急性肝衰竭大鼠肠道菌群和内毒素的动态研究
摘要
前言
重型肝炎是我国常见的急、危重疾病，死亡率高达65％～90％。感染是常见的并发症，也是造成患者死亡的重要原因之一。抗生素可以控制感染，长期大剂量使用使得细菌耐药现象不断增加。不仅如此，抗生素在杀灭病原菌的同时，也可以造成肠道菌群失调。
导致重型肝炎病人出现感染的病原菌大都来源于肠道内的正常菌群。这些细菌在生理条件下定植于肠道内，并在种群和数量上保持相对稳定。重型肝炎的发病过程中存在肠道菌群失调，表现为粪便中的双歧杆菌等有益菌下降，肠杆菌科细菌过度生长。肠道菌群失调是造成细菌易位、细菌感染的重要因素，研究肠道菌群的失调可以进一步了解细菌易位和感染的规律，建立预防细菌感染的微生态学方法。本实验采用大鼠肝衰竭模型，研究不同部位肠道菌群、内毒素的动态变化及消长关系。
浙江大学2003届硕士研究生学位论文
材料和方法
1．实验动物 雄性SD大鼠40只，体重200刁，由浙江省医学
动物中心提供。动物随机分为对照组和急性肝功能衰竭组。对照组门0
只，A组）在实验开始时处死；急性肝衰竭组分别在造模完成后24小时
（12只，B组）不 48河时（18只，C组）处死。
2．主要材料和方法 半乳糖胺购于重庆医科大学生化教研室，配制
成 20％溶液，IM的 NaOH调 pH至 7．0。按 1．4g／kg剂量间隔 12小时
腹腔注射半乳糖胺两次建立急性肝衰竭大鼠模型。按本实验室现行方法
要求下列培养基，E棚、EC、TS、EG、NBGT、LBS。
3．标本采集，细菌培养和内毒素测定 采右心室血检测肝功能和内
毒素；采门静脉血检测内毒素。无菌条件下采集空肠、回肠及结肠内容
物。稀释后用于肠道内毒素测定，并进行细菌培养计数。采用细菌三级
鉴定法将细菌鉴定到属的水平。
4．统计学分析 数据采用均数士标准差表示。根据不同情况采用X’检
验。单因素方差分析和秩和检验。
实验结果和讨论
1．大鼠死亡率观察A组大鼠全部存活。B组大鼠死亡一只，死亡
时间为 16～24小时之间，其余大鼠在处死前均有不同程度的精神萎靡，
进食减少，反应迟钝。C组大鼠在24～48小时之间死亡8只；在处死前，
l只大鼠出现狂躁，激惹等症状，处于兴奋状态；4只大鼠在40小时后
浙江大学2003届硕士研究生学位论文
出现精神好转，嗜睡症状消失，饮食增加并伴随活动增加：其余大鼠处
死前也伴有不同程度的精神萎靡，进食减少和反应迟钝。
2．肝功能检查结果 肝功能检测结果见表一：
表一 肝功能检测结果
A组（n－10） B组…＝11）C组（n—10）
总蛋白（g／L）62．7i4．5 48．9t3．7“”51．8t9．7＊＊
白蛋白（g／L）343t2．4 27．8t2刀’”303t34＊＊
球蛋白（gb）28．6f2厂 ZI．113二’”ZI．5t8月＊＊
ALT山几）60．6士门 4798对土3341刀“”3183土3973”“
AST（U／L）138刀“24．8 5033刀“32010“”29280“2666刀””
碱性磷酸酶（U／L）272二土引．5 639二1179．8＊＊747t421＊＊
总胆汁酸（umol／U．8比j425．0土994’“262．0川83．O““人
总胆红素（umol／L）7if．5 40石t24石””63二t58二““
注：＊＊表示与A组有显著差异（p＜001）人表不与B组差异（p＜005）
肝功能检测显示，造模后B、C两组大鼠肝脏出现严重损伤：总蛋
白、白蛋白和球蛋白明显下降：＊ST、＊LT、碱性磷酸酶、总胆红素和
总胆汁酸出现明显升高。实验中，C组大鼠因肝衰竭大量死亡，存活的
部分大鼠在处死时临床症状有所好转。结合肝功能结果综合进行判断后
认为：C组大鼠的肝功能开始出现好转。
3．肠道菌群分析
3．l肠杆菌科细菌计数见表H：
表二 肠道肠杆科细菌定量分析
A组（n＝10）B组（n＝11）C组（n。10）
空肠 （lgCfu／肠段）3．610．5（80％）5．110厂（73％）‘”4．310．9（80％）面
回肠 （lgCfu／肠段）5．3ti刀（90％）6．911．4（100％）””6．311．5（90％）
结肠内容物（lgCfu／粪便）7名土04（100％）8．7f0厂（100％）“7．gll刀（100％）凸
3．2肠球菌计数见左：
4
浙江大学2003届硕士研究生学位论工
表三 肠道肠球菌细菌定量分析
A组（n一川）B绢（n＝11）C组（n＝10）
空肠门gC呐肠段）3．肚0石（80％）3．9土队5（82％）3石出．7（80％）
回肠（lgCfuj肠段）4．gll．l（100％）5．710．9（100％）5二土14（90％）
Background
Fulminant Hepatic Failure (FHF) is a serious disease in our country with 65-90% death rate. Infection is the commonest complication and one of factors that lead patients to death. Although antibiotics can cure infection, we can't ignore the fact that long-time and large-quantity use of them makes the increase of drug resistance. Moreover, at the same time that antibiotics wipe of pathogen, they also disturb the equilibrium of intestinal flora. Most of pathogens, leading to infections in the FFIF patients, come from intestinal flora, which physiologically keep in equilibrium in quantity. In FHF patients, intestinal flora is disordered, manifested by the decrease of Bifdobaterium and overgrowth of Enterobacteriacea. The disorder of intestinal flora plays
