青蒿琥酯抗鸭乙型肝炎病毒的实验研究
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摘要
目的:建立后天感染鸭乙型肝炎病毒(DHBV)福州麻鸭模型,制备原代鸭肝细胞,以DHBV DNA和cccDNA TaqMan荧光定量聚合酶链反应(FQ-PCR)检测法,观察青蒿琥酯(Artesunate,ART)在体内对DHBV DNA和DHBV cccDNA及其在原代鸭肝细胞中对DHBV DNA的抑制作用,并对其可能的作用机制进行初步探讨。
方法:(1)筛选1日龄DHBV DNA(-)雏鸭,经颈静脉途径接种DHBV DNA强阳性血清,建立福州麻鸭后天感染乙型肝炎动物模型;将DHBV阳性麻鸭随机分为空白对照组、青蒿琥酯大、中、小剂量组和拉米夫定组,分别给予相应药物进行干预。用药前、用药第7、14、21、28d及停药第7d,取静脉血用实时荧光定量PCR法检测血清DHBV DNA含量。停药7天后取肝组织以UNIQ-10柱式基因组DNA提取法提取肝组织内DNA和cccDNA,并用实时荧光定量PCR法检测。(2)建立一种简单易行的原代鸭肝细胞分离培养方法,分离雏鸭肝细胞(50万/ml)于37℃、5%CO2条件下培养24h存活后,随机分为空白对照、干扰素(IFN)α-2b组和ART实验组,ART组分为15、30、60μg/ml 3个剂量浓度,每48h换药1次,连续加药6d,观察细胞形态和存活情况,8d后收集细胞并测定DHBV DNA的含量。
结果:(1)拉米夫定组于用药后,血清DHBV DNA水平迅速降低,停药后立即反跳。青蒿琥酯大剂量组第21、28天DHBV DNA抑制率与拉米夫定组相似,停药后仍有一定的抑制作用。中、小剂量组与空白对照组比较有一定的病毒抑制作用。(2)实验组15、30、60μg/ml 3种浓度ART对DHBV DNA均有明显的抑制作用,浓度越大抑制作用越明显。3种浓度ART对鸭肝细胞无毒性,各药物组细胞存活率均在95%以上。
结论:所建原代鸭肝细胞分离培养方法简单易行,TaqMan荧光定量PCR检测方法是一种灵敏度高、特异性强的DNA定量检测技术。ART在麻鸭体内和原代鸭肝细胞中对DHBV DNA均具有抑制作用,且存在量效关系。一定剂量的ART对鸭肝组织无明显毒性、安全、经济,但其抗鸭乙肝病毒的作用机制仍需要进一步的研究。
Objective: With the fluorescence quantitative polymerase chain reaction (FQ-PCR) for quantification of duck hepatitis B virus(DHBV) DNA and cccDNA (covalently closed circular DNA) based on TaqMan chemistry, the model of FuZhou duckling DHBV infected and the culture model of the primary duck hepatocytes were established to investigate the inhibitory ability on DHBV DNA and DHBV cccDNA of artesunate(ART) in vivo and in vitro. And the mechanism of this antiviral effect was also discussed.
Methods: (1). FuZhou ducklings of DHBV DNA(-) were selected 1 day after hatched and inoculated intravenously(i.v.) with DHBV DNA (+)serum, those ducklings with a successful inoculation were used as the animal models of acquired infection of DHBV 1 week after inoculation. Ducks with congenitally infection of DHBV were randomly divided into five groups: untreated group, high、medium、low-dose artesunate treated group、lamivudine-treated group. The ducks in the lamivudine-treated group were fed lamivudine with a dose of 50mg/kg once. Ducks in the three-dose artesunate-treated groups were treated with different doses of decoction of this medicine for 28 days respectively. The serum content of DHBV-DNA was determined by FQ-PCR method before and 7 days after the treatment, and on the 7th, 14th, 21st and 28th day of the treatment. The histopathological changes of liver were evaluated before and after the treatment. (2). The primary duck hepatocytes were cultured in the entironment of 37℃、5%CO2, arfter 24 hours the live duck hepatocytes were divided into five groups: untreated group, high、medium、low-dose artesunate treated group、IFNα-2b group. The conformation and livability of duck hepatocytes were observed, and the DNA of the primary duck were collected by the UNIQ-10 method and were taken to detect the inhibition of ART on DHBV.
