低分子肝素对新生鼠肝细胞先天性感染人巨细胞病毒的影响

目的 研究低分子肝素( LMWH)对新生鼠肝细胞先天性感染人巨细胞病毒(HCMV)病毒学和病理学的影响.方法 取健康纯系清洁级BALB/c鼠60只,10周龄,雌雄各半,均分为5组,每组6对.LMWH干预组和阳性对照组腹腔接种6.0 lg半数组织培养感染剂量(TCID50)的HCMV AD169,空白对照组腹腔注射高糖改良Eagles培养液0.5 mL/只,然后交配.LMWH干预Ⅰ组的雌鼠在孕期给予皮下注射LMWH 1000 U/kg×10 d;LMWH干预Ⅱ组的雌鼠在孕期给予皮下注射LMWH 1000 U/kg×5d,待生产后再给新生鼠皮下注射LMWH 1000 U/kg×5 d;LMWH干预Ⅲ组在生产后予新生鼠皮下注射LMWH 1000 U/kg×10d.产后10 d处死新生鼠,取肝脏,进行病毒分离、干湿质量测定、病理学检查和荧光定量PCR检测.多组间比较采用方差分析.结果 阳性对照组新生鼠肝组织悬液中10d可分离到HCMV,而LMWH干预的3组新生鼠需24 d分离到HCMV.阳性对照组新生鼠肝组织有浊肿、空泡变性,核内有嗜碱性包涵体,部分肝细胞坏死,而LMWH干预组肝组织仅有轻度肝细胞浊肿变性,炎性反应轻微,类似空白对照组.LMWH干预组肝组织含水量较阳性对照组明显降低.LMWH干预的Ⅰ、Ⅱ、Ⅲ组50 mg肝组织HCMV DNA载量对数值分别为(3.26±0.43)、(3.26±0.41)和(3.32±0.51) lg拷贝,较阳性对照组的(7.38±0.53) 1g拷贝显著降低(F=314.620,P＜0.01),但LMWH干预组间比较差异无统计学意义(P＞0.05).结论 LMWH在宫内和出生后干预能显著减少肝细胞HCMV复制,减轻肝组织炎性反应.

Effects of low molecular weight heparin on newborn mouse liver cells congenitally infected with human cytomegalovirus

Objective To explore the effects of low molecular weight heparin (LMWH) on the virological and pathological changes of newborn mouse liver congenitally infected with human cytomegalovirus (HCMV).Methods Sixty healthy pure line clean level BALB/c mice which were about 10 weeks old (half were female) were divided into five groups (six pairs in each group).The mice in LMWH intervention group and positive control group were intraperitoneally inoculated with 6.0 lg tissue culture infective dose50 (TCID50) of HCMV AD169; those in blank control group were intraperitoneally injected with 0.5 mL dulbecco's modified Eagles medium (DMEM) ; then all the mice were paired to mate.The pregnant mice in LMWH intervention group Ⅰ were subcutaneously injected with LMWH 1000 U/kg daily for 10 days; those in group Ⅱ were subcutaneously injected with LMWH 1000 U/kg daily for 5 days and their newborn mice were subcutaneously injected with LMWH 1000 U/kg daily for 5 days; those in group Ⅲ were subcutaneously injected with LMWH 1000 U/kg daily for 10 days in their newborn mice.All these newborn mice were sacrificed at day 10 of birth.The liver was removed for virus isolation,dry-wet weight determination,pathology examination and quantitative fluorescence-polymerase chain reaction (PCR) detection.The comparison among groups was done by analysis of variance.Results HCMV was isolated from the supernatant of liver tissue homogenate in 10-day positive control newborn mice,while HCMV was isolated in 24-day newborn mice of the other three groups of LMWH intervention.Pathology confirmed that positive control liver tissue had inflammatory changed,liver cell inflammatory swelling degeneration,vacuoles degeneration,specific HCMV inclusion body in nuclear,and portion of liver cell necrosis,while liver pathological results of LMWH intervention group showed mild liver cell inflammatory changes and slight cell inflammatory swelling degeneration,which were similar to the blank control group.The moisture of liver tissue contents in LMWH intervention group decreased more obviously than positive control group.The HCMV DNA loads in 50 mg liver tissues of LMWH intervention groups Ⅰ,Ⅱand Ⅲ were (3.26±0.43),(3.26±0.41) and (3.32±0.51) lg copy,respectively,which were significantly lower than that of positive control group [(7.38 ± 0.53) lg copy; F =314.620,P0.01],while there were no significant differences among LMWH intervention groups (P＞0.05).Conclusion LMWH intrauterine and postnatal interventions can significantly reduce HCMV DNA replication in hepatocytes,and relieve inflammatory changes in liver tissue.