拉米夫定和干扰素α对孕期降低HBV转基因小鼠血中HBV载量有效性和安全性的初步研究
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摘要
目的：既往研究证实阻断乙肝病毒宫内传播的关键之一是降低母亲外周血中病毒含量；拉米夫定(3TC)、干扰素α(IFNα)是抗HBV药物中目前在临床上使用最广泛、也是抗病毒效果较好的药物，但它们的妊娠药物分级均为C级，即尚未对妊娠妇女或动物进行研究。本研究在国家自然科学基金(No30070668)的资助下，首次应用可高水平表达HBV的转基因小鼠，在不同妊娠期中，进行3TC、IFNα抗病毒治疗，检测母血中HBsAg、HBV DNA等感染指标变化，同时观察其对母胎生长发育的影响，以期探讨这两种药物对降低母血HBV含量的作用和母胎安全性；通过评价3TC和IFNα对妊娠合并乙型肝炎病毒携带状态的疗效与安全性的探索性实验研究，为进一步的HBsAg阳性孕妇妊娠期，尤其是在妊娠早期是否选择这两种药物进行抗病毒治疗以阻断宫内传播提供一定的动物实验依据。另外，对HBV转基因小鼠作为研究HBV宫内传播动物模型的可能性做初步探索。
方法：1．收集昆明种小鼠的单细胞受精卵，将纳入合格的胚胎细胞随机分组，根据培养液的不同，分成含不同浓度3TC或IFNα的试验组和普通培养液的对照组。采用微滴培养法连续培养72h，对比观察不同浓度3TC和IFNα对体外胚胎细胞早期发育的直接影响。
2．将稳定、高水平表达HBV抗原的21只HBV转基因母鼠随机分成试验组1、试验组2及对照组。试验组1于孕第5天开始口服3TC
第四军医大学博士学位论文
10Omg·kg一，·天一，直至分娩，试验组2于孕第10天开始口服3TC
100mg·kg一，·天一，直至分娩，对照组于孕第5天开始口服生理盐水
0.5ml.天一‘直至分娩，同时为了观察HBV转基因小鼠和普通小鼠在
某些观察指标上是否存在差别，另选12只普通正常C57BL/6J母鼠，
随机分为2组做为普通鼠试验组与对照组。
3.选21只HBV转基因小鼠和12只普通正常C57BL/6J母鼠，采用
IFNQ作为试验药物，肌注IFN Q SMU.m一，.2d一，，试验组与对照组的设
立与3TC相同。
4.所有试验组和对照组统一用固相放免方法定量检测用药前后母
鼠血清HBsAg，用荧光PCR定量分析HBV DNA浓度变化。
5.各组孕鼠每天专人观察妊娠期的外观及活动等一般生理学和反
应生育情况的指标;母鼠分娩采用24h连续观察法;每组母鼠在妊
娠第19天时随机取3只，系统解剖，记录各组的平均胎仔重、黄
体重、平均胎盘重等反应生育情况的指标，计算各组平均死胎率(死
胎数/胎仔总数)和母体死胎率(出现死胎的母体数/妊娠母体总
数)，并作统计学检验;记录各组母鼠的主要脏器重量、脏器系数;
对收集的母鼠主要脏器做病理学和免疫组织化学检查。
6.各组所取活胎采用系统解剖观察法做外观畸形、内脏畸形及骨
骼畸形检查;纪录每组新生鼠出生存活情况，T检验统计各组新生
鼠存活只数，新生鼠出生后每5天称重一次，观察一般发育情况，
记录张耳天数、出毛天数和开眼天数等指标。从母鼠、胎鼠及新生
鼠几方面同时观察3TC及IFNQ对孕鼠及其子代的影响。
7.对收集的胎盘做病理学和免疫组织化学检测;新生鼠生长至一
月时，抽取血清用ELISA和PCR方法检测HBsAg和HBV DNA。
结果:1.在IFNQ浓度高达1000工U.ml一，时，单细胞胚胎至8-
细胞和桑堪胚的发育率有下降趋势;当3TC浓度高达500“mol·L一，
时，除了2一细胞发育率与对照组无明显差异外，其余各期的胚胎细
胞发育率均明显下降(P<0.05)，而且培养中胚胎细胞变性、退化
第四军医大学博士学位论文
和不规则现象增加。说明这两种抗病毒药物，在高浓度时，有抑制
胚胎生长的可能，或对胚胎有一定毒性作用，而且在着床前的8-
细胞期和桑堪胚期，其抑制作用更加明显(P<0.01)。
2.IFNa浓度(800IU.ml一，，3TC浓度(300 p mol.L一，时，对小鼠
早期胚胎细胞发育基本无抑制作用。
3.9只口服3TC的HBV DNA阳性转基因小鼠，在用药巧天或
10天后，DNA浓度均明显下降(P<0.01)，有3只则完全阴转。而口
服生理盐水的小鼠，DNA浓度无明显变化。HBsAg定量结果除连续
服用3TC巧天的转基因小鼠，用药后的血清HBsAg浓度明显比用
药前低(P<0 .05)，余各组变化则不明显(P>.05)。
4.12只HBV DNA阳性肌注IFNa或生理盐水的小鼠，HBV DNA
和HBsAg浓度均无明显变化(P>0.05)。
5.各组小鼠给药后，外观、活动未见异常变化，未见小鼠出
现死亡。小鼠体重随孕期增加均有正常增长，各组间体重增幅经比
较，差异无显著性意义(P>0.05);各组间小鼠的饮水量、进食量
比较，亦无明显差异(P>0.05)。各组小鼠剖检均未见异常，肝、
肾、脾脏器重量和脏器系数各组间无显著差异(P>0 .05)，停药后4
周，各组小鼠的肝、肾、脾脏器重量和脏器系数仍无显著差异
(P>0.05)。未见与药物毒性相关的其它特征病变。常规病理学检
查结果各组小鼠肝、肾、脾、卵巢等组织结构正常，未见任何的病
理损伤:正常小鼠与转基因小鼠各组织无明显差异。
6.各组平均死胎率、母体死胎率无显著差异(P>0 .05)。平均
胎仔重、黄体数、平均胎盘重量等指标，T检验结果各组间均无明显
差别。各组所取活胎亦未发现任何畸形。
7.各组新生鼠存活只数无显著差异。新生鼠一般发育情况，各
组比较均无显著性差异。
Objective In previous studies, HBV DNA level in maternal serum was an impotent factor for intrauterine infection; Lamifudine(STC) and interferon a (IFN a ) are clinically employed extensively now, and their antiviral effect is good, but they were categarized as grade C in the use of drugs during pregnancy, whose application for pregnant women or animals has not been studied, which prevented using of them durings pregnancy. So in this study supported by the National Nature Science Foundation of China (No.30070668), the transgenic mice with high levels of