登革熱中醫(yī)濕熱證動(dòng)物模型的建立
發(fā)布時(shí)間：2018-08-12 15:13
【摘要】： 目的和意義: 登革病毒(DV)屬黃病毒屬,是一種以蚊蟲(chóng)為主要傳播媒介的RNA病毒,可以引起登革熱(DF)、登革出血熱(DHF)以及登革休克綜合征(DSS)。近年來(lái),由于全球氣候變暖,蚊媒分布區(qū)域擴(kuò)大,登革熱流行呈不斷上升趨勢(shì),已分布在全球100多個(gè)國(guó)家和地區(qū),成為世界上在疾病、死亡和經(jīng)濟(jì)損失方面最嚴(yán)重的蟲(chóng)媒傳染病。登革病毒致病機(jī)理、疫苗或抗病毒藥物研究一直進(jìn)展緩慢,其中一個(gè)重要難點(diǎn)在于沒(méi)有與感染登革病毒的人體發(fā)病一致的實(shí)驗(yàn)動(dòng)物模型,無(wú)法直接對(duì)致病因子、疫苗或藥物進(jìn)行驗(yàn)證。中醫(yī)藥在辨證治療病毒感染性疾病方面積累了大量經(jīng)驗(yàn),而且中醫(yī)溫病濕熱證動(dòng)物模型研究已取得很大進(jìn)展,本研究就以中醫(yī)理論為指導(dǎo),采用復(fù)合因素,模擬自然感染方式,綜合環(huán)境氣候、體質(zhì)及病原微生物等因素建立合適的登革病毒感染小鼠模型,使其更加接近于臨床實(shí)際情況,為研究登革熱的疾病本質(zhì)奠定基礎(chǔ)。 方法: 以中醫(yī)理論為指導(dǎo),采用復(fù)合因素,模擬自然感染方式,綜合環(huán)境氣候、體質(zhì)及病原微生物等因素建立合適的登革病毒感染小鼠模型。 1.復(fù)合因素:高糖高脂飼料+高溫倉(cāng)+感染因子登革病毒(DV)。 2.中藥選擇甘露消毒丹:飛滑石30g、淡黃芩20g、綿茵陳20g、石菖蒲12g、川貝母10g、木通10g、藿香10g、連翹10g、白蔻仁10g、薄荷10g、射干10g 3.分組:SPF級(jí)BALB/c小鼠60只,隨機(jī)分為6組,即正常組(不施與任何條件)、病毒組(混合方式:腹腔注射與皮下多點(diǎn)注射)接種登革2型病毒GZ14株(10~4TCID_(50)),0.8ml/只,每日1次,連續(xù)3天)、濕熱組(給予高脂高糖飲食:在普通飼料中混入12%豬油、8%蜂蜜),并置于高溫氣候倉(cāng)(溫度33±2℃,相對(duì)濕度60%～65%,1次/天,2h/次,連續(xù)8天)、濕熱毒組(同時(shí)給與內(nèi)外濕熱環(huán)境及病毒感染)、中藥治療組(在濕熱毒組的基礎(chǔ)上給與甘露消毒丹,0.47g/20g,1次/天,連續(xù)7天)、陽(yáng)性藥物組(在濕熱毒組的基礎(chǔ)上給與病毒唑灌胃治療,0.003g/20g,1次/天,連續(xù)7天),每組10只。 4.觀察指標(biāo):全身情況的觀察觀察造模前后小鼠的全身情況,即精神、飲食、活動(dòng)度、體溫、大小便等變化。 血小板(PLT)、血脂(TC、TG)、肝功(AST、ALT)、水通道蛋白2(AQP2)、胃腸激素(MTL、GAS)。 熒光定量PCR檢測(cè)登革病毒(DV)。 組織學(xué)檢查動(dòng)物處死后取肝臟、腦組織,10%的多聚甲醛固定,脫水后石蠟包埋切片,HE染色,光鏡觀察病理變化。 5.統(tǒng)計(jì)方法 用SPSS軟件(13.0版)處理,實(shí)驗(yàn)結(jié)果的數(shù)據(jù)類型均為計(jì)量資料,各數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示。體溫、血小板(PLT)屬重復(fù)測(cè)量資料,采用重復(fù)測(cè)量的方差分析進(jìn)行比較,若滿足球形檢驗(yàn)則無(wú)需校正;若不滿足球形檢驗(yàn),則用Greenhouse-Geisserε校正系數(shù)來(lái)校正自由度;采用LSD法進(jìn)行多重比較。AST、ALT、AQP2、TC、TG、GAS及MTL含量屬完全隨機(jī)設(shè)計(jì)的多組均數(shù)比較,采用One-Way ANOVA對(duì)各組實(shí)驗(yàn)結(jié)果進(jìn)行方差分析,分析前首先考察方差齊性,組間多重比較用LSD法檢驗(yàn),以P＜0.05為有顯著性差異。 結(jié)果: 1.