褪黑素对胶质瘤干细胞的影响及机制研究

发布时间：2018-04-20 13:25

本文选题：褪黑素 + GSCs　；参考：《山东大学》2017年博士论文

【摘要】：胶质细胞瘤是一种颅内原发性肿瘤,其发病率高,占所有颅内肿瘤的50%以上。由于胶质瘤呈浸润性生长,侵袭性强,所以手术难以完全切除,治疗效果差,往往预后不良。WHO将胶质瘤分为4级,级别越高则恶性程度就越高。其中级别最高的胶质母细胞瘤(GBM)为WHO Ⅳ级,该肿瘤发病率高,约占到所有颅内肿瘤的15%。在胶质母细胞瘤尽可能手术切除的前提下,再辅以标准的放化疗,病人的中位生存期仍然不超过15个月。近年来的研究表明,胶质瘤,特别是恶性程度最高的胶质母细胞瘤中含有一种细胞亚群,这种细胞亚群拥有部分正常干细胞的特性,被称为胶质瘤干细胞(glioblastoma stem-like cells,GSCs)。GSCs起到了极强的促进胶质母细胞瘤增殖的作用,同时GSCs在促进肿瘤向颅内周围脑组织浸润、扩散的过程中起到了关键作用。目前手术综合放化疗并不能有效的杀灭GSCs,这导致肿瘤往往会较快复发,预后不良,所以亟需探索能够有效杀灭GSCs的治疗手段。褪黑素是一种由松果体分泌的激素。长期以来,褪黑素一直被认为是一种调节生物节律的激素,而近年来越来越多的研究证明,褪黑素对多种肿瘤均具有抑制作用,其中便包括胶质瘤。然而褪黑素对GSCs的作用还少有研究,其机制也尚不明确。EZH2具有组蛋白甲基化转移酶作用,是多梳蛋白复合体2的组分之一。在包括胶质瘤、乳腺癌、胃癌、结肠癌在内的多种肿瘤中,均发现了 EZH2的异常升高。并且EZH2的表达量越高,肿瘤往往恶性程度越高,预后越发不良,而抑制EZH2的表达能够延缓肿瘤的发生和发展。同时已经有确切证据表明,EZH2是GSCs的干性指标之一。NOTCH受体的过表达也存在于包括胶质瘤在内的多种恶性肿瘤中。EZH2-NOTCH1信号轴已经被证明存在于胰腺癌中,而EZH2-NOTCH1信号轴是否也存在于GSCs中,同时是否参与了褪黑素对GSCs的作用还尚没有报道。本实验主要研究了褪黑素对GSCs的作用及具体的作用机制。目的:1、研究褪黑素对GSCs增殖、自我更新及干性的作用;2、研究EZH2-NOTCH1信号轴在褪黑素起效过程中发挥的具体作用;3、研究EZH2对NOTCH1调控中的具体作用机制。方法:第一部分:褪黑素对GSCs增殖、自我更新及干性的影响1、U251和T98细胞系中GSCs的培养及验证1.1在干细胞富集培养基(无血清,加入B27,bFGF和EGF因子)培养U251和T98G细胞系,培养至少三周,建立GSCU251和GSCT98G细胞模型。1.2 qRT-PCR检测GSCs干细胞标记物CD133和SOX2的含量,验证GSCU251和GSCT98G细胞模型是否建立成功。2、检测不同浓度褪黑素对GSCs的生存、增殖能力的影响2.1配制含有不同浓度褪黑素(0.1-1000μM)的培养基,培养GSCU251和GSCT98G。2.2 CCK-8实验检测不同浓度褪黑素对GSCU251和GSCT98G增殖能力的影响。3、检测褪黑素其对GSCs自我更新能力的影响通过成球实验检测不同浓度褪黑素对GSCU251和GSCT98G自我更新能力的影响。4、检测褪黑素对GSCs干性的影响qRT-PCR和Western Blot检测褪黑素对GSCU251和GSCT98G中干性标记物CD133和SOX2表达的影响。第二部分:EZH2在褪黑素对GSCs的抑制过程中的作用探究1、检测褪黑素对GSCs中EZH2表达的影响。qRT-PCR和Western Blot检测褪黑素对GSCs中EZH2表达的影响。2、EZH2敲除GSCU251模型的建立2.1通过EZH2 shRNA转染GSCU251,建立EZH2敲除GSCU251模型。2.2 Western Blot检测EZH2敲除GSCU251模型建立是否成功。3、检测EZH2敲除后GSCU251的增殖能力、自我更新能力及干性的变化敲除GSCU251中的EZH2后,CCK-8实验检测GSCU251的生长、增殖能力变化;成球实验检测GSCU251的自我更新能力变化;qRT-PCR检测GSCU251中CD133分子表达的变化。4、EZH2过表达GSCU251模型的建立4.1通过GV230-EZH2质粒转染GSCU251,中建立EZH2过表达GSU251模型。4.2 Western Blot检测EZH2过表达GSCU251模型建立是否成功。5、检测EZH2过表达后GSCU251的增殖能力、自我更新能力及干性的变化GSCU251中的EZH2过表达后,CCK-8实验检测GSCU251的生长、增殖能力变化;成球试验检测GSCU251的自我更新能力变化;qRT-PCR检测GSCU251中CD133分子表达的变化。6、检测褪黑素加入后,对照组和EZH2过表达组GSCU251的增殖能力、自我更新能力及干性的变化6.1在对照组GSCU251中加入褪黑素进行培养,CCK-8、成球实验检测GSCU251的增殖能力、自我更新能力变化;qRT-PCR检测GSCU251中CD133分子表达的变化。6.2在EZH2过表达组GSCU251中加入褪黑素进行培养,CCK-8、成球实验检测GSCU251的增殖能力、自我更新能力变化;qRT-PCR检测GSCU251中CD133分子表达的变化。第三部分:NOTCH1在EZH2起效过程中的作用及机制1、检测褪黑素对GSCs中NOTCH1表达的影响1.1 qRT-PCR检测褪黑素干预后GSCU251和GSCT98G中NOTCH1及下游因子CCND1,CCNE2和HES1等的mRNA表达量变化。1.2 Western Blot 检测褪黑素干预后 GSCU251 和 GSCT98G 中 NICD1(NOTCH1的胞内功能区域)和HES1的蛋白表达量变化。2、检测EZH2对NOTCH1的调控作用2.1通过双荧光素酶报告实验检测EZH2敲除和过表达GSCU251中NOTCH1转录活性的变化。2.2 Western Blot检测EZH2敲除和过表达GSCU251中NICD1和HES1的蛋白表达量变化。2.3染色质免疫共沉淀实验检测GSCU251中NOTCH1的启动子位置EZH2、H3K27me3和SUZ12的聚集情况。第四部分:EZH2-NOTCH1信号轴在临床样本中的初步探究1、检测胶质母细胞瘤样本和正常脑组织样本中EZH2和NICD1表达的相关性1.1选取67例胶质母细胞瘤样本和12例正常脑组织样本进行EZH2和NICD1指标的免疫组化染色。1.2统计学分析胶质母细胞瘤样本中EZH2和N1CD1的表达相关性。结果:第一部分:褪黑素对GSCs增殖、自我更新及干性的影响1、U251和T98细胞系中GSCs的培养及验证通过干细胞富集培养基培养U251和T98G细胞系三周后,建立GSCU251和GSCT98G细胞模型。