hepatitis B virus were employed for the first time to study the antiviral effect of 3TC and IFN a during the different gestation, and the change of serologic HBV markers were detected. At the same time, the effects of perinatal exposure to antiviral drugs on the mother and offspring were observed. We try to probe into the efficacy of two anti-HBV drugs for decreasing the amount of HBV DNA in maternal blood and the safety of mother and offspring, and provide s
ome animal experiment basis for the use of two drugs
in antiviral therapy of HBsAg positive mother during pregnancy, especially those in early pregnancy by evaluating the efficacy and safety of 3TC and IFN a therapy. In additon, we try to probe initially into the possibility of HBV transgenic mice as an animal model for study of HBV intrauterine infection. Methods:
1. One-cell mouse embryos were collected from the super ovulated Kunming mice, and perfect embryos were randomly allocated into experiment groups and control group depending on various concentration of 3TC and IFN a , then embryos were cultured in micro drops of medium for 72 hours. The effects of 3TC and IFN a in various concentrations on development of embryos were observed.
2. Twenty-one HBV transgenic female mice with steady expression of high-level HBsAg were randomly allocated into 3 groups: experiment group 1, experiment group 2 and control group. Mice in experiment group 1 received 3TC 100mg kg-1 d-1 from pregnancy day 5 to delivery, Mice in experiment group 2 received the same dosage of 3TC from pregnancy day 10 to delivery, and mice in control group received normal saline 0. 5ml d-1 from pregnancy day 5 to delivery. At the same time, 12 C57BL/6J female mice were randomly allocated into 2 groups (experiment group and control group of common mice) for observing the possible difference in some observed markers between HBV transgenic mice and common mice.
3. Another twenty-one HBV transgenic female mice and 12 C57BL/6J female mice were randomly chosen for studying effect
of IFN (5MU m-2 per two day). The grouping method was the same as that of 3TC.
4. HBsAg and HBV DNA content before and after the treatment in the mouse sera were analysed by using RIA and HBV fluorescence PCR.
5. Daily observations of the pregnant mice during pregnancy were performed, and delivery was observed 24h a day. On pregnant day 19, 3 mice from every group were randomly chosen for complete necropsy. The index that showed the situation of procreate, such as the number of luteal, the average weight of placenta and fetus were recorded; the rate of dead fetus and the rate of maternal dead fetus (the number of mice that had dead offspring/ the number of all pregnant mice) were calculated and statistically analyzed; The organ weight and organ coefficient were recorded, and the main organ structure of every mouse was examined by histological observation and immunohistochemical method.
