【作者】 曾瀚庆；

【导师】 娄世锋；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 硕士

【摘要】 目的探讨低功率超声联合微泡靶向、可逆的开放大鼠脑血脑屏障(BBB),增强脑组织阿糖胞苷(Ara-C)药物浓度的可行性。方法1、实验组45只成年雄性SD大鼠,根据不同辐照时间(10s、30s、60s)和超声功率(0.8W、1.9W、2.7W)组合,分为9组,利用MRI增强扫描测定靶点信号增强值,通过静注伊文氏蓝后脑组织切片以观察靶点蓝染情况,从而判断血脑屏障是否开放,然后通过组织切片在光镜下观察脑组织受损程度,寻找大鼠血脑屏障开放的最适参数。2、常规剂量组25只SD大鼠,在超声联合微泡开放血脑屏障后的不同时间点(0h、2h、4h、6h、24h)经尾静脉注射常规剂量Ara-C(4mg/kg),通过MRI增强扫描测定靶点信号增强值,高效液相色谱法(HPLC)测定靶点和非靶点脑组织在不同时间点的Ara-C浓度。从而推测最适参数下超声联合微泡开放BBB后的BBB通透性的变化规律。3、大剂量组5只SD大鼠按上述方法2中相同的步骤经尾静脉注射微泡,但不进行超声辐照,模拟全身大剂量化疗注射Ara-C(50mg/kg),利用MRI增强扫描来测定靶点信号强度增强值, HPLC测定脑组织内的Ara-C浓度。4、分析实验组、对照组、大剂量组脑组织内Ara-C浓度的差异。测得的数值全部表示为均值±标准差(x±s),用SPSS13.0统计分析软件,各组数据之间的比较采用t检验,P<0.05为有统计学差异,P<0.01为有显著性差异。结果1.大鼠BBB开放的最适参数1.1靶点MRI增强扫描结果:对各组大鼠脑组织行MRI增强扫描发现,0.8W+10s组、0.8W +30s组、0.8W+60s组、1.9W+10s组、2.7W+10s组的增强值与对照组相比无显著性差异;1.9W+30s组、1.9W+60s组、2.7W+30s组、2.7W+60s组的增强值明显高于对照组(P<0.01)。1.2靶点EB染色结果:0.8W+10s组、0.8W +30s组、0.8W+60s组、1.9W+10s组、2.7W+10s组靶点均未见明显蓝染。1.9W+30s组、1.9W+60s组、2.7W+30s组、2.7W+60s组可见靶点蓝染,并且蓝染范围及程度随着幅照时长和超声功率的增加而增加。1.3 HE染色组织学检查结果:0.8W+10s组、0.8W +30s组、0.8W+60s组、1.9W+10s组、2.7W+10s组光镜下没有看到明显的靶点组织损伤或红细胞渗出,与靶点外的组织无显著区别;1.9W+30s组可见微血管周围少量红细胞的渗出,以及血管内皮细胞的轻微肿胀,但神经细胞形态没有明显异常;1.9W+60s组、2.7W+30s组见红细胞明显渗出,微血管明显破坏,内皮细胞破损,细胞碎片可见,实质细胞也有不同程度受损,周围组织可见水肿带;2.7W+60s组血管破裂以及红细胞广泛的渗出,可见血管内血栓形成以及少量炎细胞,神经细胞明显肿胀,组织液化坏死。2.最适参数下超声联合微泡开放BBB后其通透性变化规律超声功率1.9W辐照30s后0h、2h、4h靶点的MRI信号增强值和脑组织的Ara-C浓度明显高于非靶点处(P<0.01),2h达到峰值,而8h、24h组的测定值与对照组比较无显著性差异。3. HPLC测得超声实验组的脑组织Ara-C浓度为7.69±1.65ug/g脑组织,常规剂量Ara-C组为1.75±0.76 ug/g脑组织,大剂量阿糖胞苷组为7.04±2.76ug/g脑组织。超声实验组与大剂量阿糖胞苷组无显著性差异(P>0.05),而超声实验组明显高于常规剂量Ara-C组,有显著性差异(P<0.01)。结论1、在适宜的辐照时间和功率参数条件下(30s+1.9W),低功率超声联合微泡能可逆性开放BBB,而脑组织损伤很小。2、利用低功率超声联合微泡可逆开放BBB技术,能够显著增加脑组织Ara-C浓度,给予常规剂量即可达到大剂量Ara-C静脉给药相同效果,又避免了大剂量给药可能带来的全身副作用,效果明显优于单纯静脉给药,这种新型给药方式可能为防治中枢神经系统白血病提供一个新思路。更多还原

