人参皂苷Rh2通过下调Notch1信号通路调控前列腺癌干细胞生物学行为的实验研究

发布时间：2018-04-28 13:54

本文选题：雷帕霉素 + Notch1　；参考：《北京中医药大学》2017年博士论文

【摘要】：恶性肿瘤是危害人类健康的主要慢性疾病之一,在祖国医学中,属"症瘕""积聚""岩""瘤""噎膈"等范畴。林洪生教授致力于中西医结合防治恶性肿瘤科研与临床近40载,认为肿瘤是整体属虚,局部属实的全身疾病的局部反应,"虚""毒""瘀"是肿瘤致病的主要病理因素。林洪生教授在全国中医肿瘤工作者对中医肿瘤病因病机,理法方药不断研究的基础上,继承总结既往"扶正培本"理论,并进一步凝练提出了更能针对肿瘤"虚" "毒" "瘀"致病特点的"固本清源"新理论。"固本"是顾护机体正气以扶正,提高患者的防病抗病能力,纠正正气不足的病理状态;"清源"是祛除肿瘤发生发展的致病因素,从源头上控制肿瘤。林洪生教授通过循证医学研究证实"固本清源"理论指导下的扶正活血解毒法可以延长晚期非小细胞肺癌患者的生存期,提高患者的生活质量,证实了该理论指导下的扶正活血解毒法治疗肿瘤的临床有效性。为了进一步明确固本清源理论的生物学内涵,以及探明扶正活血解毒中药对肿瘤的作用机制,林洪生课题团队以肿瘤微环境和肿瘤干细胞为切入点,对扶正活血解毒法指导下的中药干预肿瘤千细胞的生物学行为做了深入研究。在既往师兄师姐博士课题中,从"固本"和"清源"两方面证实扶正活血解毒中药复方对肿瘤干细胞生物学行为的调控作用。为了深入研究不同治则中药在"固本"和"清源"中医肿瘤理论中所扮演的角色,以及他们对肿瘤干细胞生物学行为的干预作用机制,课题组针对单一治则中药进行研究,目前已经初步证实活血中药、解毒中药对肿瘤干细胞在"清源"理论指导下的调控作用。但课题组仍未对扶正中药干预肿瘤干细胞的生物学行为做以系统研究,所以为填补课题组的研究空白,毕业课题特选用扶正中药人参的提取物人参皂苷Rh2(Ginenoside Rh2,GRh2)作为研究药物,以前列腺癌DU145细胞系干细胞作为研究对象,探索扶正治则代表中药人参的提取物GRh2对肿瘤干细胞生物学行为在"清源"理论方面的调控机制,以期进一步明确扶正活血解毒法对肿瘤发生发展的干预作用,在丰富固本清源理论科学内涵的同时,也为中医药多通路多靶点调控肿瘤干细胞提供实验室依据。研究目的:1.明确GRh2通过下调Notch1信号通路调控肿瘤干细胞生物学行为,进而抑制肿瘤增殖生长的作用机制。2.探索GRh2联合mTOR抑制剂提高肿瘤抑瘤效果的作用机制。研究方法:1.体内实验:采用前列腺癌DU145细胞系,通过悬浮球状培养的方法分离富集、培养纯化出肿瘤干细胞微球(Tumor Spheres)。通过流式细胞仪鉴定干细胞表型,并采用动物成瘤实验,将不同数量级的DU145和Tumor Spheres接种到NOD/SCID小鼠双侧腋下,观察成瘤率和成瘤速度,证实Tumor Spheres的肿瘤干细胞特性。采用异种移植实验将Tumor Spheres接种到NOD/SCID小鼠腋下建立荷瘤小鼠模型,用不同剂量的GRh2尾静脉注射干预荷瘤小鼠模型,监测小鼠体重、瘤体体积,4周处死小鼠称量瘤重,明确最佳药物剂量。对瘤体的Notch1,HES1,Sox2,HIF-1α,jagged1等指标进行Western blot,免疫组化,Real-time PCR检测,明确不同药物剂量下对相关蛋白和基因的调控作用。在最佳药物剂量的给药基础上,进一步探索GRh2联合化疗药物环磷酰胺,以及联合mTOR抑制剂雷帕霉素对抑瘤效果的增强作用。2.体外实验:对分离富集、培养纯化的Tumor Spheres进行诱导分化、增殖水平、活性氧簇、细胞周期以及特异性蛋白和基因等检测实验,进一步明确Tumor Spheres的肿瘤干细胞特性。用CCK8法检测GRh2在不同时间点,不同药物浓度对DU145或肿瘤干细胞的增殖影响。用相应的试剂盒检测GRh2对肿瘤干细胞的周期影响、凋亡诱导作用、活性氧簇调控作用以及表面标志物的影响。为了进一步探索GRh2调控肿瘤干细胞生物学行为机制,并对动物实验结果加以验证,收集药物干预的不同浓度、不同时间点的肿瘤干细胞,采用 Western blot 和 Real-time PCR 技术对 Notch1,HES1,Sox2,HIF-1α,jagged1等指标进行检测。采用CCK8法观察GRh2联合mTOR抑制剂或PI3K抑制剂对肿瘤干细胞增殖的影响。并于细胞层面,进一步探索GRh2联合mTOR抑制剂对肿瘤干细胞的抑制作用机制。对动物实验结果进行验证。研究结果1.采用悬浮球状培养的方法在无血清培养基中可以使对数生长期DU145细胞分离富集形成Tumor Spheres,培养3代可达到纯化的目的,流式细胞技术鉴定其高表达前列腺癌干细胞表型 CD44(+)CD24(-/low)(p0.05)。2.动物成瘤实验,接种Tumor Spheres侧肿瘤成瘤速度和成瘤率均高于接种DU145细胞侧(p0.05)。3.不同剂量的GRh2干预下的荷瘤小鼠,体重无统计学差异(p0.05),动物瘤体体积测量和取材剥瘤的瘤重称量提示0.5mg/kg小鼠体重的尾静脉给药剂量,对肿瘤的抑瘤率最高(p0.05)。