登革热中医湿热证动物模型的建立
发布时间：2018-08-12 15:13
【摘要】： 目的和意义: 登革病毒(DV)属黄病毒属,是一种以蚊虫为主要传播媒介的RNA病毒,可以引起登革热(DF)、登革出血热(DHF)以及登革休克综合征(DSS)。近年来,由于全球气候变暖,蚊媒分布区域扩大,登革热流行呈不断上升趋势,已分布在全球100多个国家和地区,成为世界上在疾病、死亡和经济损失方面最严重的虫媒传染病。登革病毒致病机理、疫苗或抗病毒药物研究一直进展缓慢,其中一个重要难点在于没有与感染登革病毒的人体发病一致的实验动物模型,无法直接对致病因子、疫苗或药物进行验证。中医药在辨证治疗病毒感染性疾病方面积累了大量经验,而且中医温病湿热证动物模型研究已取得很大进展,本研究就以中医理论为指导,采用复合因素,模拟自然感染方式,综合环境气候、体质及病原微生物等因素建立合适的登革病毒感染小鼠模型,使其更加接近于临床实际情况,为研究登革热的疾病本质奠定基础。 方法: 以中医理论为指导,采用复合因素,模拟自然感染方式,综合环境气候、体质及病原微生物等因素建立合适的登革病毒感染小鼠模型。 1.复合因素:高糖高脂饲料+高温仓+感染因子登革病毒(DV)。 2.中药选择甘露消毒丹:飞滑石30g、淡黄芩20g、绵茵陈20g、石菖蒲12g、川贝母10g、木通10g、藿香10g、连翘10g、白蔻仁10g、薄荷10g、射干10g 3.分组:SPF级BALB/c小鼠60只,随机分为6组,即正常组(不施与任何条件)、病毒组(混合方式:腹腔注射与皮下多点注射)接种登革2型病毒GZ14株(10~4TCID_(50)),0.8ml/只,每日1次,连续3天)、湿热组(给予高脂高糖饮食:在普通饲料中混入12%猪油、8%蜂蜜),并置于高温气候仓(温度33±2℃,相对湿度60%～65%,1次/天,2h/次,连续8天)、湿热毒组(同时给与内外湿热环境及病毒感染)、中药治疗组(在湿热毒组的基础上给与甘露消毒丹,0.47g/20g,1次/天,连续7天)、阳性药物组(在湿热毒组的基础上给与病毒唑灌胃治疗,0.003g/20g,1次/天,连续7天),每组10只。 4.观察指标:全身情况的观察观察造模前后小鼠的全身情况,即精神、饮食、活动度、体温、大小便等变化。 血小板(PLT)、血脂(TC、TG)、肝功(AST、ALT)、水通道蛋白2(AQP2)、胃肠激素(MTL、GAS)。 荧光定量PCR检测登革病毒(DV)。 组织学检查动物处死后取肝脏、脑组织,10%的多聚甲醛固定,脱水后石蜡包埋切片,HE染色,光镜观察病理变化。 5.统计方法 用SPSS软件(13.0版)处理,实验结果的数据类型均为计量资料,各数据以均数±标准差((?)±s)表示。体温、血小板(PLT)属重复测量资料,采用重复测量的方差分析进行比较,若满足球形检验则无需校正;若不满足球形检验,则用Greenhouse-Geisserε校正系数来校正自由度;采用LSD法进行多重比较。AST、ALT、AQP2、TC、TG、GAS及MTL含量属完全随机设计的多组均数比较,采用One-Way ANOVA对各组实验结果进行方差分析,分析前首先考察方差齐性,组间多重比较用LSD法检验,以P＜0.05为有显著性差异。 结果: 1.一般表现小鼠有轻微的精神困倦,嗜睡,对外界刺激反应迟钝,毛色枯槁,饮水量增加,个别小鼠肛周污秽,呼吸、饮食未见明显改变。 2.体温检测各组小鼠肛温在36.1℃-38.1℃之间,组间差异无统计学意义(F=2.814,P=0.099)。 3.各小组主要指标的检测经DV感染后,小鼠PLT变化情况相似,均有不同程度的降低,各组的区别具有统计学意义(F=77.578,P=0.000);小鼠的ALT及AST较正常组普遍升高,尤其是AST变化显著,这与国外报道相似;内外湿热干预后,小鼠体内肾内髓AQP2的表达含量减少,以湿热毒组最具代表;TC水平增加,TG则变化不大,各组间差异无统计学意义(F~=1.619,P=0.185);各组MTL、GAS水平升高,说明内外湿热及病毒的干预造成了小鼠脾胃功能的紊乱。经药物治疗后,上述指标有一定程度的恢复,以中药组变化最具有统计学意义。 4、脏器病理变化 4.1肝脏病理变化正常肝组织镜下可见肝细胞索结构清晰,肝小叶结构正常,未见肝细胞变性、坏死等病理变化,网状纤维结构正常。接种DV后,打开小鼠腹腔后,肝组织体积明显增大,颜色变淡。光镜下,肝细胞显著肿胀,胞浆疏松、呈气球样变并出现DV感染典型的细胞病变(CPE),即细胞破碎、融合,形成大小不等的空泡和网状结构,失去正常的肝板结构,窦隙消失;湿热条件干预后,小鼠肝脏镜检可见到有大量的炎症细胞浸润、灶性坏死及碎屑样坏死;药物治疗后肝细胞状态有所好转。 