【作者】 李翠云；

【导师】 段小娴；

【作者基本信息】 广西医科大学 ， 肿瘤学， 2007， 硕士

【摘要】 一、目的:根据广西肿瘤防治研究所病理研究室80年代已成功建立的AFB1致大鼠肝癌前病变短期体内实验动物模型,探索用二乙基亚硝胺(DEN)致大鼠肝癌前病变的最佳剂量;观察金花茶(Camellia chrysantha,CCT)茶花及不同浓度的金花茶浓缩液对DEN诱发大鼠肝癌前病变的作用。二、方法:1、致肝癌前病变DEN剂量实验:37只健康雄性Wistar大鼠,6周龄,体重115±10g,随机分为四组。A组:9只,DEN剂量150mg/kg.bw,2-AAF浓度0.015%(二乙基氨基芴,100g饲料中含0.015g的2-AAF,下同);B组:10只,DEN剂量100mg/kg.bw,2-AAF浓度0.015%;C组:9只,DEN剂量150mg/kg.bw,2-AAF浓度0.010%(100g饲料中含0.010g的2-AAF,下同);D组:9只,DEN剂量100mg/kg.bw,2-AAF浓度0.010%。各组动物进入实验室适应环境1周后,腹腔注射一剂不同剂量的DEN,间隔2周后饲以含不同浓度2-AAF的饲料2周。并于饲该饲料的第7天在乙醚全麻下行肝脏大部分(2/3)切除术做为促癌程序,停2-AAF饲料后喂食基础饲料2天,然后禁食24小时,处死所有动物。取出肝脏,在肝特定部位取出5块组织,厚约2mm,立即固定于冷(-4℃)酒精-丙酮(1:1/V:V)固定液,24h之后进行固定、低温(52～54℃)石蜡包埋、切片并进行γ-谷氨酰转肽酶(γ-GT)染色,观察并统计每张切片中每个γ-GT阳性灶的面积(mm~2/个)、单位面积中灶的个数(个/cm~2)、单位面积中灶的总面积(mm~2/cm~2)。2、金花茶实验:91只健康雄性Wistar大鼠,6周龄,体重115±10g,随机分组法分为7组,每组13只,A、B、C、D、E、F、G组分别为茶叶渣组、5%茶花组、2.5%、5%、7.5%、10%的金花茶浓缩液组、DEN对照组。动物进入实验室1周适应环境后,A、B两组分别连续给含有不同受试物的饲料3周,同时C、D、E、F组进行金花茶浓缩液灌胃3周,对照组G组一直喂正常饲料3周;在实验的第3周每只动物腹腔注射一剂DEN,剂量为100mg/kg.bw,注射2周后开始喂含0.015%的2-AAF饲料2周,并于饲此饲料的第7天在乙醚麻醉下行肝大部分切除术作为促癌程序,大鼠停2-AAF饲料后换食正常饲料2天,然后禁食24小时,断颈处死所有动物。实验过程中肝切时各组随机选取一只大鼠的肝切组织进行固定、包埋、切片。实验结束时处死的每只动物取五块肝组织进行固定、包埋、切片,做γ-谷氨酰转肽酶(γ-GT)酶染色,观察并统计每个γ-GT阳性灶的面积(mm~2/个)、单位面积灶的个数(个/cm~2)、单位面积灶的总面积(mm~2/cm~2)。同时各组的肝切和处死组织切片做增殖细胞核抗原(PCNA)免疫组织化学染色,观察各切片中PCNA阳性细胞的特点,并与γ-GT阳性细胞灶作比较。三、结果:1、DEN剂量实验:A、B、C、D组γ-GT阳性灶总面积(mm~2/cm~2)分别为9.85±4.17、5.72±3.83、8.81±3.70、6.33±2.97;单位面积阳性灶个数(个/cm~2)分别为56.39±17.80、36.90±23.09、53.97±16.17、40.40±11.82;每个阳性灶的面积(mm~2/个)分别为0.16±0.07、0.15±0.05、0.13±0.03、0.14±0.05。各组灶总面积及单位面积阳性灶个数以B组为最少。2、金花茶实验:A、B、C、D、E、F、G组大鼠肝脏的γ-GT阳性灶总面积(mm~2/cm~2)分别为:5.23±2.99、1.59±1.16、1.94±1.34、1.85±1.19、2.59±2.53、1.65±1.18、6.39±2.76,单位面积阳性灶个数(个/cm~2)分别为:37.08±17.34、16.24±8.80、17.74±9.80、14.57±8.11、26.19±22.00、12.89±9.08、45.58±13.01,B、C、D、E、F组灶总面积(mm~2/cm~2)及单位面积阳性灶个数(个/cm~2)均明显小于对照组G组及茶叶渣A组(p＜0.05)。每个阳性灶的面积(mm~2/个)分别为:0.13±0.03、0.10±0.02、0.10±0.03、0.10±0.04、0.10±0.05、0.11±0.03、0.12±0.04;各组之间无显著差异(p＞0.05)。A、B、C、D、E、F、G组的肝重体重比分别为:1.97±0.46、2.30±0.34、2.45±0.24、2.64±0.15、2.50±0.30、2.46±0.25、1.94±0.19,金花茶茶花及不同浓度的金花茶浓缩液组肝重体重比明显大于茶叶渣组及DEN对照组(p＜0.05)。增殖细胞核抗原(PCNA)阳性细胞增生灶与r-GT阳性增生灶基本重叠,两种增生灶的个数经统计学分析无显著性差异(p＞0.05)。四、结论:1、二乙基亚硝胺诱发大鼠肝癌前病变的最适剂量为DEN剂量100mg/kg.bw,2-AAF浓度0.015%。2、金花茶茶花及其制成品金花茶浓缩液对DEN诱导的肝细胞癌前病变有抑制作用,可用于肝癌的早期预防。3、不同浓度金花茶浓缩液对大鼠肝癌前病变均有抑制作用,并且随着浓度的增加抑制作用增强,呈现一定的剂量—效应趋势,金花茶的有效成分主要是水溶性的物质。4、增殖细胞核抗原(PCNA)细胞增生灶与r-谷氨酰转肽酶(r-GT)阳性细胞灶结果基本一致,可能会成为一种新的衡量肝癌前病变的指标之一。5、金花茶茶花及浓缩液有抵抗DEN对大鼠肝脏的毒性作用,对肝脏具有一定的保护作用。更多还原