Results: (1)Lamivudine showed a rapid inhibiting effect on DHBV-DNA, but this effect didn’t last long, and the serum level of DHBV-DNA increased again after treatment. The serum level of DHBV-DNA dropped markedly in the high-dose artesunate-treated group on the 21st and 28th day. Low-dose and medium-dose artesunate also showed certain inhibiting effect on DHBV-DNA. The features of liver histopathological changes in the ART treated groups showed no statistical significance as compared with that in the untreated group. (2)The livabilities of duck hepatocytes treated with low-dose、medium-dose and high-dose artesunate were all over 95%.The DHBV DNA levels in cytoplasm of duck hepatocytes treated with ART(15、30、60μg/ml) were significantly lower than those of control cells(P < 0. 05).
Conclusions: The TaqMan FQ-PCR established for quantification of DNA is a highly sensitive and specific method. ART has the antiviral effect of DHBV DNA replication in vivo and in vitro, and has some inhibitory effect on DHBV cccDNA in liver. Otherwise the study indicated the treatment with suitable dosage of ART have no toxicity to the duckling’s liver. Thus this herbal medicine is an effective,safe and economical drug for hepatitis B. Future studies are needed to explore the active mechanism of the antiviral effect on duck hepatitis B virus of artesunate.
方法:(1)筛选1日龄DHBV DNA(-)雏鸭,经颈静脉途径接种DHBV DNA强阳性血清,建立福州麻鸭后天感染乙型肝炎动物模型;将DHBV阳性麻鸭随机分为空白对照组、青蒿琥酯大、中、小剂量组和拉米夫定组,分别给予相应药物进行干预。用药前、用药第7、14、21、28d及停药第7d,取静脉血用实时荧光定量PCR法检测血清DHBV DNA含量。停药7天后取肝组织以UNIQ-10柱式基因组DNA提取法提取肝组织内DNA和cccDNA,并用实时荧光定量PCR法检测。(2)建立一种简单易行的原代鸭肝细胞分离培养方法,分离雏鸭肝细胞(50万/ml)于37℃、5%CO2条件下培养24h存活后,随机分为空白对照、干扰素(IFN)α-2b组和ART实验组,ART组分为15、30、60μg/ml 3个剂量浓度,每48h换药1次,连续加药6d,观察细胞形态和存活情况,8d后收集细胞并测定DHBV DNA的含量。
结果:(1)拉米夫定组于用药后,血清DHBV DNA水平迅速降低,停药后立即反跳。青蒿琥酯大剂量组第21、28天DHBV DNA抑制率与拉米夫定组相似,停药后仍有一定的抑制作用。中、小剂量组与空白对照组比较有一定的病毒抑制作用。(2)实验组15、30、60μg/ml 3种浓度ART对DHBV DNA均有明显的抑制作用,浓度越大抑制作用越明显。3种浓度ART对鸭肝细胞无毒性,各药物组细胞存活率均在95%以上。
结论:所建原代鸭肝细胞分离培养方法简单易行,TaqMan荧光定量PCR检测方法是一种灵敏度高、特异性强的DNA定量检测技术。ART在麻鸭体内和原代鸭肝细胞中对DHBV DNA均具有抑制作用,且存在量效关系。一定剂量的ART对鸭肝组织无明显毒性、安全、经济,但其抗鸭乙肝病毒的作用机制仍需要进一步的研究。
Objective: With the fluorescence quantitative polymerase chain reaction (FQ-PCR) for quantification of duck hepatitis B virus(DHBV) DNA and cccDNA (covalently closed circular DNA) based on TaqMan chemistry, the model of FuZhou duckling DHBV infected and the culture model of the primary duck hepatocytes were established to investigate the inhibitory ability on DHBV DNA and DHBV cccDNA of artesunate(ART) in vivo and in vitro. And the mechanism of this antiviral effect was also discussed.