4.2脑组织病理变化:正常脑组织神经细胞完整,细胞核较大,核仁清楚,细胞及间质无水肿,施加复合因素后,多数神经细胞核固缩,核仁显不清,细胞外空隙明显增宽、间质疏松,显示细胞周围及间质水肿明显,经中药治疗后得到很好的改善。 5.病毒测定 荧光定量方法检测小鼠脑组织中的病毒核酸,除正常组和湿热组以外,其他均呈阳性,其中湿热毒组含量最高,其次是病毒组,估计是因为施加湿热条件后,机体平衡状态被打破,更有利于病毒的生长繁殖。中药组与阳性药物组病毒含量下降,以中药组最明显。 结论: 本实验以中医理论为指导,采用复合因素,模拟自然感染方式,综合环境气候、体质及病原微生物等因素建立DV感染的小鼠模型,符合登革热病的特点,未改变登革热模型的关键指标(PLT下降,ALT及AST较正常组普遍升高,肝细胞出现典型的病变,病毒检测阳性),同时小鼠出现明显湿热证客观指标的变化(AQP2蛋白的表达含量减少,TC水平增加,MTL、GAS水平升高),经中药复方甘露消毒丹(清热解毒,利湿化浊)治疗后,上述指标得到明显的改善,说明可以建立登革热中医湿热证动物模型,为以后研究登革热致病机理及临床疗效奠定了基础。
[Abstract]:Purpose and significance:
Dengue virus (DV), a flavivirus, is a RNA virus mainly transmitted by mosquitoes. It can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In recent years, due to global warming, the distribution of mosquito vectors has expanded, and the prevalence of dengue fever is on the rise. It has been distributed in more than 100 countries and regions worldwide. Dengue virus pathogenesis, vaccines or antiviral drugs have been progressing slowly. One of the important difficulties is that there is no experimental animal model consistent with the human pathogenesis of dengue virus, and no direct response to pathogens, vaccines or drugs. Chinese medicine has accumulated a lot of experience in the treatment of viral infectious diseases based on syndrome differentiation and has made great progress in the study of animal models of damp-heat syndrome of febrile diseases. A suitable dengue virus infection model in mice was established to make it more close to the actual clinical situation and lay a foundation for the study of the nature of dengue fever.
Method:
Under the guidance of traditional Chinese medicine theory, a mouse model of dengue virus infection was established by using compound factors, simulating natural infection mode, integrating environmental climate, physical constitution and pathogenic microorganisms.
1. compound factors: high sugar, high fat diet + high temperature warehouse + infection factor dengue virus (DV).