通过qRT-PCR检测,我们发现,与普通U251和T98G细胞系相比,GSCU251和GSCT98G细胞模型的干细胞标记物CD133和SOX2的含量明显升高。2、检测不同浓度褪黑素对GSCs的生存、增殖能力的影响不同浓度褪黑素处理GSC251和GSCT98G后,CCK-8实验结果显示,100μM和1mM的褪黑素对GSCs的生长产生了抑制作用。3、检测褪黑素其对GSCs自我更新能力的影响不同浓度褪黑素处理GSCU251和GSCT98G后,成球实验结果显示,100μM和1mM的褪黑素对GSCs的自我更新产生了抑制作用。4、检测褪黑素对GSCs干性的影响qRT-PCR和Western Blot检测结果表明,1mM的褪黑素对GSCU251和GSCT98G中干性标记物CD133和SOX2表达有明显的抑制作用。第二部分:EZH2在褪黑素对GSCs的抑制过程中的作用探究1、检测褪黑素对GSCs中EZH2表达的影响。qRT-PCR和Western Blot检测结果表明,褪黑素的干预显著降低了 GSCU251和GSCT98G中EZH2的表达量。2、EZH2敲除GSCU25,模型的建立通过EZH2 shRNA转染GSCU251,建立EZH2敲除GSCU251模型后,Western Blot实验表明,EZH2敲除GSC251模型中的EZH2蛋白表达量明显降低。3、检测EZH2敲除后GSCU251的增殖能力、自我更新能力及干性的变化敲除GSCU251中的EZH2后,CCK-8实验表明,GSCU251的生长、增殖能力显著降低;成球实验表明,GSCU251的自我更新能力显著降低;qRT-PCR检测显示,GSCU251中CD133分子表达量显著下降。4、EZH2过表达GSCU251模型的建立通过GV230-EZH2转染GSCU251,建立EZH2过表达GSCU251模型后,Western Blot实验表明,EZH2过表达GSCU251模型中的EZH2蛋白表达量明显升高。5、检测EZH2过表达后GSCU251的增殖能力、自我更新能力及干性的变化GSCU251中的EZH2过表达后,CCK-8实验表明,GSCU251的生长、增殖能力显著增强;成球实验表明,GSCU251的自我更新能力显著增强;qRT-PCR检测显示,GSCU251中CD133分子表达量显著升高。6、检测褪黑素加入后,对照组和EZH2过表达组GSCU251的增殖能力、自我更新能力及干性的变化6.1对照组GSCU251中加入褪黑素进行培养后,CCK-8、成球实验结果显示,GSCU251的增殖能力、自我更新能力显著下降;qRT-PCR检测表明,GSCU251中CD133的表达量明显降低。6.2 EZH2过表达组GSCU251中加入褪黑素进行培养后,CCK-8、成球实验结果显示,GSCU251的增殖能力、自我更新能力显著下降;qRT-PCR检测表明,GSCU251中CD133的表达量明显降低。第三部分:NOTCH1在EZH2起效过程中的作用及机制1、检测褪黑素对GSCs中NOTCH1表达的影响1.1 qRT-PCR检测结果显示,褪黑素干预后GSCU251和GSCT98G中NOTCH1及下游因子CCND1,CCNE2和HES1等的mRNA表达量显著下降。1.2 Western Blot检测结果也表明,褪黑素干预后GSCU251和GSCT98G中NICD1和HES1的蛋白表达量显著下降。2、检测EZH2对NOTCH1的调控作用2.1通过双荧光素酶报告实验,我们发现,GSCU251中的EZH2敲除和过表后,NOTCH1转录活性随之减弱和增强。2.2 Western Blot结果表明,GSCU251中EZH2敲除和过表达后,NICD1和HES1的蛋白表达量也随之升高和降低。2.3染色质免疫共沉淀实验检测发现,GSCU251中NOTCH1的启动子位置可以检测到EZH2的显著聚集,而并未检测到H3K27me3和SUZ12的聚集。同时,褪黑素的干预显著降低了 EZH2在NOTCH1的启动子区域的聚集。第四部分:EZH2-NOTCH1信号轴在临床样本中的初步探究1、检测胶质母细胞瘤样本和正常脑组织样本中EZH2和NICD1表达的相关性1.1免疫组化染色结果显示,73%的EZH2高表达胶质母细胞瘤样本中也存在着NICD1的高表达。1.2统计学分析发现,胶质母细胞瘤样本中EZH2和NICD1的表达存在高度的正相关。结论:1、特定浓度的褪黑素对GSCs增殖、自我更新能力具有显著地抑制作用,并能显著降低GSCs的干性。2、通过机制实验,我们发现,褪黑素通过抑制EZH2-NOTCH1信号轴发挥了抑制GSCs的作用。3、EZH2-NOTCH1信号轴中,EZH2通过直接结合NOTCH1的启动子区域调控NOTCH1的表达。
[Abstract]:Glioma is a primary intracranial tumor with high incidence of all intracranial tumors, accounting for more than 50% of all intracranial tumors. Because glioma is infiltrative and aggressive, the operation is difficult to complete resection, and the treatment is poor. Often the prognosis is bad.WHO divides the glioma into 4 grade, the higher the grade, the higher the malignant degree. The highest grade of glioma is the glioma. Mother cell tumor (GBM) is WHO IV, and the tumor has a high incidence of 15%. in all intracranial tumors under the premise of surgical removal of glioblastoma, supplemented by standard radiotherapy and chemotherapy, and the patient's median survival is still not more than 15 months. Recent studies have shown that gliomas, especially the highest malignant glioblastoma, have been shown. The tumor contains a cell subgroup, which has some characteristics of normal