SCID小鼠人脑胶质瘤原位模型建立及蒿甲醚治疗作用研究

发布时间：2018-04-13 06:13

本文选题：青蒿素 + SCID小鼠　；参考：《昆明医科大学》2017年硕士论文

【摘要】：目的:首先观察蒿甲醚在体外对U251人脑胶质瘤细胞增殖的影响,随后建立SCID小鼠人脑胶质瘤原位模型,观察蒿甲醚在体内对胶质瘤的抑制及其抗血管生成的作用,探讨蒿甲醚抑制胶质瘤增殖的机制,为胶质瘤的临床治疗提供一种新的思路和方法。方法:1.体外细胞基中培养人脑U251胶质瘤细胞,采用MTT(四甲基偶氮唑蓝)法测定蒿甲醚对U251胶质瘤细胞的生长抑制作用,并计算半数抑制浓度(IC50);采用ELISA法检测蒿甲醚抑制U251胶质瘤细胞的VEGF表达。2.构建SCID小鼠人脑胶质瘤原位模型,采用立体定位仪定位麻醉状态小鼠,剪开头皮后,在距矢状缝与冠状缝交点处分别向右向后2mm定位注射点,钻孔后,使用10ul微量注射器垂直于颅骨在小鼠脑部定位点(右侧顶叶区)深5mm处,注入10ulU251人脑胶质瘤细胞,骨蜡填封骨孔,可吸收线缝合头皮,观察小鼠生活状态,在小鼠出现癫痫症状后,经活体左心室灌注后取脑组织肉眼观察肿瘤大体外观及切面,并行HE染色确证肿瘤模型成功构建。3.构建24只SCID小鼠U251人脑胶质瘤原位模型,随机分为3组,分别为对照组(生理盐水0.4ml/10g/d)、蒿甲醚组(蒿甲醚50.0mg/kg/d)、替莫唑胺组(20mg/kg/d)。替莫唑胺采用其粉剂制成混悬液灌胃,量为1.65mg/只/d。接种后标记小鼠,每天定时定量给药,并记录生存时间。约接种20天后,当SCID小鼠濒临死亡时行活体左心灌注,取出并固定全部脑组织,分别行肉眼观察、HE切片染色、免疫组化,证实SCID小鼠胶质瘤情况。结果:1.蒿甲醚可抑制人脑U251胶质瘤细胞增殖,其抑瘤作用呈时间和剂量依赖性;蒿甲醚作用后的U251胶质瘤细胞上清液中VEGF含量和生理盐水组相比明显降低,差异有统计学意义(P0.05)。2.成功构建SCID小鼠U251人脑胶质瘤原位模型,经活体左心灌注后取脑组织发现均有肿瘤生长。3.蒿甲醚组和替莫唑胺组SCID小鼠的中位生存期较对照组有明显延长(P0.05);脑组织HE切片染色可见对照组的U251细胞核大深染,呈梭型或不规则形,高倍镜下可见U251细胞向正常脑组织浸润,蒿甲醚组及替莫唑胺组肿瘤细胞密集程度均明显低于对照组。结论:1、蒿甲醚可抑制人脑U251胶质瘤细胞增殖,其抑瘤效应呈剂量-时间依赖性。2、蒿甲醚可能通过抑制VEGF表达而发挥其抗肿瘤作用。3、蒿甲醚可抑制SCID小鼠颅内胶质瘤增殖。
[Abstract]:Objective: To observe the first effect of artemether on proliferation of U251 glioma cells in vitro, and then establish a SCID mouse glioma orthotopic model, observe the effect of artemether in vivo inhibition of glioma and its anti angiogenesis, investigate the mechanism of artemether inhibiting glioma proliferation, provides a new idea and method for the clinical treatment of glioma. Methods: Cultured U251 glioma cells in vitro 1. medium, using MTT (four methyl thiazolyl tetrazolium) method for the determination of inhibition effect of artemether on U251 glioma cell growth, and calculate the half inhibitory concentration (IC50); detection of artemether inhibition of U251 glioma cells by ELISA method the expression of VEGF.2. to construct SCID mouse glioma orthotopic model using stereotaxic apparatus positioning anesthesia mice, cut the scalp, from the sagittal suture and coronal suture respectively at the intersection of right back 2mm injection point positioning And after drilling, using 10ul micro syringe perpendicular to the skull in the mouse brain location (right parietal area) deep 5mm, injection of 10ulU251 human glioma cells, bone wax filling bone hole, absorbable suture scalp, living conditions in mice, epilepsy symptoms in mice after in vivo perfusion via the left ventricle after brain tissue visual observation, gross appearance and section, HE staining confirmed the tumor model of.3. is constructed successfully constructed 24 SCID mice U251 glioma orthotopic model, randomly divided into 3 groups, including control group (saline group (0.4ml/10g/d), artemether artemether 50.0mg/kg/d), temozolomide group (20mg/kg/d) temozolomide. Using the powder made of suspension gavage, weight of 1.65mg/ /d. after inoculation were labeled, regular daily dosing, the survival time was recorded. About 20 days after inoculation, when SCID mice died of left heart in vivo during reperfusion, take Out and fixed all of the brain, were observed by gross, HE staining, immunohistochemistry, confirmed SCID mouse glioma. Results: 1. artemether could inhibit the proliferation of human U251 glioma cells, the inhibitory effect was time and dose dependent effect of artemether; after U251 glioma cells supernatant the content of VEGF and saline group was significantly decreased, the difference was statistically significant (P0.05).2. was successfully constructed SCID mice U251 glioma orthotopic model by in vivo left ventricular perfusion after brain tissue found that the median survival time of tumor growth were.3. artemether group and temozolomide group in SCID mice than in the control group obviously extended (P0.05); brain tissue HE staining was observed in control group U251 nucleus anachromasis, spindle shaped or irregular shape, visible at high magnification U251 cell infiltration to normal brain tissue, artemether group and temozolomide group of tumor cell intensive process Conclusion: 1, artemether can inhibit the proliferation of human brain U251 glioma cells, and its anti-tumor effect is dose time dependent.2. Artemether can inhibit the expression of VEGF and exert its antitumor effect.3, artemether can inhibit the growth of intracranial glioma in SCID mice.

【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.41;R-332

【参考文献】