黄芩、柴胡及其配伍的提取物对CVB_（3m）增殖及心肌组织细胞凋亡干预的比较研究
|
摘要
目的:
研究黄芩、柴胡及其配伍提取物体外抗CVB_(3m)病毒及对细胞活性影响,探讨不同药物的作用机制和靶点;研究黄芩、柴胡及其配伍提取物对CVB_(3m)诱导Balb/c小鼠心肌炎的治疗作用,并初步探讨其作用机制。
方法:
1.采用微量细胞培养法,以细胞病变法和MTT法判定细胞活性,观察黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂体外抗CVB_(3m)病毒和对细胞活性的影响,确定黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂抑制病毒指数和药物对细胞的预防和治疗保护率。
2.采用腹腔注射CVB_(3m)病毒诱导Balb/c小鼠心肌炎,应用RT-PCR技术检测病原核酸,结合光镜和电镜病理观察方法,鉴定小鼠病毒性心肌炎模型。
3.将黄芩、柴胡及其配伍的提取物以灌胃给药途径治疗CVB_(3m)心肌炎小鼠14天,观察三组药物对小鼠一般状态、体重、14天存活率、心脏质量/体质量、心脏组织CVB_(3m)病毒滴度及光镜和电镜病理改变的影响。
4.采用RT-PCR技术,检测各组小鼠给药后不同时间(3天、5天、7、10天、14天)心肌组织中CVB_(3m)—RNA的复制和IFN-mRNA的表达情况,探讨黄芩、柴胡及其配伍提取物干预病毒复制和诱生干扰素的作用。
5.分别采用TUNEL、免疫组化和RT-PCR技术,检测小鼠给药后不同时间点心肌组织细胞凋亡情况、Bcl-2和Bax的蛋白及mRNA表达情况,探讨黄芩、柴胡及其配伍提取物对病毒性心肌炎心肌组织细胞凋亡的干预。
结果:
一、体外实验
1.黄芩提取物浓度为1.563mg/ml时,抑制病毒指数大于2,而柴胡组和配伍组的抑病毒指数均小于2;三组药物都具有不同程度的预防保护作用,而柴胡组和配伍组的预防保护作用尤为明显,其预防保护率明显高于黄芩组,有显著性差异(P<0.05);在病毒感染后,三
Objeetive1. To study the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on Coxsackievixus B_3a. (CVB_3m) in vitro, and to explore the antiviral mechanism, and to observe the influence on cell infected by CVB_3m.2. To observe the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on the 3alb/c mice myocarditis infected by CVB_3 and to exnlore the mechanism of the effect . Methods1. We used microcell culture method to observe the effect of the skullcap extract、 the Bupleurum extract and their compatibility extract on antivirus and protecting cell. We detected the restrained indexes of the three kinds of drugs on CVB-3m and the cell protection rate of the three kinds of drugs when the cell was infected by CVB_3M2. Balb/c mice were inoculated intraperitoneally with CVB_3m and batch sacrificed late. On the base of the models , following experiment were carried.(1)The CVB_3m-RNA were detected by reverse transcription - polymerase chain reaction (RT-PCR), the heart histopathologic (macro- and ultramicro) were observed by the light microscope and electron microscope.(2)Mice were randomly divided into 5 groups, including the model group、 the control group、 the skullcap extract group、 the Bupleurum extract group and the compatibility extract group. We cured the myocarditis of mice by intragastric administration for 14 days, and observed the body of weigh of mice, appetite, the
symptom and the death of mice. We detected the tite of CVB_3m, in heart、 rate between the heart mass and weigh of the body and macro-histopathologic and ultrandcro changes of hearts.(3) The CVB_3m-RNA and IFN mRNA in heart were examed by RT-PCR method to explore the effect of antivirus and induction of IFN by the three kinds of drugs.(4)The apoptosis in heart were examed by TDT/Dutp Nick End labeling (TUNBL), and the apoptosis related protein and gene expression of Bc1-2 /Bax were detected by RT-PCR method and immunohistochemistry method .Result Experiments in vitro.1. The skullcap extract can directly kill CVB_3m. in vitro when his density is 1. 563 mg/ml, but the Bupleurum extract and their compatibility extract hadn' t this effect. The three kinds of drugs can protect cell infected by CVB3.. The effect of the Bupleurum group and the compatibility group is better than the skullcap groups when the drugs were dministered in advance. The effect of the skullcap group and the compatibility group were better than the Bupleurum group when the drugs were dministered after infection.2. The restrained indexes of the crude extracting skullcap was more than 2 when his density is TD_0; The restrained indexes of the fine extracting skullcap was less than 2 when his density is TD_0、 1/2TD_O and 1/4TD_0. The two kinds of drugs can protecte cell infected by CVB_3m , and the effect of the crude extracting skullcap was better than the fine extracting skullcap. Experiments on animal.