Methods: (1). FuZhou ducklings of DHBV DNA(-) were selected 1 day after hatched and inoculated intravenously(i.v.) with DHBV DNA (+)serum, those ducklings with a successful inoculation were used as the animal models of acquired infection of DHBV 1 week after inoculation. Ducks with congenitally infection of DHBV were randomly divided into five groups: untreated group, high、medium、low-dose artesunate treated group、lamivudine-treated group. The ducks in the lamivudine-treated group were fed lamivudine with a dose of 50mg/kg once. Ducks in the three-dose artesunate-treated groups were treated with different doses of decoction of this medicine for 28 days respectively. The serum content of DHBV-DNA was determined by FQ-PCR method before and 7 days after the treatment, and on the 7th, 14th, 21st and 28th day of the treatment. The histopathological changes of liver were evaluated before and after the treatment. (2). The primary duck hepatocytes were cultured in the entironment of 37℃、5%CO2, arfter 24 hours the live duck hepatocytes were divided into five groups: untreated group, high、medium、low-dose artesunate treated group、IFNα-2b group. The conformation and livability of duck hepatocytes were observed, and the DNA of the primary duck were collected by the UNIQ-10 method and were taken to detect the inhibition of ART on DHBV.
Results: (1)Lamivudine showed a rapid inhibiting effect on DHBV-DNA, but this effect didn’t last long, and the serum level of DHBV-DNA increased again after treatment. The serum level of DHBV-DNA dropped markedly in the high-dose artesunate-treated group on the 21st and 28th day. Low-dose and medium-dose artesunate also showed certain inhibiting effect on DHBV-DNA. The features of liver histopathological changes in the ART treated groups showed no statistical significance as compared with that in the untreated group. (2)The livabilities of duck hepatocytes treated with low-dose、medium-dose and high-dose artesunate were all over 95%.The DHBV DNA levels in cytoplasm of duck hepatocytes treated with ART(15、30、60μg/ml) were significantly lower than those of control cells(P < 0. 05).
Conclusions: The TaqMan FQ-PCR established for quantification of DNA is a highly sensitive and specific method. ART has the antiviral effect of DHBV DNA replication in vivo and in vitro, and has some inhibitory effect on DHBV cccDNA in liver. Otherwise the study indicated the treatment with suitable dosage of ART have no toxicity to the duckling’s liver. Thus this herbal medicine is an effective,safe and economical drug for hepatitis B. Future studies are needed to explore the active mechanism of the antiviral effect on duck hepatitis B virus of artesunate.
引文
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2. Hepatitis B Foundation. What is hepatitis B?Statistics,2005:18.
3. Lavanchy D. Hepatitis B virus epidemiology,diseas burden,treatment,current and emerging prevention an control measures:a review. J Viral Hepat,2004,1(2) :97-107.
4. De Clereg E. Antivirals and antiviral strategies. Nat Rev Microbiol,2004,2:704-720.
5.谈 博,张奉学,操红缨,等.鸭乙型肝炎病毒感模型及其方法学研究进展[J].热带医学杂志,2005,2(5):111-115.
6. 邓学龙,朱宇同,郭兴伯,等.先天和后天感染鸭乙型肝炎病毒的广州麻鸭外周血中病毒血症的动态比较及应用.广州中医药大学学报,1999,16(1):56-60.
7. Eun A J,Seob M,Wong S,et al.Simultaneous quantitation of two orchid viruses by the TaqMan real-time RT-PCR.J Virol Methods,2000,87(1-2):151-160.
8. 邹 文,杨 旭,杨立平,等.SYBR 实时定量聚合酶链反应检测鸭乙型肝炎病毒的研究.中华肝脏病杂志,2004,12(7):444.
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