抗骨桥蛋白抗体抑制肝泡型棘球蚴侵袭性生长和转移

Inhibition of Invasive Growth and Metastasis of Hepatic Alveolar Echinococcosis by Anti-Osteopontin Antibody

目的外源性给予抗骨桥蛋白(osteopontin,OPN)抗体,观察其对肝泡型棘球蚴生长和转移的抑制作用。方法采用开腹直视下肝脏穿刺接种泡型棘球蚴组织混悬液的方法(0.1 ml/只,约含原头节400个),建立肝泡型棘球蚴沙鼠模型180只,随机分为3组。实验组和对照组于接种后当天经尾静脉分别注射0.15 ml抗OPN抗体(效价1∶32)和沙鼠注射用的兔血清,共注射17次,前7次每次间隔2 d,后10次每次间隔1周。模型组未作处理。上述3组沙鼠分别于感染后1、20、40、60、80和100 d随机各处死10只沙鼠,观察泡型棘球蚴的生长和转移情况,采用免疫组织化学SP法观察各组沙鼠肝泡型棘球蚴组织中OPN的表达情况。于感染后100 d乙醚轻度麻醉沙鼠后,摘眼球取血,分离血清,用ELISA测定沙鼠血清中OPN的含量。结果感染后1、20、40、60和80 d,实验组与对照组和模型组囊湿重、胸腔淋巴结转移率间的差异均无统计学意义(P>0.05)。感染后100 d,实验组囊湿重、胸腔淋巴结转移率[(7.28±0.38)g、20%]均低于对照组[(9.70±0.61)g、70%]和模型组[(9.32±0.73)g、70%](P<0.05)。免疫组化SP法结果显示,在肝泡型棘球蚴组织中有OPN不同程度的表达,均定位于细胞浆,呈黄色、棕黄色或棕褐色,主要分布在肝泡型棘球蚴纤维囊壁的生发层。感染后1、20、40、60和80 d,实验组与对照组和模型组比较,骨桥蛋白在肝泡型棘球蚴组织中的阳性表达率间的差异无统计学意义(P>0.05)。感染后100 d,实验组骨桥蛋白在肝泡型棘球蚴组织中的阳性表达率(20%)明显低于对照组(80%)和模型组(80%)(P<0.05),且实验组血清中OPN含量[(30.90±2.25)ng/μl]显著低于对照组[(41.03±2.76)ng/μl]和模型组[(42.39±2.85)ng/μl](P<0.05)。结论抗OPN抗体可以降低沙鼠肝泡型棘球蚴组织和血清中OPN的水平,阻断OPN的功能,可抑制泡型棘球蚴的侵袭性生长和转移。

Objective To observe the inhibitive effect on invasive growth and metastasis of Echinococcus multolocularis by exogenous anti-osteopontin(OPN)antibody.Methods 180 gerbils were infected with 20% E.multolocularis suspension(approximately 400 protoscoleces in 0.1 ml per gerbil)through abdominal opening injection in liver,and then divided into model group,experiment group and control group.Experiment group and control group each with 60 gerbils were injected via the tail vein with 0.15 ml of anti-OPN antibody(1 ∶ 32)and rabbit serum,respectively.All gerbils in the two groups received injections,with 2-day interval for the first seven injections,and then at 7-day interval for the remaining injections.Model group were without any treatment.The three groups were subdivided into six groups each with 10 gerbils.The gerbils from each group were sacrificed on day 1,20,40,60,80,and 100 after infection,respectively.Hepatic echinococcus cyst and metastasis tissue were observed.The expression of OPN was measured by immunohistochemistry staining(SP method).Serum samples were collected at 100 d post-infection,and the content of OPN in sera was measured by ELISA.Results There were no significant difference in cyst weight and metastatic rate of thoracic lymph node among the three groups at 1,20,40,60,and 80 d post-infection(P>0.05),while at 100 d post-infection,cyst weight and metastatic rate of thoracic lymph node in experiment group[(7.28±0.38) g,20%]were lower than that of control group[(9.70±0.61) g,70%]and model group[(9.32±0.73) g,70%](P<0.05).Expression of OPN was found at different levels in echinococcus cysts.OPN was located in the cytoplasm,and mainly distributed in the germinal layer.The OPN positive expression levels were not significantly different between experiment group and other groups on day 1,20,40,60,and 80 afer infection(P>0.05).At 100 d post-infection,OPN positive expression rate and serum OPN content in experimental group[20% and(30.90±2.25) ng/μl,respectively]was lower than that of control group[80% and(41.03±2.76) ng/μl]and model group[80% and(42.39±2.85) ng/μl](P<0.05).Conclusion Anti-osteopontin antibody can reduce OPN concentration in hepatic echinococcus cyst and serum,and inhibit the invasive growth and metastasis of E.multolocularis.