【作者】 王刚；

【导师】 曾南；

【作者基本信息】 成都中医药大学 ， 中药学， 2012， 博士

【摘要】 目的：以槐米为原药材,通过水解法提取并精制槲皮素,采用乳化蒸发-低温固化法制备槲皮素纳米脂质体(QUE-NL),考察其在大鼠体内口服吸收特性、小鼠脑靶向性及药动学参数；考察QUE-NL在大鼠血浆中存在的物质形式及大鼠脑靶向性。方法：采用乳化蒸发-低温固化法,山嵛酸甘油酯、大豆卵磷脂和胆固醇为油相,泊洛沙姆、聚乙二醇-二硬脂酰磷脂酰乙醇胺和吐温-80作为水相,以正交试验法优化的处方与制备工艺制备槲皮素纳米脂质体(QUE-NL);透射电镜观察其微观形态,并测定其粒径分布、包封率和载药量；建立高效液相色谱法测定QUE-NL在大鼠胃肠道内容物及粪样混合物中的槲皮素含量的方法,计算其在大鼠胃肠道口服吸收百分率；采用高效液相色谱法测定小鼠血浆及脑组织中槲皮素浓度,计算药物靶向指数(DTI)；采用高效液相色谱法测定口服QUE-NL在大鼠体内不同时间点的血药浓度,用3p97药动学软件处理数据,计算相关药动学参数；采用高效液相色谱法检测大鼠血浆中槲皮素存在形式及脑组织中槲皮素浓度,计算药物靶向指数(DTI)。结果：以乳化蒸发-低温固化法和优选的制备工艺制得的QUE-NL呈球状或类球状,平均粒径为172.63nm,包封率为85.90%,载药量为7.25%；QUE-NL在大鼠体内吸收优于槲皮素原料药和槲皮素普通脂质体；QUE-NL在大鼠体内的血药浓度显著高于槲皮素原料,其药时曲线下面积较槲皮素原料大,表观分布容积较槲皮素原料小,血浆清除率较槲皮素慢；QUE-NL在小鼠脑内吸收分布优于槲皮素原料药和槲皮素普通脂质体,QUE-NL药物靶向指数(DTI)均大于1；QUE-NL在大鼠血浆中主要是以槲皮素苷元和异鼠李素存在,且其在大鼠脑内吸收分布优于槲皮素原料药和普通脂质体,QUE-NL药物靶向指数(DTI)均大于1。结论：乳化蒸发-低温固化法适于制备槲皮素纳米脂质体,以正交法优化法制备的槲皮素纳米脂质体,能显著提高大鼠对槲皮素的口服吸收量,延长槲皮素在血浆中的循环时间,代谢半衰期更长,AUC更大,表明其具有长循环特征；槲皮素纳米脂质体能显著促进槲皮素在小鼠、大鼠脑内的吸收,具有良好的脑靶向性；吸收进入血浆中的槲皮素主要是以结合形式而非单体形式存在,为进一步应用于脑胶质瘤的治疗提供了实验依据。目的：研究槲皮素纳米脂质体(QUE-NL)对体外培养的大鼠C6胶质瘤细胞增殖与凋亡的调控作用,探讨其诱导C6胶质瘤细胞凋亡作用的线粒体途径与JAK2/STAT3信号通路机制。方法：采用体外培养大鼠C6胶质瘤细胞,采用MTT比色法检测不同药物(QUE、QUE-NL、QUE-L、替莫唑胺等)作用于大鼠C6胶质瘤细胞12,24,48和72h后的增殖抑制情况,流式细胞术(FCM)检测不同药物作用24h后大鼠C6胶质瘤细胞凋亡及死亡情况；LDH活性法检测对C6脑胶质瘤细胞的毒性作用。检测C6脑胶质瘤细胞活性氧(reactive oxygen species, ROS)、 SOD和MDA水平,比色法测定C6胶质瘤细胞内caspase-8、 caspase-9及caspase-3的活性；线粒体膜电位试剂盒(JC-1)检测线粒体膜电位变化；流式细胞分析技术检测C6脑胶质瘤细胞ROS水平；Western-blot法检测C6胶质瘤细胞caspase-8、 caspase-9及caspase-3的蛋白表达；检测C6脑胶质瘤细胞ATP和LDH释放水平,探讨其诱导凋亡及促坏死的可能线粒体信号传导途径机制。进一步,采用AG490(STAT3信号抑制剂)干预,从检测细胞毒,细胞凋亡,细胞因子IL-6、TNF-α的分泌,及STAT3信号通路JAK2、STAT3蛋白、p53与抗凋亡蛋白Bcl-2的表达等方面,探讨其通过JAK2/STAT3信号传导途径诱导C6胶质瘤细胞凋亡的可能机制。结果：与空白对照组比较,QUE-NL处理组随药物浓度增加和作用时间的延长,对C6细胞的增殖抑制作用增强,具有剂量依赖性；与QUE比较,QUE-NL给药组凋亡细胞及坏死细胞所占比例显著增多,尤以坏死细胞比例明显增多,QUE-NL能显著诱导C6胶质瘤凋亡和促进坏死；QUE-NL组的LDH释放量、ROS水平较空白对照组显著增加。QUE-NL显著提高C6胶质瘤凋亡细胞的caspase-3、 caspase-9的活性,促进C6胶质瘤细胞LDH释放、诱导C6胶质瘤细胞坏死,同时显著降低线粒体膜电位。QUE-NL能使C6胶质瘤细胞cytochromeC蛋白表达加强,对坏死caspase-9与caspase-3的蛋白表达无明显影响,显著升高C6脑胶质瘤细胞ATP和LDH的释放水平。QUE-NL使前凋亡蛋白Bax表达明显升高,抗凋亡蛋白Bcl-2表达降低,促进P53蛋白表达和抑制Bcl-2蛋白表达。与空白对照组比较,QUE-NL高、中剂量组JAK2和STAT3蛋白表达显著增加,AG490则显著抑制QUE-NL的促C6胶质瘤细胞坏死作用,显著抑制STAT3和p-STAT3的蛋白表达,但AG490对QUE-NL上调C6胶质瘤细胞p53蛋白表达的作用无显著影响。结论：QUE-NL能诱导大鼠C6胶质瘤细胞的凋亡,并促进肿瘤细胞的坏死,具有细胞毒性作用,表现出浓度、时间依赖性。其诱导细胞凋亡和促进细胞坏死作用机制与调节C6脑胶质瘤细胞活性氧(reactive oxygen species, ROS)、 SOD和MDA水平有关；QUE-NL增加凋亡的C6脑胶质瘤细胞中caspase-3和caspase-9的相对活性,对caspase-8活性影响不显著,提示槲皮素通过激活caspase活性诱导肿瘤细胞凋亡。QUE-NL促坏死的机制可能通过线粒体途径调节C6脑胶质瘤细胞ROS水平、降低线粒体膜电位与调节CytochromeC活性两种方式,促进大鼠C6胶质瘤细胞坏死；其通过活化线粒体信号传导途径caspase-9与caspase-3的活性诱导C6胶质瘤细胞凋亡,达到抗大鼠C6胶质瘤作用。不同浓度的QUE-NL可能从ROS/线粒体途径促进C6脑胶质瘤细胞凋亡或坏死,还可能经JAK2/STAT3信号转导途径调控C6脑胶质瘤细胞凋亡或坏死,主要调节JAK2/STAT3信号转导途径的关键信号分子(TNF-α、IL-6)和关键基因蛋白(JAK2、 STAT3、 Bax等)。两种途径交汇作用于线粒体,可能是其抗C6胶质瘤细胞的主要信号机制。目的：研究槲皮素纳米脂质体(QUE-NL)体内对C6胶质瘤的抑制作用,比较槲皮素原料药与QUE-NL的抗胶质瘤作用,评价QUE-NL在脑肿瘤组织的脑靶向性及其毒副作用。方法：利用大鼠脑立体定位仪进行C6胶质瘤细胞近皮层原位接种建立体内模型,分别给予槲皮素原料药、槲皮素纳米脂质体及替莫唑胺等药。给药治疗三周后,测定其肿瘤直径、计算抑瘤率,观察大鼠的一般生长情况。末次给药2小时后断头取血测定肝功能。按照高效液相色谱法测定血浆与肿瘤组织槲皮素浓度,测定脑组织中槲皮素含量,并计算药物靶向指数(DTI)；另取肿瘤组织固定后,常规石蜡包埋标本,石蜡切片,HE染色。免疫组织化学染色法检测大鼠脑肿瘤组织JAK2、STAT3、p53和survivin蛋白表达情况,并取大鼠心、肝、肾做常规病理切片。结果：QUE-NL高、低剂量组(50mg.kg-1、25mg.kg-1)能明显减小肿瘤体积。免疫组织化学染色结果显示,空白对照组肿瘤组织JAK2和survivin蛋白表达呈强阳性,核外STAT3蛋白表达呈强阳性,肿瘤细胞核内p-STAT3蛋白表达明显；QUE-NL治疗组肿瘤组织JAK2、STAT3和survivin蛋白表达相对较弱、p-STAT3蛋白表达减少,p53蛋白表达呈强阳性。与槲皮素原料药相比,QUE-NL能显著增加C6胶质瘤大鼠血浆和脑组织的槲皮素浓度,表明聚山梨酯-80包衣的QUE-NL显著促进槲皮素进入血脑屏障。QUE-NL高、低剂量组的脑肿瘤组织DTI均大于1,显示出较好的肿瘤靶向性。槲皮素原料与QUE-NL对C6胶质瘤大鼠血清ALT, AST, γ-GT均无显著影响,仅替莫唑胺组荷瘤大鼠的ALT指标明显升高,替莫唑胺组显示有轻度肝肾损害,其余各组肝肾功能均无明显变化。结论：本研究制备的QUE-NL具有提高槲皮素抗脑胶质瘤生长的效果,具有肿瘤靶向性,并能降低药物的毒性作用。更多还原

