【作者】 王涛；

【导师】 韦安阳；

【作者基本信息】 南方医科大学 ， 外科学（泌尿外科）（专业学位）， 2016， 博士

【摘要】 研究背景和目的灵芝药用在我国已有2000多年的历史,被历代医药家视为滋补强壮、扶正固本的神奇珍品。已知灵芝属(Ganoderma)真菌约100余种,分布最广的为赤芝(G. lucidum),其次为紫芝(G. japonicum),还有树舌(G. applanatum)、松杉灵芝(G. tsugae)和簿树芝(G. capense)等均供药用。其主含氨基酸、多肽、蛋白质、真菌溶菌酶(fungal lysozyme),以及糖类(还原糖和多糖)、麦角甾醇、三萜类、香豆精甙、挥发油、硬脂酸、苯甲酸、生物碱、维生素B2及C等；孢子还含甘露醇、海藻糖(trehalose)。经大量临床研究显示,灵芝对神经衰弱、高脂血症、冠心病心绞痛、心律失常、克山病、高原反应不适症、肝炎、出血热、消化不良、气管炎等各有不同程度的疗效。统计研究表明,目前我国已报道的灵芝科真菌有98种,隶属4个种属。现已被开发利用的有十余种,大多是灵芝属,而目前对灵芝的研究多见于灵芝属的赤芝种,而其他的灵芝属的大量品种尚未得到研究开发。灵芝一般单生或丛生于枯树干或木桩上,它能分解木材中的木质素作为养分而生长,灵芝属真菌类大多生长在有散射阳光、树林较稀疏的地方或空旷地带。灵芝属真菌分布广泛,在全世界各大洲均有分布,已有250多个种属被描述,其中绝大部分生长在热带、亚热带、温带区。灵芝在我国主要分布在河北、山西、辽宁、吉林、福建、江苏、河南、广东、广西、四川、海南等地的山林中。灵芝中除了含有三萜类化合物和多糖类化合物外,还有生物碱类、核苷类、呋喃衍生物、氨基酸多肽类、甾醇类、无机元素和脂肪酸等化学成分,而其中的灵芝三萜和灵芝多糖是主要的活性物质。灵芝富含糖类、多种氨基酸、三萜类化合物、有机锗等成分,具有滋补强身、扶正固本、安神止痛等作用。现被用作消化道恶性肿瘤及术后化疗引起的消瘦、失眠、疼痛、厌食、白细胞减少、软弱疲乏等症的康复作用,起到增强抵抗力的效果。以往的研究表明,灵芝精粉和孢子粉能增加小鼠的免疫功能,对小鼠肿瘤有一定的抑制作用,并能增强丝裂霉素的抗肿瘤作用。有研究表明,灵芝口服液对人鼻咽癌细胞在裸小鼠体内的成瘤性有明显的抑制作用。灵芝多糖可引起体外培养的小鼠脾细胞核DNA、RNA含量显著增加,细胞内超微结构改变,细胞质和细胞核的平均截面积明显增加,核质比呈下降趋势,表明灵芝多糖在促进脾细胞和蛋白质合成的同时,促进细胞增殖。自1982年日本学者Kubota首次从灵芝实体中分离得到三萜类化合物灵芝三萜A和灵芝三萜B后,各国都展开了对灵芝三萜类化合物的研究,目前已先后从灵芝的子实体、菌丝体和孢子粉中分离到140多种三萜类化合物。研究发现传统中药灵芝含有122种三萜类成分,灵芝三萜具有抗高血压、抗肿瘤、抑制组胺释放、降血糖等许多药理活性。灵芝三萜可明显降低高脂性脂肪肝大鼠的血脂含量,并可能通过降低脂质过氧化反应,维护体内自由基稳态与平衡,改善肝组织损伤,起到阻遏高脂性脂肪肝发生和发展的作用。研究发现灵芝对结核分枝杆菌具有抑菌作用,而有效成分是灵芝三萜。当灵芝三萜浓度为500 ug/mL时,对结核分枝杆菌的标准菌株和耐药菌株表现出明显的抑菌作用。Su等报道了ganodermic acid S对TXA2拟似物U46619诱导的人源硅胶过滤处理的血小板聚集有明显的抑制作用,还可延迟血小板形态改变的时间,并减少血小板的初始聚集,降低其聚集的百分率,该抑制作用是剂量依赖型。另外,灵芝还被报道有抑制胆固醇合成的作用。Komoda等报道,将灵芝菌丝体加入到大鼠的高胆固醇饲料中,可显著地降低肝脏和血清中甘油三酯和胆固醇的含量,并指出其中的有效成分是三萜类化合物。灵芝中的活性成分灵芝醇A,灵芝醇B,灵芝醛A,灵芝三萜Y这类物质具有抑制人肝细胞株合成胆固醇的生物效应,灵芝中氧合的固醇抑制胆固醇的生物合成是通过胆固醇的前体醋酸或甲羟戊酸的转变实现的。因此,灵芝也有降血脂的功效。灵芝三萜中的某些化合物对由中性粒细胞等释放的炎症介质有抑制作用。在哮喘的鼠模型中,富含灵芝三萜的提取物显著减弱了气道高反应性,减少了总体粒细胞和嗜酸性粒细胞的浸润。减少了炎症介质的水平。除上述作用外,灵芝三萜对多种肿瘤细胞的生长和转移都有抑制作用。研究表明,灵芝对很多疾病都具有良好的治疗效果,尤其是对肿瘤的预防和治疗作用,已经得到了大量的实验和临床结果的验证。有研究报道灵芝三萜具有一定的抗肿瘤作用,比如乳腺癌和口腔癌。主要机理是灵芝三萜是通过影响机体的免疫系统而起作用的。灵芝醇F、灵芝酮二醇、灵芝酮三醇能有效地抑制补体激活的经典途径。灵芝三萜能促使肺癌的猪体内IL-2的含量上升,并提高起NK细胞的免疫活性,具有免疫促进功能。灵芝三萜通过阻断人肝瘤细胞株BEL7402细胞周期G1期向S期转变,使细胞周期停滞从而抑制癌细胞的生长等。迄今为止,采用中药提取物治疗癌症的研究报道较多,且多数研究显示出理想结果,关于灵芝三萜的抗肿瘤研究也正逐日升温,但灵芝三萜抗前列腺增生和前列腺癌的研究却少有报道。灵芝三萜是从灵芝中提取出的一种活性成分,具有广泛的药理学作用。近年来的研究表明,灵芝三萜能在体内外抑制多种肿瘤细胞的生长,并诱导肿瘤细胞凋亡。目前国家大力提倡对祖国传统医学宝库中的传统中草药进行开发研究,采用分子生物学手段对其药理学作用给予新的解释致力于研究开发出新的抗癌药物来大力弘扬祖国传统医学,已成为当前中医药研究的发展趋势。在这样的大背景下深入探讨灵芝三萜抑制肿瘤的作用揭示其抗癌作用的分子机制无疑是一项很有前景和研究价值的课题,是非常有意义的。本研究拟对灵芝三萜的工业化提取纯化工艺进行再研究,以期建立更加快速、稳定、简便的工业化提取纯化工艺,为其后续产品研发提供技术支撑。明确灵芝三萜是否具有抗前列腺癌细胞的作用并探讨其机制。首先观测灵芝三萜对前列腺癌细胞生长增殖的影响,分析灵芝三萜是否具有诱导前列腺癌细胞凋亡的作用；然后通过观察灵芝三萜对前列腺癌细胞周期及其周期调控蛋白等的影响,同时检测了其对前列腺癌细胞凋亡因子Caspase 3和Caspase 9蛋白表达水平的影响,以期找出灵芝三萜诱导前列腺癌细胞凋亡的相关信号转导通路或靶点,阐明灵芝三萜抗前列腺癌的可能机制,为其临床应用和产品研发提供科学依据。方法1灵芝三萜的提取工艺研究按照药典标准,对实验用灵芝子实体进行鉴定。对符合标准的原药材进行粉碎。按照渗漏装置,取各子实体粗粉,入大小适宜层析柱,使柱高比在10:1～15:1之间,加入8-12倍量石油醚,浸泡24h,收集渗漏液,而后依次加入8-12倍量乙醇、水,收集渗漏液。合并石油醚和乙醇部分部分渗漏液,加水至20%,摇匀,静置分层,得有机层、水层。其中有机层回收溶剂,得脂溶性成分(提取物1)；水层回收溶剂,得乙醇提取物部分(提取物2)。渗漏液水层,加乙醇至80%,摇匀,置4℃冷藏24 h,过滤,取沉淀得到粗多糖部位(提取物3)。经HPLC色谱分析并结合相关文献报道,确定提取物2中主要成分为灵芝三萜(Total Triterpenoids from Ganoderma Lucidum, GLT)。2灵芝三萜的纯化工艺研究以C18填料装柱,提取物2上柱,以不同浓度甲醇洗脱,收集各部分洗脱液,HPLC检测跟踪,以确定最佳洗脱纯化工艺。以大孔树脂为填料,提取物2上柱,以不同浓度乙醇洗脱,收集各部分洗脱液,HPLC检测跟踪,以确定最佳洗脱纯化工艺。