【作者】 唐华非；

【导师】 刘松青；

【作者基本信息】 第三军医大学 ， 药剂学， 2010， 硕士

【摘要】 背景:羟基喜树碱(Hydroxycamptothecin,HCPT)是从珙桐科植物喜树(Camptotheca acuminata Decne)中提取得到的一种吲哚类生物碱,具有广泛的抗肿瘤活性,且与其它抗肿瘤药物无交叉耐药性。HCPT可选择性抑制拓朴异构酶而干扰DNA的复制,为拓扑异构酶Ⅰ特异性抑制剂。HCPT的抗癌作用与抗代谢药及烷化药不同,HCPT作用于S期细胞,为细胞周期性特异药物。对S期的作用较G1,G2期明显,对G0期细胞无作用。在较高浓度时对核分裂有抑制作用,阻止细胞进入分裂期。脑胶质瘤(Glioma)是神经外科难治性疾病之一,术后常易复发,因此术后化疗成为脑胶质瘤辅助治疗的重要手段。由于血脑屏障(blood brain barrier, BBB)的存在,多数化疗药物常规静脉给药后,难以在脑内达到有效浓度,若加大给药量又会导致全身性毒副作用的增加。近年来,间质化疗在脑胶质瘤治疗领域引起了广泛的关注,已成为脑胶质瘤治疗的重要手段。脑植入剂作为脑部间质化疗的一种新型制剂,不仅可以使化疗药物避开血脑屏障,直接作用于肿瘤组织,减少对正常组织的毒副作用,还具有缓控释特性,使药物能够在病变部位较长时间维持在有效治疗浓度,取得了较好的临床效果。本课题组前期采用聚乳酸(polylactic acid, PLA)为缓释材料,研制了HCPT脑内植入缓释片,并对其治疗大鼠实验脑胶质瘤的疗效进行了研究,显示HCPT缓释片脑内植入治疗能明显延长荷瘤大鼠的生存期。但HCPT脑内植入缓释片植于脑内后,在抑制肿瘤细胞增殖的同时,对大鼠的毒性的大小,特别是对脑组织的损伤程度是决定是否对该制剂进一步研究的重要因素,本实验拟对HCPT脑内植入缓释片的毒性进行研究,为该制剂进一步开发奠定基础。实验目的:(1)探讨大鼠大脑皮层神经细胞的体外培养方法,并对其形态学进行观察;(2)HCPT缓释片对体外培养大鼠大脑皮层神经细胞进行干预,观察是否具有细胞毒性作用;(3)在体考察HCPT缓释片对大鼠大脑组织结构和功能的损伤;(4)在体考察HCPT缓释片对大鼠心脏、肝脏、脾脏、肺以及肾脏组织的损伤;(5)在体考察HCPT缓释片对大鼠骨髓的抑制作用。(6)在体考察HCPT缓释片对胎鼠的致畸毒副作用及对胎鼠脑组织结构的影响。实验方法:(1)取新生SD乳大鼠的大脑皮层组织,采用胰酶消化、筛网过滤、离心等技术获取大脑皮层神经细胞并进行培养;(2)通过倒置显微镜、免疫细胞化学等方法对SD乳大鼠大脑皮层神经细胞进行定性和定量鉴定;(3)采用CCK-8检测HCPT对体外培养大鼠大脑皮层神经细胞的毒性;(4)采用Hoechst33258染色检测HCPT致体外培养大鼠大脑皮层神经细胞凋亡的程度;(5)将不同剂量的HCPT缓释片植入大鼠脑内,分别取30d和120d的实验大鼠,灌注后,取脑、心脏、肝脏、脾脏、肺以及肾脏组织,通过石蜡切片、HE染色观察各组织的形态结构变化;(6)分别对术后30d和120d实验大鼠脑组织标本进行Nissl染色,观察神经元胞浆内尼氏体的数量,评价神经元蛋白合成的能力;(7)分别对术后30d和120d实验大鼠脑组织标本进行TUNEL染色,观察受损脑组织神经元的凋亡比例;(8)取术后30d大鼠股骨骨髓,涂片后进行瑞氏染色(wright’s stain),观察有核细胞的比例。(9)将HCPT缓释片植入孕鼠脑内,在分娩1d时取孕大鼠及胎鼠脑组织,通过神经元体外原代培养、HE染色观察脑组织神经元的形态结构变化;实验结果:(1)经形态学、免疫细胞化学(NSE)鉴定所培养的大鼠大脑皮层神经元并鉴定其纯度大于95%;(2)CCK-8检测结果显示,①PLA对体外原代培养的大脑皮层神经元和星形胶质细胞生长影响不明显;②体外原代培养的大鼠大脑皮层神经元经以40μg/ml HCPT处理24h后,与正常对照组比较,细胞活性显著降低(P<0.05);HCPT浓度达到320μg/ml时,降低程度十分显著(P<0.01);③体外原代培养的大鼠大脑皮层星形胶质细胞经20μg/ml HCPT处理24h后,与正常对照组比较,细胞活性显著降低(P<0.05);浓度达到80μg/ml时,降低程度十分显著(P<0.01);④体外模拟HCPT缓释片处理神经细胞较单独使用HCPT,对细胞的毒性更明显。(3)Hoechst33258染色结果显示,①PLA致体外原代培养大鼠大脑皮层神经元和星形胶质细胞凋亡不明显;②体外原代培养的大鼠大脑皮层神经元经80μg/ml HCPT处理24h后,与正常对照组比较,细胞活性显著降低(P<0.05);HCPT浓度达到160μg/ml时,降低程度十分显著(P<0.01);③体外原代培养的大鼠大脑星形胶质细胞经40μg/ml HCPT处理24h后,与正常对照组比较,细胞活性显著降低(P<0.05);HCPT浓度达到80μg/ml时,降低程度十分显著(P<0.05);④体外模拟HCPT碱缓释片处理神经细胞较单独使用HCPT,细胞凋亡比例无显著差异。(4)经大鼠体内实验,大脑皮层组织免疫组化染色结果显示,PLA对皮层组织神经细胞的数量和活性没有显著的影响;HCPT缓释片对皮层组织神经细胞的数量和活性有一定的影响;低剂量HCPT缓释片植入大脑皮层后,与假手术组比较,脑组织未见病理学改变;高剂量HCPT缓释片对大脑皮层组织神经元的数量和活性具有显著影响。(5)经大鼠体内实验,HE染色显示,高剂量HCPT缓释片对肝脏具有一定的损伤,出现肝水肿,而心脏、脾脏、肺以及肾脏组织未见病理学改变。(6)经大鼠体内实验,瑞氏染色显示,未见HCPT缓释片骨髓抑制。(7)经神经元体外原代培养,模型组胎鼠神经元体外发育良好,与正常对照组比较未见形态学差异;经大鼠体内实验,HE染色显示,模型孕鼠脑组织有一定的炎症反应,而胎鼠脑组织未见病理学改变。结论:(1)采用无血清培养方法成功培养出了数量多、纯度高的大脑皮层神经元;(2)PLA对体外原代培养的大鼠大脑皮层星形胶质细胞和大鼠大脑皮层神经元毒性作用不显著。(3)HCPT对体外原代培养的大鼠大脑皮层星形胶质细胞和大鼠大脑皮层神经元均表现出一定的毒性作用,且对大鼠大脑皮层星形胶质细胞的毒性强于对大鼠大脑皮层神经元的毒性;(4)经大鼠体内实验,PLA空白片对脑组织结构和功能的损伤不显著;(5)经大鼠体内实验,HCPT缓释片对脑组织有一定的毒性,且随HCPT剂量的增加,毒性增大;低剂量和中剂量HCPT缓释片对心脏、肝脏、脾脏、肺及肾脏未见病理学改变;高剂量HCPT缓释片除对肝脏有一定的损伤外,对其它脏器组织未见病理学改变;(6)经大鼠体内实验,未见HCPT缓释片骨髓抑制。(7)经大鼠体内实验,HCPT缓释片对胎鼠脑组织未见毒性;更多还原

【Abstract】 BackgroundHydroxycamptothecin(HCPT) is an indole alkaloid extracted from Camptotheca acuminata Decne, which have a wide range of anti-tumor activity, and no cross-resistance to other anticancer drugs. The anticancer of HCPT is different from anti-metabolite and alkylating drugs. As the specific drug for cell periodic, HCPT can effect the cells which in S phase, more significantly than those in G1, G2 phase, and HCPT have no effect on the G0 phase cells. Cell mitosis can be inhibited by HCPT at higher concentrations. In addition, HCPT is the specific inhibitor of topoisomeraseⅠwhich can interfere with DNA replication by selectively inhibiting