【作者】 池菲；

【导师】 姜达；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2009， 硕士

【摘要】 目的:本实验通过构建癌性恶病质动物模型,观察经β2受体激动剂福莫特罗干预后,癌性恶病质小鼠一般状态及去瘤体重、生化指标、血清细胞因子、骨骼肌中Myostatin蛋白、生存期的改变,初步探讨β2受体激动剂早期抗恶病质作用及可能机制。方法:选取健康雄性BALB/c小鼠82只随机分为5组,健康对照组(HC) :10只,生理盐水组(CC) :18只,甲羟孕酮组(MPA) :18只,预防用药组(PG):18只,福莫特罗组(FG):18只。将鼠结肠腺癌26(colon26)细胞株接种于CC组、MPA组、PG组、和FG组小鼠右腋窝皮下,构建癌性恶病质实验动物模型,HC组小鼠接种等量生理盐水,随机从各荷瘤组小鼠取出8只用于观察生存期。3-5天后各荷瘤小鼠皮下均可触及瘤结节,PG组开始腹腔注射福莫特罗(2mg·kg-1·d-1)。荷瘤第15天小鼠进入癌性恶病质阶段后,FG组开始腹腔注射福莫特罗(2mg·kg-1·d-1),CC组给予等量生理盐水腹腔注射,MPA组甲羟孕酮(120mg·kg-1·d-1)灌胃,连续给药1周后,处死全部研究药效小鼠并留取标本,其余用于观察生存期的小鼠继续给药,记录自然死亡时间。应用全自动生化分析仪测定各组营养代谢指标,放射免疫法测定血清肿瘤坏死因子α(TNF-α)、胰岛素样生长因子-1(IGF-1)的水平,普通光学显微镜下观察腓肠肌纤维萎缩情况,免疫组化方法测定小鼠腓肠肌中肌肉生长抑制素(Myostatin)蛋白的表达情况,观察比较这些指标在各组小鼠之间的差异。用SPSS13.0统计学软件进行数据处理,各组数据以均数±标准差(x±s)的形式表示,多组间均数差异性比较采用单因素方差分析(one-way ANOVA)。结果:1动物模型:将colon26癌细胞接种于各组小鼠皮下,3-5天后各荷瘤组均可触及瘤结节,一般情况无明显变化。荷瘤15天,荷瘤小鼠出现皮毛粗糙、厌食、去瘤体重下降10%以上,即进入癌性恶病质状态。2一般状况及去瘤体重:试验初,各组小鼠体重无明显差异(Ρ>0.05)。荷瘤第15天CC组小鼠饮食量及一般状况恶化最明显,MPA组、PG组、FG组摄食量较CC组略多;MPA组、PG组、FG组较CC组的终末去瘤体重增加(P<0.05);PG组较MPA组、FG组能显著增加终末去瘤体重(P<0.05);FG组去瘤体重大于MPA组,但无统计学意义(Ρ>0.05)。3生化指标:各荷瘤组小鼠呈现不同程度的代谢紊乱。CC组小鼠血糖低于其余各组(Ρ<0.05);药物干预后:MPA组、PG组、FG组较CC组能提高血清中血糖含量,但无统计学意义(Ρ>0.05);MPA组血清中总胆固醇较CC组下降(Ρ<0.05);CC组小鼠血浆总蛋白水平较HC组小鼠无明显降低(P>0.05);各荷瘤组小鼠血浆总蛋白水平与HC组相比亦无明显差别(P>0.05)。4血清细胞因子:各组荷瘤小鼠血清TNF-α显著高于HC组(Ρ<0.05);药物干预后MPA组、PG组、FG组小鼠血清TNF-α较CC组显著降低(Ρ<0.05);PG组小鼠血清中TNF-α水平明显低于MPA组(Ρ<0.05);FG组小鼠血清TNF-α水平低于MPA组,无统计学意义(Ρ>0.05);PG组较FG组能明显降低血清TNF-α(Ρ<0.05)。各组荷瘤小鼠血清IGF-1水平明显低于HC组(Ρ<0.05);药物干预后MPA组、PG组、FG组小鼠血清IGF-1水平与CC组无明显差别(Ρ>0.05)。5小鼠腓肠肌湿重:各组荷瘤小鼠腓肠肌湿重较HC组有明显下降(Ρ<0.05);PG组、FG组小鼠腓肠肌湿重明显大于CC组、MPA组(Ρ<0.05);PG组小鼠腓肠肌湿重明显大于FG组(Ρ<0.05);MPA组小鼠腓肠肌湿重与CC组之间无明显差别(Ρ>0.05)。6普通光学显微镜下观察腓肠肌萎缩程度:各组荷瘤小鼠腓肠肌均有不同程度萎缩,横截面最大横径均小于HC组(Ρ<0.05),其中CC组最明显;药物干预后PG组、FG组小鼠腓肠肌纤维横截面最大横径明显大于CC组、MPA组(Ρ<0.05);PG组小鼠腓肠肌纤维横截面最大横径大于FG组,有显著差别(Ρ<0.05);CC组与MPA组比较无明显差别(Ρ>0.05)。7腓肠肌中Myostatin蛋白表达:各组荷瘤小鼠Myostatin蛋白表达水平均明显高于HC组(Ρ<0.05);药物干预后PG组、FG组小鼠腓肠肌中Myostatin蛋白表达量较CC组明显降低(Ρ<0.05);MPA组与CC组比较无明显差别(Ρ>0.05) ;PG组小鼠腓肠肌中Myostatin蛋白表达明显低于FG组(Ρ<0.05)。8各组生存期:治疗后MPA组、PG组、FG组生存时间较CC组明显延长(Ρ<0.05):CC组8只小鼠中位生存期为23.38天;MPA组8只小鼠中位生存期为26.13天;PG组8只小鼠中位生存期为26.25天;FG组8只小鼠中位生存期为28.50天。MPA组小鼠的生存时间与PG组无明显差别(Ρ>0.05);FG组较PG组能明显延长生存时间(Ρ<0.05)。结论:1本试验成功构建了癌性恶病质动物模型,癌细胞造成宿主动物进食减少、体重下降,骨骼肌消耗表现更为突出。为研究癌性恶病质中骨骼肌蛋白降解的机制及其早期防治提供了的实验基础。2癌性恶病质小鼠体内细胞因子水平发生改变:TNF-α是癌性恶病发生机制中一重要细胞因子;药物干预不能改变血清IGF-1水平。3甲羟孕酮可增加恶病质小鼠终末去瘤体重,降低血清TNF-α水平,调节代谢,但对于骨骼肌的增重作用不明显。4β2受体激动剂福莫特罗可以抑制骨骼肌蛋白的降解而发挥抗恶病质作用;对调节代谢无效。应用该药还能减缓腓肠肌重量的下降,从而增加去瘤体重,与甲羟孕酮机制不同。5癌性恶病质骨骼肌分泌的Myostatin对泛素相关基因有上调的作用,最终通过泛素蛋白酶途径降解骨骼肌蛋白,通过研究β2受体激动剂对Myostatin表达的调控,可以使逆转恶病质发生在更早阶段,达到预防的目的。6醋酸甲羟孕酮和福莫特罗均能够延长恶病质小鼠的生存期;但预防性应用β2受体激动剂延长恶病质小鼠生存时间的优势较弱。更多还原

【Abstract】 Objective:The experiment is to study the influence ofβ2-agonist formoterol on cancer cachexia by establishing an experimental cancer cachectic model,observing the influence on condition,removed tumor weight,metabolism indicators,serum cytokine,survival time and the regulation of myostatin in skeletal muscle.Studying the mechanism of formoterol in anticachectic effects in the early stage.Methods:82 healthy BALB/c mice were randomized into 5 groups:healthy control group(HC) 10,cancer cachexia with physiologic solution group(CC) 18,cancer cachexia with Medroxyprogesterone group(MPA)18,cancer cachexia with prophylactic group(PG)18,cancer cachexia with Formoterol group(FG)18."CC"group、"MPA"group、"PG"groupand" FG"group received a inoculum of colon26 cells on right armpit to establish cancer cachexia model."HC"group received a corresponding volume of physiologic solution.8 mice were randomized selected