6. Abnormalities of live fetus were investigated by examining their appearance, gut and skeleton. Live pups were counted, and the postnatal survival was statistically analyzed. The data on developmental performance such as weight gain, activity, the time of opening the eyes or ears and emergence of hair were observed. Drug effects on adult mice and offspring were assessed.
7. One month after delivery, HBV DNA and HBsAg in the newborn mice sera were detected. The placental tissue was collected to detect HBsAg in the cells by immunohistochemical method. Results:
1. Compar
方法：1．收集昆明种小鼠的单细胞受精卵，将纳入合格的胚胎细胞随机分组，根据培养液的不同，分成含不同浓度3TC或IFNα的试验组和普通培养液的对照组。采用微滴培养法连续培养72h，对比观察不同浓度3TC和IFNα对体外胚胎细胞早期发育的直接影响。
2．将稳定、高水平表达HBV抗原的21只HBV转基因母鼠随机分成试验组1、试验组2及对照组。试验组1于孕第5天开始口服3TC
第四军医大学博士学位论文
10Omg·kg一，·天一，直至分娩，试验组2于孕第10天开始口服3TC
100mg·kg一，·天一，直至分娩，对照组于孕第5天开始口服生理盐水
0.5ml.天一‘直至分娩，同时为了观察HBV转基因小鼠和普通小鼠在
某些观察指标上是否存在差别，另选12只普通正常C57BL/6J母鼠，
随机分为2组做为普通鼠试验组与对照组。
3.选21只HBV转基因小鼠和12只普通正常C57BL/6J母鼠，采用
IFNQ作为试验药物，肌注IFN Q SMU.m一，.2d一，，试验组与对照组的设
立与3TC相同。
4.所有试验组和对照组统一用固相放免方法定量检测用药前后母
鼠血清HBsAg，用荧光PCR定量分析HBV DNA浓度变化。
5.各组孕鼠每天专人观察妊娠期的外观及活动等一般生理学和反
应生育情况的指标;母鼠分娩采用24h连续观察法;每组母鼠在妊
娠第19天时随机取3只，系统解剖，记录各组的平均胎仔重、黄
体重、平均胎盘重等反应生育情况的指标，计算各组平均死胎率(死
胎数/胎仔总数)和母体死胎率(出现死胎的母体数/妊娠母体总
数)，并作统计学检验;记录各组母鼠的主要脏器重量、脏器系数;
对收集的母鼠主要脏器做病理学和免疫组织化学检查。
6.各组所取活胎采用系统解剖观察法做外观畸形、内脏畸形及骨
骼畸形检查;纪录每组新生鼠出生存活情况，T检验统计各组新生
鼠存活只数，新生鼠出生后每5天称重一次，观察一般发育情况，
记录张耳天数、出毛天数和开眼天数等指标。从母鼠、胎鼠及新生
鼠几方面同时观察3TC及IFNQ对孕鼠及其子代的影响。
7.对收集的胎盘做病理学和免疫组织化学检测;新生鼠生长至一
月时，抽取血清用ELISA和PCR方法检测HBsAg和HBV DNA。
结果:1.在IFNQ浓度高达1000工U.ml一，时，单细胞胚胎至8-
细胞和桑堪胚的发育率有下降趋势;当3TC浓度高达500“mol·L一，
时，除了2一细胞发育率与对照组无明显差异外，其余各期的胚胎细
胞发育率均明显下降(P<0.05)，而且培养中胚胎细胞变性、退化
第四军医大学博士学位论文
和不规则现象增加。说明这两种抗病毒药物，在高浓度时，有抑制
胚胎生长的可能，或对胚胎有一定毒性作用，而且在着床前的8-
细胞期和桑堪胚期，其抑制作用更加明显(P<0.01)。
2.IFNa浓度(800IU.ml一，，3TC浓度(300 p mol.L一，时，对小鼠
早期胚胎细胞发育基本无抑制作用。
3.9只口服3TC的HBV DNA阳性转基因小鼠，在用药巧天或
10天后，DNA浓度均明显下降(P<0.01)，有3只则完全阴转。而口
服生理盐水的小鼠，DNA浓度无明显变化。HBsAg定量结果除连续
服用3TC巧天的转基因小鼠，用药后的血清HBsAg浓度明显比用
药前低(P<0 .05)，余各组变化则不明显(P>.05)。
4.12只HBV DNA阳性肌注IFNa或生理盐水的小鼠，HBV DNA
和HBsAg浓度均无明显变化(P>0.05)。
5.各组小鼠给药后，外观、活动未见异常变化，未见小鼠出
现死亡。小鼠体重随孕期增加均有正常增长，各组间体重增幅经比
较，差异无显著性意义(P>0.05);各组间小鼠的饮水量、进食量
比较，亦无明显差异(P>0.05)。各组小鼠剖检均未见异常，肝、
肾、脾脏器重量和脏器系数各组间无显著差异(P>0 .05)，停药后4
周，各组小鼠的肝、肾、脾脏器重量和脏器系数仍无显著差异
(P>0.05)。未见与药物毒性相关的其它特征病变。常规病理学检
查结果各组小鼠肝、肾、脾、卵巢等组织结构正常，未见任何的病
理损伤:正常小鼠与转基因小鼠各组织无明显差异。
6.各组平均死胎率、母体死胎率无显著差异(P>0 .05)。平均
胎仔重、黄体数、平均胎盘重量等指标，T检验结果各组间均无明显
差别。各组所取活胎亦未发现任何畸形。
7.各组新生鼠存活只数无显著差异。新生鼠一般发育情况，各
组比较均无显著性差异。
Objective In previous studies, HBV DNA level in maternal serum was an impotent factor for intrauterine infection; Lamifudine(STC) and interferon a (IFN a ) are clinically employed extensively now, and their antiviral effect is good, but they were categarized as grade C in the use of drugs during pregnancy, whose application for pregnant women or animals has not been studied, which prevented using of them durings pregnancy. So in this study supported by the National Nature Science Foundation of China (No.30070668), the transgenic mice with high levels of hepatitis B virus were employed for the first time to study the antiviral effect of 3TC and IFN a during the different gestation, and the change of serologic HBV markers were detected. At the same time, the effects of perinatal exposure to antiviral drugs on the mother and offspring were observed. We try to probe into the efficacy of two anti-HBV drugs for decreasing the amount of HBV DNA in maternal blood and the safety of mother and offspring, and provide s