1. The positive percent of CVB_3m-BNA in heart is 100% during 3th ~14th day after infected. The heart histopathologic change were inflammatory cell infiltration、 dropsy and necrosis of heart cell.2. The three kinds of drugs can increcse the body weight of myocarditis mice, appetite and the rate of survival; In increasing the rate of survival and the body weight, the difference of the three groups is not significant . The three kinds of drugs can inhibit replication of CVB_3m. in heart. The viral titer in heart of the skullcap group were lower than the model group in the3th、 5th、 7th and 10th day: The viral titer in heart of the Bupleurnm. group were lower than the model group in the 3th、 5th day: The viral titer in heart of the compatibility group were lower than the model group in the 5th、 7th day: The macro-, histopathologic and ultramicro changes of mice hearts of the three drugs were better than the model group too, but the effect of the Bupleurum group and the compatibility group was better than the skullcap group.3. The content of CVB_3m-RNA in the hearts of the three drugs
研究黄芩、柴胡及其配伍提取物体外抗CVB_(3m)病毒及对细胞活性影响,探讨不同药物的作用机制和靶点;研究黄芩、柴胡及其配伍提取物对CVB_(3m)诱导Balb/c小鼠心肌炎的治疗作用,并初步探讨其作用机制。
方法:
1.采用微量细胞培养法,以细胞病变法和MTT法判定细胞活性,观察黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂体外抗CVB_(3m)病毒和对细胞活性的影响,确定黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂抑制病毒指数和药物对细胞的预防和治疗保护率。
2.采用腹腔注射CVB_(3m)病毒诱导Balb/c小鼠心肌炎,应用RT-PCR技术检测病原核酸,结合光镜和电镜病理观察方法,鉴定小鼠病毒性心肌炎模型。
3.将黄芩、柴胡及其配伍的提取物以灌胃给药途径治疗CVB_(3m)心肌炎小鼠14天,观察三组药物对小鼠一般状态、体重、14天存活率、心脏质量/体质量、心脏组织CVB_(3m)病毒滴度及光镜和电镜病理改变的影响。
4.采用RT-PCR技术,检测各组小鼠给药后不同时间(3天、5天、7、10天、14天)心肌组织中CVB_(3m)—RNA的复制和IFN-mRNA的表达情况,探讨黄芩、柴胡及其配伍提取物干预病毒复制和诱生干扰素的作用。
5.分别采用TUNEL、免疫组化和RT-PCR技术,检测小鼠给药后不同时间点心肌组织细胞凋亡情况、Bcl-2和Bax的蛋白及mRNA表达情况,探讨黄芩、柴胡及其配伍提取物对病毒性心肌炎心肌组织细胞凋亡的干预。
结果:
一、体外实验
1.黄芩提取物浓度为1.563mg/ml时,抑制病毒指数大于2,而柴胡组和配伍组的抑病毒指数均小于2;三组药物都具有不同程度的预防保护作用,而柴胡组和配伍组的预防保护作用尤为明显,其预防保护率明显高于黄芩组,有显著性差异(P<0.05);在病毒感染后,三
Objeetive1. To study the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on Coxsackievixus B_3a. (CVB_3m) in vitro, and to explore the antiviral mechanism, and to observe the influence on cell infected by CVB_3m.2. To observe the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on the 3alb/c mice myocarditis infected by CVB_3 and to exnlore the mechanism of the effect . Methods1. We used microcell culture method to observe the effect of the skullcap extract、 the Bupleurum extract and their compatibility extract on antivirus and protecting cell. We detected the restrained indexes of the three kinds of drugs on CVB-3m and the cell protection rate of the three kinds of drugs when the cell was infected by CVB_3M2. Balb/c mice were inoculated intraperitoneally with CVB_3m and batch sacrificed late. On the base of the models , following experiment were carried.(1)The CVB_3m-RNA were detected by reverse transcription - polymerase chain reaction (RT-PCR), the heart histopathologic (macro- and ultramicro) were observed by the light microscope and electron microscope.(2)Mice were randomly divided into 5 groups, including the model group、 the control group、 the skullcap extract group、 the Bupleurum extract group and the compatibility extract group. We cured the myocarditis of mice by intragastric administration for 14 days, and observed the body of weigh of mice, appetite, the
symptom and the death of mice. We detected the tite of CVB_3m, in heart、 rate between the heart mass and weigh of the body and macro-histopathologic and ultrandcro changes of hearts.(3) The CVB_3m-RNA and IFN mRNA in heart were examed by RT-PCR method to explore the effect of antivirus and induction of IFN by the three kinds of drugs.(4)The apoptosis in heart were examed by TDT/Dutp Nick End labeling (TUNBL), and the apoptosis related protein and gene expression of Bc1-2 /Bax were detected by RT-PCR method and immunohistochemistry method .Result Experiments in vitro.1. The skullcap extract can directly kill CVB_3m. in vitro when his density is 1. 563 mg/ml, but the Bupleurum extract and their compatibility extract hadn' t this effect. The three kinds of drugs can protect cell infected by CVB3.. The effect of the Bupleurum group and the compatibility group is better than the skullcap groups when the drugs were dministered in advance. The effect of the skullcap group and the compatibility group were better than the Bupleurum group when the drugs were dministered after infection.2. The restrained indexes of the crude extracting skullcap was more than 2 when his density is TD_0; The restrained indexes of the fine extracting skullcap was less than 2 when his density is TD_0、 1/2TD_O and 1/4TD_0. The two kinds of drugs can protecte cell infected by CVB_3m , and the effect of the crude extracting skullcap was better than the fine extracting skullcap. Experiments on animal.
