抗精神病药喹硫平治疗胶质瘤的实验研究

发布时间：2018-03-17 02:16

本文选题：胶质瘤干细胞　切入点：喹硫平　出处：《第三军医大学》2015年博士论文　论文类型：学位论文

【摘要】：恶性胶质瘤细胞对放疗、化疗不敏感,导致其临床治愈率低,术后复发率居高不下,患者5年存活率不到5%。因此,探寻该疾病行之有效的治疗途径,发现相应的生物学治疗途径至关重要。胶质瘤干细胞(glioma stem-like cells,GSLCs)是胶质瘤组织中存在的极少数具有高度抵抗放疗和化疗的细胞,具有无限增殖和多向分化能力,可以分化为神经元表型细胞,星形胶质细胞表型细胞以及少突胶质细胞表型细胞。GSLCs概念的提出为胶质瘤治疗提供了重要靶点,因此寻找能够促进GSLCs定向分化药物可能为临床治疗胶质瘤提供新的途径。流行病学调查发现一个有趣的现象:精神分裂症患者的肿瘤发病率比正常人群明显减少。研究人员发现多种抗精神病药如氯丙嗪、奥氮平、利培酮等,均能明显抑制胶质母细胞瘤细胞系IMR32细胞增生,且对正常细胞影响甚微。喹硫平(Quetiapine,QUE)是继氯氮平、利培酮和奥氮平之后的第4个非典型抗精神病药物。本课题组前期研究首次发现喹硫平能显著增加神经干细胞(neuronal stem cells,NSCs)分化为少突胶质细胞的比例,并能进一步促进少突胶质细胞的成熟及髓鞘形成。我们在实验中还发现QUE能促进少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)分化成熟,考虑GSLCs与NSCs,OPCs关系密切,我们提出QUE可能具有抑制GSLCs增殖及促进GSLCs定向分化为少突胶质细胞表型的潜能。本研究利用GSLCs培养和移植成瘤模型,研究了QUE对胶质瘤发生发展的作用,及其与胶质瘤一线治疗药物替莫唑胺(Temozolomide,TMZ)的联合药效,探讨了QUE对GSLCs增殖分化的调节作用及可能的分子机制研究总共分为三个部分:第一部分:喹硫平抑制胶质瘤生长的作用利用无血清条件培养系统从胶质母细胞瘤细胞系GL261中筛选获得胶质瘤干细胞,建立胶质瘤原位及皮下成瘤模型,分别采用QUE、TMZ以及二者联合治疗等方式,运用荧光素酶活体成像技术、动物行为学、HE染色、免疫组织化学和Western blot等方法检测各种治疗方式对胶质瘤发生和生长特性的作用。最后为了进一步检验QUE可能作用于GSLCs而抑制胶质瘤的产生或生长。我们分别对GSLCs移植裸鼠皮下成瘤以及脑室成瘤后使用TMZ 21天来杀死肿瘤细胞,停药后再分别给予生理盐水和QUE治疗,利用荧光素酶活体成像技术观察胶质瘤的生长情况。主要结果如下:1.免疫荧光染色显示无血清培养条件下筛选GL261获得的细胞表达肿瘤干细胞(cancer stem cells,CSCs)标记物CD133、Sox2、NG2和Nestin,加入10%胎牛血清分化10天后大部分细胞表达神经胶质酸性蛋白(glial fibrillary acidic protein,GFAP),少部分表达髓鞘碱性蛋白(myelin basic protein,MBP)。以上结果表明所获得细胞具有胶质瘤干细胞特性,为胶质瘤干细胞GSLCs。2.GSLCs皮下移植裸后7天后可见小的瘤体形成,21天后瘤体平均直径达到1.5cm,而QUE处理组、TMZ处理组及QUE联合TMZ组,成瘤时间均有所推迟,瘤体平均体积也小于对照组,其中尤以QUE联合TMZ疗效最明显。组织学病理检测发现,治疗组明显减少了胶质瘤细胞的分裂潜能,即核分裂像减少,增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)阳性细胞明显减少。而同对照组相比,治疗组,尤其是QUE和TMZ联合治疗组,Olig2阳性细胞明显减少,而MBP阳性细胞则明显增多(P0.01)。Western blot结果显示QUE和QUE联合TMZ治疗组MBP表达明显增多,而Vimentin,GFAP表达下降(P0.05)。3.GSLCs脑室移植后,活体荧光成像显示小鼠于1周后出现肿瘤,而QUE,TMZ治疗后肿瘤大小有所减少,而二者联合用药后肿瘤最小,动物成活率增加。行为学旷场实验显示,药物治疗组小鼠较原位脑胶质瘤模型小鼠活动总路程明显提高(P0.05),提示其活动能力保留较好。QUE对胶质瘤生长有抑制作用,而且QUE联合替莫唑胺能更好抑制胶质瘤生长,降低其恶性程度。4.TMZ治疗21天后肿瘤生长明显抑制,但停药后1周后,残留的胶质瘤干细胞开始生长至瘤。而QUE处理组,肿瘤生长明显减慢,大小明显减小(P0.05)。说明QUE可能抑制GSLCs增殖或促进其分化。本部分实验说明QUE对胶质瘤的生长有一定的抑制作用,而且QUE联合替莫唑胺能更好抑制胶质瘤生长,降低其恶性程度。这些结果提示QUE可能对GSLCs生长分化有一定的影响。第二部分:喹硫平对肿瘤干细胞增殖及分化的作用为了证明QUE可能作用于GSLCs而抑制胶质瘤干细胞的产生或生长。我们利用无血清条件培养系统从胶质母细胞瘤细胞系GL261中筛选获得胶质瘤干细胞,用MTT,RT-PCR,流式细胞检测等技术观察不同浓度QUE对胶质瘤干细胞增殖作用。用免疫荧光及Western blot来检测不同浓度QUE对胶质瘤干细胞分化作用。主要结果如下:1.体外实验显示QUE浓度50、100μmol/L能明显抑制GSLCs的生长,悬浮的球团明显小于对照组(P0.01)。MTT实验也证实了QUE的抑制效应(P0.01)。流式细胞检测中QUE明显减少S期细胞(P0.05),而且能显著减少胶质瘤干细胞中Sox2及Ki67的基因表达(P0.01)。2.以1%胎牛血清诱导GSLCs分化过程中,QUE处理可显著提高少突胶质细胞成熟蛋白MBP及CC1阳性细胞数;而且浓度依赖性降低干细胞因子Sox2,Olig2蛋白表达(P0.05),而升高少突胶质细胞相关因子MBP,Olig1蛋白表达(P0.05)。本部分实验结果表明QUE能抑制胶质瘤干细胞增殖并且能促进其向类似少突胶质细胞分化。第三部分:喹硫平促进胶质瘤干细胞分化的分子机制初探由于Wnt/β-catenin通路在GSLCs增殖分化中发挥了至关重要的作用,而且QUE的抗精神病的药物机制也涉及该途径,所以我们利用免疫组化及Western blot等技术检测了离体在体中QUE对胶质瘤干细胞分化中Wnt/β-catenin信号通路的影响。主要结果如下:1.在体实验中,免疫组化显示对照组β-catenin在细胞质表达较多并且有入核现象,而QUE组中β-catenin在细胞质表达较少。Western blot显示QUE组中p-GSK-3β(ser9)减少(p0.01),促进β-catenin降解,导致β-catenin表达减少(p0.01)。2.在胶质瘤干细胞分化过程中,QUE可以减少p-GSK-3β(ser9)的表达,提高磷酸化β-catenin(P-β-catenin)而降低β-catenin水平(P0.05)。这种作用呈时间浓度依赖性。而且这种作用可以被Wnt/β-catenin通路激活剂QS11和Li Cl拮抗。本部分实验说明QUE可能通过激活GSK-3β,使β-catenin降解,进而抑制Wnt/β-catenin通路而促进GSLCs向少突胶质样细胞分化。以上所有结果提示QUE有可能成为与TMZ联合用药的候选,为恶性胶质瘤的治愈提供新的策略。