【Abstract】 OBJECTIVETo investigate the effects of reversible and targeted opening the blood–brain barrier (BBB) by MRI-guided focused ultrasound (FUS) Asssociated with Microbubbles and delivering cytrarabine(Ara-C) to the rat brain. Then to explore a effective administration route of curing central nervous system leukemia(CNS-L).METHODS1. According to different exposure times(10s,30s,60s) and different ultrasonic frequency(0.8W,1.9W,2.7W) respectively, 45 SD rats sonicated as experimental group(EG) by MRI-guided FUS were divided into 9 groups. then Evans blue extravasation , contrast-enhanced MRI scans and histologic examination were performed to evaluate the optimum condition for opening BBB with minimal damage.2. 25 SD rats were injected with Normal doses of Ara-c(4mg/kg) through the tail vein at every time point(0h,2h,4h,6h,24h) after FUS with microbubbles. The target as experimental group and the non-target ascontrol group. The Ara-c concentration was analyzed by high– performance liquid chromatography (HPLC) and the Ara-c concentration and signal intensity enhancement of MRI was detected on every time point to determine the change tendency of permeability of BBB after sonication.3. Five SD rats were injected with Microbubbles through the tail vein without sonication to model general chemotherapy by large doses of Ara-c(50mg/kg) as large doses group(LDG). The MRI signal intensity enhancement in the target locations was detected the specimens of the brain tissue were and the concentration of Ara-c in all the specimens was determined by HPLC.4. To analyze and compare the concentration of Ara-c in the brain tissue specimens of 3 groups. Results are presented as mean±SD. Statistical differences in the experimental groups were analyzed with a t test, where P < .01 was considered statistically significant.RESULTS1. The optimum condition for opening BBB of rats.1.1 Contrast-enhanced MRI: Significance difference of Ga retention was observed in 1.9W+30s group, 1.9W+60s group, 2.7W+30s group and 2.7W+60s group compared with the control group (P < 0.01).1.2 Evans blue staning: Immediately performed after sonication. No visible blue stain was noted in the brain specimens exposed to 0.8W+10s group, 0.8W+30s group, 0.8W+60s group, 1.9W+10s group and 2.7W+10s group; While the EB stained positively in 1.9W+30s group, 1.9W+60s group, 2.7W+30s group, and 2.7W+60s group. EB extravasation was more broadly distributed and darker when sonication was performed for longer durations and higher power.1.3. Hematoxylin(HE) and eosin histologic analysis at the targeted focal locations: Immediately after sonication, there was no damage and RBCs extravasation in the sonicated sites of 0.8W+10s group, 0.8W+30s group, 0.8W+60s group, 1.9W+10s group and 2.7W+10s group, and the findings of these sites differed little from those of the nonsonicated sites. In the sites of 1.9W+30s group, a few scattered erythrocytes were observed, and no obvious signs of parenchymal damage were detected. Sites of 1.9W+60s group and 2.7W+30s group performed extravasation of erythrocytes around microvessels, and signs of vasodilatation, stasis, and destruction were noted in the vessel walls. Parenchymal damage was mostly in the form of ischemic changes, and slight vacuolation of the neuropil was seen. In the sites of 2.7W+60s group, multiple areas of blood vessel destruction and extensive erythrocyte extravasations into brain parenchyma were noted, the brain tissue was edematous with fragmented axons, the vacuolation, local tissue necrosis, and neutrophil infiltration were observed.2. The change tendency of permeability of BBB after sonication with Microbubbles:The MRI signal intensity enhancement and the concentration of Ara-c in brain tissue of specimens in sites of 0h, 2h and 4h was significant higher than that of nontargeted sites. It reached peak at 2h while it had no significant difference with control group at 8h and 24h(P<0.01).3. Ara-c concentrations for all the groups: the Ara-c levels of EG, NDG and LDG were 7.69±1.65ug/g tissue, 1.75±0.76ug/g tissue and 7.04±2.76ug/g tissue respectively. No significant differences were observed between the EG and LDG(P>0.05). Ara-c concentration in the brain tissue of EG was significantly higher than that of NDG(P < 0.01).CONCLUTION1. MRI-guided focused ultrasound can open the BBB targeted and reversible with the minimal damage of brain tissue on the optimum condition (30s+1.9W).2. MRI-guided FUS can open the BBB reversibly and deliver the intravenously administered Ara-c to the targeted brain locations; the application of this method brings about a substantial increase in the drug level in the targeted brain tissue and has similar effect to large drug doses delivery. In this way side-effect large drug doses delivery could be avoided. Accordingly, this effective administration route may hew out a new perspective of curing CNS-L.更多还原