1. The positive percent of CVB_3m-BNA in heart is 100% during 3th ~14th day after infected. The heart histopathologic change were inflammatory cell infiltration、 dropsy and necrosis of heart cell.2. The three kinds of drugs can increcse the body weight of myocarditis mice, appetite and the rate of survival; In increasing the rate of survival and the body weight, the difference of the three groups is not significant . The three kinds of drugs can inhibit replication of CVB_3m. in heart. The viral titer in heart of the skullcap group were lower than the model group in the3th、 5th、 7th and 10th day: The viral titer in heart of the Bupleurnm. group were lower than the model group in the 3th、 5th day: The viral titer in heart of the compatibility group were lower than the model group in the 5th、 7th day: The macro-, histopathologic and ultramicro changes of mice hearts of the three drugs were better than the model group too, but the effect of the Bupleurum group and the compatibility group was better than the skullcap group.3. The content of CVB_3m-RNA in the hearts of the three drugs
引文
1.王雪峰,严文初,郭振武,等.柴胡清心饮对小儿病毒性心肌炎左心功能及T细胞亚群的影响.中国中西医结合杂志,1997:17(2):73～75.
2.王雪峰,王永梅.小柴胡汤及其分解剂对柯萨奇B3m病毒感染乳鼠心肌保护及细胞免疫调节作用.中国中西医结合杂志,2000;20(8):599～602.
3. Renes J,Helin M, Vaino P, et al. Clinical outcome and left ventricular function 23years after acute coxsackie virus myopericarditis. Eur Heart J, 1990; 11: 182～188.
4. See DM and Tilless JG. Viral myocarditis. Rev Infect Dis, 1991; 13: 951～956.
5. Mcmanus BM, Chow LH, Wilson JE, et al. Direct myocarditis injury by enterovirus: a central role in the evolution of murine myocarditi. Clin Immunol Immunopathol, 1993; 68 (2): 159～169.
6.李延文,杨英珍,何梅先,等.肠道病毒感染与扩张型心肌病发病关系的探讨.中华内科杂志,1997;36(6):377～379.
7. 37. Sole M J.Liu P.V.Virol myocarditis:a paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy.J Am Coll Cardiol. 1993;22:99～105.
8. Kyu BS.Matsumori A.Sato Y, et al.Cardiac persistence of cardioviral rna detected by polymerase chain reaction in a murine model of dilated cardiomyopathy. Circulation. 1992;86:522～530.
9. Obrador D, Ballerter M, Carrio I, et al. Active myocardial damage without attending inflammatory response in dilated cardiomyopathy. J Am coll cardiol. 1993;21:1667～1671.
10.王盛春,党峻英,贾旭东.柴胡与黄芩伍用清热与抗病毒作用.中草药,1997;29(1):27～29.
11.蒋金法,邓南伟,杨英珍.急性病毒性心肌炎与扩张型心肌病关系的探讨.中华心血管病杂志,1992:20(1):4～6.
12. Bowles NE, Rose ML, Taylor P, et al. End-stage dilated cariomyopathy persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response. Circulation, 1989; 80: 1128～1136.
13.高中洪,黄开勋,徐辉碧.黄芩中黄酮类生物活性的研究进展.中国药学杂志,1998;33(12):705～707.
14. Baylor NW, Fu T, Yaan YD, et al. Inhibition of human T cell leukemia virus by the plant flavonoid baicalin(7-glucuronic acid, 5,6-dihydroxyflavone). J infect dis 1992:165 (3): 433.