3灵芝三萜对前列腺癌细胞生长的影响应用不同浓度的灵芝三萜(10,50,100μg/mL)作用于前列腺增生上皮细胞系(BPH)和4种人前列腺癌(LNCaP、22Rv1、PC-3、DU-145)细胞48h,收集细胞,进行Cell counting kit-8增殖实验检测灵芝三萜对前列腺癌细胞增殖的影响。同时应用Cell Death Detection ELISA kit检测灵芝三萜对前列腺癌细胞调亡的影响。4灵芝三萜诱导前列腺癌细胞凋亡的研究应用不同浓度的灵芝三萜(10,50或100 μg/mL)作用于前列腺增生上皮细胞系(BPH)和4种人前列腺癌(LNCaP、22Rv1、PC3、DU-145)细胞48h,收集细胞,应用流式细胞仪检测灵芝三萜对前列腺癌细胞调亡的影响。5灵芝三萜对前列腺癌细胞凋亡相关基因的影响应用不同浓度(10,50或100 μg/mL)灵芝三萜作用于前列腺增生上皮细胞系(BPH)和4个人前列腺癌(LNCaP、22Rv1、PC-3、DU-145)细胞0,12,24和48h,收集细胞,分别提取LNCaP、22Rv1、PC-3、DU-145前列腺癌细胞的总RNA。利用primer 5.0软件设计凋亡相关蛋白p21,CDK4,E2F1和Cyclin D1引物。应用RT-PCR、qPCR和Western Blot检测LNCaP、22Rv1、PC3、DU-145前列腺癌细胞中这些基因和蛋白的表达水平。6灵芝三萜对前列腺癌细胞凋亡因子Caspase 3和Caspase 9表达水平的影响采用试剂盒--分光光度法检测前列腺癌细胞中Caspase-3、Caspase-9活性,初步探讨灵芝三萜发挥抑杀前列腺癌细胞作用的可能分子机制。结果1灵芝三萜提取纯化工艺实验采用渗漉法,石油醚-乙醇-水溶剂系统,“一步法”提取得到灵芝全组分,其中回收乙醇部位,得到灵芝三萜粗提物。灵芝三萜粗提物经C18、大孔树脂吸附纯化得到灵芝三萜各组分,各组分经细胞活性评价,药效活性差异不明显。综合考虑后,取中段洗脱部位即为灵芝三萜纯提取物。2灵芝三萜对前列腺癌细胞生长的影响为研究GLT对前列腺癌细胞生长的影响,实验取对数生长期的前列腺上皮细胞(BPH)和LNCaP,22Rv1, PC-3, DU-145等4种前列腺癌细胞系。分别给予10、50、100μg/ml的灵芝三萜提取物,cell counting kit-8检测细胞增殖活力。我们发现GLT抑制细胞生长在所有的5种细胞系呈剂量依赖的方式抑制生长。有趣的是,与比前列腺增生上皮细胞系(BPH),4种前列腺癌细胞系(LNCaP, 22Rv1, PC-3, DU-145)对GLT更敏感(p<0.05)。当给药浓度达到50μg/ml时,4种细胞系的细胞数量下降了大约55%到45%(p<0.05),而对BPH只减少12%(p>0.05)3灵芝三萜诱导前列腺癌细胞凋亡的研究为了观察GLT诱导前列腺癌细胞凋亡作用,我们测量未经处理和50μg/mlGLT处理组前列腺癌细胞核小体以及凋亡蛋白PARP的表达情况。结果显示,与对照组比较,50μg/ml GLT处理组,细胞核小体数量显著增加(p<0.05),PARP的表达也显著增加(p<0.05),且呈剂量依赖性。为评估GLT是否能诱导细胞周期的阻滞,使用流式细胞仪分析了G1(G0),S,G2(M)期各阶段细胞百分比。结果发现10μg/ml的GLT,能显著增加ⅠNCaP、22Rv1细胞在G1/G0的百分比,但显著降低在S期的百分比细胞(p<0.01)。而对于PC-3和DU-145细胞,100μg/ml,GLT处理组能显著增加细胞在G1/G0期的百分比,相应地减少S期细胞百分比,有一些效果,但其差异在统计学上没有显著,尤其在低剂量GLT处理组(p>0.05)；不过在高剂量如100μg/ml,GLT处理组能显著增加细胞在G1/G0期的百分比,相应地减少S期细胞百分比。以上结果显示GLT主要抑制细胞G1/S的过渡,从而抑制细胞增殖,但还需进一步实验验证。4灵芝三萜对前列腺癌细胞凋亡相关基因的影响实验采用PCR和Western blot等方法,检测了细胞周期调节器(p21、CDK4、 E2F1和细胞周期蛋白D1)等的表达情况,实验所用4种前列腺癌细胞采用50 μg/ml GLT处理48 h后,p21表达水平显著增加(p<0.05),而CDK4、E2F1和Cyclin D1表达水平则显著减少。5灵芝三萜对前列腺癌细胞凋亡因子Caspase 3和Caspase 9蛋白表达水平的影响实验使用酶联免疫试剂盒检测细胞凋亡因子Caspase-3和Caspase-9的活性。在50或100 μg/ml的GLT治疗24 h后,Caspase-3的水平在所有4种前列腺癌细胞系中,均显著升高(p<0.05)。在50或100μg/ml的GLT处理后24 h,Caspase-9的活性水平也表现出与Caspase-3同样的趋势。结论本实验采用“一步溶剂”渗漏法,提取得到灵芝全组份,结合柱色谱纯化得到灵芝三萜提取部位,建立了从灵芝中快速高效提取分离灵芝三萜的方法,初步建立了灵芝三萜质量控制方法。灵芝三萜能明显抑制前列腺癌细胞生长,其主要通过调控细胞周期和减少凋亡蛋白,从而抑制肿瘤细胞增殖及促进细胞凋亡。但是具体作用效果及其机制,还需要进一步的在体实验和临床实验验证。更多还原

【Abstract】 Background and ObjectivesGanoderma lucidum has been used in our country for more than 2000 years. It can bring about nourishing and strengthening, strengthening the body. It is well known that there are about more than 100 species of Ganoderma lucidum. The most widely distributed specie is Ganoderma lucidum, followed by Ganoderma japonicum (G. japonicum, Ganoderma applanatum, Ganoderma tsugae and Bu Shuzhi. It consists amino acids, peptides, proteins, fungal lysozyme (fungal lysozyme), and sugar (reducing sugars and polysaccharides), ergosterol, triterpene, coumarin glycosides, essential oil, stearic acid, benzoic acid, alkaloid, vitamin B2 and C. Spores also contain mannitol, trehalose (trehalose). A large number of clinical studies show that Ganoderma lucidum have varying degrees of efficacy for neurasthenia, high fat, hyperlipidemia, coronary heart disease angina pectoris, arrhythmia, Keshan disease and plateau of discomfort