2. Choose manna disinfection pill: flying talcum 30g, pale yellow Scutellaria 20g, Mianyin 20g, Acorus tatarinowii 12g, Fritillaria japonica 10g, Mutong 10g, Pogostemon 10g, Forsythia suspensa 10g, Nutmeg 10g, Mint 10g, Shegan 10g
3. Grouping: 60 SPF BALB/c mice were randomly divided into 6 groups: normal group (without any conditions), virus group (mixed mode: intraperitoneal injection and subcutaneous multi-point injection) inoculated with dengue 2 virus GZ14 strain (10-4TCID_ (50)), 0.8ml/mouse, once a day for 3 days, damp-heat group (given high fat and high sugar diet: mixed with 12% lard and 8% honey in common feed). They were placed in high temperature climate warehouse (temperature 33 The rats were treated with 0.003g/20g, 1 times per day, 7 days in each group, 10 rats in each group.
4. Observing indexes: Observing the whole body condition of the mice before and after the modeling, namely, the changes of spirit, diet, activity, body temperature, urine and urine.
Platelet (PLT), blood lipid (TC, TG), liver function (AST, ALT), aquaporin 2 (AQP2), gastrointestinal hormones (MTL, GAS).
Detection of dengue virus (DV) by fluorescent quantitative PCR.
Histological examination showed that the liver and brain tissues were taken from the animals after death, fixed with 10% paraformaldehyde, embedded with paraffin after dehydration, stained with HE, and pathological changes were observed under light microscope.
5. statistical methods
SPSS software (version 13.0) was used to process the experimental data. The data types of the experimental results were all measurements. The mean (?) + standard deviation (?) + s was used to represent the data. Body temperature and platelet (PLT) were repeated measurements. The results were compared by repeated measurements of variance analysis. If the spherical test was satisfied, no correction was needed. If the spherical test was not satisfied, Greenhouse-Geisser epsilon was used to calibrate the data. AST, ALT, AQP2, TC, TG, GAS and MTL contents were completely randomly designed multi-group mean comparisons. One-Way ANOVA was used to analyze the variance of each group of experimental results. The homogeneity of variance was first examined before the analysis. The multiple comparisons between groups were tested by LSD, and the significant difference was P<0.05.
Result:
1. Generally speaking, the mice were slightly tired, drowsy, slow to respond to external stimuli, hairy, increased water intake, some mice perianal dirty, breathing, diet has not changed significantly.
2. There was no significant difference in the anal temperature between 36.1 and 38.1 degrees centigrade (F = 2.814, P = 0.099).
3. The changes of PLT in mice infected with DV were similar, and the difference was statistically significant (F = 77.578, P = 0.000); ALT and AST in mice were generally higher than those in normal mice, especially AST, which was similar to that reported abroad; AQP2 in renal medulla in mice was significantly increased after damp-heat intervention at home and abroad. The expression level of MTL and GAS in each group increased, indicating that the disturbance of spleen and stomach function was caused by damp-heat and virus. After treatment, the above indexes were restored to a certain extent, with Chinese herbal medicine. Group changes were statistically significant.
4, visceral pathological changes
4.1 The pathological changes of the liver were normal. The structure of hepatic cords was clear, the structure of hepatic lobules was normal, there were no pathological changes such as hepatocyte degeneration and necrosis, and the structure of reticular fibers was normal. The typical cell pathological changes (CPE) of DV infection were broken and fused, forming vacuoles and reticular structures of different sizes, losing the normal structure of hepatic plate and disappearing of sinus space. After intervention of damp and hot conditions, a large number of inflammatory cell infiltration, focal necrosis and debris-like necrosis were observed in the liver of mice. To become better.
4.2 Pathological changes of brain tissues: The normal brain tissues have complete nerve cells, large nuclei, clear nucleoli, no edema in cells and stroma. After applying complex factors, most of the nerve cells have nucleus pyknosis, unclear nucleoli, significantly widened extracellular space, interstitial loose, showing obvious pericellular and interstitial edema, which has been improved very well after treatment with traditional Chinese medicine.
