常规超声联合血清甲胎蛋白评估改良二乙基亚硝胺给药法诱导大鼠肝细胞肝癌模型的可行性

Feasibility of conventional ultrasound combined with serum alpha-fetoprotein in the evaluation of modified diethylnitrosamine administration induced hepatocellular liver cancer model in rats

目的探讨改良二乙基亚硝胺(DEN)给药法诱导大鼠肝细胞肝癌(HCC)模型及常规超声联合血清甲胎蛋白(AFP)评估HCC模型的可行性。方法将175只无特定病原体级雄性Sprague-Dawley大鼠随机分为空白对照组(n=8)、对照组(n=69)和模型组(n=98)。对照组大鼠给予单一剂量DEN(50 mg·kg^(-1))腹腔注射制备HCC模型。模型组大鼠采用改良DEN给药法诱导大鼠HCC,即根据体质量变化动态调整DEN给药剂量:初始剂量50 mg·kg^(-1),然后每周根据体质量变化调整给药剂量,若大鼠体质量未下降或下降幅度小于每周5 g时,按50 mg·kg^(-1)给药;若每周大鼠体质量下降5~20 g,按25 mg·kg^(-1)给药;若大鼠体质量下降超过每周20 g,暂停给药1周。空白对照组大鼠腹腔注射等量的生理盐水。第1~4周每周给药2次,第5~15周每周给药1次,连续给药15周。造模完成后,记录并比较造模期间3组大鼠死亡率;对存活大鼠行肝脏超声、血清AFP水平检查。随机抽取空白对照组7只大鼠、对照组14只大鼠和模型组14只大鼠,麻醉后处死并解剖,观察肝脏成瘤情况,记录肝脏质量并计算肝脏指数,苏木精-伊红染色观察大鼠肝脏病理学变化。结果造模期间,空白对照组、对照组、模型组大鼠死亡率分别为0.0%(0/8)、34.8%(24/69)、19.4%(19/98);对照组大鼠死亡率显著高于空白对照组(χ^(2)=4.043,P<0.05),模型组与空白对照组大鼠死亡率比较差异无统计学意义(χ^(2)=1.890,P>0.05),模型组大鼠死亡率显著低于对照组(χ^(2)=5.019,P<0.05)。空白对照组大鼠均未见癌结节或肝硬化表现,超声影像正常。对照组30只、模型组48只大鼠肝脏可见癌结节,癌结节超声检出率分别为66.7%(30/45)、60.8%(48/79);对照组与模型组大鼠的肝脏癌结节超声检出率比较差异无统计学意义(χ^(2)=0.278,P>0.05)。对照组与模型组大鼠血清AFP水平显著高于空白对照组(t=0.728、0.771,P<0.05);对照组与模型组大鼠血清AFP水平比较差异无统计学意义(t=0.043,P>0.05)。超声对肝脏癌结节诊断的敏感度为62.9%(78/124),血清AFP对肝脏癌结节诊断的敏感度为58.1%(50/86),血清AFP联合超声对肝脏癌结节诊断的敏感度为87.2%(73/86)。超声与血清AFP对肝脏癌结节诊断的敏感度比较差异无统计学意义(χ^(2)=1.014,P>0.05);血清AFP联合超声检查对肝脏癌结节诊断的敏感度显著高于超声和血清AFP单独检测(χ^(2)=35.342、21.271,P<0.05)。空白对照组大鼠肝脏均表面光滑,颜色红润细腻,未见结节;对照组和模型组大鼠肝脏增大,质地变硬,弥漫着大小不等的癌结节,部分癌结节伴出血坏死。对照组和模型组大鼠体质量显著低于空白对照组,肝质量、肝脏指数显著高于空白对照组(P<0.05);对照组和模型组大鼠体质量、肝质量、肝脏指数比较差异无统计学意义(P>0.05)。空白对照组大鼠肝小叶结构清晰,肝细胞索排列整齐,细胞核明显,未见水肿及炎症细胞浸润;对照组和模型组大鼠肝小叶结构不完整,出现假小叶,癌细胞排列成巢状,可见纤维组织增生,部分区域可见局灶性的细胞坏死,癌细胞核大、深染,偶见双核或多核,具有明显的细胞异形性,部分肿瘤细胞侵犯血管。2组大鼠的成瘤率均为100.0%。结论根据体质量变化动态调整DEN给药剂量诱导HCC大鼠模型,既可保证成瘤率又可降低造模过程中大鼠死亡率,肝脏超声联合血清AFP检测可作为评估大鼠HCC模型的方法。