mononucleosis, hepatitis, hemorrhagic fever, indigestion, tracheitis.Statistics show that there are 98 kinds of Ganoderma lucidum fungi in China, belonging to 4 genera. More than ten species, mostly Ganoderma lucidum, have been developed and utilized. However, the current research of Ganoderma lucidum are more common in Ganoderma lucidum Ganoderma species, a large number of species of the genus Ganoderma has not yet been developed. Ganoderma lucidum generally grow solitary or clustered in tree stem or stakes. The decomposition of wood lignin is a nutrient and growth. Ganoderma lucidum fungi of the genus grow mostly in a sparse scattering sunlight, the trees, local or open ground. Ganoderma lucidum is widely distributed in all continents of the world, there are more than 250 species are described, most of them grow in tropical, subtropical, temperate zone. Ganoderma in China are mainly distributed in Hebei, Shanxi, Liaoning, Jilin, Fujian, Jiangsu, Henan, Guangdong, Guangxi, Sichuan, Hainan and other places in the mountains.Ganoderma lucidum contains mainly triterpenoid compounds and polysaccharides compounds and sterols, and other chemical composition such as nucleosides, alkaloids, furan derivatives, amino acid polypeptide, inorganic elements and fatty acids. The ganoderma lucidum triterpenes and ganoderma lucidum polysaccharides are the main active substances in Ganoderma lucidum. Ganoderma lucidum has the effect of nourishing the body, supporting the healthy energy and alleviating pain. They have been used for a malignant tumor of digestive tract and postoperative chemotherapy induced weight loss, insomnia, pain, anorexia, neutropenia, fatigue weakness in rehabilitation and health care, to get a good effect. Previous studies showed that Ganoderma lucidum spore powder can increase the immune function of mice and had some inhibitory effect on tumor growth in mice, and can enhance the mitomycin antitumor effects. Some studies showed that Ganoderma oral liquid has obvious inhibitory effect on human nasopharyngeal carcinoma cells in nude mice tumor. Ganoderma lucidum polysaccharide can induce cultured mouse spleen cell nuclear DNA, RNA content increased significantly and intracellular ultrastructural changes, in both the cytoplasm and nucleus of the average cross-sectional area increased significantly, nucleo cytoplasmic ratio was decreased trend, indicating that Ganoderma lucidum polysaccharides in promoting spleen cells and protein synthesis at the same time, promote cell proliferation.Since 1982, Ganoderma lucidum such as Ganoderma lucidum acid A and Ganoderma lucidum acid B were firstly isolated by Kubota. Then, the research on the three kinds of Ganoderma lucidum were carried out. At present, more than 140 kinds of triterpenes compounds have been isolated from the fruiting body, mycelium and spore powder of Ganoderma lucidum. The study found that the traditional Chinese medicine Ganoderma lucidum contains more than 122 components of triterpenes compounds Ganoderma lucidum, which can inhibit the release of histamine, blood sugar, anti hypertension, anti-tumor and many other pharmacological activities. Ganoderma