【作者】 张娟；

【导师】 王元银；

【作者基本信息】 安徽医科大学 ， 口腔临床医学， 2008， 硕士

【摘要】 目的1.建立一种操作更加简单、成活率高、生物相似性高的口腔鳞状细胞癌动物模型,为口腔肿瘤实验研究奠定基础;2.采用光动力学疗法( photodynamic therapy,PDT )联合5-氟尿嘧啶(5-fluorouracil,5-FU)缓释剂治疗口腔鳞癌,研究其在促进肿瘤细胞凋亡中是否具有协同和强化作用。方法采用局部植入瘤块法建立100只兔Vx-2口腔鳞癌动物模型,成功后随机分为5组:A组,空白对照组;B组,静脉给药组;C组,缓释剂组;D组,PDT组;E组,缓释剂联合PDT组。A组:不予任何处理;B组:全麻后耳缘静脉注射2.5mg/ml氟尿嘧啶溶液30ml(75mg),注射速度控制为0.5ml/min;C组:将5-FU缓释剂用专用植药针按时钟4个位点植入到肿瘤中,单点给药不超过20mg,总剂量为75mg;D组:先从耳缘静脉注入稀释为1mg/ml的HPD0.3mg/kg,避光养殖24h后麻醉。照射时术区外用生理盐水纱布覆盖保护,光纤末端距离肿瘤约2cm,波长630nm,光斑覆盖肿瘤,激光功率密度为200mW/cm3,照射10min,能量密度为100J/cm2;E组:先局部植入5-FU缓释剂(方法剂量同C组)然后予以激光照射,照射前24h静脉注射HPD。接下来对各实验组兔子精神、饮食、活动等行为学进行观察,连续五周测量肿瘤体积,五周后处死动物取肿瘤组织做组织学观察和免疫组化、流式细胞仪等来评价肿瘤抑瘤率、生长状态、肿瘤细胞的凋亡率及肿瘤组织中p53基因蛋白表达情况来初步研究PDT联合5-FU缓释剂的疗效,各组数据比较用SPSS11.0统计软件进行统计分析,以P<0.05为具有显著性差异。结果1. Vx-2肿瘤移植成功率较高,成瘤率百分之百,操作简单,具有可重复性。2.治疗后连续观察五周并测肿瘤体积,发现治疗后第1周各组间肿瘤体积无明显差异,第2周起空白对照组、静脉给药组和PDT组肿瘤生长较缓释剂组和联合治疗组较快。联合治疗组兔子精神状态、饮食、活动等均较其他各组稍显优势。3.治疗后第五周观察并计算抑瘤率得:静脉给药组(39.9±15.9)%;缓释剂组(73.8±22.3)%;PDT组(50.6±21.8)%;联合治疗组(93.6±19.5)%,联合治疗组抑瘤率明显低于其他各组,与其他各组间比较具有显著性差异(P<0.05)。4. HE染色镜下观察肿瘤组织为鳞状细胞癌,联合治疗组较其他组肿瘤细胞增殖能力下降、核分裂像罕见、细胞大量死亡或凋亡、出现空泡样变、核固缩等现象。5.免疫组化测得各组p53基因蛋白阳性表达率为:空白对照组(60%)、静脉给药组(50%)、缓释剂组(30%)、PDT组(45%)、联合组(15%),联合组明显低于其他组,各组间比较具有显著性差异(P<0.05)。6.流式细胞仪检测肿瘤细胞凋亡率结果:空白对照组(5.05±1.19)%、静脉给药组(11.81±0.89)%、缓释剂组(25.80±13.78)%、PDT组(20.32±11.24)%、联合组(32.51±15.32)%,联合组明显高于其他各组,与其他组间比较具有显著性差异(P<0.05),缓释剂组高于静脉给药组及空白对照组,组间比较均具有显著性差异(P <0.05),PDT组与空白对照组和静脉给药组比较具有显著性差异(P<0.05),而缓释剂组与PDT组比较则无显著性差异(P>0.05)。结论采用Vx-2肿瘤组织块移植法建立口腔鳞状细胞癌操作简单,成活率高,是一种较好的可供口腔肿瘤实验研究的动物模型。在口腔鳞癌治疗上5-FU缓释剂较常规静脉给药在维持抗瘤有效浓度,治疗疗效上具有一定的优势,与PDT联合应用时在抑制肿瘤生长、促进肿瘤细胞凋亡上较其单独应用效果显著。因此我们可以得出5-FU与PDT联合应用可能成为治疗口腔鳞癌的一种新的临床治疗方法。更多还原