from "CC"group、"MPA"group、"PG"group、and "FG"group were used to observe survival time.3-5 days later,the tumor could be touched in each tumor bearing group ."PG "group being administered a daily intraperitoneal dose of formoterol (2mg/kg body weight).On the 15th day, the other tumor bearing mice got into cancer cachexia."FG"group being administered a daily intraperitoneal dose of formoterol (2mg/kg body weight),"CC"group administered a corresponding intraperitoneal dose of physiologic solution."MPA"group theraped with Medroxyprogesterone (120mg/kg body weight/day).All groups were treated one week later,the animal were killed and collected tissue and serum.The rest mice which to observe survival time had daily drug therapy and write down the survival time.The serum levels of biochemical indicators were measured by omni-automatic biochemistry analyzer. The serum levels of cytokine TNF-αand IGF-1 were measured by RIA. The atrophy of gastrocnemius was observed by optical microscope.The expression of myostatin in gastrocnemius was investigated by immunohistochemistry.The differences in all indicators of each group were observed and contrasted. All data were analyzed with the spss13.0 software and expressed by ( x±s), the comparison among multitude simple mean by One-Way analysis of variance (One-Way ANOVA).Results:1 Animal model:The tumor could be touched on the tumor bearing mice when received inoculum of tumor cell 3-5 days later. The general condition had no obviously change.All tumor bearing mice got into cancer cachexia characterized coarse fur, anorexia and weight decreased more than 10% 15 days later.2The condition and removed tumor weight : In the begining,the body weight of each group had no notable difference (Ρ>0.05).15 days after tumor bearing,the mice in"CC"group had least food intake and worst condition.The food intake in "MPA"group,"PG"group and "FP"group was more than "CC"group ( P<0.05 ) .The last removed tumor weight in"MPA"group,"PG"group and "FG"group was higher than in"CC"group ( P<0.05 ) ;the last removed tumor weight in"PG"group was higher than in "MPA"group and "FG"group;the last tumor removed weight in "FG"was higher than in"MPA"group,with no significant difference(Ρ>0.05).3 Biochemical indicators:All tumor bearing mice were characterized by metabolic disorders.The glucose in "CC"group was lower than other groups(P<0.05);with drug intervened,the serum level of glucose in"MPA"group,"PG"group and "FG"group was higher than in "CC"group,with no significant difference(P>0.05);there was significant difference of the serum level of cholesterol between "MPA"group and"CC"group(P<0.05);there was no significant difference of the serum level total protein between "CC"group and "HC"group(P>0.05);the serum level of total protein in all tumor bearing mice and "HC"group has no significant difference(P>0.05).4 Cytokines:The serum level of TNF-αin each tumor bearing mice group was higher than in "HC"group;with drug intervened,the serum level of TNF-αin"MPA"group,"PG"gorup and "FG"group was lower obviously than in "CC"group(P<0.05);the serum level of TNF-αin "PG"group was lower than in "MPA"(P<0.05);the serum level of TNF-αin "FG"group was lower than in "MPA"group with no significant difference(P>0.05);the serum level of TNF-αin "PG"group was obviously lower than in "FG"group(P<0.05).The serum level of IGF-1 in each tumor bearing mice group was lower than in "HC"group(P<0.05);with drug intervened,there was no significant difference of the serum level of IGF-1 between each tumor bearing