triterpenoids decreased significantly rats with hyperlipidemia fatty liver lipid content, and possibly by reducing lipid peroxidation, maintaining the in vivo free radical homeostasis and balance, improve the injury of liver tissue, to repression of hyperlipidemic fatty liver and the role of development. It is found that the Ganoderma lucidum has the antimicrobial effect to the mycobacterium tuberculosis, and the effective ingredient is the Ganoderma triterpenoids. When the total concentration of Ganoderma triterpenoids was 500 ug/ml, the growth of the standard strains and drug resistant strains of mycobacterium tuberculosis were inhibited. Su et al. reported that Ganodermic acids on TXA2 quasi U46619 induced source of silica gel filtration treatment of platelet aggregation significantly inhibited, platelet shape change of the time delay, and reduce the initial platelet aggregation, reduce the aggregate percentage and the inhibition effect is dose dependent.In addition, It has also been reported that Ganoderma lucidum can inhibit cholesterol synthesis. Komoda et al. found that Ganoderma lucidum mycelium was added to rat high cholesterol diet can significantly reduce the content of triglyceride and cholesterol in serum and liver, and it is pointed out that the active ingredient is triterpenoid compounds.. this substances active ingredients of Ganoderma lucidum alcohol A, Ganoderma lucidum alcohol B, Ganoderma lucidum aldehyde A, ganoderic acid Y in Ganoderma lucidumbiological have the effect of inhibiting human liver cell lines synthesize cholesterol and sterol in Ganoderma lucidum inhibit cholesterol biosynthesis by changing the precursors of cholesterol acetate or methyl hydroxy acid. Therefore, Ganoderma lucidum also has the effect of decreasing blood lipids. Some compounds in ganoderma triterpenes have inhibitory effects on the inflammatory mediators released by neutrophils. In the mouse model of asthma, the extract of Ganoderma lucidum triterpenes significantly attenuated the airway high reactivity, reducing the overall granulocyte and eosinophilic infiltration and the level of inflammatory mediators.Ganoderma lucidum triterpenes can inhibit the growth and metastasis of a variety of tumor cells. A lot of experiments and clinical studies have indicated that Ganoderma lucidum has a good therapeutic effect on many diseases, especially for the prevention and treatment of cancer. Many studies have reported that Ganoderma lucidum triterpenes has a certain anti-tumor effect, such as breast cancer and oral cancer. The main mechanism is that Ganoderma lucidum triterpenes can activate the immune system of the body. Glossy ganoderma alcohol F, Ganoderma lucidum ketone, glycol, ganodermanontriol can effectively inhibit complement activation of the classical pathway. Ganoderma lucidum acid can increased the content of IL-2 in pigs of lung cancer, and activate the immune activity of NK cells, with enhanced immune function. Ganoderic Acid B can block the cell cycle of human hepatoma cell line BEL7402 from phase G1 to phase S transition, and inhibit the growth of cancer cells.Nowadays, the use of traditional Chinese