纳米细菌与动脉粥样硬化关系的初步探讨
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摘要
背景与目的：动脉粥样硬化的病因尚未完全明了，有关发病机制的学说众多，但任何一种学说均不能单独地全面地解释动脉粥样硬化的发病机制。近年来，感染学说重新受到广大学者的关注，越来越多的研究支持炎症反应学说。纳米细菌是近年来发现的新菌属，具有在菌体周围产生矿化外壳的特性，与许多骨骼外钙化或硬化性疾病有关。动脉粥样硬化斑块中存在大量的钙化物质，目前尚无圆满的解释。本文通过对动脉粥样硬化斑块中纳米细菌的检测、分离、培养和鉴定，同时进行纳米细菌感染人脐静脉内皮细胞系ECV-304的实验，以期发现动脉粥样硬化斑块中纳米细菌存在的证据，证明纳米细菌具有感染内皮细胞的能力，为研究动脉粥样硬化形成过程及其发病机制提供新的切入点。
方法：1．对19例动脉粥样硬化斑块和19例对照组动脉壁进行纳米细菌单克隆抗体免疫荧光和免疫组化染色，阳性者进行透射电子显微镜检测。2．对6例腹主动脉瘤粥样斑块标本进行纳米细菌的分离、培养和鉴定。3．用分离培养获得的纳米细菌在体外进行感染人脐静脉内皮细胞系ECV-304的实验。
结果：1．动脉粥样硬化斑块的纳米细菌单克隆抗体免疫荧光和免疫组化染色阳性部分主要分布在动脉内膜下层，外膜层其次，中膜层最少。2．动脉粥样硬化斑块中纳米细菌感染率为63.2％，对照组标本中感染率为10.5％，两者之间差异有显著性(p＜0.05)。3．阳性标本透射电镜观察见细胞间质和细胞浆内均有较多的细菌，细菌表面有一层致密的外衣。4．6例腹主动脉瘤粥样斑块标本中有5例分离培养出纳米细菌，阳性率为83.3％，并予以透射电镜鉴定。6．人脐静脉内皮细胞系ECV-304与分离培养的纳米细菌共同混合培养后发现内皮细胞胞浆内免疫荧光染色阳性。7．混合培养后的内皮细胞透射电镜观察见细胞间、
小南大学 啡士学位沦文
细胞内均什纳米纲菌存在，9！1胞核内也可见纳米细菌，细胞空泡变，膜
性结构类似坏死，部分线粒体水肿，空泡变，溶酶体呈现髓鞘图像。
结论：1．介动脉粥样硬化斑块标本中发现有纳米细菌存在的证据。
2．腹主动脉瘤粥杆斑块小能分离培养出纳米细菌。3．纳米细菌具有感
染人脐静脉内皮细胞系＊CV304的能力。4．内皮别胞在感染纳米细菌
厂发生结构炳仰什改变。5．纳米细｝ ＊能参’5动脉粥样硬化的 炳理过
W，互尚须进·儿互以i且人门
Background and Aims: The etiology of atherosclerosis is still not very clear. There are lots of theories about pathogenesis, but none of them can elucidate the pathogenesis of atherosclerosis alone and entirely. The theory of infection has been pay close attention to recently. The theory of inflammation has been supported by more and more research report. Nanobacteria is a novel apatite mineral-forming bacteria involved in the pathological calcification or sclerosis. There is a large amount of calcification material which is not perfectly explained in atherosclerotic plaques. The nanobacteria was tested, isolated, cultured and identified in the atherosclerotic plaques. Experiment of vascular endothelial cell line EC V-304 infection with nanobacteria was done. The aim of this study is to discover the evidence of nanobacteria in the atherosclerotic plaques and to identify the ability of the nanobacteria infection with cell and to offer a new researchful field to formation and pathogenesis of atherosclerosis.
Methods:1. Nanobacteria monoclonal antibody immunohistochemical staining and immunofluorescence staining were performed in 19 atherosclerotic plaques and 19 contrast artery. The positive samples were checked by transmission electron microscopy. 2. The nanobacteria was isolated and cultured and identified in 6 atherosclerotic plaques of abdominal aortic aneurysm. 3. The experiment of the vascular endothelial cell line ECV-304 infection with nanobacteria was done.
