小鼠肝多房棘球蚴中血管新生与病程发展的相关性研究

Correlation between angiogenesis and disease progression of hepatic Echinococcus multilocularis in C57BL/6 mice

目的初步探讨小鼠肝多房棘球蚴中血管新生与疾病发生发展的相关性。方法将60只6~8周龄的C57BL/6雌性小鼠随机分成实验组和对照组,每组30只。小鼠经5%水合氯醛溶液麻醉后开腹,实验组小鼠肝脏注射多房棘球蚴原头节混悬液100μl (约400个原头节),对照组注射等量PBS,无菌缝合关腹。分别于感染后30、 60、 90、 120 d,肝脏灌注法观察病变组织血管分布和新生情况;尾静脉采血, ELISA检测小鼠血清中血管内皮生长因子A (VEGFA)的表达;实验组取棘球蚴组织和棘球蚴周围肝组织,对照组取相应肝组织,制备石蜡切片,苏木素-伊红(HE)染色观察病理组织学改变;免疫组织化学染色法观察不同感染时间、不同部位肝脏组织中VEGFA、 CD34和CD31的表达情况。结果肝脏灌注法结果显示,随着感染时间的延长,实验组小鼠肝脏棘球蚴组织逐渐增大,周围可见明显的血管分布。ELISA检测结果显示,感染后30、 60、 90和120 d,实验组小鼠血清中VEGFA浓度分别为269.00、 420.62、 539.00和271.73 pg/ml,差异有统计学意义(P <0.01),且均高于相应对照组的VEGFA浓度(194.00、 173.00、 234.00和127.00 pg/ml)(P <0.05)。HE染色结果显示,随着感染时间的延长,实验组小鼠棘球蚴组织体积逐渐增大,并伴有嗜酸性粒细胞浸润、结核样肉芽组织形成和纤维组织增生,病变内部区域可见原头节和钙化灶。免疫组织化学结果显示,感染后棘球蚴组织中均可见VEGFA、 CD34和CD31不同程度的表达,阳性染色定位于棘球蚴组织内皮细胞。感染后30、 60、 90和120 d,棘球蚴组织中VEGFA免疫组织化学评分分别为(1.60±0.52)、(3.10±0.87)、(4.80±1.32)和(2.40±1.07)分,其中感染后30 d与感染后60 d、 90 d比较差异均有统计学意义(P <0.05, P <0.01);棘球蚴组织与棘球蚴周围组织(VEGFA评分均为0)和对照组(VEGFA评分均为0)比较差异均有统计学意义(P <0.01)。感染后30、 60、 90和120 d,棘球蚴组织中微血管密度(CD34-MVD)分别为(38.70±11.06)/HP、(65.50±8.46)/HP、(109.90±9.40)/HP和(56.86±6.64)/HP,差异有统计学意义(P <0.001),且均高于棘球蚴周围组织和对照组(P <0.01)。棘球蚴组织中CD31-MVD分别为(19.80±3.12)/HP、(30.70±2.50)/HP、(47.90±4.77)/HP和(31.10±3.84)/HP,差异有统计学意义(P <0.01),且均低于Em周围组织和对照组(P <0.001)。结论多房棘球蚴浸润性生长中伴有血管新生, VEGFA的分泌刺激了血管新生,新生血管促进棘球蚴的生长。

Objective To investigate the relationship between angiogenesis and disease progression in hepatic Echinococcus multilocularis(Em) in mice. Methods Total 60 C57 BL/6 female mice with 6-8 weeks old were randomly divided into two groups: the experimental group and the control group with 30 for each. Each mouse in the experimental group was infected by direct injection into liver with 100 μl of suspension containing 400 Em protoscoleces under anesthesia of 5% chloral hydrate. In the control group mice were injected with the same volume of PBS. Some of mice were euthanized and the liver blood vessel was filled with silicone to show the distribution of blood vessels and angiogenesis 30 d, 60 d, 90 d and 120 d after infection. The vascular endothelial cell growth factor A(VEGFA) in the sera of mice were measured by ELISA. The mouse livers were collected and subjected to hematoxylin and eosin(HE) staining for observing pathological changes. The dynamic changes of VEGFA, CD31 and CD34 expression in infected liver was analyzed by immunohistochemical staining. Results The liver blood vessel filled with silicone showed that Em gradually grew in the livers of mice, surrounded by the increased blood vessels as the infection continued in the experimental group. The levels of VEGFA in the sera of infected mice were269.00 pg/ml at 30 d, 420.62 pg/ml at 60 d, 539.00 pg/ml at 90 d and 271.73 pg/ml at 120 d after infection,which were significant higher than that in un-infected control mice(194.00 pg/ml at 30 d, 173.00 pg/ml at 60 d,234.00 pg/ml at 90 d and 127.00 pg/ml at 120 d after infection) with statistical difference( P < 0.05). HE staining of infected liver tissue demonstrated that the growing Em tissue surrounded by the filtration of eosinophils, granuloma and fibrosis. The protoscoleces and calcification could be observed in the middle of the lesion. Immunohistochemical staining demonstrated different degrees of expression of VEGFA, CD34 and CD31 in the endothelial cells of Em tissue. The VEGFA immunohistochemical scores of Em tissues were 1.60 ± 0.52 at 30 d, 3.10 ± 0.87 at 60 d, 4.80 ± 1.32 at 90 d and 2.40 ± 1.07 at 120 d after infection, which were significant higher than that in the surrounding normal liver tissue of infected mice or livers of control mice(0)( P < 0.01). Among the infected mice, the VEGFA immunohistochemical scores at 60 d and 90 d were significant higher than that at 30 d after infection( P < 0.05,P < 0.01, respectively). The expression of CD34-MVD in the Em tissues were 38.70 ± 11.06 at 30 d, 65.50 ± 8.46 at 60 d, 109.90 ± 9.40 at 90 d, and 56.86 ± 6.64 at 120 d after infection, which were significant higher than those expressed in normal liver cells( P < 0.01). The expression of CD34-MVD at the different time of infection was also significantly different(P < 0.01). Expression of CD31-MVD in the Em tissues were 19.80 ± 3.12 at 30 d, 30.70 ± 2.50 at 60 d, 47.90 ± 4.77 at 90 d and 31.10 ± 3.84 at 120 d after infection with significant difference among different infection time points(P < 0.01), but significant lower than that expressed in normal liver tissues( P < 0.01).Conclusion E. multilocularis invasively grows in infected liver along with significant angiogenesis and expression of CD31 possibly due to the stimulated secretion of VEGFA. The secretion of VEGFA stimulated angiogenesis,neovascularization induced by Em infection in liver of mouse.