【Abstract】 Objective:To prepare quercetin nanoliposomes (QUE-NL) and investigate its properties and the oral absorption character in rat. To investigate the absorption character of QUE-NL in brain tissue and plasma in mice and rat, and measure the pharmacokinetic parameters in rats and the existence form of QUE-NL in plasma and absorption character in rat brain tissue.Methods:Preparation of QUE-NL by the emulsification-evaporation and low temperature curing method and the optimum formulation with the optimal conditions which oil phase were mainly made of Glyceryl behenate (ATO), soy lecithin and cholesterol, and aqueous phase was made of poloxamer, PEG2000-DPSE and Tween80. The morphology of QUE-NL was examined by transmission electron microscope (TEM) and the particle diameter distribution was determined by laser particle size analyzer, and the drug loading and encapsulation efficiency of QUE-NL was determined by HPLC. The determination methods of quercetin in the nanoliposomes and in the mixture of gastrointestinal contents and feces were established by HPLC. Determination of oral absorption rate of QUE-NL in gastrointestinal in rat by HPLC. The serial blood samples were collected at designed time points and determination of quercetin in plasma and drug targeting index (DTI) of brain tissue in mice by HPLC. The pharmacokinetic parameters were calculated with the software3P97a. The determination of quercetin existence form in plasm and drug targeting index (DTI) of brain tissue in rat by HPLC. Result:The mean diametre of QUE-NL was172.63nm, and the nanoparticles morphology of QUE-NL was sphere or spherical.The entrapment efficiency was85.90%and the drug loading rate was7.25%. The oral absorption of QUE-NL in rat was better than that of quercetin suspension and common liposomes. The absorption of quercetin and common liposomes all increased with concentration of quercetin and showed linear correlation. Meanwhile, the absorption of QUE-NL with the increase concentration of quercetin and showed no linear correlation. The distribution of QUE-NL in mice brain tissue was better than that of quercetin suspension and common liposomes, and drug targeting index (DTI) of QUE-NL values were more than1in brain tumor tissue. The plasma concentration and area under the curve (AUC) of QUE-NL in rat was obviously higher than that of quercetin suspension. Volume of distribution (Vd) and plasma clearances of QUE-NL were lower than that of free quercetin. Quercetin mainly was in faglycone and isorhamnetin state, and the distribution of QUE-NL in rat brain tissue was better than that of quercetin suspension and common liposomes and drug targeting index (DTI) of QUE-NL values were more than1in brain tumor tissue.Conclusion:QUE-NL can improve the oral absorption