Results: 1. The positive of monoclonal antibody immunohistochemical staining and immunofluorescence staining of nanobacteria in the atherosclerotic plaques mainly located under intima. Few located in
vascular smooth muscle cells. 2. The positive rate of nanobacteria was significantly higher in the atherosclerotic plaques than contrast group (63.2% VS 10.5%). 3. A great deal of bacteria was found intracellularly and extracellularly in these positive samples under transimission electron microscopy. 4. The nanobacteria which was identified by transimission electron microscopy was isolated from 5 atherosclerotic plaque samples. The positive rate was 83.3%(5/6). 5. The immunofluorescence staining of vascular endothelial cell line ECV-304 was discovered positively after co-culture with nanobacteria. 6. There were nanobacteria in the nucleus and intracellar and extracellar endothelial cells under transmission electron microscopy after co-culture. The cell vacuolar degeneration, membrane structural necrosis, mitochondrin edema, mitochondrin vacuolar degeneration and lysosome myelinization were found in the endothelial cells.
Conclusions: 1. The evidence of nanobacteria is discovered in the atherosclerotic plaques. 2. The nanobacteria is isolated from the atherosclerotic plaques of abdominal aortic aneurysm. 3. The nanobacteria has the ability of infection to the vascular cell line ECV-304. 4. The endothelial cells have pathological structural alteration after infected with nanobacteria. 5. The nanobacteria may be involved in the pathological mechanism of atherosclerosis.
方法：1．对19例动脉粥样硬化斑块和19例对照组动脉壁进行纳米细菌单克隆抗体免疫荧光和免疫组化染色，阳性者进行透射电子显微镜检测。2．对6例腹主动脉瘤粥样斑块标本进行纳米细菌的分离、培养和鉴定。3．用分离培养获得的纳米细菌在体外进行感染人脐静脉内皮细胞系ECV-304的实验。
结果：1．动脉粥样硬化斑块的纳米细菌单克隆抗体免疫荧光和免疫组化染色阳性部分主要分布在动脉内膜下层，外膜层其次，中膜层最少。2．动脉粥样硬化斑块中纳米细菌感染率为63.2％，对照组标本中感染率为10.5％，两者之间差异有显著性(p＜0.05)。3．阳性标本透射电镜观察见细胞间质和细胞浆内均有较多的细菌，细菌表面有一层致密的外衣。4．6例腹主动脉瘤粥样斑块标本中有5例分离培养出纳米细菌，阳性率为83.3％，并予以透射电镜鉴定。6．人脐静脉内皮细胞系ECV-304与分离培养的纳米细菌共同混合培养后发现内皮细胞胞浆内免疫荧光染色阳性。7．混合培养后的内皮细胞透射电镜观察见细胞间、
小南大学 啡士学位沦文
细胞内均什纳米纲菌存在，9！1胞核内也可见纳米细菌，细胞空泡变，膜
性结构类似坏死，部分线粒体水肿，空泡变，溶酶体呈现髓鞘图像。
结论：1．介动脉粥样硬化斑块标本中发现有纳米细菌存在的证据。
2．腹主动脉瘤粥杆斑块小能分离培养出纳米细菌。3．纳米细菌具有感
染人脐静脉内皮细胞系＊CV304的能力。4．内皮别胞在感染纳米细菌
厂发生结构炳仰什改变。5．纳米细｝ ＊能参’5动脉粥样硬化的 炳理过
W，互尚须进·儿互以i且人门
Background and Aims: The etiology of atherosclerosis is still not very clear. There are lots of theories about pathogenesis, but none of them can elucidate the pathogenesis of atherosclerosis alone and entirely. The theory of infection has been pay close attention to recently. The theory of inflammation has been supported by more and more research report. Nanobacteria is a novel apatite mineral-forming bacteria involved in the pathological calcification or sclerosis. There is a large amount of calcification material which is not perfectly explained in atherosclerotic plaques. The nanobacteria was tested, isolated, cultured and identified in the atherosclerotic plaques. Experiment of vascular endothelial cell line EC V-304 infection with nanobacteria was done. The aim of this study is to discover the evidence of nanobacteria in the atherosclerotic plaques and to identify the ability of the nanobacteria infection with cell and to offer a new researchful field to formation and pathogenesis of atherosclerosis.
Methods:1. Nanobacteria monoclonal antibody immunohistochemical staining and immunofluorescence staining were performed in 19 atherosclerotic plaques and 19 contrast artery. The positive samples were checked by transmission electron microscopy. 2. The nanobacteria was isolated and cultured and identified in 6 atherosclerotic plaques of abdominal aortic aneurysm. 3. The experiment of the vascular endothelial cell line ECV-304 infection with nanobacteria was done.