an important role in bacterial translocation and infections. So, researches on the intestinal flora can uncover more about bacterial translocation and infections, and help us find Microbial Interference Therapies (MIT) to treat them. Now, using FHF rat models, we study on the dynamical viabilities of intestinal flora and endotoxin.
Materials and Methods
1. Experiment Animals
Forty male SD rats (Zhejiang Medical Animal Central), weighting 200-300g, are randomly divided into the control group and the FHF group. The control group (10 rats, Group A) was killed at the beginning of experiment while the FHF group was killed at 24 (12 rats, Group B) and 48 (18 rats, Group C) hours respectively after GlaN injection.
2. Materials and Methods
D-galactosamine (GlaN) (Biochemical Institute, Chongqing Medical University) was freshly dissolved in physiological saline (1:4) and adjusted to pH 7.0. After fasting 12 hours, the rats of the group B and C were administered GlaN intraperitioneally two times at a dose of 1.4g/kg with 12 hours interval. Following culture mediums were used in our experiment: EMB EC TS EG NBGT LBS.
3. Specimen Collection, Bacterial Culture and Endotoxin Detecting
Collecting blood from right ventricle for measuring liver function parameters and endotoxin. Collecting blood from portal vein for measuring endotoxin. Collecting jejunum, ileum and feces in colons and diluting these samples for measuring endotoxin and bacterium culture. The bacteria were identified by morphology, Gram reaction, API Fermentation Tests. 4. Statistical Analysis
Results are expressed as Mean±SD. Statistical significance among the groups of parametric data was evaluated using x2 test, ANOVA or rank sum test. Differences were considered significant when possibility less then 0.05.
Experiment Result and Discussion
1. Death Rats All control rats survival. One rat in the Group B died between 16 and 24 hours after the second injection and the rest were depressed with the decrease of diet and reaction at the time of killing. In the Group C, eight rats died between 24 and 48 hours after the second injection, at the time of killing, one rat was excited with craziness and exasperation, four rats got some recover with turnover of spirit, disappearance of somnolence and increase of diet and activity, the rest were depressed with decrease of diet and reaction.