mice group and "HC"group (P>0.05).5The wet weight of grastrocnemius:The wet weight of gastrocnemius in each tumor bearing mice group was lower obviously than "HC"group(P<0.05);the wet weight of grastrocnemius in "PG"group and "FG"group was higher than "MPA"group and "CC"group(P<0.05);the wet weight of grastrocnemius in "PG"group was higher obviously than in "FG"group(P<0.05);there was no obviously difference of the wet weight of grastrocnemius between "CC"group and "MPA"group(P>0.05).6The atrophy of grastrocnemius observed by optical microscope:All tumor bearing mice characterized variable degrees of atrophy.The maximum cross sectional diameter in grastrocnemius of each tumor bearing group was shorter than in "HC"group(P<0.05);with drug intervened,the maximum diameter in "PG"group and "FG"group was longer obviously than "CC"group and "MPA"group(P<0.05);the maximum diameter in "PG"group was obviously longer than in "FG"group(P<0.05);there was no significant difference of maximum diameter between "CC"group and "MPA"grouop (P>0.05).7The activity of myostatin in grastrocnemius:The activity of myostatin in grastrocnemius in each tumor bearing group was more strengthened than in "HC"group(P<0.05);"CC"group was the most strenghtened;with drug intervened,the activity of myostatin in grastrcnemius in "PG"grouop and "FG"group was weaker than in "CC"group(P<0.05);there was no significant difference in activity of myostatin in grastracnemius between "MPA"group and "CC"group(P>0.05);the activity of myostatin in grastracnemius in "PG"group was weaker than in"FG"group,with significant difference(P<0.05).8The survival time:With drug intervened,the survival time in "MPA"group,"PG "group and "FG"group was longer than "CC"group(P<0.05);the survival time of "CC"group,"MPA"group,"PG"groupand"FG"groupwas23.38days,26.13days,26.25days and 28.50days;the survival time between "MPA"group and "PG"group had no difference(P>0.05);the survival time in "FG"group was longer than in "PG"group(P<0.05).Conclusions:1The study had successfully established an experimental animal model .Carcinoma cell caused in the host rapid loss of body weight gradually,particularly in skeletal muscle. This model provides contribution to study the mechanism of skeletal muscle depletion in cancer cachexia and the prevention and cure of cancer cachexia in the early stage.2In cancer cachectic model, there were cytokine disorder such as TNF-αand IGF-1.TNF-αhad an important role in the mechanism of cancer cachexia; with drug intervened,the serum level of IGF-1 had no significant changed.3Medroxyprogesterone increased the last tumor removed weight ,lowed the serum level of TNF-αand regulated the metabolic disorders.However,it had no effect on skeletal muscle.4Asβ2-agonist,formoterol atagonized the skeletal muscle depletion to take anticachectic effects. However,formoterol had no effect on metabolic disorders.With different mechanism compared to medroxyprogesterone,mice intervened with formoterol resulted in an reversal of the grastrocnemius muscle-wasting process.Formoterol increased the last tumor removed weight .5Myostatin,secreted from skeletal muscle in cancer cachexia ,up-regulated ubiquitin related genes to proteolysis in skeletal muscle. On the regulation of myostatin, the study indicatedβ2-agonist as a potential therapeutic tool in the early stage of cancer cachexia.6Both medroxyprogesterone and formoterol prolonged the survival time of cancer cachectic mice;preventive use of formoterol in prolong the survival time was the weakest.更多还原