Results: 1. The positive of monoclonal antibody immunohistochemical staining and immunofluorescence staining of nanobacteria in the atherosclerotic plaques mainly located under intima. Few located in
vascular smooth muscle cells. 2. The positive rate of nanobacteria was significantly higher in the atherosclerotic plaques than contrast group (63.2% VS 10.5%). 3. A great deal of bacteria was found intracellularly and extracellularly in these positive samples under transimission electron microscopy. 4. The nanobacteria which was identified by transimission electron microscopy was isolated from 5 atherosclerotic plaque samples. The positive rate was 83.3%(5/6). 5. The immunofluorescence staining of vascular endothelial cell line ECV-304 was discovered positively after co-culture with nanobacteria. 6. There were nanobacteria in the nucleus and intracellar and extracellar endothelial cells under transmission electron microscopy after co-culture. The cell vacuolar degeneration, membrane structural necrosis, mitochondrin edema, mitochondrin vacuolar degeneration and lysosome myelinization were found in the endothelial cells.
Conclusions: 1. The evidence of nanobacteria is discovered in the atherosclerotic plaques. 2. The nanobacteria is isolated from the atherosclerotic plaques of abdominal aortic aneurysm. 3. The nanobacteria has the ability of infection to the vascular cell line ECV-304. 4. The endothelial cells have pathological structural alteration after infected with nanobacteria. 5. The nanobacteria may be involved in the pathological mechanism of atherosclerosis.
引文
[1] Burch GE, Harb JM, Hiramoto Y. Coxsackie viral infection of the aorta of man. South Med J, 1974, 67(2): 1661～69
[2] Fabricant CG, Fabricant J, Litrenta MM, et al. Virus-induced atherosclerosis. J Exp Med, 1978,148(1):335～340
[3] Fabricant CG, Fabricant J, Minick CG, et al. Herpesvirus-induced atherosclerosis in chickens. Fed Proc, 1983, 42(8): 2476～2479
[4] Hajjar DP, Fabricant CG, Minick CG, et al. Virus-induced atherosclerosis: herpesvirus infection alter aortic cholesterol metabolism and accumulation. Am J Pathol, 1986, 122(1):62～70
[5] Wissler RW. Update on the pathogenesis of atherosclerosis. Am J Med, 1991, 91 (Suppl 113): 113～135.
[6] Sutter MC. Lessons for atherosclerosis research from tuberculosis and peptic ulcer. Can Med Assoc J, 1995, 152(5): 667～670.
[7] Cook PJ, Lip GY. Infection agents and atherosclerotic vascular disease. QJM, 1996, 89(10): 727～735.
[8] Melnick SL, Shahar E, Foisom AR, et al. Past infection by chlamydia pneumonia strain TWAR and asymptomatic carotid atherosclerosis. Atherosclerosis risk in communities (ARIC) study investigation. Am J Med, 1993, 95(5): 499～504.
[9] Ciflcioglu N, Kuronen I, Akerman K, et al. A New Potential Treat in Antigen and Antibody Products' Nanobacteria Vaccines 97, Brown & al ed, Cold Spring Harbor Laboratory Press, New York, 1997, 99～103.
[10] Kajander EO, Clfcioglu N. Nanobacteria: a alternative mechanism for pathogenic intra-and extra cellular calcification and stone formation. Proc Nalt Acad USA, 1998, 95(14): 8274～8279.
[11] Cuerpo GE, Kajander EO, Cifcioglu N, et al. Nanobacteria: an experimental neo-lithogenesis medol. Arch Esp Urol, 2000, 53:1893～1898
[12] Holmberg M. Prevalence of Human Anti-Nanobacteria Antibodies Suggest Possible Zoonosis. International Nanobacteria Minisymposium. www.nanobac.com.
[13] Kajander EO, Tahvanainen E, Kuronen I, et al. Comparison of Staphylococci and Novel Bacteria-Like Particles from Blood. Zbl Bakt, 1994, suppl, 26: 105.
[14] Cificioglu N, Kajander EO. Interaction of nanobacteria with cultured mammalian cells. Pathophysiology, 1998, 4(3): 259～270.
[15] Dennis A, Carson. An infections origin of extraskeletal calcification. Proc Natl Acad Sci USA, 1998, 95(7): 7846～7847.