medicine extract in the treatment of prostate cancer has been extensively investigated, the research about triterpenoids of Ganoderma lucidum is gradually increasing. However, the studies on benign prostatic hyperplasia and prostate cancer have rarely been reported. Ganoderma lucidum triterpenoids can inhibit the growth of a variety of tumor cells in vitro and in vivo, and induce the apoptosis of tumor cells. At present, the country vigorously promotes the development of traditional Chinese herbal medicine. By means of molecular biology gives a new explanation is committed to research and development of new drugs to vigorously carry forward the traditional Chinese medicine for its pharmacological effects, has become the development trend of Chinese medicine research. It is undoubtedly a promising and valuable topic to study the molecular mechanism of the anti-tumor effect of Ganoderma lucidum triterpenoids.This study aimed to study on the extraction and purification of triterpenoids to establish a more rapid, stable and convenient industrialized extraction and purification process and provide technical support For the subsequent product development. To clarify whether Ganoderma lucidum triterpenoids has the effect of inhabiting prostate cancer cells and to explore its mechanism. First, the effect of Ganoderma lucidum triterpenoids on the growth and proliferation of prostate cancer cells were observed, and the effect of Ganoderma lucidum three on the apoptosis of prostate cancer cells were analyzed. Second, the effect of Ganoderma lucidum triterpenoids on the cell cycle of prostate cancer cell cycle and the regulation of the cell cycle were observed, then, the influence of the most Caspase-3 and Caspase-9 levels were detected. In order to find out the related signal transduction pathway or target of the apoptosis of prostate cancer cell induced by Ganoderma lucidum triterpenoids, to elucidate the possible mechanism of prostate cancer, and to provide scientific basis for its clinical application and product development.Methods1. The study of extraction and purification technics of Ganoderma lucidum triterpenoidsThe fruiting body of Ganoderma lucidum was identified according to the pharmacopoeia standards. The original medicinal herbs which meet the standards were crushed. The edible fungus coarse powder were put in some size suitable chromatography column of leakage device. The column ratio was between the 10:1-15:1.8-12 times amount of petroleum ether were put in and soak for 24 h, and then, collect fluid leakage by adding 8 to 12 times the amount of ethanol and water to collect fluid leakage. The petroleum ether and ethanol part leakage liquid were mixed, added water to 20%, then shaking and layering, organic layer and water layer were observed. The liposoluble constituent were extracted from Organic layer recovery solvent (extract 1). The alcohol extracts were extracted from water recovery solvent (extract 2). The ethanol is added into leakage of liquid water to 80%. They were shaked and cold storage at 4℃ for 24 h. The crude polysaccharide were filtered and obtained (extract 3). The main components of the