一般表現(xiàn)小鼠有輕微的精神困倦,嗜睡,對(duì)外界刺激反應(yīng)遲鈍,毛色枯槁,飲水量增加,個(gè)別小鼠肛周污穢,呼吸、飲食未見(jiàn)明顯改變。 2.體溫檢測(cè)各組小鼠肛溫在36.1℃-38.1℃之間,組間差異無(wú)統(tǒng)計(jì)學(xué)意義(F=2.814,P=0.099)。 3.各小組主要指標(biāo)的檢測(cè)經(jīng)DV感染后,小鼠PLT變化情況相似,均有不同程度的降低,各組的區(qū)別具有統(tǒng)計(jì)學(xué)意義(F=77.578,P=0.000);小鼠的ALT及AST較正常組普遍升高,尤其是AST變化顯著,這與國(guó)外報(bào)道相似;內(nèi)外濕熱干預(yù)后,小鼠體內(nèi)腎內(nèi)髓AQP2的表達(dá)含量減少,以濕熱毒組最具代表;TC水平增加,TG則變化不大,各組間差異無(wú)統(tǒng)計(jì)學(xué)意義(F~=1.619,P=0.185);各組MTL、GAS水平升高,說(shuō)明內(nèi)外濕熱及病毒的干預(yù)造成了小鼠脾胃功能的紊亂。經(jīng)藥物治療后,上述指標(biāo)有一定程度的恢復(fù),以中藥組變化最具有統(tǒng)計(jì)學(xué)意義。 4、臟器病理變化 4.1肝臟病理變化正常肝組織鏡下可見(jiàn)肝細(xì)胞索結(jié)構(gòu)清晰,肝小葉結(jié)構(gòu)正常,未見(jiàn)肝細(xì)胞變性、壞死等病理變化,網(wǎng)狀纖維結(jié)構(gòu)正常。接種DV后,打開(kāi)小鼠腹腔后,肝組織體積明顯增大,顏色變淡。光鏡下,肝細(xì)胞顯著腫脹,胞漿疏松、呈氣球樣變并出現(xiàn)DV感染典型的細(xì)胞病變(CPE),即細(xì)胞破碎、融合,形成大小不等的空泡和網(wǎng)狀結(jié)構(gòu),失去正常的肝板結(jié)構(gòu),竇隙消失;濕熱條件干預(yù)后,小鼠肝臟鏡檢可見(jiàn)到有大量的炎癥細(xì)胞浸潤(rùn)、灶性壞死及碎屑樣壞死;藥物治療后肝細(xì)胞狀態(tài)有所好轉(zhuǎn)。 4.2腦組織病理變化:正常腦組織神經(jīng)細(xì)胞完整,細(xì)胞核較大,核仁清楚,細(xì)胞及間質(zhì)無(wú)水腫,施加復(fù)合因素后,多數(shù)神經(jīng)細(xì)胞核固縮,核仁顯不清,細(xì)胞外空隙明顯增寬、間質(zhì)疏松,顯示細(xì)胞周圍及間質(zhì)水腫明顯,經(jīng)中藥治療后得到很好的改善。 5.病毒測(cè)定 熒光定量方法檢測(cè)小鼠腦組織中的病毒核酸,除正常組和濕熱組以外,其他均呈陽(yáng)性,其中濕熱毒組含量最高,其次是病毒組,估計(jì)是因?yàn)槭┘訚駸釛l件后,機(jī)體平衡狀態(tài)被打破,更有利于病毒的生長(zhǎng)繁殖。中藥組與陽(yáng)性藥物組病毒含量下降,以中藥組最明顯。 結(jié)論: 本實(shí)驗(yàn)以中醫(yī)理論為指導(dǎo),采用復(fù)合因素,模擬自然感染方式,綜合環(huán)境氣候、體質(zhì)及病原微生物等因素建立DV感染的小鼠模型,符合登革熱病的特點(diǎn),未改變登革熱模型的關(guān)鍵指標(biāo)(PLT下降,ALT及AST較正常組普遍升高,肝細(xì)胞出現(xiàn)典型的病變,病毒檢測(cè)陽(yáng)性),同時(shí)小鼠出現(xiàn)明顯濕熱證客觀指標(biāo)的變化(AQP2蛋白的表達(dá)含量減少,TC水平增加,MTL、GAS水平升高),經(jīng)中藥復(fù)方甘露消毒丹(清熱解毒,利濕化濁)治療后,上述指標(biāo)得到明顯的改善,說(shuō)明可以建立登革熱中醫(yī)濕熱證動(dòng)物模型,為以后研究登革熱致病機(jī)理及臨床療效奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R-332;R259
本文編號(hào)：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R-332;R259
【引證文獻(xiàn)】
相關(guān)期刊論文 前1條
1 張守平;汪明;;登革熱研究進(jìn)展[J];中國(guó)獸醫(yī)雜志;2012年10期
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