15. Li BQ, Fu T, Dongyan Y et al. Flavonoid baicalin inhibits HIV-1 infection at the level of viral entry. Biochem Biophys Res Commun, 2000; 276(2): 534～538.
16. Wu J A, Attele A S, Zhang L et al. Anti-HIV activity of medicinal herbs: Usage and potential development. Am J Chin Med, 2001; 29(1): 69～81.
17.孙国贤,林久治,王蕙瑛.柯萨奇B_(3m)病毒所致Balb/c鼠急性心肌炎模型.中华微生物与免疫学杂志,1987;7(1):35.
18.杨英珍,金佩英,郭棋,等.上海地区柯萨奇B组病毒感染的研究.中华传染病杂志,1989;7(1):4～7.
19.崔小岱,Ricardo Gomez,马连华,等.柯萨奇B_3病毒亲心肌株与非亲心肌株感染小鼠心肌细胞的实验比较.中华实验和临床病毒学杂志,1997;11(3):274～276
20. Huber S A, Job L P. Differences in myocarditic T cell response of Balb/c mice infected with myocarditic and non-myocarditic strains of coxsackievirus group B type 3. Infect Immun.1983; 39: 1419.
21.杨英珍.病毒性心脏病.上海科学技术出版社,第一版,2001.10:352.
22.杨英珍,金佩英,李志善,等.成人型柯萨奇病毒性心肌炎的临床观察、治疗及随访.中华心血管病杂志,1981:9:152.
23. Gowrishanker K, Rajajee S. Varied manifestation ofvirial myocarditis. Indian J Pediatr, 1994; 61: 263.
24. Easton AJ, Eglin RP. The detection of coxsackievirus RNA in cardic tissue by in situ hybridization. Gen virol Journal, 1988:69:285～291.
25. Griffin LD, Keamey D, Ni J, et al. Analysis of formalin-fixed and frozen myocardial autopsy samples for viral genome in childhood myocarditis and dilated cardimyopathy with endocardial fibroelastosis using polymerase chain reaction(PCR). Cardiovas Pathol, 1995;4(1)3～11.
26.彭天庆,杨英珍,Kandolf,等.黄芪对柯萨奇B_3病毒核糖核酸作用的研究及其机理探讨,1994;14(11):664～666.
27.杨英珍,金佩英,等.小鼠急慢性柯萨奇B_3病毒性心肌炎的病理改变.中华医学杂志,1991,71(11):639～640.
28. Hayakawa M, Inoh T, Yokota Y, et al. A long term follow-up study of acute viral and idiopathic myocarditis.Japan Circulation Journal,1983; 47 (1): 1304～1309.
29. Simth WG. Coxsackie B myopericarditis in adults. American Heart Journal, 1970; 80:251～253.
30.王雪峰,郭津津,魏克伦,等小柴胡汤分解剂抗柯萨奇B_3病毒感染及其对心肌炎防治作用的研究.中国医科大学学报,2000;29(2):115～117.
31.王雪峰,张小梅.从三焦枢机论治小儿肠道病毒性心肌炎探要.中医函授通讯,1995;3:43。
32.王雪峰,刘芳,魏克伦,等.小柴胡汤及其分解剂对柯萨奇B_(3m)病毒感染小鼠心肌保护及细胞免疫调节作用.中国中西医结合杂志,20(8):599～602.
33.史青,聂淑琴,黄璐琦.柴胡属植物化学成分及药理研究新进展,2002;8(5):53～55.
34.杨英珍,郭棋,金佩英,等.大鼠搏动心肌细胞培养感染柯萨奇B2病毒后超微结构的研究.中华医学杂志,1985;63:524.
35. Mcmanus BM, Chow LH, Wilson JE, et al. Direct myocarditis injury by enterovirus: a central role in the evolution of murine myocarditis. Clin Immunopathol, 1993; 68(2): 159～169.
36.杨英珍,Dyke JW.人白细胞干扰素对coxsackieB-2病毒感染培养大鼠搏动心肌细胞的作用.中国药理学报,1985;6(2):102～106.
37. Yamamoto N, Shibamori M, Ogura M, et al. Effects of intranasal administration of recombinant murine interferon-gamma on murine acute myocarditis caused by encephalomyoarditis virus. Circulation, 1998; 17: 1017-1023.
38. 徐安平,崔若兰.柴胡在肾小球疾病中治疗作用的研究进展国外,医药植物药分册, 1996; 11 (2): 55-58.
39.Adams JM , Cory S.The Bcl-2 protein family :arbiters of cell surv ival.Science. 1998;281; 1322-1326.
