利用BALB/C小鼠及其原代培养心肌细胞研究川金丝猴源CVB3的致病性
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摘要
柯萨奇病毒(Coxsackievirus,CV)属于小RNA病毒科,肠道病毒属的成员,自1948年发现以来,在人群中的感染普遍存在,是近年来引起关注的人类病毒性心肌炎的一种主要病原之一,而且在国内外人群中的发病呈上升趋势。CV可分为A、B两个型,即CVA和CVB,其中,CVB又有6个血清型(CVB1~CVB6)。除人类外,CVB还可感染猩猩和比格犬等动物,并导致死亡。2005年,本课题组对临床死亡的金丝猴进行系统的诊断,证实其死于CVB3感染,这是国内外首次有该病原感染金丝猴死亡的报道。多数资料报道CVB3具有嗜心肌性,主要侵犯心肌并造成其损伤,但是,有的毒株还可导致胰腺、肾脏、神经以及子宫等组织器官损伤,也有部分毒株甚至没有致病性。由此可见,不同的分离毒株感染后其致病性不尽相同。为了明确川金丝猴源CVB3(CVB3/SGM-05)这一新分离毒株的致病特性,本研究以BALB/C小鼠为实验动物,开展相关的致病性研究。
本研究以新生BALB/C小鼠的心脏为材料,分离培养其心肌细胞,经形态学观察及其心肌生长特性鉴定,确定已成功建立了一种原代心肌细胞培养方法。为研究具有嗜心肌性病毒的致病机制研究提供良好的实验体系。将CVB3/SGM-05毒株接种原代培养的小鼠心肌细胞,染毒细胞出现明显的细胞病变,而且失去心肌细胞特有的节律收缩现象。将该染毒细胞制作超薄切片后电镜观察发现,在胞浆内有大量的CVB3病毒粒子形成的晶格状结构,说明该病毒可感染原代培养的小鼠心肌细胞,并可在其中进行大量的复制,初步明确CVB3/SGM-05毒株对原代心肌细胞的超微病变。
将CVB3/SGM-05毒株通过腹腔注射途径接种健康的BALB/C小鼠后,观察该病毒对小鼠的致病性。小鼠于接毒后60h开始死亡,发病小鼠均出现典型的结膜炎,死亡率高达80%以上。对死亡小鼠剖检可见:最明显的病变为腹腔内脂肪的大量坏死;肝脏明显肿大,表面可见针尖大灰白小点;部分小鼠的脾脏肿大、边缘与坏死的脂肪发生粘连。石蜡病理组织切片显示,脂肪坏死尤其明显;心、肝、肾等实质器官均有不同程度的细胞变性,肝细胞坏死严重;脑组织可见轻度淤血;胃、肠黏膜上皮坏死、脱落。说明CVB3分离毒株感染BALB/C可引起多脏器的损伤。对死亡小鼠的心肌制作组织超薄切片,电镜观察显示,无明显的病变。
将接种CVB3/SGM-05毒株的BALB/C小鼠于不同时间采集血液,分离血清后检测其中的心肌酶、肝功、肾功、血糖以及胰淀粉酶的变化。结果显示,血清中被检的酶类随着病程的发展呈现出一定的变化特点,如心肌酶CK于接毒后第2天即升高到正常值的2倍左右,7天后可达到3倍以上,之后呈逐渐下降趋势;而HBD于接毒后第2天即升高到正常值的4倍以上,以后逐渐下降。小鼠肝功能相关酶类变化不显著。肾功能相关的BUN无显著变化等。这些检测结果与CVB3标准毒株感染BALB/C小鼠后的血清酶学指标存在一定的差异,提示临床诊断CVB3感染时,除酶学指标外应综合分析。该项研究从细胞病理、组织病理及血液病理等多个方面研究了CVB3/SGM-05毒株对BALB/C小鼠的致病特性,不仅为CVB3感染的临床综合诊断提供一定的参考,而且为下一步建立CVB3/SGM-05毒株感染的实验动物模型奠定了坚实的基础。
Backgroud:
Coxsackievirus belongs to micro-RNA viraceae, the member of Enterovirus genus, since 1948 discovered, is widespread in the crowd, is one of the major pathogeny of human viral myocarditis which people pay close attention to for the past few years. CV can be separated to two subtypes, A and B. CVB has six serotypes (CVB1~CVB6). Besides human, CVB can infect orangutan and beagle, even to death. Most information report CVB3 encroachment myocardium, cause myocardium damage, especially in children. Except myocardiophilic CVB3 can cause typical myocarditis, some strain can cause pancreas, kidney, nerves, uterus damage, some strain has no pathogenicity. It can be seen that different strain can cause different pathological changes. In order to identify the pathopoiesis characteristics of CVB3 Strain from Sichuan Golden Monkey, this study by means of BALB/C experimental animal, carry out correlative study of pathogenicity .
