【作者】 苏静；

【导师】 孙连坤；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的：1.特异性抑制C1C-3表达对人胶质瘤U251细胞生物学特性的影响；2.抑制U251细胞C1C-3表达观察其对顺铂敏感性变化及探讨机制。方法：1.MTT法检测细胞生存率；2.构建pSH1Si-C1C-3重组质粒；3.细胞转染；4.细胞凋亡检测；5.AO染色检测细胞内酸性小泡；6.溶酶体示踪红染色；7.电镜检测细胞超微结构变化；8.MDC检测自噬泡形成；9.RT-PCR检测mRNA表达变化；10Western-blot检测蛋白表达变化；11.DCFH-DA检测细胞内ROS产生；12.数据进行统计学分析。结果：1.用特异性反义寡核苷酸有效抑制U251细胞C1C-3基因和蛋白表达(抑制率>50%),对U251细胞生物学特性影响：①抑制U251细胞增殖,诱导U251细胞发生凋亡；②降低U251细胞体外侵袭力和迁移力,抑制MMPs基因表达；③抑制内吞体-溶酶体酸化；④增加胞浆中氯离子浓度。2.抑制氯通道C1C-3表达增加人胶质瘤U251细胞对顺铂的敏感性：①顺铂对U251细胞生长抑制作用具有时间依赖性和剂量依赖性,U251细胞对顺铂具有相对较高耐药性；②抑制ClC-3表达能明显增加U251细胞对顺铂的敏感性；③顺铂作用于U251细胞诱导细胞发生自噬,抑制C1C-3表达能抑制顺铂诱导的U251细胞自噬；④顺铂诱导细胞凋亡具有时间依赖性,抑制C1C-3表达能明显增加顺铂诱导U251细胞凋亡,可使细胞凋亡窗口前移；⑤顺铂可诱导U251细胞Akt活化增加,抑制C1C-3表达可明显抑制顺铂诱导Akt活化。3.抑制氯通道C1C-3表达增加人胶质瘤U251细胞对顺铂敏感性机制探讨：①抑制ClC-3表达通过NADPH氧化酶依赖途径降低顺铂诱导ROS生成增多。②抑制C1C-3表达能明显抑制顺铂诱导的Nox2、Nox4 mRNA表达增加,并能降低hVps34 mRNA表达；③抑制C1C-3表达能明显抑制顺铂诱导的Akt/mTOR途径活化；NADPH氧化酶抑制剂DPI可抑制顺铂诱导的Akt/mTOR途径活化；用3-MA抑制hVps34,对顺铂诱导的Akt/mTOR途径活化无明显影响；④顺铂可同时诱导U251细胞发生凋亡和自噬,应用自噬抑制剂3-MA能明显增加顺铂诱导细胞凋亡；DPI抑制NADPH氧化酶活性,可增强顺铂诱导细胞自噬,但由于其抑制Akt/mTOR途径活化,故也增加顺铂诱导细胞凋亡；抑制ClC-3表达可抑制顺铂诱导自噬发生,增加凋亡。结论：1.特异性反义寡核苷酸可有效抑制U251细胞C1C-3 mRNA和蛋白表达,抑制C1C-3表达可影响内吞体和溶酶体酸化,改变细胞内氯离子浓度；抑制U251细胞生长,诱导U251细胞发生凋亡,但程度不高,即难以依靠单独抑制C1C-3基因表达的作用达到肿瘤治疗的目的；降低MMPs基因表达,抑制U251细胞体外侵袭力和迁移力。2.构建pSH1Si-C1C-3表达质粒能显著抑制U251细胞C1C-3 mRNA和蛋白表达(抑制率＞80%)；抑制ClC-3表达能明显增加U251细胞对顺铂的敏感性,原因包括：顺铂可诱导U251细胞发生自噬和凋亡,抑制ClC-3表达通过抑制自噬增加顺铂诱导的细胞凋亡；Akt激活需要ClC-3,抑制ClC-3表达可明显抑制顺铂诱导Akt活化。3.抑制ClC-3表达影响由NADPH氧化酶介导的顺铂诱导U251细胞ROS产生,从而抑制顺铂诱导Akt/mTOR途径活化；抑制ClC-3表达影响细胞自噬主要通过影响内吞体-溶酶体酸化和Beclinl/Vps34途径。更多还原

【Abstract】 Neurospongioma is one of the most frequent cerebral tumor, it was account 40%-50% in intracranial tumor, invasive growth, because its limit is not so clear between tumor tissue and nomal tissue, so it is so difficult to eliminate by operation, and it is very ease to recur; Otherwise neurospongioma can resist the routine antitumor drug, and its prognosis is not good, the survival rate of the neurospongioma is not enough 12 month. Recently, following the deeply research on tumor molecular biology, tumor gene therapy has been the most interest therapy.CLC-3 is one of the most important channel protein of CLC family, now as research on CIC-3, found CIC-3 was expressed in many kinds of tumor cells, include neurogliocytoma. CIC-3 participate intracellular internalization-cytolysosome acidation、chloridion disposition regulation some anti-tumor drug resistance form. CIC-3 as a intracellular chloridion pathway, normally condition mainly location in internalization-cytolysosome system and nerves cell synaptic vesicle, few expressed on cytolemma. It is considered that CIC-3 protein transportation through the cytomembrane, at least partly CIC-3 protein transportation pass cytomembrane after it was synthesis, then expressed in internalization-cytolysosome system, participate alveolar acidation and NADPH oxidase generate ROS.Autophagy is conservative intracellular long life protein,and large protein aggregate and cell organ, it can maintenance cell stable in normal and stress condition, that is very important in normal development process and changed environment stress. There are some dispute about autophagy role in tumor development and cure therapeutic reaction, it need further approach. In physiology、pathology and pharmacology condition stimulate, autophagy can extend cells life. It is very complicate between autophagy and apoptosis, still exit argument. A hyothesis is autophagy is to inhibit apoptosis for cells.The CLC-3 specificity antisense oligonucleotides and CLC-3 siRNA recombinant plasmid were used in this study, they can utility inhibited express of CLC-3 protein, observe the influence on the biological features of U251 after CIC-3 was been efficiently inhibited. Further approach the mechanism and the possibility of CIC-3 as a new gene cure target.Objective:1. The Influence on the biological features of glioma cells induced by supression of Intracellular chloride channel CLC-3 in human neurogliocytoma cell U251.2.Establish stable transfection CLC-3 siRNA recombinant plasmid U251 cells, to observed