of quercetin in mice and rat with optimal preparation of QUE-NL in this experiment. QUE-NL can prolong the circle time of quercetin in plasma for longer half-life and larger AUC and the results show that QUE-NL has long circulating effects. Quercetin was obsorbde in plasma in a combinative form but not in free state, QUE-NL can improve the absorption of quercetin in mice and rat brain tissue, which showed good brain targeting in mice and rat, so as to provide experimental foundation for further application for treatment of glioma. Objective:To study on the effects of quercetin nanoliposomes (QUE-NL) on proliferation and apoptosis on C6glioma cells in vitro. To study the role of mitochondrial pathway in the apoptosis or necrosis in C6glioma cell lines induced by QUE-NL. To investigate the effects and mechanism of QUE-NL induced apoptosis on C6glioma cells through of JAK2/STAT3signaling pathway. Furthermore, provided the evidence for further development and supplement of quercetin using for anti-glioma.Methods:Using emulsification-evaporation and low temperature curing method to prepare quercetin nanoliposomes.The cultured cells were divided into QUE and QUE-NL treatment groups according to the concentrations of QUE, blank control and DMSO as control groups. MTT assay was used to observe the proliferation on the cells treated for12,24,48and72h.Given the concentration of QUE-NL50、100、200、400umol/L,taking quercetin and temozolomide as control. The apoptosis and necrosis of C6glioma cells was observed by flow cytometry (FCM) after the cells were respectively treated with QUE and QUE-NL for24h. Detected the activity of LDH to study the cytotoxicity effect of quercetin on rat C6glioma cell. Detected the level of ROS,SOD and MDA and the activity of Caspase-3、8、9on glioma C6cell to explore the possible mechanism of rat C6glioma cell apoptosis and necrosis.To detect the effect of quercetin nanoparticles promoting on rat C6glioma cell apoptosis and necrosis by flow cytometry analysis technical. Detected the activity of LDH to study the cytotoxicity effect of quercetin on rat C6glioma cell. Detected the change of mitochondrial membrane potential by flow cytometry, and the proteins express of Caspase-3,8,9by western-blot methods to explore the possible mechanism of apoptosis and necrosis on rat glioma C6cell induced by QUE-NL through mitochondrial pathway.Given the concentration of quercetin nanoliposomes (the final concentrations were50,100,200and400μmol.L-1respectively), taking quercetin and temozolomide as control. The supernatant was collected to measure the secretion of cytokine TNF-a, IL-6and IL-8by ELISA, and measure expression of JAK2. The changes of the protein p53and Bcl-2were detected respectively after C6glioma cells were treated with QUE and QUE-NL for24h200μmol.L-1were detected by Western-blot methods, and to explore the possible mechanism of rat glioma C6cell apoptosis and necrosis induced by QUE-NL through JAK2/STAT3pathway.Results:With the augmentation of quercetin nanoliposomes and the extension of the treated time, the C6cell growth was inhibited, the OD values decreased (P<0.05) and