5. virus determination
Fluorescence quantitative method was used to detect the viral nucleic acid in mouse brain tissues. Except normal group and damp-heat group, the content of viral nucleic acid in damp-heat group was the highest, followed by viral group. It was estimated that the balance state of the body was broken after applying damp-heat condition, which was more conducive to the growth and reproduction of the virus. The Chinese medicine group is most obvious.
Conclusion:
Under the guidance of traditional Chinese medicine theory, a mouse model of DV infection was established by using compound factors, simulating natural infection mode, integrating environmental climate, physical constitution and pathogenic microorganisms. The model accorded with the characteristics of dengue fever, and did not change the key indicators of the model (PLT decreased, ALT and AST increased generally compared with the normal group, and typical diseases of hepatocytes appeared. At the same time, the changes of objective indexes (AQP2 protein expression decreased, TC level increased, MTL, GAS level increased) were observed in mice with dampness-heat syndrome. After treatment with Manlu disinfectant pill (clearing away heat and detoxification, removing dampness and turbidity), the above indexes were obviously improved, indicating that the animal model of dampness-heat syndrome of traditional Chinese medicine for dengue fever could be established. This will provide a basis for further study of the pathogenesis and clinical efficacy of dengue fever.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R-332;R259
本文编号：2179444
[Abstract]:Purpose and significance:
Dengue virus (DV), a flavivirus, is a RNA virus mainly transmitted by mosquitoes. It can cause dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In recent years, due to global warming, the distribution of mosquito vectors has expanded, and the prevalence of dengue fever is on the rise. It has been distributed in more than 100 countries and regions worldwide. Dengue virus pathogenesis, vaccines or antiviral drugs have been progressing slowly. One of the important difficulties is that there is no experimental animal model consistent with the human pathogenesis of dengue virus, and no direct response to pathogens, vaccines or drugs. Chinese medicine has accumulated a lot of experience in the treatment of viral infectious diseases based on syndrome differentiation and has made great progress in the study of animal models of damp-heat syndrome of febrile diseases. A suitable dengue virus infection model in mice was established to make it more close to the actual clinical situation and lay a foundation for the study of the nature of dengue fever.
Method:
Under the guidance of traditional Chinese medicine theory, a mouse model of dengue virus infection was established by using compound factors, simulating natural infection mode, integrating environmental climate, physical constitution and pathogenic microorganisms.
1. compound factors: high sugar, high fat diet + high temperature warehouse + infection factor dengue virus (DV).
2. Choose manna disinfection pill: flying talcum 30g, pale yellow Scutellaria 20g, Mianyin 20g, Acorus tatarinowii 12g, Fritillaria japonica 10g, Mutong 10g, Pogostemon 10g, Forsythia suspensa 10g, Nutmeg 10g, Mint 10g, Shegan 10g
3. Grouping: 60 SPF BALB/c mice were randomly divided into 6 groups: normal group (without any conditions), virus group (mixed mode: intraperitoneal injection and subcutaneous multi-point injection) inoculated with dengue 2 virus GZ14 strain (10-4TCID_ (50)), 0.8ml/mouse, once a day for 3 days, damp-heat group (given high fat and high sugar diet: mixed with 12% lard and 8% honey in common feed). They were placed in high temperature climate warehouse (temperature 33 The rats were treated with 0.003g/20g, 1 times per day, 7 days in each group, 10 rats in each group.
4. Observing indexes: Observing the whole body condition of the mice before and after the modeling, namely, the changes of spirit, diet, activity, body temperature, urine and urine.
Platelet (PLT), blood lipid (TC, TG), liver function (AST, ALT), aquaporin 2 (AQP2), gastrointestinal hormones (MTL, GAS).
Detection of dengue virus (DV) by fluorescent quantitative PCR.
Histological examination showed that the liver and brain tissues were taken from the animals after death, fixed with 10% paraformaldehyde, embedded with paraffin after dehydration, stained with HE, and pathological changes were observed under light microscope.