topoisomerase.Glioma is one of the hardest remedial diseases, which often recurs after surgery, therefore, the chemotherapy after surgery is essential. Most chemotherapeutics are difficult to achieve therapeutic level within brain tissue due to the Blood Brain Barrier(BBB) via vein injection. Serious side effect may be caused by increasing dose. In recent years, interstitial chemotherapy through local embbeding, which wrapped the chemotherapy drugs into biodegradable polymer, has brought widespread attention. However, implant as a new dosage form, it not only bypass the BBB to administer directly into the brain tissue, but also extend the functional time of drug with high concentration result from sustained-release.Using Polylactic acid (PLA) as release material, we have prepared the HCPT sustained-release tablet. Its effect on Glioma have been studied. It showed that HCPT sustained-release tablet can prolong survival time of tumor-bearing rats. The experiment imitate to stduy the toxicity of the HCPT sustained-release tablet which implanted into rat brain. The foundation would be expected to lay for the further development of the preparation.Objective(1)To isolate and to identify the cerebral cortical neurons in vitro methods. (2)To observe the cytotoxicity of HCPT sustained-release tablet through intervening the cultured nerve cells in vitro.(3)To study the damage of HCPT sustained-release tablet on brain organization and function in vivo.(4)To study the damage of HCPT sustained-release tablets on heart, liver, spleen, lung and kidney in vivo.(5)To study the suppression of HCPT sustained-release tablets on the bone marrow in vivo.(6)To study the teratogenic effect of HCPT sustained-release tablets to fetal rat, and evaluate the change of the fetal brain tissue in vivo.Methods(1)The cerebral cortex nerve cells of SD newborn rats were prepared by filtering through a mesh, centrifugation and trypsogen digestion.(2)Neurons were affirmed by immunocytochemical method and morphologies under Light Microscope.(3)The toxicity of HCPT on cerebral cortex nerve cells were evaluated by CCK-8 assay.(4)The cerebral cortex nerve cells’apoptosis were detected by Hoechst33258 staining.(5)Obtain the brain, heart, liver, spleen, lung and kidney tissue of rats after reperfusion at the 30 day and the 120 day from experimental points, then observe the morphological changes in the organization through paraffin and HE staining.(6)The number of Nissl bodies in neurons’cytoplasm were viewed to evaluate the capacity of protein synthesis by Nissl staining at the 30 day and the 120 day from experimental points.(7)The neuronic apoptosis ratio of damaged brain tissue was observated by TUNEL at the 30 day and the 120 day from experimental points.(8)The bone marrow suppression of HCPT sustained-release tablet was observated by bone marrow smear and Wright stain at the 30 day from experimental points.(9)we implant HCPT sustained-release tablets to the brain of pregnant mice, taking Brain of pregnant rats and fetal rats after 1d delivery. Neurons’primary culture in vitro, HE staining to Observation neurons morphological changes. Results(1)The purity of cerebral cortex neurons were more than 95% based on the morphology and immunocytochemistry(NSE) identification in vitro.(2)The results of CCK-8 Test show that:①There was no significantly cytotoxicity of polylactic acid on primary cultured cerebral cortex neurons and astrocytes.