stem cells and is called glioblastoma stem-like cells (GSCs).GSCs, which plays a very strong role in promoting the proliferation of glioblastoma, and GSCs plays a key role in promoting the infiltration and diffusion of the tumor to the brain tissue around the cranium. At present, combined chemotherapy and chemotherapy can not effectively kill GSCs, which causes the tumor to relapse quickly and have poor prognosis, so it is urgent to explore the treatment that can effectively kill GSCs. Melatonin is a hormone secreted by the pineal body. More and more studies have shown that melatonin has an inhibitory effect on a variety of tumors, including glioma. However, the role of melatonin in GSCs is still less studied. Its mechanism is not yet clear that.EZH2 has a histone methyltransferase, which is one of the components of the multi comb protein complex 2. It includes glioma, breast cancer, and gastric cancer. The higher the expression of EZH2, the higher the expression of EZH2, the higher the expression of the tumor, the higher the malignancy, the worse the prognosis, and the inhibition of the expression of EZH2 can delay the occurrence and development of the tumor. At the same time, there is a definite evidence that EZH2 is one of the dry indexes of GSCs, the overexpression of.NOTCH receptor is also The.EZH2-NOTCH1 signal axis in a variety of malignant tumors, including gliomas, has been shown to exist in pancreatic cancer, and whether the EZH2-NOTCH1 signal axis is also in GSCs, and whether melatonin has been involved in the effect of melatonin on GSCs has not yet been reported. This experiment mainly studied the effect of melatonin on GSCs and the specific mechanism of action. Objective: 1, to study the effect of melatonin on GSCs proliferation, self renewal and dry action; 2, to study the specific role of EZH2-NOTCH1 signal axis in the process of melatonin, and 3, to study the specific mechanism of EZH2 in the regulation of NOTCH1. Methods: the first part: the effects of melatonin on GSCs proliferation, self renewal and dry effect 1, GSC in U251 and T98 cell lines. Culture and verification of s 1.1 in the culture medium of stem cell enrichment (serum free, B27, bFGF and EGF factor) culture U251 and T98G cell lines, culture for at least three weeks, and establish GSCU251 and GSCT98G cell model.1.2 qRT-PCR to detect GSCs stem cell marker CD133 and content. Effects of melatonin on the survival and proliferative ability of GSCs 2.1 prepared medium containing different concentrations of melatonin (0.1-1000 M), culture GSCU251 and GSCT98G.2.2 CCK-8 to detect the effect of melatonin on the proliferation of GSCU251 and GSCT98G at different concentrations. The effects of melatonin on the self-renewal ability of GSCs were tested by a ball test. The effects of melatonin on the self-renewal capacity of GSCU251 and GSCT98G.4,.4, the effect of melatonin on GSCs's