[Abstract]:Radiotherapy for malignant glioma cells, not sensitive to chemotherapy, the clinical cure rate and low recurrence rate is high, with 5 year survival rate of less than 5%. so the search for the effective way of disease, found crucial biological treatment corresponding approaches. Glioma stem cells (glioma stem-like cells, GSLCs) is one of the few it is highly resistant to radiotherapy and chemotherapy of glioma cells exist in the organization, with unlimited proliferation and multi differentiation capacity and can differentiate into neuron cell phenotype, propose astrocytic phenotype cells and phenotype of oligodendrocytes.GSLCs concept provides an important target for glioma therapy, therefore can promote the differentiation of GSLCs for drugs may provide a new way for the clinical treatment of glioma. Epidemiological survey found an interesting phenomenon: the schizophrenia patients tumor incidence rate ratio The normal population decreased significantly. The researchers found that many antipsychotic drugs such as chlorpromazine, olanzapine, risperidone, can significantly inhibit glioblastoma cell line IMR32 cell proliferation, and has little effect on normal cells. Quetiapine (Quetiapine, QUE) is the second after clozapine, risperidone and olanzapine, fourth atypical antipsychotics. Our previous study found for the first time quetiapine can significantly increase the neural stem cells (neuronal stem cells, NSCs) to differentiate into oligodendrocytes and proportion, to further promote the formation of mature and myelin oligodendrocyte. We also found that in the experiment of QUE can promote oligodendrocyte precursor cells (oligodendrocyte progenitor cells, OPCs) differentiation and maturation, consider GSLCs and NSCs, OPCs are closely related, we proposed that QUE may inhibit GSLCs proliferation and promote the differentiation of GSLCs into oligodendrocytes form Type potential. This study culture and transplantation tumor model by using GSLCs, QUE to study the occurrence and development of glioma cells and glioma, and first-line therapy of temozolomide (Temozolomide, TMZ) of the combined effect, discuss the molecular mechanism of regulation of QUE on the proliferation and differentiation of GSLCs and the total is divided into three parts: the first part: quetiapine inhibited culture system from glioblastoma cell line GL261 in vitro glioma stem cells in serum-free conditions using glioma growth, establish glioma orthotopic and subcutaneous tumor model, respectively QUE, TMZ and the combination of the two treatment methods, the use of in vivo imaging of luciferase, animal behavior, HE staining, immunohistochemistry and Western blot methods to detect various treatments of glioma and growth characteristics. Finally, in order to further test QUE Effect on GSLCs and inhibit the production or growth of glioma. We were on GSLCs transplanted subcutaneous tumor formation and tumor ventricle using TMZ 21 days to kill tumor cells, after