【Abstract】 Objective:we use an animal model induced by AFB1 which was established in the 80 times by our institute to explore the best dose of diethylnitrosamine(DEN) and 2-acetylaminofluorene,which can induce liver cancer of rats.we study the anti-tumor effect and its mechanisms of different concentration Camellia chrysantha(Hu)Tuyama and it’s flower in the rat’s liver preneoplastic nodules induced by DEN。Methods:1.diethylnitrosamine concentration experiment:The male rats,6 weeks old, were used for establishing a short-term preneoplastic model:37 healthy male wistar rats were divived into four group at random at the bases of diethylmitro samine(DEN)doses of abdomen injection and 2-AAF solution which was mixed into the normal food.9 rats for group A,150mg/kg.bw of DEN and 0.015% 2-AAF,10 rats for group B,100mg/kg.bw of DEN and 0.015%2-AAF;9 rats for group C,150mg/kg.bw of DEN and 0.010%2-AAF;9 rats for group D, 100mg/kg.bw of DEN and 0.010%2-AAF.The rats received DEN by abdomen injection.Then 2-AAF solution which was mixed into the normal food was administered by the rat for two weeks.Two-thirds partial hepatectomy was per formed after seven days for taking 2-AAF to promote tumor process.Stop giving 2-AAF solution food then feeding the normal food two days.Prohibit to give food for 24 hours later all the rats were executed.Five pieces of liver were taken from each rat.The preneoplastic nodules forγ-GT positive foci in liver sections were measured,and the total areas of the positive foci per square centimeter(mm~2/cm~2),the average acreage per positive foci(mm~2/entries),and the amount of positive foci per square centimeter(entries/cm~2)were calculated respectively.2.Camellia chrysantha(Hu)Tuyama experiment:The male rats,6 weeks old,were used for establishing a short term experiment model:91 healthy male wistar laboratory rats were randomly devived into seven groups,eath of which contains 13 animals,being consecutively fed with animal food containing different objiects or filled stomach along with treatment by abdomen injection of one dose diethylnitrosamine 100mg/kg.bw,respectively.Then 0.015%2-AAF solution which was mixed into the normal food was administered to the rat for two weeks.Two-thirds partial hepatectomy was performed after seven days for taking 2-AAF.13 rats for A、B、C、D、E、F、G,the leaves residues group、the flowers of Camellia chrysantha(Hu)Tuyama group、2.5%、5%、7.5%、10%the concentrated liquids of CCT and the positive control group.Five pieces of liver were taken from each rat.The preneoplastic nodules forγ-GT positive foci in liver sections were measured,then the total