dry property, qRT-PCR and Western Blot were used to detect the effects of melatonin on CD133 and SOX2 expression of dry markers in GSCU251 and GSCT98G. Part second: the role of EZH2 in the inhibition process of melatonin 1, detection of melatonin The influence of.QRT-PCR and Western Blot on the expression of EZH2 in GSCs,.QRT-PCR and Western Blot were used to detect the effect of melatonin on the expression of EZH2 in GSCs. After the ability, self renewal ability and dry change to knock out EZH2 in GSCU251, CCK-8 test detected the growth of GSCU251 and the change of proliferation ability; the ball test detected the change of self renewal ability of GSCU251; qRT-PCR detected the change of CD133 molecule expression in GSCU251, and the establishment of GSCU251 model of EZH2 over expression 4.1 was transfected through GV230-EZH2 plasmids. 51, we established EZH2 overexpressed GSU251 model.4.2 Western Blot to detect EZH2 over expression GSCU251 model to establish a successful.5, detect the proliferation ability of GSCU251 after EZH2 overexpression, self renewal ability and the EZH2 over expression in the dry change GSCU251. Changes in self renewal ability; qRT-PCR detection of the changes in the expression of CD133 in GSCU251,.6, after the addition of melatonin, the proliferation ability of GSCU251 in the control group and EZH2 overexpression group, the ability of self renewal and the change of dry nature 6.1 were added in the control group GSCU251 with melatonin to be cultured, CCK-8, and the ball test to detect the proliferating ability of GSCU251, and the self more Changes in new capacity; qRT-PCR detection of the changes in the expression of CD133 molecules in GSCU251,.6.2 added melatonin in EZH2 overexpression group GSCU251 to culture, CCK-8, test of CCK-8, test of GSCU251's proliferation ability, change of self-renewal ability, and qRT-PCR detection CD133 molecule expression in GSCU251. The third part: the role of NOTCH1 in the initiation process. And mechanism 1, detection of the effect of melatonin on the expression of NOTCH1 in GSCs 1.1 qRT-PCR detection of NOTCH1 and NOTCH1 and downstream factors CCND1, CCNE2, HES1 and other mRNA expressions in GSCU251 and GSCT98G after the intervention of melatonin Quantitative change.2 and detection of EZH2's regulatory role on NOTCH1 2.1 detection of EZH2 knockout and overexpressed GSCU251 NOTCH1 transcriptional activity by double luciferase reporter assay.2.2 Western Blot detection of.2.2 Western Blot detection EZH2 knockout and overexpression GSCU251 NICD1 and protein expression changes The aggregation of promoter location EZH2, H3K27me3 and SUZ12. Part fourth: preliminary inquiry into the EZH2-NOTCH1 signal axis in clinical samples 1, the correlation of the expression of EZH2 and NICD1 in glioblastoma samples and normal brain tissue samples 1.1 selected 67 cases of glioblastoma samples and 12 normal brain