[16] Cisar JO, Xu DQ, Thompson J, et al. An alternative interpretation of nanobacteriainduced biomineralization. Proc Natl Acad Sci USA, 2000, 97(21): 11511～11515.
[17] Kajander EO, Cifcioglu N. Nanobacteria: a alternative mechanism for pathogenic intra-and extra cellular calcification and stone formation. Proc Nalt Acad USA, 1998, 95(14): 8274～8279.
[18] Cifcioglu N, Bjorklund M, Kuorikoski K, et al. Nanobacteria: an infectious cause for kidney stone formation. Kidney Int, 1999, 56(5):1893～1898
[19] Hjelle JT, Miller-Hjelle MA, Poxtou IR, et al. Endotoxin and nanobateria in polycystic kidney disease. Kidney Int, 2000, 57(6): 2360～2374
[20] Sommer AP, Hasstnen HI, Kajander EO. Light-induced replication of nanobacteria., a preliminary report. J Clin Laser Med Surg, 2002, 20(5): 241～244
[21] Lusis AJ. Atherosclerosis. Nature, 2000, 407: 233～241.
[22] Ikeda U, Takahashi M, Shimada K, et al. Monocyte- endothelial cell interaction in atherogenesis and thrombosis. Clin Cardiol, 1998, 21: 11～14.
[23] Visser MR, Tracy PB, Vercellotti GM, et al. Enhanced thrombin generation and platelet binding on herpes simplex virus-infected endothelium. Proc Natl Acad Sci USA, 1988, 85: 8227～8230
[24] Key NS, Vercellotti GM, Winkeiman JC, et al. Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression. Proc Natl Acad Sci USA, 1990, 87: 7095～7099
[25] Etingin OR, Silverstein RL, Friedman HM, et al. Viral activation of the coagulation cascade: molecular interactions at the surface of infected endothelial cells. Cell, 1990, 61:657～662
[26] Kol A, Sukhoova GK, Lichtman AH, et al. Chlamydial heatshock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression. Circulation, 1998, 98: 300～307
[27] Dechend R, Maass M, Gieffers J, et al. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cell activates NF-κB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation, 1999, 100: 1369～1373
[28] Molestina RE, Miller RD, Ramirez JA, et al. Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrphils and monocytes. Infect Immun, 1999, 67:1323～1330
[29] Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis, 1999, 180: 780～790
[30] Kol A, Bourcier T, Lichtman AH, et al. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth cells, and macrophages. J Clin Invest, 1999, 103: 575～577
[31] Summergill JT, Molestina RE, Miller RD, et al. Interactions of Chlamydia pneumoniae with human endothelial cells. J Infect Dis, 2000, 181 Suppl 3:S479～482
[2] Fabricant CG, Fabricant J, Litrenta MM, et al. Virus-induced atherosclerosis. J Exp Med, 1978,148(1):335～340
[3] Fabricant CG, Fabricant J, Minick CG, et al. Herpesvirus-induced atherosclerosis in chickens. Fed Proc, 1983, 42(8): 2476～2479
[4] Hajjar DP, Fabricant CG, Minick CG, et al. Virus-induced atherosclerosis: herpesvirus infection alter aortic cholesterol metabolism and accumulation. Am J Pathol, 1986, 122(1):62～70
[5] Wissler RW. Update on the pathogenesis of atherosclerosis. Am J Med, 1991, 91 (Suppl 113): 113～135.
[6] Sutter MC. Lessons for atherosclerosis research from tuberculosis and peptic ulcer. Can Med Assoc J, 1995, 152(5): 667～670.
[7] Cook PJ, Lip GY. Infection agents and atherosclerotic vascular disease. QJM, 1996, 89(10): 727～735.
[8] Melnick SL, Shahar E, Foisom AR, et al. Past infection by chlamydia pneumonia strain TWAR and asymptomatic carotid atherosclerosis. Atherosclerosis risk in communities (ARIC) study investigation. Am J Med, 1993, 95(5): 499～504.
[9] Ciflcioglu N, Kuronen I, Akerman K, et al. A New Potential Treat in Antigen and Antibody Products' Nanobacteria Vaccines 97, Brown & al ed, Cold Spring Harbor Laboratory Press, New York, 1997, 99～103.
[10] Kajander EO, Clfcioglu N. Nanobacteria: a alternative mechanism for pathogenic intra-and extra cellular calcification and stone formation. Proc Nalt Acad USA, 1998, 95(14): 8274～8279.