extract were determined to be the Ganoderma lucidum triterpenes by using HPLC chromatography and the relevant literature.2. The study of extraction and purification technics of triterpenoids from Ganoderma lucidumC18 were packed into the column, extract 2 were added into the columns, and eluted with different concentrations of methanol. the eluent were collected. HPLC was used to detect and track to determine the best elution purification process. Macroporous resin were filled into the column, extract 2 were added into the column, and eluted with different concentrations of ethanol. Each part of the elution liquid were collected. HPLC was used to detect and track to determine the best elution purification process.3. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rvl, PC-3, DU-145) cells for 48 hours. The cells were harvested and the effect of growth inhibitory of prostate cancer cells were assessed with cell counting kit-8 proliferation assay. Death Detection ELISA kit Cell was used to detect the effect of Ganoderma lucidum triterpenoids on the apoptosis of prostate cancer cells.4. The study on the apoptosis of prostate cancer cells induced by Ganoderma lucidum triterpenesThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rvl, PC-3, DU-145) cells for 48 hours. The flow cytometry was used to detect the effect of Ganoderma lucidum triterpenoids on the cell cycle of prostate cancer cells.5. Effect of Ganoderma lucidum triterpenoids on apoptosis related genes in prostate cancer cell lineThe different concentrations of triterpenoids from Ganoderma lucidum (50,100 UG/ml) were used to treat normal prostate epithelial cell line (BPH) and four human prostate cancer (LNCaP and 22Rv1, PC-3, DU-145) cells for 0,12,24,48 hours. Cells were collected, and the total RNA of LNCaP,22Rv1, DU-145 and PC-3 prostate cancer cells were extracted respectively. Apoptosis related proteins p21, CDK4, E2F1 and D1 Cyclin primers were designed by primer 5 software. Western, qPCR and Blot RT-PCR were used to detect the expression levels of these genes and proteins in LNCaP,22Rvl, PC3 and DU-145 prostate cancer cells.6. Effect of Ganoderma lucidum triterpenoids on the expression of apoptosis factor Caspase 3 and Caspase 9 protein in prostate cancer cell lineThe activity of Caspase-3 and Caspase-9 in prostate cancer cells was detected by ELISA, and the possible molecular mechanism of the effect of Ganoderma lucidum triterpenoids were further explored.Results1. The extraction and purification technology of Ganoderma lucidum triterpenoidsThe protocol was carried out with the percolation method and petroleum ether and ethanol water solvent system. All ingredients of Ganoderma lucidum were extracted by "one step" extraction. The crude extract of Ganoderma lucidum triterpenes were recycled and extracted from the ethanol extract.The crude extract of Ganoderma lucidum triterpenes were obtained, adsorbed and purified by C18 and macroporous resin, and the three components of Ganoderma lucidum were divided into. The ingredients were not significant among the different groups. The middle part of the extract was taken as the pure extract of Ganoderma lucidum triterpenes.2. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe different concentrations of Ganoderma lucidum triterpenes extract (10,50,100 g/ml) were treated cells. Cell viability of prostate cancer cells was detected by cell counting kit-8. The results showed that GLT inhibited cell growth in all 5 cell lines in a dose-dependent manner and inhibited growth. Compare with the normal prostate epithelial cell line (BPH), the other four prostate cancer cell line (LNCaP,22Rv1, PC-3, DU-145) were more sensitive for of GLT (p<0.05 for all). When the concentration reached 50 ug/ml (p<0.05 for all), the number of cells in the four cell lines decrease by 55% to 45%, However, the BPH only decreased by 12%(p>0.05 for all).3. Effect of Ganoderma lucidum triterpenes on the growth of prostate cancer cellsThe Morphologic changes of apoptosis nuclei and the expression of apoptosis protein PARP were observed. The results showed that compared with the control group, the 50 ug/ml GLT treatment group significantly increased the number of nuclear bodies (p<0.05), the PARP expression was also significantly increased in a does dependent manner (p<0.05).The percentage of phase G1 (G0), S, and G2 (M) cell were analyzed by flow cytometry. Results showed that 10g/ml GLT can significantly increase the percentage of LNCaP,22Rvl cells in the phase G1/G0, but significantly reduced the percentage of the S phase cells. For PC-3 and DU-145 cells,100 g/ml GLT treatment group significantly increased the percentage of cells in the G1/G0 phase, while decrease in the percentage of phase S cells.4. The study on the apoptosis of prostate cancer cells induced by Ganoderma lucidum triterpenesThe expression of modulators of cell cycle regulation (p21, CDK4, E2F1 and cyclin Dl) was detected by Western blot and PCR. The results showed that after 48 h treatment with 50 ug/ml GLT, the expression of p21 was significantly increased in four prostate cancer cells (p<0.05 for all), while the levels of CDK4, E2F1 and D1 Cyclin were significantly decreased (p<0.05 for all).5. Effect of Ganoderma lucidum triterpenoids on the expression of apoptosis factor Caspase 3 and Caspase 9 protein in prostate cancer cell lineThe activity of Caspase-3 and Caspase-9 were detected by ELISA kit. The results showed that after 48h, treatment with 50 and 100 ug/ml GLT, the expression of Caspase-3 in all four prostate cancer cell line were significantly increased (p< 0.05) and the activity of caspase-9 level also showed the same tendency.ConclusionThe method of one-step solvent-penetration was used in this experiment to extract all components from Ganoderma lucidum. Solvent extraction followed by column chromatographic purification was processed to obtain the GLT. Thus, we established methods of rapid and efficient extraction from Ganoderma lucidum and the quality control method of GLT. GLT can significantly inhibit the growth of prostate cancer cells. The mechanism may involve the regulation of cell cycle, which can affect cell proliferation and apoptosis. However, the precise mechanism needs to be further verified by using in-vitro and in-vivo experiments.更多还原