the cell numbers were cut down. Quercetin nanoliposomes test groups have higher proportion of apoptotic cells and dead cells than blank control group and the proportion of dead cell increased significantly. QUE-NL groups have higher proportion of apoptotic and necrosIs cells than blank test groups and the proportion of necrosis cell increased significantly. The quantities of LDH released by rat C6glioma cells are significantly increased. The level of ROS, SOD and MDA all changed significantly. The proportion of apoptotic cells with Caspase-3、9activity increased, indicating that QUE-NL induced the apoptotic cells via mitochondrial pathway. QUE-NL induced cell death(necrosis) in C6glioma cells in a dose-and time-dependent manner, high concentrations of quercetin nanoliposome (QUE-NL) can induce necrotic cell death of which was distinct from apoptosis and autophagy. QUE-NL-induced mitochondrial membrane potential loss, cytochrome C released and had no effects on caspase activations, however, reductions in ATP levels and increases in LDH activity indicated that QUE-NL stimulated necrotic cell death.The proapoptotic protein of Bax and p53protein increased evidently, at the same time Bcl-2protein expression decreased after cultivated with QUE-NL by western blotting. The decreased expression of Bcl-2protein and the increased expression of p53protein were also observed after treatment with QUE. We also detected the activation of (JAK2/STAT3) and the inhabitation of JAK2regulator STAT3inhibited QUE-NL-induced C6glioma necrotic cell death upstream of the mitochondria. In addition, application of STAT3-specific inhibitors for defined periods showed that inhabited p53protein and activation of JAK2between24and36h. Furthermore, STAT3inhibitors failed to significantly inhibit QUE-NL-induced down-regulating of STAT3and p-STAT3protein on C6glioma cell death. Conclusion:Quercetin nanoliposomes have obviously cytotoxic on glioma C6cell and higher proportion of apoptotic cells and the proportion of necrosis cells increased significantly with different concertration of QUE-NL than blank control groups. Inhibitory effect of QUE-NL on C6cells was proved to be dependent on the treated time and the dose of QUE-NL. The possible mechanism of QUE-NL induced apoptosis and necrosis of C6glioma cells may regulate the level of ROS,SOD and MDA, and the induced apoptosis effect of C6glioma cells was implemented by activating mitochondrial pathway. C6glioma cells which treated with QUE-NL exhibited a cellular pattern related with necrosis and were not apoptosis and was characterized by caspase independence.Quercetin nanoliposomes can induce rat glioma C6cell apoptosis and promote cell necrosis, mitochondrial and JAK2/STAT3pathway are essential for QUE-NL-induced C6glioma necrosis and the JAK2/STAT3cascade has a crucial role in QUE-NL-induced C6glioma cell death. Quercetin nanoliposomes regulated JAK2/STAT3pathway resulted in down regulation of JAK2, STAT3, p-STAT3and Bcl-2protein expression, up-regulation of p53protein expression and ROS throug mitochondrial pathway may be the mained mechanisms for anti-glioma. Objective:To