the sensibility for cis-diamminedichloroplatinum and approach the mechanism.Method:(1)Use MTT to detect the cells survival rate:(2)Build the pSH1Si-CIC-3 recombinant plasmid; (3)Cells transfection; (4)Detect the cells apoptosis; (5)Detect the intracellular acidity bullule; (6) cytolysosome tagging red staining; (7)Use electron microscope to detect the changing of the ultramicrostructure; (8)Detect MDC autophagic vacuole formation with MDC method; (9)Use RT-PCR to detect the changing of mRNA; (10)Use Western-blot to detect the changing of protein expression; (11)Use DCFH-DA staining to detect the generation of ROS intracellar; (12)Statistics analysis; Results:1.Use special antisense oligonucleotides can inhibited CLC-3 gene and protein expression in U251 cell(inhibition rate>50%), the Influence on the biological features of U251 cells; (1)The MTT results show that inhibit CLC-3 expression can inhibit the cell proliferation in U251cells;(2)TUNEL and PI/Annexin V double staining results show that inhibit CLC-3 expression can be induced U251 cells to apoptosis;(3)Tanswell results show that inhibit CLC-3 expression can be down regulated the invasiveness and immigration of U251 cells, and inhibited the expression of MMPs gene;(4)AO staining and cytolysosome tagging red staining results show that the inhibition of CLC-3 expression can be inhibited internalization internalization-cytolysosome acidation; (5)MQAE fluorescent probe signal show that chloridion concentration can be up regulation in endochylema through inhibit CLC-3 expression; All of these results hint that CLC-3 played important biological role in U251 cells, the expression inhibition of CLC-3 can be effected the concentration of C1 in U251 cells, influence the function of internalization and cytolysosome, and so CLC-3 can be effected the biological feature of U251 tumor cells. Even though CLC-3 inhibition can induced U251 cell to apoptosis, inhibit cell proliferation, but it was not enough, it was so hard to cure tumor through out inhibited CLC-3 expression.2.Up regulated the sensitive of human neurogliocytoma U251 cell for DDP through out inhibit CLC-3 protein expression:(1)Successful build recombination plasmid pSH1Si-CIC-3, transfection U251 cells, build stable transfection cell line;(2)RT-PCR and Western-blot results show that CIC-3 mRNA and protein expression down regulate>80% in stable transfection pSH1 Si-scramble plasmid U251 cells;(3)Survive rate of every group were detected by MTT method, calculate cell inhibition ratio, results show that the role of DDP in inhibition for U251 cells were time dependence and dose dependent;(4)according to the calculating IC50 results of MTT for DDP in U251 cells that this kind of inhibition was relative dependence;(5)MTT results show that the inhibition of CIC-3 expression can significant up regulate the sensitive for DDP in U251 cells;(6)The labeling protein LC3-II was detected by Western-blot method, use electron microscope to observe the ultramicrostructure of U251 cells, associate with MDC staining, AO staining to detect cell autophagia, the results show that U251 cells were induced to take place cell autophagia by DDP, this kind of role can be inhibited through out inhibited CIC-3 expression.(7)Use P1/Annexin V double staining to detect cells apoptosis, Flow cytometry results show that the apoptosis induced by DDP was time dependent, the inhibition of CIC-3 expression can significantly up regulate inducing apoptosis for U251 cells, and it can also be moved front in the apoptosis plot.(8)Detect the activity of Akt by Western-blot, the results show that DDP can be induced U251 cells to active Akt, inhibit CIC-3 express can significant suppress this kind of activity for Akt, so CIC-3 was needed for active Akt.3. Study on up regulated the sensitive of human neurogliocytoma U251 cell for DDP through out inhibit CLC-3 protein expression:(1)Use DCFH-DA to detect ROS generation, observe that inhibit CIC-3 and pre-treat with NADPH oxidase inhibitor DPI for U251 cell can significant down regulate ROS generation that induced by DDP, inhibite NADPH oxidase can decrease ROS generation through inhibit CIC-3 expression.