【作者】 赵丹；

【导师】 梁斌；

【作者基本信息】 华中科技大学 ， 影像医学与核医学， 2019， 硕士

【摘要】 第一部分透明质酸酶及阿霉素细胞毒性实验(CCK8实验)目的:通过细胞实验研究透明质酸酶及阿霉素对Hep G2肝癌细胞的毒性,优选药物浓度和作用时间。材料与方法:将对数生长期的Hep G2细胞分别以空白培养基和含不同浓度透明质酸酶或阿霉素的培养基进行不同时间的培养。细胞预定培养时间结束后使用酶标仪测定450 nm光吸收值,以溶剂处理的细胞为对照组,不含细胞的培养基为空白组,按公式计算药物对细胞的抑制率。结果:透明质酸酶不同药物浓度及作用时间对Hep G2细胞的抑制率无明显差异,0、30、60、120min的IC50分别为536.743μM、483.112μM、425.019μM和416.206μM。阿霉素不同药物浓度及作用时间对Hep G2肝癌细胞的的抑制率结果显示0、30、60、90、120 min的IC50分别为4622.767μM、1273.328μM、678.042μM、329.50μM和183.767μM。结论:不同透明质酸酶药物浓度及作用时间情况下,对Hep G2肿瘤细胞基本无毒性作用;结合实验结果,后继细胞实验采用透明质酸酶药物浓度为160μM,作用时间为60min。同时由于后续实验涉及多细胞球形体内阿霉素药物荧光的数量和渗透距离,要求细胞存活率在80%以上,综合阿霉素药物浓度和作用时间的优化组合因素,选择阿霉素药物浓度为160μM、作用时间为60min。第二部分透明质酸酶对肿瘤多细胞球形体内阿霉素分布的影响目的:通过细胞实验研究透明质酸酶对多细胞球形体内阿霉素药物分布的影响。材料与方法:采用转瓶琼脂法体外培养人Hep G2肝癌细胞多细胞球形体,分为两组,分别接受阿霉素、阿霉素+透明质酸酶处理,透明质酸酶药物浓度均为160μM,干预时间60min,处理后采用荧光显微镜观察肿瘤多细胞球形体内的阿霉素药物荧光的分布,并采用Image Pro Plus软件计算多细胞球形体内阿霉素药物荧光的数量和渗透距离。结果:Hep G2多细胞球形体直径两组间无明显差异;阿霉素+透明质酸酶组在阿霉素药物荧光面积、荧光强度和荧光渗透距离方面明显高于单纯阿霉素组。结论:透明质酸酶可明显促进阿霉素在肿瘤多细胞球体内的渗透。第三部分透明质酸酶对肿瘤间质液压的影响目的:通过不同给药途径比较透明质酸酶对肿瘤间质液压的影响,优选透明质酸酶给药方案及作用时间。材料与方法:购置新西兰大白兔15只,雌雄不限,体重2.5~3.0kg。通过移植方式建立15只实验兔VX2肝癌模型,肿瘤种植外置位于肝左外叶。术后17天采用MRI检测肿瘤大小。将15只荷瘤兔分为3组(每组5只),A组(空白对照组)未做任何处理;B组(耳缘静脉组,IV组)通过兔耳缘静脉注射透明质酸酶溶液10ml(10mg/ml);C组(肝动脉灌注组,TAI组)通过肝动脉灌注透明质酸酶溶液10ml(10mg/ml);先将压力导管埋入实验兔VX2瘤体内,实时观察压力读数变化,待压力读书稳定后开始灌注透明质酸酶并通过计算机记录间质液压,实验结束后根据相关数值绘制时间-间质液压曲线。结果:肿瘤内间质液压TAI组变化率最大,IV组次之,对照组最小,给药40min后肿瘤间质液压降至“平台期”。结论:透明质酸酶经可有效降低肿瘤内间质液压,其中通过介入的方式经肝动脉给药更具优势,给药40min后肿瘤间质液压改变趋于稳定。第四部分经导管介入治疗联合透明质酸酶治疗肝癌的疗效与机制目的:评价TACE联合透明质酸酶对肝癌经导管介入治疗药物渗透和疗效的影响,初探联合治疗机制。材料与方法:建立20只兔VX2肝癌模型,分成两组每组10只,分别接受阿霉素经肝动脉注入+PVA栓塞(c TACE组)和透明质酸酶经肝动脉注入+阿霉素经肝动脉注入+PVA栓塞(TACE联合透明质酸酶组);每组5只动物于处理后0~1小时采用微尖导管压力传感器活体检测肿瘤间质液压,并于术后4小时处死采用免疫荧光技术检测药物渗透;每组另5只动物于处理后7天处死采用HE染色评价肿瘤坏死率,比较肿瘤间质液压、组织内药物渗透及浓度和坏死率的组间差异,分析它们之间的关系。结果:肿瘤内间质液压改变两组间存在显著性差异,TACE联合透明质酸酶组(58.7%±9.8%)明显大于c TACE组(35.8%±8.3%)(P=0.004);TACE联合透明质酸酶组及c TACE组阿霉素荧光点数分别为3330.91±945.73及1564.38±633.99(P<0.01);两者阿霉素相对渗透距离分别为38.06±8.72μM及29.51±4.76μM(P<0.05),TACE联合透明质酸酶组明荧光点数及阿霉素渗透距离均明显高于单纯阿霉素组。TACE联合透明质酸酶组及c TACE的阿霉素浓度组内比较显示其术后7天肿瘤内阿霉素浓度都比术后4小时肿瘤内阿霉素浓度降低;对于同一时间点而言,TACE联合透明质酸酶组肿瘤内阿霉素浓度均明显高于c TACE组,两者比较均有统计学差异(P=0.021,P=0.0004);TACE联合透明质酸酶组的肿瘤坏死率(90.61%±5.38%)也明显高于c TACE组(77.49%±7.11%)(P=0.011)。结论:透明质酸酶可通过降低肿瘤内间质液压而促进阿霉素对肿瘤实质的渗透,改善药物分布,增加肿瘤实质内药物浓度,从而提高TACE的治疗效果。更多还原

【Abstract】 PART Ⅰ: Cytotoxicity assay of hyaluronidase and doxorubicinObjective: To study the toxicity of hyaluronidase and doxorubicin to Hep G2 cells and decide the best drug concentration and processing time.Materials and Methods: The Hep G2 cells in the logarithmic growth phase were cultured in a blank medium and a medium containing different concentrations of hyaluronidase or doxorubicin for different times.After the predetermined culture time of the cells,the 450 nm light absorption value was measured by a microplate reader.The solvent-treated cells were used as a control group,and the cell-free medium was a blank group.The inhibition rate of the drug on the cells was calculated according to the formula.Results: There were no significant differences in the inhibitory rates of hyaluronan different drug concentrations and duration of process on Hep G2 cells.The IC50 at 0,30,60,and 120 minutes were 536.743μM,483.112μM,425.019μM,and 416.206μM,respectively.The inhibitory rates of doxorubicin at different concentrations and