areas of the positive foci per square centimeter(mm~2/cm~2),the average acreage per positive foci(mm~2/foci), and the amount of positive foci per square centimeter(foci/cm~2)were calculated repectively.The expression of proliferating cell nuclear antigen protein was determined with immunohistochemistry and compared withγ-GT positive foci.Results:1.The total areas of positive foci forγ-GT in group A,B,C,D were 9.85±4.17,5.72±3.83,8.81±3.70,6.33±2.97;the positive foci per square centimeter were 56.39±17.80,36.90±23.09,53.97±16.17,40.40±11.82;the average areas per positive foci were:0.16±0.07,0.15±0.05,0.13±0.03, 0.14±0.05.the lest of the total areas of positive foci and the positive foci per square centimeter was group B.2.The total areas of positive foci forγ-GT in group A,B,C,D,E,F,G were 5.23±2.99,1.59±1.16,1.94±1.34,1.85±1.19,2.59±2.53,1.65±1.18,6.39±2.76. respectively.The positive foci per square centimeter were 37.08±17.34, 16.24±8.80,17.74±9.80,14.57±8.11,26.19±22.00,12.89±9.08,45.58±13.01;The average areas per positive foci were:0.13±0.03,0.10±0.02,0.10±0.03,0.10±0.04, 0.10±0.05,0.11±0.03,0.12±0.04.The total acreage of positive foci and the amount of positive foci per square centimeter in groupB、C、D、E、F were all less than the group G(p＜0.05)There was no difference for the average areas per positive foci between different groups(p＞0.05).From group A,B,C,D,E,F and G,the organ coefficient of liver(g/100g)were 1.97±0.46,2.30±0.34, 2.45±0.24,2.64±0.15,2.50±0.30,2.46±0.25,1.94±0.19.The organ coefficient in groupB、C、D、E、and F were more than that in group A and G,(p＜0.05).PCNA and r-GT could have same effect on weighing precancerous lesion to liver of rat.Conclusion:1.The best dose of diethylnitrosamine and 2-acetylaminofluorene,which can induce liver cancer of rats was DEN 100mg/kg.bw and 2-AAF 0.015%.2.The different concentrated liquids of CCT and the flowers of CCT could inhibit the growth of the hepatic cells in the preneoplastic nodules,CCT can be used to protective liver.3.Inhibiting the growth of the hepatic cells in the preneoplastic nodules of the different concentrated liquids of CCT show dose-effect-realationship,the effecttive element of CCT was water-solubility substance.4.PCNA and r-GT have same effect on weighing precancerous lision to liver of rat.PCNA could be an new one of signs weighing liver cancer preneoplastic nodules.5.Camellia Chrysantha(Hu)Tuyama flowers and concentrated liquids can resist toxicity action of diethylnitrosamine and have protective effect to liver.更多还原