ome animal experiment basis for the use of two drugs
in antiviral therapy of HBsAg positive mother during pregnancy, especially those in early pregnancy by evaluating the efficacy and safety of 3TC and IFN a therapy. In additon, we try to probe initially into the possibility of HBV transgenic mice as an animal model for study of HBV intrauterine infection. Methods:
1. One-cell mouse embryos were collected from the super ovulated Kunming mice, and perfect embryos were randomly allocated into experiment groups and control group depending on various concentration of 3TC and IFN a , then embryos were cultured in micro drops of medium for 72 hours. The effects of 3TC and IFN a in various concentrations on development of embryos were observed.
2. Twenty-one HBV transgenic female mice with steady expression of high-level HBsAg were randomly allocated into 3 groups: experiment group 1, experiment group 2 and control group. Mice in experiment group 1 received 3TC 100mg kg-1 d-1 from pregnancy day 5 to delivery, Mice in experiment group 2 received the same dosage of 3TC from pregnancy day 10 to delivery, and mice in control group received normal saline 0. 5ml d-1 from pregnancy day 5 to delivery. At the same time, 12 C57BL/6J female mice were randomly allocated into 2 groups (experiment group and control group of common mice) for observing the possible difference in some observed markers between HBV transgenic mice and common mice.
3. Another twenty-one HBV transgenic female mice and 12 C57BL/6J female mice were randomly chosen for studying effect
of IFN (5MU m-2 per two day). The grouping method was the same as that of 3TC.
4. HBsAg and HBV DNA content before and after the treatment in the mouse sera were analysed by using RIA and HBV fluorescence PCR.
5. Daily observations of the pregnant mice during pregnancy were performed, and delivery was observed 24h a day. On pregnant day 19, 3 mice from every group were randomly chosen for complete necropsy. The index that showed the situation of procreate, such as the number of luteal, the average weight of placenta and fetus were recorded; the rate of dead fetus and the rate of maternal dead fetus (the number of mice that had dead offspring/ the number of all pregnant mice) were calculated and statistically analyzed; The organ weight and organ coefficient were recorded, and the main organ structure of every mouse was examined by histological observation and immunohistochemical method.
6. Abnormalities of live fetus were investigated by examining their appearance, gut and skeleton. Live pups were counted, and the postnatal survival was statistically analyzed. The data on developmental performance such as weight gain, activity, the time of opening the eyes or ears and emergence of hair were observed. Drug effects on adult mice and offspring were assessed.
7. One month after delivery, HBV DNA and HBsAg in the newborn mice sera were detected. The placental tissue was collected to detect HBsAg in the cells by immunohistochemical method. Results:
1. Compar
引文
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