②Compared with the control group, significant differences emerged when 40μg/ml HCPT treated cortical neurons(P<0.05); The differences were obvious when the concentration of HCPT achieve to 320μg/ml(P<0.01).③Compared with normal control group, significant differences emerged when 20μg/ml HCPT treated cerebral cortex astrocytes(P<0.05); The differences were obvious when the concentration of HCPT achieve to 80μg/ml(P<0.01).④The cytotoxicity of simulated HCPT sustained-release tablet in vitro was more obvious than HCPT.(3)The results of Hoechst33258 staining showed that:①The apoptosis of cerebral cortex neurons and astrocytes were not obvious induced by polylactic acid;②Compared with the control group, significant variances emerged after 80μg/ml HCPT treated cortical neurons(P<0.05); The differences were obvious when the concentration of HCPT achieved to 160μg/ml(P<0.01);③Compared with normal control group, significant variances emerged after 40μg/ml HCPT treated cerebral astrocytes(P<0.05); the differences were obvious when the concentration of HCPT achieved to 80μg/ml(P<0.01);④The apoptosis ratio of cerebral cortex nerve cells were no significant difference between simulated HCPT sustained-release tablet in vitro than HCPT.(4)After the experiment of rats in vivo, the results of immunohistochemical staining of cerebral cortex showed that:①Polylactic acid have no significant impact in the number and activity of nerve cells;②The cerebral cortex damage of HCPT sustained-release tablets dependented HCPT dose;③Compared with the sham group, no significant differences emerged on the histology of Low-dose tablets of HCPT after implanted into the cerebral cortex;④High dose of HCPT sustained-release had a significant effect on the number and activity of cerebral cortex neurons.(5) After the experiment of rats in vivo, the results of HE staining showed that high doses of HCPT sustained-release tablets had some damage to the liver, and the liver edema emerged. The heart, spleen, lung and kidney did not change significantly. (6) After the experiment of rats in vivo, the results of Wright’s stain showed that HCPT sustained-release tablets had no bone marrow suppression.(7) Having a primary culture of the neurons. neurons of Model groups’fetal rats were well-developed in vitro and there were no Significant difference compared with the control groups; Through Experiment of rats in vivo, HE staining demonstrate that there are a certain degree of toxicity whith HCPT release tablets to pregnant rat’s brain, while the influence was not obvious for fetal rat’s brain tissue.Conclusions(1)A large number of cerebral cortex neurons with high purity had successfully trained used by the serum-free culture methods.(2) The toxic effect of cerebral cortex neurons and astrocytes induced by polylactic acid was not obvious.(3) There was invariably toxicity of cerebral cortex neurons and astrocytes induced by HCPT. The cerebral cortex neurons have more sensitivity to HCPT than astrocytes.(4)The damage of the structure and function of brain tissue was not obvious treated by Polylactic acid in vivo.(5)HCPT sustained-release tablets had some toxicity on the brain, and the higher dose of HCPT, the higher toxicity. Low dose and middle dose of HCPT sustained-release tablets had no clear toxicity on heart, liver, spleen, lung and kidney. In addition to some damage on the liver, high dose of HCPT sustained-release tablets had no significant toxicity to other organs on the histology in vivo.(6)HCPT sustained-release tablets had no bone marrow suppression in vivo.更多还原