the armpit of NOD/SCID mice to establish a tumor bearing mouse model by xenotransplantation, and the tumor bearing mice model was injected with different doses of GRh2 tail vein to monitor the weight of the mice and the volume of the tumor. The tumor weight of the mice was weighed at 4 weeks, and the best dose of the tumor was determined. Notch1, HES1, Sox2, HIF of the tumor body. -1 alpha, Jagged1 and other indexes were used for Western blot, immunohistochemistry, and Real-time PCR to determine the regulation of related proteins and genes under different dosage. On the basis of the best drug dosage, we further explored the combination of GRh2 combined with chemotherapeutic cyclophosphamide, and the enhanced effect of rapamycin combined with mTOR inhibitor on the anti tumor effect. In vitro experiment: the differentiation, enrichment, culture and purification of Tumor Spheres were induced and differentiated, the proliferation level, active oxygen cluster, cell cycle and specific proteins and genes were tested to further clarify the characteristics of Tumor Spheres tumor stem cells. CCK8 method was used to detect GRh2 at different time points and different drug concentrations to DU145 or tumor stem cells. The effect of GRh2 on the cycle of tumor stem cells, apoptosis induction, reactive oxygen cluster regulation and the effect of surface markers. In order to further explore the mechanism of GRh2 regulation of biological behavior of tumor stem cells, and to verify the experimental results of animal experiments and collect different concentrations of drug intervention. Western blot and Real-time PCR were used to detect Notch1, HES1, Sox2, HIF-1, and Jagged1, and CCK8 method was used to observe the effects of GRh2 mTOR inhibitor or inhibitor on the proliferation of tumor stem cells. The results of the inhibitory action of the cell were verified. Results 1. the method of suspension spheroidal culture was used to isolate and enrich the DU145 cells of the logarithmic growth phase in the serum-free medium, to form Tumor Spheres, and to cultivate the 3 generation to achieve the purpose of purification. Flow cytometry was used to identify the high expression of the phenotype of the prostate cancer stem cell CD4. 4 (+) CD24 (-/low) (P0.05).2. animal tumorigenesis experiment, the tumor growth rate and the tumor formation rate of Tumor Spheres side tumor were higher than the GRh2 intervention of DU145 cell side (P0.05).3. different doses of GRh2 intervention mice, no statistical difference (P0.05), the volume measurement of animal tumor body and the weight of the harvested tumor weighed the tail of the 0.5mg/kg mice. P0.05.4.Western blot and immunohistochemical results showed that the dose of body weight of 0.5mg/kg mice could be reduced by JAG1, HES1, Sox2, HIF-1 a, NICD, NTM, and p-mTOR protein expression. The expression level of mRNA, the mRNA expression level of Sox2, HIF-1 A and the combination of.6.GRh2 with chemotherapeutic cyclophosphamide can improve the tumor inhibition rate of.7.GRh2 with the mTOR inhibitor, and increase the tumor suppressor rate. The results of Western blot suggest that the combination of the two can improve the inhibitory effect of p-mTOR, NTM, NICD. The immunohistochemical results suggest that the combination of the two can be improved. The inhibitory effect of HES1, HIF-1 alpha on.8. tumor stem cells has a stronger self-renewal ability and differentiation potential. Cell cycle detection found that most of the tumor stem cells were in the GO/G1 phase of the cell cycle. The active oxygen cluster level detection showed that the tumor stem cells had a lower level of active oxygen cluster level.9. tumor stem cells with high expression of HES1, p-mTOR eggs. The mRNA.10.CCK8 results of high expression of Notch1, HES1, HIF-1 alpha, Sox2, Jagged 1 showed that 24h, 48h, 72h GRh2 intervened the DU145 cells of the tumor stem cells respectively. The results of 73.562uM.11. cell cycle indicate that the GRh2 of 50uM block the DU145 cell cycle in S stage, and 75uM's GRh2 block the DU145 cell cycle at G1.50uM and 75uM. .GRh2 can up regulate the level of active oxygen cluster level.14.GRh2 of DU145 and tumor stem cells. The higher the level of CD44 (+) CD24 (-/low) of tumor stem cells, the higher the dose, the more obvious the down regulation is the result of.15.Western blot. The PCR results suggest that GRh2 intervention by GRh2 and 75uM has an up-regulation effect on Notch1, HES1, HIF-1 alpha, Sox2, JAG1 mRNA. The results showed that the inhibition rates of LY in both 24h and 48h groups were higher than that of RAPA (P0.01), and the inhibition rates of RAPA+GRh2 were higher than those of RAPA (P0.01), and the inhibition rates of LY+GRh2 were all higher than LY (P0.01). The test of HES1 protein in the 18.Western blot experiment found that the expression of HES1 protein in the group GRh2, the United Group and the RAPA group had a down effect (P0.01), and the inhibition effect of the combined group was the most obvious in.19.Western blot test, and the GRh2 group, the joint group and the RAPA group had the lower expression of the protein. The effect was statistically different (P0.01). And the inhibitory effect of the combined group was the most obvious. Conclusion: the 1. animal tumor experiment and the biological function detection of tumor stem cells all confirm that Tumor Spheres has the biological characteristics of tumor stem cells, such as self renewal, differentiation and proliferation,.2. in vivo, GRh2 tail vein injection, and 2 times a weekly medication, The strongest inhibitory effect of 0.5mg/kg mice weight on the self-renewal capacity of tumor stem cells in.3. in vivo confirmed that GRh2 could regulate the biological behavior of tumor stem cells by down regulation of Notch1/HES1 signaling pathway, and then inhibit the proliferation and growth of tumor in.4. in vivo, and confirmed that GRh2 combined with chemotherapeutic cyclophosphamide could increase inhibition. .5. in vivo experiments confirmed that GRh2 combined with mTOR inhibitors can further reduce the Notch1 signaling pathway and mTOR signaling pathway, and improve the tumor suppression effect in.6. in vitro, GRh2 can inhibit the proliferation of tumor stem cells; GRh2 can promote the development of tumor stem cells from the static state of cell cycle to S and G2 phase of the cell cycle. GRh2 can induce apoptosis of tumor stem cells; GRh2 can up regulate the level of intracellular reactive oxygen species; GRh2 inhibits the proliferation of cells with the expression of surface markers of tumor stem cells,.7. in vitro experiments confirm that GRh2 can regulate the biological behavior of tumor stem cells by down regulation of Notch1/HES1 signaling pathway. The conclusion of animal experimental conclusion.8. in vitro verified real G. Rh2 combined with mTOR inhibitors can further inhibit HES1 and p-mTOR proteins and enhance the inhibition of tumor stem cells.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.25

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