duration of process on Hep G2 cells showed that IC50 at 0,30,60,90,and 120 minutes were 4622.167μM,1273.328μM,678.042μM,329.502μM,and 183.767μM,respectively.Conclusion: Under different hyaluronidase drug concentration and processing time,there is no obvious toxic effect on tumor cells;combined with the experimental results,the concentration of hyaluronidase was 160μM in subsequent cell experiments,the process time was 60 minutes.Meanwhile,because the follow-up experiment involves the amount and penetration distance of doxorubicin drug fluorescence in multi-cell spheroids,the cell survival rate is required to be above 80%.The optimal combination of doxorubicin drug concentration is 160μM,the process time was 60 minutes.PART Ⅱ: Effect of hyaluronidase on the distribution of doxorubicin in tumor multicellular spheroidsObjective: To study the effect of hyaluronidase on the distribution of doxorubicin in multicellular spheroids through cell experiments.Materials and Methods: The multi-cell spheroids of human Hep G2 hepatoma cells were cultured in vitro by using the flask agar method.They were divided into two groups and treated with doxorubicin,doxorubicin and hyaluronidase respectively.The concentration of hyaluronidase was 160μM and the processing time was 60 minutes.After treatment,the distribution of doxorubicin drug fluorescence in tumor multicellular spheroids was observed by fluorescence microscope,and the amount and penetration distance of doxorubicin drug fluorescence in multicellular spheres were calculated by Image Pro Plus software.Results: There was no significant difference in the diameter of Hep G2 multicellular spheroids between the two groups.The doxorubicin with hyaluronidase group was significantly higher than the doxorubicin group in the fluorescence area,fluorescence intensity and fluorescence penetration distance of doxorubicin.Conclusion: Hyaluronidase can significantly promote the penetration of doxorubicin in tumor multicellular spheroids.PART Ⅲ: Effect of hyaluronidase on tumor interstitial fluid pressureObjective: Compare the effects of hyaluronidase on tumor interstitial fluid pressure with different routes of administration and decides the best hyaluronidase administration schedule and processing time.Materials and Methods: Purchased 15 New Zealand white rabbits,male or female,weighing 2.5~3.0kg.Fifteen experimental rabbit VX2 liver cancer models were established by transplantation,and the tumors were externally located in the left outer lobe of the liver.Tumor size was measured by MRI on the 17 th day after surgery.15 tumor-bearing rabbits were divided into 3 groups(5 in each group),group A(blank control group);group B(IV group)was injected with hyaluronidase solution through the rabbit ear vein(10ml,10mg/ml);group C(hepatic arterial infusion group,TAI group)was perfused with hyaluronidase(10ml,10mg/ml)through the hepatic artery;At first,we embed the pressure catheter into the experimental rabbit VX2 tumor and observe the change of pressure in real time,After the pressure become stabilized,the hyaluronidase was perfused and the interstitial fluid pressure was recorded.After the experiment,the time-interstitial fluid pressure curve was drawn according to the relevant values.Results: The rate of change in the intratumoral