tissue samples for EZH2 and NICD1 indicators Immunohistochemical staining.1.2 analysis of the expression of EZH2 and N1CD1 in glioblastoma samples. Results: the first part: the effect of melatonin on GSCs proliferation, self renewal and dry effect 1, the culture and validation of GSCs in the U251 and T98 cell lines, and the establishment of GSCU251 and the development of U251 and T98G cell lines by stem cell enrichment medium for three weeks. GSCT98G cell model. Through qRT-PCR detection, we found that the content of CD133 and SOX2 in the stem cell markers of GSCU251 and GSCT98G cell models significantly increased.2 compared with the common U251 and T98G cell lines, and the effects of melatonin on the survival of GSCs and the proliferation ability of different concentrations were different from that of GSC251 and GSCT98G after the concentration of melatonin. The results showed that melatonin, 100 M and 1mM, inhibited the growth of GSCs, and the effects of melatonin on the self-renewal capacity of GSCs were detected by melatonin. The effects of melatonin on GSCU251 and GSCT98G in different concentrations were observed. The results showed that melatonin of 100 mu and 1mM produced a more inhibitory effect on the self of GSCs, and the detection of melatonin to GSCs dried. The results of qRT-PCR and Western Blot detection showed that melatonin in 1mM had an obvious inhibitory effect on the expression of CD133 and SOX2 in GSCU251 and GSCT98G. The second part: EZH2 in the inhibitory process of melatonin to GSCs 1, the effect of melatonin on EZH2 expression in GSCs and the detection results The results showed that the intervention of melatonin significantly reduced the expression of EZH2 in GSCU251 and GSCT98G, and EZH2 knocked out GSCU25. The establishment of the model was established through EZH2 shRNA to GSCU251, and EZH2 knocking GSCU251 model was established. After the ability, self renewal ability and the dry change to knock out EZH2 in GSCU251, the CCK-8 experiment showed that the growth of GSCU251 and the proliferation ability significantly decreased, and the ball test showed that the self-renewal ability of GSCU251 decreased significantly; qRT-PCR detection showed that the expression of CD133 molecules in GSCU251 decreased significantly and the GSCU251 model of EZH2 overexpression was established through GV2. After transfecting GSCU251 with 30-EZH2 and establishing EZH2 overexpressed GSCU251 model, Western Blot experiment showed that the expression of EZH2 protein in EZH2 overexpressed GSCU251 model was significantly higher than that of.5. The colonization ability was significantly enhanced, and the self-renewal ability of GSCU251 was significantly enhanced by the ball formation test; qRT-PCR detection showed that the expression of CD133 molecules in GSCU251 increased significantly by.6. After the detection of melatonin, the proliferation ability