特异性杀伤胶质瘤干细胞化合物SN38的发现及评价
摘要：
开心激情播播网目的：建立一种高效筛选特异性杀伤胶质瘤干细胞(glioma stem cell,GSC)的药物方法，筛选特异性杀伤胶质瘤干细胞的药物，为促进脑胶质母细胞瘤（Glioblastoma,GBM）治疗药物发现提供策略。
方法：将临床病人的胶质瘤干细胞T387GSC稳定转染绿色荧光蛋白，以荧光读值为指示，建立高通量药物筛选方法；筛选60个肿瘤干细胞凋亡相关信号通路抑制剂，优选强活性药物用D456GSC重复验证化合物的效果并测试其人神经胶质细胞(normal human microglia,NHA)及人小胶质细胞(human microglia,HM)的细胞毒性，考察化合物的选择性，发现SN38对胶质瘤干细胞具有选择性的杀伤作用。采用体内移植瘤模型评价SN38的体内抗肿瘤活性研究，考察其对原位瘤小鼠生存期的影响，并通过脑切片HE染色评价SN38对肿瘤增殖的抑制作用。组织病理学水平检测SN38对GSC的杀伤效果及诱导GSC的凋亡情况。并采用流式细胞术及蛋白印迹技术检测候选化合物诱导细胞凋亡情况，通过诱导敲除的方法验证SN38在GSC中的作用靶点。
结果：建立了一种高通量胶质瘤干细胞选择性杀伤药物的筛选方法；发现化合物SN38对胶质瘤干细胞细胞毒性最强。实验证明SN38选择性杀伤胶质瘤干细胞，对各株胶质瘤干细胞IC50均小于10nM。而对正常人神经胶质细胞和人小胶质细胞IC50均大于100nM。动物实验结果证明给予化合物SN38明显延长荷
瘤小鼠生存期，肿瘤增长
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减慢，肿瘤细胞凋亡增加，肿瘤干细胞比例降低。细胞免疫荧光实验证明SN38通过诱导胶质瘤干细胞凋亡，减慢肿瘤干细胞增殖。Western-blot结果显示SN38处理胶质瘤干细胞引起拓扑异构酶I蛋白降解，诱导敲低拓扑异构酶I后，胶质瘤干细胞对SN38敏感性下降。
签名英语
结论：以细胞荧光强度为指示的高通量筛选方法能够高效准确地筛选抗脑胶质母细胞瘤药物。化合物SN38在胶质瘤干细胞的作用靶点是TOPI，通过促进胶质瘤干细胞凋亡，抑制增殖来发挥抗肿瘤效果。辛亥革命影响
关键词：胶质母细胞瘤；干细胞；高通量筛选；特异性
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Establishment of a drug screening method to screen chemicals preferentially eliminated GSCs.
Hongquan Zhang(Pharmacology)
Directed by Prof.Zifen Guo and associate fellow.Xinhua He Abstract：张继楼
Objective:To provide a strategy for glioblastoma(GBM) chemotherapy chemicals discovery，we establish a high efficient drug screening method.半年工作总结>秋天的词
Methods:Patient derived T387GSC was stably transfected with green fluorescent protein,and then it was applied to establish a high efficient drug screening method in which fluorescence intensity is regarded as a readout.60chemicals related to tumor stem cell apoptosis signaling pathway were screened using this method,and then were subjected to D456GSC to reconfirm its efficiency.In which some of the chemicals are further tested to determine its toxicity for normal human astrocyte(NHA)and human microglia(HM).Chemicals,which killed glioma stem cells(GSCs)but had little influence on normal human astrocytes,were chon as a candidate for induction of the cell apoptosis study using flow cytometry assay.SN38is chon to test antitumor activity rearch on animal.Survive time was evaluated and the effect of SN38on brain tumor expansion was also evaluated by hematoxylin and eosin(HE)staining.Histopathology analysis method was applied to
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determine the ratio of GSCs in tumor tissue and its potential on inducing apoptosis after SN38treatment.Flow cytometry and western blot analysis was ud to determine whether or not apoptosis is the cau of cell death.
Results:A high throughput drug screening method was established for the discovery of chemicals with ability against GSCs;SN38and bortezomib were found to be most lethal toward GSCs.We confirm that sn38lectively kills GSCs.The half maximal concentration of SN38on all GSC cell lines we have is lower than10nanomole,while the half maximal concentration of SN38on NHA and HM is higher than100 nanomole.Animal experimental results show that sn38extend tumor bearing mou survival time and inhibit tumor growth.It also implies that SN38treatment induces glioma stem cell apoptosis and reduces the ratio of glioma stem cells in tumor tissue.Cell immunofluorescence experiment shows that SN38kills GSCs by inducing apoptosis and inhibiting proliferation.Western-blot experiment results show that GSCS topoisomera I protein level is lower after SN38treatment.Inducible knockdown topoisomera I lead to GSCs less nsitive to SN38.
贵州好玩吗Conclusion:A high throughput screening method bad on calculation of cell fluorescence intensity is able to obtain candidate chemicals targeting GSCs efficiently.TopI is the target of sn38on glioma stem cells.SN38shows antitumor effect by inducing apoptosis and inhibiting proliferation.
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Key words:GBM GSC high content screening specificity
幼儿园美术V