discontinuation were given normal saline and QUE treatment, and observed the growth of glioma by in vivo imaging of luciferase. The main results are as follows: 1. immunofluorescence staining showed that the serum-free medium GL261 cells obtained by screening the expression of tumor stem cells (cancer stem cells, CSCs) markers CD133, Sox2, NG2 and Nestin, with 10% fetal bovine serum for 10 days most of the cell differentiation and expression of glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP), the expression of myelin basic protein a small part (myelin basic protein, MBP). The results show that the obtained cells have the characteristics of glioma stem cells, glioma stem cells of GSLCs.2.GSLCs skin grafting after 7 days naked down See the small tumor formation, 21 days after the tumor diameter reached 1.5cm, and the QUE treatment group, TMZ treatment group and QUE combined with TMZ group, the time of tumor formation was delayed, the average volume of the tumor is less than that of the control group, especially in the curative effect of QUE combined with TMZ. The most obvious histopathological detection, treatment group significantly reduced glioma cell division potential, namely the mitotic reduce proliferating cell nuclear antigen (proliferating cell nuclear antigen, PCNA). The positive cells were significantly decreased compared with the control group, treatment group, especially QUE and TMZ combined treatment group, Olig2 positive cells decreased significantly, while MBP positive cells was significantly increased (P0.01).Western blot showed that QUE and QUE combined with TMZ treatment group MBP was significantly increased, while Vimentin decreased expression of GFAP (P0.05).3.GSLCs ventricle after transplantation, in vivo fluorescence imaging showed that mice in 1 weeks after the tumor, and QUE, TMZ after the treatment of goiter The tumor size decreased, and the two combination tumor minimum, animal survival rate increased. The behavior of open field test showed that the drug treatment group compared with the mouse glioma model of mice significantly increased the total distance (P0.05), suggesting that the activity of well preserved.QUE has inhibitory effect on glioma growth, and QUE combined with temozolomide can better inhibit glioma growth, reduce the malignant degree of.4.TMZ could inhibit the growth of tumor after 21 days of treatment, but after 1 weeks after stopping the growth of glioma tumor cells began to dry. The residues in QUE treated group, the tumor length was significantly decreased, size decreased significantly (P0.05) QUE. May inhibit GSLCs proliferation or promote their differentiation. The results suggested that QUE had a certain inhibitory effect on the growth of glioma, and QUE combined with temozolomide can better inhibit glioma growth, reduce the degree of malignancy. These results suggest that QUE May have a certain impact on the growth and differentiation of GSLCs. The second part: the effect of quetiapine on the proliferation and differentiation of tumor stem cells