interstitial hydraulic TAI group was the highest,followed by the IV group,and the control group was the smallest.After 40 minutes of administration,the tumor interstitial fluid pressure dropped to the “platform stage”.Conclusion: Hyaluronidase can effectively reduce the interstitial fluid pressure in the tumor.The route of applying hyaluronidase through hepatic artery by intervention is more advantageous.The tumor interstitial fluid pressure tends to be stable after 40 minutes of administrationPART Ⅳ: Efficacy and mechanism of transcatheter interventional therapy combined with hyaluronidase in the treatment of liver cancerObjective: To evaluate the effect of TACE combined with hyaluronidase on drug penetration and efficacy of transcatheter interventional therapy for hepatocellular carcinoma,and to explore the mechanism of combined therapy method.Materials and Methods: Twenty rabbit VX2 liver cancer models were established and divided into two groups(10 in each group)receiving doxorubicin via hepatic artery injection + PVA embolization(TACE alone)and hyaluronidase through hepatic artery injection + doxorubicin via hepatic artery injection + PVA Embolization(TACE combined with hyaluronidase group);5 animals in each group were tested for tumor interstitial fluid pressure by microtip catheter pressure sensor 0 to 1 hour after treatment,and were sacrificed 4 hours,then using immunofluorescence technique to detect drug penetration.The other 5 animals in each group were sacrificed 7 days after treatment.The tumor necrosis rate was evaluated by HE staining.The interstitial differences in tumor interstitial fluid pressure,tissue drug penetration and concentration and necrosis rate were compared,and the relationship between them was analyzed.Results: There was a significant difference in interstitial fluid pressure between the two groups.TACE combined with hyaluronidase group(58.7%±9.8%)was significantly greater than c TACE group(35.8%±8.3%)(P=0.004);TACE combined with hyaluronic acid The fluorescence points of doxorubicin in the enzyme group and c TACE group were 3390.91±945.73 and 1564.38±633.99,respectively(P<0.01).The relative permeation distances of doxorubicin were 38.06±8.72 μM and 29.51±4.76 μM(P<0.05),respectively.The number of fluorescent spots and the penetration distance of doxorubicin in the hyaluronidase group were significantly higher than those in the simple doxorubicin group.Comparison of TACE combined with hyaluronidase group and c TACE in the concentration of doxorubicin showed that the concentration of doxorubicin in the tumor was lower than that in the tumor at 4 hours after procedure.Meanwhile,The concentration of doxorubicin in the TACE combined with hyaluronidase group was significantly higher than that in the c TACE group,and the difference was statistically significant(P=0.021,P=0.0004);the tumor necrosis rate in the TACE combined with hyaluronidase group(90.61%±5.38%)was also significantly higher than the c TACE group(77.49%±7.11%)(P=0.011).Conclusion: The hyaluronidase can promote the penetration of doxorubicin into the tumor parenchyma by reducing the interstitial fluid pressure in the tumor,improving the drug distribution,increasing the drug concentration in the tumor parenchyma,and thereby improve the therapeutic effect of TACE.更多还原