(2)Detect Nox2、Nox4、hVps34 mRNA expressed in U251 cell by RT-PCR, Nox2 and Nox4 mRNA can be induced to up regulate by DDP, but DDP can not effect expression of hVps34 mRNA; The up regulate of Nox2 and Nox4 mRNA induced by DDP can be significantly inhibited through inhibit CIC-3 expression, the same time it can also down regulate the expression of hVps34 mRNA;(3)Use Western-blot to detect the activity condition of Akt/mTOR, results show that Akt,mTOR and mTOR substrate(p70SK) phosphorylation were induced up regulation by DDP in U251 cells, demonstrate that DDP can be used to inducing the activation of Akt/mTOR pathway; Inhibit CIC-3 can also significant inhibit this kind of activation;(4)Detect the autophagia labeling protein LC3-Ⅱaggregation, Beclin1/Atg8 protein expression, Atg5-Atg12 complex form, and associated with MDC staining、AO staining to observe intracellular acid bullule generate increased, sure it was happened autophagy; At the same time detect activity of Capase3, detect apoptosis induced by Hoechst33342;All of results account that at the same time DDP can induce U251 to apoptosis and autophagy, use autophagy inhibitor 3-MA can significantly up regulate cells apoptosis induced by DDP; DPI can inhibit activity of NADPH oxidase, it can reinforce DDP to induce apoptosis; inhibit CIC-3 can suppress DDP to induce autophagy, up regulate apoptosis.Conclusion:1. Specialized antisense oligonucleotides can efficent inhibit CIC-3 mRNA and protein expression in U251 cells; Intracellular acidation can be effected by inhibition of CIC-3; The concentration of intracellular C1 is concern with the inhibition of CIC-3; Inhibit CIC-3 would effect U251 cells biological feature; Inhibit CIC-3 expression would also inhibit U251 cell proliferation; U251 cell can be induced to apoptosis by inhibit CIC-3 expression, but the extent was not enough, so it is hard to cure tumor by inhibit CIC-3 expression singly; Inhibit CIC-3 expression can down-regulate MMPs gene expression in U251 cells, so that inhibit U251 cell invasion and immigration ability.2. Build pSH1Si-CIC-3 expression plasmid can significantly inhibit CIC-3 mRNA and protein expression in U251 cell(inhibition ration>80%); The inhibition of DDP for U251 cell was time and dose dependent; U251 has relative higher drug resistance for DDP, so U251 sensitive for DDP was changed when inhibit CIC-3 expression; DDP can be induced U251 cell to apoptosis and autophagy, and inhibit CIC-3 can significantly inhibit the apoptosis inducing role by DDP; Autophagy can postpone the apoptosis that induced by DDP, at the same time significant up regulate DDP to induce U251 to apoptosis, so it is account that autophagy can postpone the apoptosis, and this is one of the most important reason that inhibit CIC-3 can up regulate U251 sensitive for DDP; DDP can induce Akt active in U251 cell, and inhibit CIC-3 can significantly inhibit DDP active Akt, so it is account that Akt active need CIC-3. This is an other important reason for inhibit CIC-3 up regulation sensitive for DDP.3. NADPH oxidase participate the pathway that DDP induce U251 cell to up regulate the generation of ROS. NADPH oxidase participate the pathway which was induced Akt/mTOR activity by DDP, inhibit CIC-3 can also inhibit Akt/mTOR activity induced by DDP. Inihibt NADPH oxidase can up regulate autophagy that induced by DDP, and inhibit CIC-3 expression and inhibit autophagy mainly through out effected internalization body and Beclin1/Vps34 pathway, inhibit autophagy induced by DDP. The expression changing of CIC-3 can effect Akt/mTOR pathway and autophagy at the same time, to up regulate DDP cytotoxic effect for U251 cells. The effect of CIC-3 of cell autophagy mainly include two reasons:CIC-3 plays important role in internalization and cytolysosome acidation, so CIC-3 can effect autophagy function through out inhibit internalization and cytolysosome acidation; Otherwise CIC-3 can effect autophagy by direct of indirect inhibit hVps34 fuction.更多还原