2. Liver Function Parameters (see Diagram One)
Diagram One: Liver Function Parameters
Liver function parameters showed that the livers of the Group B and C were seriously injured with the decrease of TP ALB GLB and the increase of ALT AST AKP TBA TBiL. In the Group C, many rats died of liver failure while several rats of the survivals got some recovery at the time of killing. So, we thought that in the group C, liver function began to take a turn for better. 3. Intestinal Flora Analysis
3.1 Enumeration of Enterobacteriacea (see Diagram Two)
Diagram Two: Enumeration of Enterobacteriacea
3.2 Enumeration of Lactobacillus (see Diagram Three)
Diagram Three: Enumeration of Lactobacillus
3.3 Enumeration of Enterococus (see Diagram Four)
Diagram Four: Enumeration of enterococus
3.4 Enumeration of Bacteroides (see Diagram Five)
Dia
重型肝炎是我国常见的急、危重疾病，死亡率高达65％～90％。感染是常见的并发症，也是造成患者死亡的重要原因之一。抗生素可以控制感染，长期大剂量使用使得细菌耐药现象不断增加。不仅如此，抗生素在杀灭病原菌的同时，也可以造成肠道菌群失调。
导致重型肝炎病人出现感染的病原菌大都来源于肠道内的正常菌群。这些细菌在生理条件下定植于肠道内，并在种群和数量上保持相对稳定。重型肝炎的发病过程中存在肠道菌群失调，表现为粪便中的双歧杆菌等有益菌下降，肠杆菌科细菌过度生长。肠道菌群失调是造成细菌易位、细菌感染的重要因素，研究肠道菌群的失调可以进一步了解细菌易位和感染的规律，建立预防细菌感染的微生态学方法。本实验采用大鼠肝衰竭模型，研究不同部位肠道菌群、内毒素的动态变化及消长关系。
浙江大学2003届硕士研究生学位论文
材料和方法
1．实验动物 雄性SD大鼠40只，体重200刁，由浙江省医学
动物中心提供。动物随机分为对照组和急性肝功能衰竭组。对照组门0
只，A组）在实验开始时处死；急性肝衰竭组分别在造模完成后24小时
（12只，B组）不 48河时（18只，C组）处死。
2．主要材料和方法 半乳糖胺购于重庆医科大学生化教研室，配制
成 20％溶液，IM的 NaOH调 pH至 7．0。按 1．4g／kg剂量间隔 12小时
腹腔注射半乳糖胺两次建立急性肝衰竭大鼠模型。按本实验室现行方法
要求下列培养基，E棚、EC、TS、EG、NBGT、LBS。
3．标本采集，细菌培养和内毒素测定 采右心室血检测肝功能和内
毒素；采门静脉血检测内毒素。无菌条件下采集空肠、回肠及结肠内容
物。稀释后用于肠道内毒素测定，并进行细菌培养计数。采用细菌三级
鉴定法将细菌鉴定到属的水平。
4．统计学分析 数据采用均数士标准差表示。根据不同情况采用X’检
验。单因素方差分析和秩和检验。
实验结果和讨论
1．大鼠死亡率观察A组大鼠全部存活。B组大鼠死亡一只，死亡
时间为 16～24小时之间，其余大鼠在处死前均有不同程度的精神萎靡，
进食减少，反应迟钝。C组大鼠在24～48小时之间死亡8只；在处死前，
l只大鼠出现狂躁，激惹等症状，处于兴奋状态；4只大鼠在40小时后
浙江大学2003届硕士研究生学位论文
出现精神好转，嗜睡症状消失，饮食增加并伴随活动增加：其余大鼠处
死前也伴有不同程度的精神萎靡，进食减少和反应迟钝。
2．肝功能检查结果 肝功能检测结果见表一：
表一 肝功能检测结果
A组（n－10） B组…＝11）C组（n—10）
总蛋白（g／L）62．7i4．5 48．9t3．7“”51．8t9．7＊＊
白蛋白（g／L）343t2．4 27．8t2刀’”303t34＊＊
球蛋白（gb）28．6f2厂 ZI．113二’”ZI．5t8月＊＊
ALT山几）60．6士门 4798对土3341刀“”3183土3973”“
AST（U／L）138刀“24．8 5033刀“32010“”29280“2666刀””
碱性磷酸酶（U／L）272二土引．5 639二1179．8＊＊747t421＊＊
总胆汁酸（umol／U．8比j425．0土994’“262．0川83．O““人
总胆红素（umol／L）7if．5 40石t24石””63二t58二““
注：＊＊表示与A组有显著差异（p＜001）人表不与B组差异（p＜005）
肝功能检测显示，造模后B、C两组大鼠肝脏出现严重损伤：总蛋
白、白蛋白和球蛋白明显下降：＊ST、＊LT、碱性磷酸酶、总胆红素和
总胆汁酸出现明显升高。实验中，C组大鼠因肝衰竭大量死亡，存活的
部分大鼠在处死时临床症状有所好转。结合肝功能结果综合进行判断后
认为：C组大鼠的肝功能开始出现好转。
3．肠道菌群分析
3．l肠杆菌科细菌计数见表H：
表二 肠道肠杆科细菌定量分析
A组（n＝10）B组（n＝11）C组（n。10）
空肠 （lgCfu／肠段）3．610．5（80％）5．110厂（73％）‘”4．310．9（80％）面
回肠 （lgCfu／肠段）5．3ti刀（90％）6．911．4（100％）””6．311．5（90％）
结肠内容物（lgCfu／粪便）7名土04（100％）8．7f0厂（100％）“7．gll刀（100％）凸
3．2肠球菌计数见左：
4
浙江大学2003届硕士研究生学位论工
表三 肠道肠球菌细菌定量分析
A组（n一川）B绢（n＝11）C组（n＝10）
空肠门gC呐肠段）3．肚0石（80％）3．9土队5（82％）3石出．7（80％）
回肠（lgCfuj肠段）4．gll．l（100％）5．710．9（100％）5二土14（90％）
Background
Fulminant Hepatic Failure (FHF) is a serious disease in our country with 65-90% death rate. Infection is the commonest complication and one of factors that lead patients to death. Although antibiotics can cure infection, we can't ignore the fact that long-time and large-quantity use of them makes the increase of drug resistance. Moreover, at the same time that antibiotics wipe of pathogen, they also disturb the equilibrium of intestinal flora. Most of pathogens, leading to infections in the FFIF patients, come from intestinal flora, which physiologically keep in equilibrium in quantity. In FHF patients, intestinal flora is disordered, manifested by the decrease of Bifdobaterium and overgrowth of Enterobacteriacea. The disorder of intestinal flora plays