4.Western blot和免疫组化结果显示,0.5mg/kg小鼠体重的给药剂量,可下调JAG1,HES1,Sox2,HIF-1 α,NICD,NTM,p-mTOR 蛋白表达水平。5.Real-timePCR结果显示,0.5mg/kg小鼠体重的给药剂量,可显著下调Notch1,HES1,jagged1的mRNA表达水平,对Sox2,HIF-1 α的mRNA表达水平具有上调作用。6.GRh2与化疗药环磷酰胺联用,可提高抑瘤率。7.GRh2与mTOR抑制剂联用,可提高抑瘤率,Western blot结果提示两者联用可提高p-mTOR,NTM,NICD的抑制作用,免疫组化结果提示两者联用可提高HES1,HIF-1α的抑制作用。8.肿瘤干细胞具有更强的自我更新能力和分化潜能,细胞周期检测发现肿瘤干细胞大多数处于细胞周期的GO/G1期。活性氧簇水平检测发现肿瘤干细胞具有更低水平的活性氧簇水平。9.肿瘤干细胞高表达HES1,p-mTOR蛋白,高表达Notch1,HES1,HIF-1α,Sox2,jagged 1 的 mRNA。10.CCK8结果显示,24h、48h、72h的GRh2干预DU145细胞的IC50分别为62.833uM,54.746uM,51.796uM。24h,48h 的 GRh2 干预肿瘤干细胞单细胞的 IC50 分别为71.834uM,70.579uM。24h,48h的GRh2干预肿瘤干细胞球的IC50分别为83.969uM,73.562uM。11.细胞周期结果提示,50uM的GRh2将DU145细胞周期阻滞在S期,75uM的GRh2将DU145细胞周期阻滞在G1期。50uM和75uM可促使肿瘤干细胞由静止状态G1期向S期和G2期发展,100uM将肿瘤干细胞周期阻滞在G2/M期。12.75uM的GRh2可诱导肿瘤干细胞凋亡。13.GRh2可上调DU145细胞和肿瘤干细胞的活性氧簇水平。14.GRh2可下调肿瘤干细胞表面标志物CD44(+)CD24(-/low)的水平,且剂量越高,下调越明显。15.Western blot结果提示,50uM和75uM的GRh2分别干预肿瘤干细胞24h和48h,均可下调蛋白NTM,HES1,Sox2。16.Real-time PCR 结果提示,50uM 和 75uM 的 GRh2 干预 24h,对 Notch1,HES1,HIF-1α,Sox2,JAG1的mRNA均有下调作用。50uM的GRh2干预48h,对Sox2和JAG1的mRNA均有上调作用,而对Notch1,HES1,HIF-1α无显著作用。75uM的GRh2干预48h,对Notch1,HES1,HIF-1α,Sox2,JAG1的mRNA均有上调作用。17.CCK8结果显示,24h和48h组内比较,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组间比较,24h的RAPA和LY抑制率均低于48h(p0.01),而RAPA+GRh2组和LY+GRh2组在24h和48h的抑制率比较无统计学差异(p0.05)18.Western blot实验对HES1蛋白进行检测,发现GRh2组,联合组,RAPA组对HES1蛋白表达量均有下调作用,均具有统计学差异(p0.01)。且联合组抑制效果最明显。19.Western blot实验对p-mTOR蛋白进行检测,发现GRh2组,联合组,RAPA组对p-mTOR蛋白表达量均有下调作用,均具有统计学差异(p0.01)。且联合组抑制效果最明显。结论:1.动物成瘤实验和肿瘤干细胞生物学功能检测均证实Tumor Spheres具有肿瘤干细胞自我更新,分化增殖等生物学特性。2.体内实验中,GRh2尾静脉注射,每周给药2次的用药方式,0.5mg/kg小鼠体重的给药剂量对肿瘤干细胞自我更新能力抑制作用最强。3.体内研究证实GRh2可以通过下调Notch1/HES1信号通路调控肿瘤干细胞的生物学行为,进而抑制肿瘤的增殖生长。4.体内实验证实GRh2联合化疗药物环磷酰胺可以提高抑瘤效果,增加化疗疗效。5.体内实验证实GRh2联合mTOR抑制剂可以通过进一步下调Notch1信号通路和mTOR信号通路,提高抑瘤效果。6.体外实验中,GRh2可以抑制肿瘤干细胞的增殖;GRh2可促使肿瘤干细胞由细胞周期的静止状态G1期向S期和G2期发展;GRh2可诱导肿瘤干细胞凋亡;GRh2可上调细胞内活性氧簇水平;GRh2可抑制具有肿瘤干细胞表面标志物表达的细胞增殖。7.体外实验证实GRh2可以通过下调Notch1/HES1信号通路调控肿瘤干细胞的生物性行为,验证动物实验结论。8.体外实验证实GRh2联合mTOR抑制剂可以通过进一步下调HES1和p-mTOR蛋白,提高对肿瘤干细胞的抑制作用。
[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.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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