【Abstract】 Objective1. Looking for an oral squamous cell carcinoma animal model which is more simple and has high survival rate and biological similarity in order to provide convenience for the experimental study of oral tumors.2. Study sustained release 5-FU with photodynamic therapy in the treatment of oral squamous cell carcinoma ; the combination of PDT and sustained-release 5-FU may be more effective in OSCC.MethodsNew Zealand white rabbits weighing 1.5 to 2.0 kg, maintained on a standard laboratord diet were given transplants of partially homogenized Vx-2 carcinoma in oral. One hundred rabbits were randomly divided into five groups:①control group;②intravenous group③sustained release 5-FU group;④PDT group;⑤sustained release 5-FU combined with PDT group. Continuous measurement of tumor volum for five weeks, and five weeks later killed the animals for histological observation(stained with hematoxylin and eosin, H&E) , immunohistochemistry and flow cytometry, and to evaluate tumor inhibition rates, growth state, the rate of tumor cell apoptosis and expression of p53. Our purpose is to preliminary study the efficacy of sustained release 5-FU combined with PDT. The data was analyzed by SPSS11.0 software package with P <0.05 for having a significant difference. Results1 It is successful to set up rabbit Vx-2 Oral Squamous Cell Carcinoma model, and the success rate of method that tissue mass of the tumor transplanted is obviously higher than the other methods. Tumor had the similar biological features of human oral squamous cell carcinoma so that it is an option for oral cancer animal model.2 On our continuous observation and measurement of tumor size for five weeks we found that in the first week there were no significant differences between groups in tumor size. from the second week on, the tumor of the control group, the intravenous group and the PDT group were grown faster than the sustained release 5-FU group and the joint group. Rabbits of the last group had advantages in spirit, eating, active than the other groups.3 5 weeks later, we got the inhibition rate of tumor: intravenous group is (39.9%±15.9%); sustained-release group is (73.8%±22.3%); PDT group is (50.6%±21.8%); therapeutic alliance group is (93.6%±19.5%). The results showed that the inhibition rate based on tumor measurement of therapeutic alliance group was higher than the other groups, having significant difference.4 Hematoxylin and eosin(H&E) staining of tumor tissue were observed for squamous cell carcinoma. In the therapeutic alliance group we found that the proliferation of the tumor cells was relatively suppressed, rare nuclear fission, massive cell death or apoptosis, cell vacuolar changes and karyopyknosis phenomenon.5 Immunohistochemistry was measured for p53 protein expression : the control group (60%), intravenous injection group (50%), sustained-release 5-FU group (30%), the PDT group (45%), the therapeutic alliance group (15%), the therapeutic alliance group was significantly lower than that of other groups, with significant differences (P<0.05).6 FCM rate of tumor cell apoptosis shows that the control group was (5.05±1.19)%, intravenous group was (11.81±0.89)%, sustained-release 5-FU group was (25.80±13.78)%, the PDT group was (20.32±11.24)%, the therapeutic alliance group was (32.51±15.32)%. We found that the therapeutic alliance group was significantly higher than that of other groups with a significant difference (P<0.05). The sustained-release 5-FU was higher than the intravenous group and the control group, having a significant difference between groups (P<0.05). Between the PDT group, the control group and the intravenous group also had significant difference (P<0.05), but the sustained-release group was compared with the PDT group showing no significant difference (P>0.05).ConclusionVx-2 by tumor tissue transplantation is a simple operation to made oral squamous cell carcinoma with a high survival rate. It is a better way for oral cancer research in animal models. In the treatment of oral squamous cell carcinoma the sustained-release 5-FU compared with conventional intravenous administration was more effective in anti-tumor concentration. Our study demonstrates that the combination of PDT and sustained-release 5-FU was more effective at the inhibition of tumor growth, and was able to promote apoptosis of tumor cells. Therefore, we can say that 5-FU combined with PDT may become a new clinical approach on OSCC’s treatment.更多还原