an important role in bacterial translocation and infections. So, researches on the intestinal flora can uncover more about bacterial translocation and infections, and help us find Microbial Interference Therapies (MIT) to treat them. Now, using FHF rat models, we study on the dynamical viabilities of intestinal flora and endotoxin.
Materials and Methods
1. Experiment Animals
Forty male SD rats (Zhejiang Medical Animal Central), weighting 200-300g, are randomly divided into the control group and the FHF group. The control group (10 rats, Group A) was killed at the beginning of experiment while the FHF group was killed at 24 (12 rats, Group B) and 48 (18 rats, Group C) hours respectively after GlaN injection.
2. Materials and Methods
D-galactosamine (GlaN) (Biochemical Institute, Chongqing Medical University) was freshly dissolved in physiological saline (1:4) and adjusted to pH 7.0. After fasting 12 hours, the rats of the group B and C were administered GlaN intraperitioneally two times at a dose of 1.4g/kg with 12 hours interval. Following culture mediums were used in our experiment: EMB EC TS EG NBGT LBS.
3. Specimen Collection, Bacterial Culture and Endotoxin Detecting
Collecting blood from right ventricle for measuring liver function parameters and endotoxin. Collecting blood from portal vein for measuring endotoxin. Collecting jejunum, ileum and feces in colons and diluting these samples for measuring endotoxin and bacterium culture. The bacteria were identified by morphology, Gram reaction, API Fermentation Tests. 4. Statistical Analysis
Results are expressed as Mean±SD. Statistical significance among the groups of parametric data was evaluated using x2 test, ANOVA or rank sum test. Differences were considered significant when possibility less then 0.05.
Experiment Result and Discussion
1. Death Rats All control rats survival. One rat in the Group B died between 16 and 24 hours after the second injection and the rest were depressed with the decrease of diet and reaction at the time of killing. In the Group C, eight rats died between 24 and 48 hours after the second injection, at the time of killing, one rat was excited with craziness and exasperation, four rats got some recover with turnover of spirit, disappearance of somnolence and increase of diet and activity, the rest were depressed with decrease of diet and reaction.
2. Liver Function Parameters (see Diagram One)
Diagram One: Liver Function Parameters
Liver function parameters showed that the livers of the Group B and C were seriously injured with the decrease of TP ALB GLB and the increase of ALT AST AKP TBA TBiL. In the Group C, many rats died of liver failure while several rats of the survivals got some recovery at the time of killing. So, we thought that in the group C, liver function began to take a turn for better. 3. Intestinal Flora Analysis
3.1 Enumeration of Enterobacteriacea (see Diagram Two)
Diagram Two: Enumeration of Enterobacteriacea
3.2 Enumeration of Lactobacillus (see Diagram Three)
Diagram Three: Enumeration of Lactobacillus
3.3 Enumeration of Enterococus (see Diagram Four)
Diagram Four: Enumeration of enterococus
3.4 Enumeration of Bacteroides (see Diagram Five)
Dia
引文
[1] Barker P J, Sheehan R. Impact of gemifloxacin on the normal human intestinal microflora [J]. J Chemother, 2001, 13(1): 47-51.