Objective To investigate the feasibility of modified diethylnitrosamine(DEN)administration induced hepatocellular liver cancer(HCC)in rats and conventional ultrasound combined with serum alpha-fetoprotein(AFP)in the evaluation of HCC model.Methods One hundred and seventy-five specific pathogen free male Sprague-Dawley rats were randomly divided into blank control group(n=8),control group(n=69)and model group(n=98).The rats in the control group were given a single dose of DEN(50 mg·kg^(-1))intraperitoneally to prepare HCC model.The rats in the model group were treated with modified DEN administration to induce HCC,the dose of DEN was adjusted dynamically according to the change of body mass:the initial dose was 50 mg·kg^(-1),and then the dose was adjusted weekly according to the change of body mass,if the body weight of rats did not decrease or the decrease was less than 5 g in one week,the drug was administered at the dose of 50 mg·kg^(-1);if the body mass of rats decreased in the range of 5-20 g in one week,the drug was administered at 25 mg·kg^(-1);if the body mass of rats decreased and the rate of decrease of body mass of rats exceeded 20 g in one week,the drug was suspended for one week.Rats in the blank control group were injected intraperitoneally with an equal amount of saline.The rats were administered twice a week from the 1^(st) to the 4^(th) week,and once a week from the 5^(th) to the 15^(th) week,and continuously for 15 weeks.After the completion of modeling,the mortality rate of the rats in the three groups during the modeling period was recorded and compared;liver ultrasound and serum AFP levels were examined in the surviving rats.Seven rats in the blank control group,14 rats in the control group and 14 rats in the model group were randomly selected,they were anesthetized and executed,and the liver tumor formation of the rats were observed,the liver weight of the rats was recorded and the liver index was calculated,and the pathological changes of liver of the rats were observed by hematoxylin-eosin staining.Results During the modeling period,the mortality rates of rats in the blank control group,control group and model group were 0.0%(0/8),34.8%(24/69)and 19.4%(19/98),respectively;the mortality rate of rats in the control group was significantly higher than that in the blank control group(χ^(2)=4.043,P<0.05),there was on significant difference in the mortality rate of rats between the model group and the blank control group(χ^(2)=1.890,P>0.05),the mortality rate of rats in the model group was significantly lower than that in the control group(χ^(2)=5.019,P<0.05).None of the rats in the blank control group had tumor nodules in liver and cirrhosis,and ultrasound images were normal.Cancer nodules were visible in the liver of 30 rats in the control group and 48 rats in the model group,and the detection rates of cancer nodules by ultrasound in the control group and model group were 66.7%(30/45)and 60.8%(48/79),respectively;there was no significant difference in the detection rates of liver cancer nodules by ultrasound between the control group and model group(χ^(2)=0.278,P>0.05).The serum AFP level of rats in the control group and model group were significantly higher than that in the blank control group(t=0.728,0.771;P<0.05);there was no significant difference in the serum AFP level of rats between the control group and model group(t=0.043,P>0.05).The sensitivity of ultrasound in the diagnosis of liver cancer nodules was 62.9%(78/124),the sensitivity of serum AFP in the diagnosis of liver cancer nodules was 58.1%(50/86),and the sensitivity of serum AFP combined with ultrasound detection in the diagnosis of liver cancer nodules was 87.2%(73/86).There was no significant difference in the sensitivity of the diagnosis of liver cancer nodules between the ultrasound and serum AFP(χ^(2)=1.014,P>0.05);the sensitivity of serum AFP combined with ultrasound in the diagnosis of liver cancer nodules was significantly higher than that of ultrasound and serum AFP alone(χ^(2)=35.342,21.271;P<0.05).The liver of rats in the blank control group had smooth surface,ruddy and delicate color,and no nodule was found;the liver of the rats in the control group and the model group increased,the texture became hard,and nodules of different sizes were diffused,and some nodules were accompanied with hemorrhage and necrosis.The body mass of rats in the control group and model group was significantly lower than that in the blank control group,and the liver mass and liver index were significantly higher than those in the blank control group(P<0.05);there was no significant difference in the body mass,liver mass and liver index of rats between the control group and model group(P>0.05).In the blank control group,the liver lobule structure of rats was clear,the hepatocyte cords were arranged neatly,the nuclei were obvious,and there was no edema and inflammatory cell infiltration;in the control group and model group,the liver lobule structure was incomplete,there was pseudolobules,cancer cells were arranged in a nest,fibrous tissue proliferation could be seen,focal cell necrosis could be seen in some areas,cancer cells showed pleomorphism with large and hyperchromatic nuclei,the binuclear or polynuclear cells could be seen occasionally,and some tumor cells invaded blood vessels.The tumor formation rate of rats in the both groups was 100.0%.Conclusion Dynamic adjustment of DEN dose according to body mass changes to induce HCC model in rats can ensure the tumorigenic rate and reduce the mortality of rats during the modeling process.Conventional ultrasound combined with serum AFP detection can be used as a method to evaluate HCC model in rats.