investigate anti-tumor effects of quercetin nanoliposomes on the growth of C6glioma in vivo. To compare the anti-glioma and toxic effect between QUE-NL and quercetin suspension, and evaluate the character of brain targeting.Methods:Utility of stereodirected instrument to establish the in situ in vivo C6glioma model by inoculate C6glioma cells into the cerebral cortex of SD rat, and investigate the effect of quercetin nanoliposomes on the rat C6glioma model. The C6glioma rats were divided into different groups by random selection,which were treated with different doses of QUE-NL or quercetin suspension (at dose of25mg/kg, and50mg/kg, every day for3weeks), temozolomide, blank nanoliposomes respectivyly after established the rat C6glioma model for1week. The size of tumor was measured and the living state and weight of C6glioma rats were observed. The concentration of quercetin in plasma and DTI of brain tumor tissues was determined by HPLC. The protein expression of JAK2、STAT3、p53and survivin was detected by immunohistochemistry methods. The parameters concerning heart, liver and kidney function were measured and the morphology of organ tissues was observed pathologically.Results:Compared with the control groups, the tumor volume was reduced obviously (P<0.05) in groups of QUE-NL and quercetin suspension at dose of25and50mg/kg can inhibit the growth of glioma in rat, and temozolomide also can inhibit the growth of glioma. There are significant different inhibit growth of tumor between quercetin nanoliposomes and quercetin suspension at dose of25,50mg/kg (P<0.05), and QUE-NL can inhibit the growth of glioma in rat more effective than quercetin suspension.There are stronger growth inhibition against C6glioma and no significant difference (P>0.05) in the groups of QUE-NL and temozolomide at dose of25and50mg/kg.The result of immunohistochemistry showed that the protein expression of JAK2STAT3and survivin was strongly positive in rat C6glioma model group, and the protein expression of p-STAT3increased significantly. The protein expression of STAT3and p-STAT3decreased significantly and protein expression of p53increased significantly in QUE-NL group.QUE-NL can increase significantly quercetin content and drug targeting index (DTI) values were more than1in brain tumor tissue, there are significant difference compared with quercetin suspension (P<0.05), which indicated that QUE-NL modified by polysorbate80can enhance quercetin pass the blood-brain barrier.QUE-NL and quercetin suspension have no obviouly effect on the activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (y-GT) in C6glioma rats, there were no changes in liver and kidneys function. While the activity of serum ALT increased significantly and liver damage were observed in temozolomide adminwastrated group. QUE-NL also showed made no morphologic changes to heart, liver, kidneys and spleen in C6glioma rats.Conclusion:It was demonstrated that QUE-NL can enhance effect of anti-glioma with lower dosage. The side-effect of QUE-NL was less than the same dosage of quercetin. QUE-NL had good targeting efficiency and DTI in C6glioma rats.更多还原