Cultivate Primary Cardiac Muscle Cells of BALB/C successfully by means of primary cell culture . Inoculate the virus in the mouse myocardial cell of primary culture, we can see the apparente pathological changes in the poison cell, including pyknosis, desquamation, lost rhythmic shrink which is myocardial cell characteristic. Collect CPE cell, centrifugate, fix in 2.5% glutaraldehyde, make virus ultramicrocut, there are a lot of crystal lattice structure caused by CVB3 nucleocapsid in electron microscope, which demonstrate the virus was massive duplicate in myocardial cell, endochylema vacuolization , most organoid lost its internal structural feature.
Inoculate the virus in healthy BALB/C mouse by peritoneal injection, observe the pathogenicity to mouse. The mouse began to die after inoculate the virus 60h, death rate is 80%. Appearing typical conjunctivitis. Analyze the death mouse, we can find the most apparente pathological changes is abdominal cavity fat necrosis、liver swell, needlepoint hoariness tittle in the surface, some mouse spleen mouse、conglutination in balance and necroticfat . Select the heart, liver, spleen, lung, kidney, brain and fat pathology tissue, make paraffin section, observe its pathological change characteristic in light microscope, we can find adiponecrosis is obvious; The parenchymal organ such as heart, liver, kidney have different extent of Cell degeneration, liver neorobiosisis very severe; brain tissue has light blood clot; stomach intestine mucosa epithelium necrosis and desquamate. which indicated BALB/C Mice infected with a CVB3 Strain from Sichuan Golden Monkey can cause most organ damage. Immunity class result show, virus positive reaction in myocardial cell. make myocardial cell ultramicrocut, there is no apparente ultramicro pathological changes in electron microscope.
Collect the blood of Mice infected with a CVB3 Strain from Sichuan Golden Monkey, separate the serum, detect the myocardial enzyme, liver duty, kidney duty, blood sugar, amylopsin. Result demonstrate the Erzymes in serum show different extent changing characteristic. BALB/C mouse of peritoneal injection inoculated CVB3/SGM-05, every myocardial enzyme apparentely heighten, compared to control group P<0.01, the difference extremely significant. Oral nasal way is the same as peritoneal injection, but the rangeability is lower. Except individual mouse liver duty has marked change (P<0.05), most has no significant deviation. Compared to control group, group of peritoneal injection mouse BUN significantly decrease after virus inoculation, then return to normal gradually; Oral nasal group BUN most lower thannormal; CRE higher than control group,but no significant deviation. The two groups AMY heighten significantly, P<0.01, difference extremely significant. These detection is different from CVB3infected BALB/C, suggest that clinical diagnosis of CVB3 infection, should aggregate analysis except enzymology.
This study initial identify pathology characteristic of CVB3/SGM-05 to BALB/C mouse and in vitro cardiac infection,not only settle the grounding for further construct animal model of this strain,but also offer the reference for clinical hematology diagnosis of this strain infection .
本研究以新生BALB/C小鼠的心脏为材料,分离培养其心肌细胞,经形态学观察及其心肌生长特性鉴定,确定已成功建立了一种原代心肌细胞培养方法。为研究具有嗜心肌性病毒的致病机制研究提供良好的实验体系。将CVB3/SGM-05毒株接种原代培养的小鼠心肌细胞,染毒细胞出现明显的细胞病变,而且失去心肌细胞特有的节律收缩现象。将该染毒细胞制作超薄切片后电镜观察发现,在胞浆内有大量的CVB3病毒粒子形成的晶格状结构,说明该病毒可感染原代培养的小鼠心肌细胞,并可在其中进行大量的复制,初步明确CVB3/SGM-05毒株对原代心肌细胞的超微病变。
将CVB3/SGM-05毒株通过腹腔注射途径接种健康的BALB/C小鼠后,观察该病毒对小鼠的致病性。小鼠于接毒后60h开始死亡,发病小鼠均出现典型的结膜炎,死亡率高达80%以上。对死亡小鼠剖检可见:最明显的病变为腹腔内脂肪的大量坏死;肝脏明显肿大,表面可见针尖大灰白小点;部分小鼠的脾脏肿大、边缘与坏死的脂肪发生粘连。石蜡病理组织切片显示,脂肪坏死尤其明显;心、肝、肾等实质器官均有不同程度的细胞变性,肝细胞坏死严重;脑组织可见轻度淤血;胃、肠黏膜上皮坏死、脱落。说明CVB3分离毒株感染BALB/C可引起多脏器的损伤。对死亡小鼠的心肌制作组织超薄切片,电镜观察显示,无明显的病变。
将接种CVB3/SGM-05毒株的BALB/C小鼠于不同时间采集血液,分离血清后检测其中的心肌酶、肝功、肾功、血糖以及胰淀粉酶的变化。结果显示,血清中被检的酶类随着病程的发展呈现出一定的变化特点,如心肌酶CK于接毒后第2天即升高到正常值的2倍左右,7天后可达到3倍以上,之后呈逐渐下降趋势;而HBD于接毒后第2天即升高到正常值的4倍以上,以后逐渐下降。小鼠肝功能相关酶类变化不显著。肾功能相关的BUN无显著变化等。这些检测结果与CVB3标准毒株感染BALB/C小鼠后的血清酶学指标存在一定的差异,提示临床诊断CVB3感染时,除酶学指标外应综合分析。该项研究从细胞病理、组织病理及血液病理等多个方面研究了CVB3/SGM-05毒株对BALB/C小鼠的致病特性,不仅为CVB3感染的临床综合诊断提供一定的参考,而且为下一步建立CVB3/SGM-05毒株感染的实验动物模型奠定了坚实的基础。
Backgroud:
Coxsackievirus belongs to micro-RNA viraceae, the member of Enterovirus genus, since 1948 discovered, is widespread in the crowd, is one of the major pathogeny of human viral myocarditis which people pay close attention to for the past few years. CV can be separated to two subtypes, A and B. CVB has six serotypes (CVB1~CVB6). Besides human, CVB can infect orangutan and beagle, even to death. Most information report CVB3 encroachment myocardium, cause myocardium damage, especially in children. Except myocardiophilic CVB3 can cause typical myocarditis, some strain can cause pancreas, kidney, nerves, uterus damage, some strain has no pathogenicity. It can be seen that different strain can cause different pathological changes. In order to identify the pathopoiesis characteristics of CVB3 Strain from Sichuan Golden Monkey, this study by means of BALB/C experimental animal, carry out correlative study of pathogenicity .
Cultivate Primary Cardiac Muscle Cells of BALB/C successfully by means of primary cell culture . Inoculate the virus in the mouse myocardial cell of primary culture, we can see the apparente pathological changes in the poison cell, including pyknosis, desquamation, lost rhythmic shrink which is myocardial cell characteristic. Collect CPE cell, centrifugate, fix in 2.5% glutaraldehyde, make virus ultramicrocut, there are a lot of crystal lattice structure caused by CVB3 nucleocapsid in electron microscope, which demonstrate the virus was massive duplicate in myocardial cell, endochylema vacuolization , most organoid lost its internal structural feature.
Inoculate the virus in healthy BALB/C mouse by peritoneal injection, observe the pathogenicity to mouse. The mouse began to die after inoculate the virus 60h, death rate is 80%. Appearing typical conjunctivitis. Analyze the death mouse, we can find the most apparente pathological changes is abdominal cavity fat necrosis、liver swell, needlepoint hoariness tittle in the surface, some mouse spleen mouse、conglutination in balance and necroticfat . Select the heart, liver, spleen, lung, kidney, brain and fat pathology tissue, make paraffin section, observe its pathological change characteristic in light microscope, we can find adiponecrosis is obvious; The parenchymal organ such as heart, liver, kidney have different extent of Cell degeneration, liver neorobiosisis very severe; brain tissue has light blood clot; stomach intestine mucosa epithelium necrosis and desquamate. which indicated BALB/C Mice infected with a CVB3 Strain from Sichuan Golden Monkey can cause most organ damage. Immunity class result show, virus positive reaction in myocardial cell. make myocardial cell ultramicrocut, there is no apparente ultramicro pathological changes in electron microscope.
Collect the blood of Mice infected with a CVB3 Strain from Sichuan Golden Monkey, separate the serum, detect the myocardial enzyme, liver duty, kidney duty, blood sugar, amylopsin. Result demonstrate the Erzymes in serum show different extent changing characteristic. BALB/C mouse of peritoneal injection inoculated CVB3/SGM-05, every myocardial enzyme apparentely heighten, compared to control group P<0.01, the difference extremely significant. Oral nasal way is the same as peritoneal injection, but the rangeability is lower. Except individual mouse liver duty has marked change (P<0.05), most has no significant deviation. Compared to control group, group of peritoneal injection mouse BUN significantly decrease after virus inoculation, then return to normal gradually; Oral nasal group BUN most lower thannormal; CRE higher than control group,but no significant deviation. The two groups AMY heighten significantly, P<0.01, difference extremely significant. These detection is different from CVB3infected BALB/C, suggest that clinical diagnosis of CVB3 infection, should aggregate analysis except enzymology.
This study initial identify pathology characteristic of CVB3/SGM-05 to BALB/C mouse and in vitro cardiac infection,not only settle the grounding for further construct animal model of this strain,but also offer the reference for clinical hematology diagnosis of this strain infection .
引文
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