[11] Cuerpo GE, Kajander EO, Cifcioglu N, et al. Nanobacteria: an experimental neo-lithogenesis medol. Arch Esp Urol, 2000, 53:1893～1898
[12] Holmberg M. Prevalence of Human Anti-Nanobacteria Antibodies Suggest Possible Zoonosis. International Nanobacteria Minisymposium. www.nanobac.com.
[13] Kajander EO, Tahvanainen E, Kuronen I, et al. Comparison of Staphylococci and Novel Bacteria-Like Particles from Blood. Zbl Bakt, 1994, suppl, 26: 105.
[14] Cificioglu N, Kajander EO. Interaction of nanobacteria with cultured mammalian cells. Pathophysiology, 1998, 4(3): 259～270.
[15] Dennis A, Carson. An infections origin of extraskeletal calcification. Proc Natl Acad Sci USA, 1998, 95(7): 7846～7847.
[16] Cisar JO, Xu DQ, Thompson J, et al. An alternative interpretation of nanobacteriainduced biomineralization. Proc Natl Acad Sci USA, 2000, 97(21): 11511～11515.
[17] Kajander EO, Cifcioglu N. Nanobacteria: a alternative mechanism for pathogenic intra-and extra cellular calcification and stone formation. Proc Nalt Acad USA, 1998, 95(14): 8274～8279.
[18] Cifcioglu N, Bjorklund M, Kuorikoski K, et al. Nanobacteria: an infectious cause for kidney stone formation. Kidney Int, 1999, 56(5):1893～1898
[19] Hjelle JT, Miller-Hjelle MA, Poxtou IR, et al. Endotoxin and nanobateria in polycystic kidney disease. Kidney Int, 2000, 57(6): 2360～2374
[20] Sommer AP, Hasstnen HI, Kajander EO. Light-induced replication of nanobacteria., a preliminary report. J Clin Laser Med Surg, 2002, 20(5): 241～244
[21] Lusis AJ. Atherosclerosis. Nature, 2000, 407: 233～241.
[22] Ikeda U, Takahashi M, Shimada K, et al. Monocyte- endothelial cell interaction in atherogenesis and thrombosis. Clin Cardiol, 1998, 21: 11～14.
[23] Visser MR, Tracy PB, Vercellotti GM, et al. Enhanced thrombin generation and platelet binding on herpes simplex virus-infected endothelium. Proc Natl Acad Sci USA, 1988, 85: 8227～8230
[24] Key NS, Vercellotti GM, Winkeiman JC, et al. Infection of vascular endothelial cells with herpes simplex virus enhances tissue factor activity and reduces thrombomodulin expression. Proc Natl Acad Sci USA, 1990, 87: 7095～7099
[25] Etingin OR, Silverstein RL, Friedman HM, et al. Viral activation of the coagulation cascade: molecular interactions at the surface of infected endothelial cells. Cell, 1990, 61:657～662
[26] Kol A, Sukhoova GK, Lichtman AH, et al. Chlamydial heatshock protein 60 localizes in human atheroma and regulates macrophage tumor necrosis factor-α and matrix metalloproteinase expression. Circulation, 1998, 98: 300～307
[27] Dechend R, Maass M, Gieffers J, et al. Chlamydia pneumoniae infection of vascular smooth muscle and endothelial cell activates NF-κB and induces tissue factor and PAI-1 expression: a potential link to accelerated arteriosclerosis. Circulation, 1999, 100: 1369～1373
[28] Molestina RE, Miller RD, Ramirez JA, et al. Infection of human endothelial cells with Chlamydia pneumoniae stimulates transendothelial migration of neutrphils and monocytes. Infect Immun, 1999, 67:1323～1330
[29] Kalayoglu MV, Hoerneman B, LaVerda D, et al. Cellular oxidation of low-density lipoprotein by Chlamydia pneumoniae. J Infect Dis, 1999, 180: 780～790
[30] Kol A, Bourcier T, Lichtman AH, et al. Chlamydial and human heat shock protein 60s activate human vascular endothelium, smooth cells, and macrophages. J Clin Invest, 1999, 103: 575～577
[31] Summergill JT, Molestina RE, Miller RD, et al. Interactions of Chlamydia pneumoniae with human endothelial cells. J Infect Dis, 2000, 181 Suppl 3:S479～482