[2] Edlund C, Beyer G. Comparative effects of moxifloxacin and clarithromycin on the normal intestinal microflora [J]. Scand J Infect Dis, 2000, 32(1): 81-85.
[3] Hooper L V, Wong M H. Molecular analysis of commensal host-microbial relationships in the intestine [J]. Science, 2001, 291(5505): 881-884.
[4] Goris H, de-Boer F. Oral administration of antibiotics and intestinal flora associated endotoxin in mice [J]. Scand J Infec Dis, 1986, 18(1): 55-63.
[5] Wang XD, Andersson AR. The influence of surgical induced acute liver
failure on the intestinal in the rat [J]. Scand J Gastroenterol, 1993, 28:31-40.
[6] Wang XD, Soltesz V. Cholecystokinin increase small instestinal motility anf reduces enteric bacterial overgrowth and translocation in rats with surgically induced acute liver failure [J]. Digestion. 1996, 57: 67-72.
[7] WU Zhongwen, LI Lanjua, et al (吴仲文，李兰娟，等). Study on the intestinalmicrobial colonization resistance in patients with chronic severe hepatitis[J]. Chin J hepatol(中华肝脏病杂志) [J].2001, 9(6):329-331. (in Chinese)
[8] Wang XD, Soltesz V. Bacteria translocation in acute liver failure induced by 90 per cent hepatectomy in the rates [J]. B J Surg, 1993, 80(1): 66-71.
[9] Casafont F, de las Heras G. Small intestinal bacterial overgrowth in patients with alcoholic cirrhosis [J]. Dig Dis Sci, 1995, 40:1252-1256.
[10] Guarner C, Runyon BA. Small intestinal dysmotility and bacterial overgrowth in cirrhotic patients with apontaneous bacterial peritonitis [J]. Hepatology, 2000,31:858-863.
[11] Albillos A, Freile I. Effect of propranolol on the factors promoting bacterial translocationin cirrhotic rats with ascites[J]. Hepatology, 2000, 31:43-48.
[12] Shito M. A fulminant hepatic failure model in the rat: involvement of
interleukin-lbeta and tumor necrosis factor-alpha[J]. Dig-Dis-Sci, 2001, 46(8): 1700-1708.
[13]Robert F, Stachiewitz, Vitor Seabra. Glycine and uridine prevent D-galactosamine hepatotoxicity in the rat: role of Kupffer cell[J]. Hepatology, 1999, 29(3): 737-745.
[14]Grill J P, Perrin S.Bile salt toxicity to some bifidobacteria strains: role of Conjugated bile salt hydrolase and pH [J]. Can J Microbiol, 2000, 46(10): 878-884.
[15]S J H Van Deventer, J W Ten Cate. Intestinal endotoxemia clinical significance [J]. Gasteroenterology, 1988, 94(3):825-831.
[2] Edlund C, Beyer G. Comparative effects of moxifloxacin and clarithromycin on the normal intestinal microflora [J]. Scand J Infect Dis, 2000, 32(1): 81-85.
[3] Hooper L V, Wong M H. Molecular analysis of commensal host-microbial relationships in the intestine [J]. Science, 2001, 291(5505): 881-884.
[4] Goris H, de-Boer F. Oral administration of antibiotics and intestinal flora associated endotoxin in mice [J]. Scand J Infec Dis, 1986, 18(1): 55-63.
[5] Wang XD, Andersson AR. The influence of surgical induced acute liver
failure on the intestinal in the rat [J]. Scand J Gastroenterol, 1993, 28:31-40.
[6] Wang XD, Soltesz V. Cholecystokinin increase small instestinal motility anf reduces enteric bacterial overgrowth and translocation in rats with surgically induced acute liver failure [J]. Digestion. 1996, 57: 67-72.
[7] WU Zhongwen, LI Lanjua, et al (吴仲文，李兰娟，等). Study on the intestinalmicrobial colonization resistance in patients with chronic severe hepatitis[J]. Chin J hepatol(中华肝脏病杂志) [J].2001, 9(6):329-331. (in Chinese)
[8] Wang XD, Soltesz V. Bacteria translocation in acute liver failure induced by 90 per cent hepatectomy in the rates [J]. B J Surg, 1993, 80(1): 66-71.
[9] Casafont F, de las Heras G. Small intestinal bacterial overgrowth in patients with alcoholic cirrhosis [J]. Dig Dis Sci, 1995, 40:1252-1256.
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