of GSCU251 in the control group and EZH2 overexpressed group, the self-renewal ability and the changes of dry nature were added to the 6.1 control group GSCU251 to add melatonin into the GSCU251. After the culture, CCK-8, the results of the ball formation test showed that the proliferation ability of GSCU251 and self renewal ability decreased significantly, and qRT-PCR detection showed that the expression of CD133 in GSCU251 was obviously reduced by.6.2 EZH2 overexpression group GSCU251 added melatonin in the culture, CCK-8, and the result showed that the proliferation ability and self-renewal ability of GSCU251 were significant. The qRT-PCR detection showed that the expression of CD133 in GSCU251 was obviously reduced. Third part: the role and mechanism of NOTCH1 in the process of EZH2, the effect of melatonin on the expression of NOTCH1 in GSCs 1.1 qRT-PCR detection results showed that after the intervention of melatonin and GSCT98G NOTCH1 and downstream factors CCND1, etc. The.1.2 Western Blot detection results also showed that the protein expression of NICD1 and HES1 in GSCU251 and GSCT98G decreased significantly after the intervention of melatonin, and the regulatory role of EZH2 on NOTCH1 was 2.1 through the double Luciferase Report experiment, we found that the transcriptional activity of EZH2 knocking and passing the table was weakened and enhanced. 2 Western Blot results showed that after EZH2 knockout and overexpression in GSCU251, the protein expression of NICD1 and HES1 also increased and decreased the.2.3 chromatin immunoprecipitation test, and found that the promoter location of NOTCH1 in GSCU251 could detect the significant aggregation of EZH2, but did not detect the accumulation of H3K27me3 and SUZ12. Meanwhile, the dry of melatonin. Pre significantly reduced the aggregation of EZH2 in the promoter region of NOTCH1. Fourth part: preliminary inquiry into EZH2-NOTCH1 signal axis in clinical samples 1, correlation of EZH2 and NICD1 expression in glioblastoma samples and normal brain tissue samples 1.1 immuno histochemical staining results showed that 73% of EZH2 high expression glioblastoma samples were also found NICD1 high expression.1.2 statistical analysis found that there was a high positive correlation between the expression of EZH2 and NICD1 in the samples of glioblastoma. Conclusion: 1, the specific concentration of melatonin has a significant inhibitory effect on GSCs proliferation and self-renewal capacity, and can significantly reduce the dry.2 of GSCs. Through mechanism experiments, we found that melatonin passes through the mechanism. Inhibition of EZH2-NOTCH1 signal axis plays a role in inhibiting GSCs..3, in EZH2-NOTCH1 signal axis, EZH2 regulates NOTCH1 expression by directly combining NOTCH1 promoter region.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R739.41

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