in order to prove the possible role of QUE on GSLCs and inhibit the cell production or growth of glioma stem. We use the training system from the glioblastoma cell line GL261 was obtained from human glioma stem cells. With MTT, RT-PCR in serum-free conditions, flow cytometry were used to evaluate the different concentrations of QUE on glioma stem cell proliferation. By immunofluorescence and Western blot to detect different concentrations of QUE on glioma stem cell differentiation. The main results are as follows: 1. in vitro experiments showed that QUE concentration of 50100 mol/L could significantly inhibit the growth of GSLCs the suspension, the pellet was significantly less than the control group (P0.01).MTT experiments also confirmed the inhibitory effect of QUE (P0.01). Flow cytometry QUE significantly reduced S phase cells (P0.05), and can significantly reduce glioma The expression of Sox2 and Ki67 in stem cell gene (P0.01).2. with 1% fetal bovine serum to induce GSLCs differentiation, QUE treatment can significantly improve the oligodendrocyte maturation protein MBP and the number of CC1 positive cells; and concentration dependent decrease of stem cell factor Sox2, the expression of Olig2 protein (P0.05), and the increase of oligodendrocyte cell related cytokines MBP, Olig1 protein expression (P0.05). The experimental results show that QUE can inhibit the proliferation of glioma stem cells and can promote its similar to oligodendrocyte differentiation. The third part: the differentiation of glioma stem cells into the mechanism due to Wnt/ beta -catenin pathway play a crucial role in the proliferation and differentiation of GSLCs in the promotion of quetiapine, and antipsychotic drug mechanism of QUE is also involved in the way, so we use immunohistochemistry and Western blot were detected in vitro in QUE on glioma stem cell differentiation in W Effect of nt/ beta -catenin signal pathway. The main results are as follows: 1. in vivo experiment, control group P -catenin in the cytoplasm and more nuclear phenomena, and beta -catenin in the QUE group in the cytoplasm less.Western blot display QUE group p-GSK-3 beta (ser9) decreased (P0.01), promote beta the degradation of -catenin, resulting in decreased expression of beta -catenin (P0.01).2. in glioma stem cell differentiation process, QUE can reduce the p-GSK-3 beta (ser9) expression, increase the phosphorylation of beta -catenin (P- beta -catenin) decreased beta -catenin level (P0.05). This effect is dose dependent and time dependent. And this effect can. Wnt/ beta -catenin pathway activation agent QS11 and Li. The results suggested that Cl antagonist QUE may activate GSK-3 beta, the beta -catenin degradation, and inhibition of Wnt/ beta -catenin pathway and promote GSLCs to differentiate into oligodendrocytes. All of the above results. QUE may be a candidate for combination with TMZ, providing a new strategy for the cure of malignant gliomas.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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