5. statistical methods
SPSS software (version 13.0) was used to process the experimental data. The data types of the experimental results were all measurements. The mean (?) + standard deviation (?) + s was used to represent the data. Body temperature and platelet (PLT) were repeated measurements. The results were compared by repeated measurements of variance analysis. If the spherical test was satisfied, no correction was needed. If the spherical test was not satisfied, Greenhouse-Geisser epsilon was used to calibrate the data. AST, ALT, AQP2, TC, TG, GAS and MTL contents were completely randomly designed multi-group mean comparisons. One-Way ANOVA was used to analyze the variance of each group of experimental results. The homogeneity of variance was first examined before the analysis. The multiple comparisons between groups were tested by LSD, and the significant difference was P<0.05.
Result:
1. Generally speaking, the mice were slightly tired, drowsy, slow to respond to external stimuli, hairy, increased water intake, some mice perianal dirty, breathing, diet has not changed significantly.
2. There was no significant difference in the anal temperature between 36.1 and 38.1 degrees centigrade (F = 2.814, P = 0.099).
3. The changes of PLT in mice infected with DV were similar, and the difference was statistically significant (F = 77.578, P = 0.000); ALT and AST in mice were generally higher than those in normal mice, especially AST, which was similar to that reported abroad; AQP2 in renal medulla in mice was significantly increased after damp-heat intervention at home and abroad. The expression level of MTL and GAS in each group increased, indicating that the disturbance of spleen and stomach function was caused by damp-heat and virus. After treatment, the above indexes were restored to a certain extent, with Chinese herbal medicine. Group changes were statistically significant.
4, visceral pathological changes
4.1 The pathological changes of the liver were normal. The structure of hepatic cords was clear, the structure of hepatic lobules was normal, there were no pathological changes such as hepatocyte degeneration and necrosis, and the structure of reticular fibers was normal. The typical cell pathological changes (CPE) of DV infection were broken and fused, forming vacuoles and reticular structures of different sizes, losing the normal structure of hepatic plate and disappearing of sinus space. After intervention of damp and hot conditions, a large number of inflammatory cell infiltration, focal necrosis and debris-like necrosis were observed in the liver of mice. To become better.
4.2 Pathological changes of brain tissues: The normal brain tissues have complete nerve cells, large nuclei, clear nucleoli, no edema in cells and stroma. After applying complex factors, most of the nerve cells have nucleus pyknosis, unclear nucleoli, significantly widened extracellular space, interstitial loose, showing obvious pericellular and interstitial edema, which has been improved very well after treatment with traditional Chinese medicine.
5. virus determination
Fluorescence quantitative method was used to detect the viral nucleic acid in mouse brain tissues. Except normal group and damp-heat group, the content of viral nucleic acid in damp-heat group was the highest, followed by viral group. It was estimated that the balance state of the body was broken after applying damp-heat condition, which was more conducive to the growth and reproduction of the virus. The Chinese medicine group is most obvious.
Conclusion:
Under the guidance of traditional Chinese medicine theory, a mouse model of DV infection was established by using compound factors, simulating natural infection mode, integrating environmental climate, physical constitution and pathogenic microorganisms. The model accorded with the characteristics of dengue fever, and did not change the key indicators of the model (PLT decreased, ALT and AST increased generally compared with the normal group, and typical diseases of hepatocytes appeared. At the same time, the changes of objective indexes (AQP2 protein expression decreased, TC level increased, MTL, GAS level increased) were observed in mice with dampness-heat syndrome. After treatment with Manlu disinfectant pill (clearing away heat and detoxification, removing dampness and turbidity), the above indexes were obviously improved, indicating that the animal model of dampness-heat syndrome of traditional Chinese medicine for dengue fever could be established. This will provide a basis for further study of the pathogenesis and clinical efficacy of dengue fever.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R-332;R259
【引证文献】
相关期刊论文 前1条
1 张守平;汪明;;登革热研究进展[J];中国兽医杂志;2012年10期
相关博士学位论文 前1条
1 黄仕营;登革热湿热证动物模型构建及清热祛湿法作用机理探讨研究[D];广州中医药大学;2010年
相关硕士学位论文 前1条
1 马丹娟;登革病毒感染湿热证小鼠模型建立及清热祛湿法作用机理探讨研究[D];南方医科大学;2011年
，本文编号：2179444
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