40. Hanada M, Aime-Sempe C, Sato T , et al. Structure function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J Biol Chem. 1995;270:1962-1969
41. Tamaru Y, Miyawaki T, Iwai K, et al. Absence of bcl-2 express by activated CD45RO+ T lymphocyte in acute infet alious mononucleosis supporting their susceptibility to programmed cell death. Blood.l993;82:521~527.
42. Chen S. In vitro mechanism of PC SPES. Urology, 2001; 58 (2suppl-1): 28-35.
2.王雪峰,王永梅.小柴胡汤及其分解剂对柯萨奇B3m病毒感染乳鼠心肌保护及细胞免疫调节作用.中国中西医结合杂志,2000;20(8):599～602.
3. Renes J,Helin M, Vaino P, et al. Clinical outcome and left ventricular function 23years after acute coxsackie virus myopericarditis. Eur Heart J, 1990; 11: 182～188.
4. See DM and Tilless JG. Viral myocarditis. Rev Infect Dis, 1991; 13: 951～956.
5. Mcmanus BM, Chow LH, Wilson JE, et al. Direct myocarditis injury by enterovirus: a central role in the evolution of murine myocarditi. Clin Immunol Immunopathol, 1993; 68 (2): 159～169.
6.李延文,杨英珍,何梅先,等.肠道病毒感染与扩张型心肌病发病关系的探讨.中华内科杂志,1997;36(6):377～379.
7. 37. Sole M J.Liu P.V.Virol myocarditis:a paradigm for understanding the pathogenesis and treatment of dilated cardiomyopathy.J Am Coll Cardiol. 1993;22:99～105.
8. Kyu BS.Matsumori A.Sato Y, et al.Cardiac persistence of cardioviral rna detected by polymerase chain reaction in a murine model of dilated cardiomyopathy. Circulation. 1992;86:522～530.
9. Obrador D, Ballerter M, Carrio I, et al. Active myocardial damage without attending inflammatory response in dilated cardiomyopathy. J Am coll cardiol. 1993;21:1667～1671.
10.王盛春,党峻英,贾旭东.柴胡与黄芩伍用清热与抗病毒作用.中草药,1997;29(1):27～29.
11.蒋金法,邓南伟,杨英珍.急性病毒性心肌炎与扩张型心肌病关系的探讨.中华心血管病杂志,1992:20(1):4～6.
12. Bowles NE, Rose ML, Taylor P, et al. End-stage dilated cariomyopathy persistence of enterovirus RNA in myocardium at cardiac transplantation and lack of immune response. Circulation, 1989; 80: 1128～1136.
13.高中洪,黄开勋,徐辉碧.黄芩中黄酮类生物活性的研究进展.中国药学杂志,1998;33(12):705～707.
14. Baylor NW, Fu T, Yaan YD, et al. Inhibition of human T cell leukemia virus by the plant flavonoid baicalin(7-glucuronic acid, 5,6-dihydroxyflavone). J infect dis 1992:165 (3): 433.
15. Li BQ, Fu T, Dongyan Y et al. Flavonoid baicalin inhibits HIV-1 infection at the level of viral entry. Biochem Biophys Res Commun, 2000; 276(2): 534～538.
16. Wu J A, Attele A S, Zhang L et al. Anti-HIV activity of medicinal herbs: Usage and potential development. Am J Chin Med, 2001; 29(1): 69～81.
17.孙国贤,林久治,王蕙瑛.柯萨奇B_(3m)病毒所致Balb/c鼠急性心肌炎模型.中华微生物与免疫学杂志,1987;7(1):35.
18.杨英珍,金佩英,郭棋,等.上海地区柯萨奇B组病毒感染的研究.中华传染病杂志,1989;7(1):4～7.
19.崔小岱,Ricardo Gomez,马连华,等.柯萨奇B_3病毒亲心肌株与非亲心肌株感染小鼠心肌细胞的实验比较.中华实验和临床病毒学杂志,1997;11(3):274～276
20. Huber S A, Job L P. Differences in myocarditic T cell response of Balb/c mice infected with myocarditic and non-myocarditic strains of coxsackievirus group B type 3. Infect Immun.1983; 39: 1419.
21.杨英珍.病毒性心脏病.上海科学技术出版社,第一版,2001.10:352.
22.杨英珍,金佩英,李志善,等.成人型柯萨奇病毒性心肌炎的临床观察、治疗及随访.中华心血管病杂志,1981:9:152.
23. Gowrishanker K, Rajajee S. Varied manifestation ofvirial myocarditis. Indian J Pediatr, 1994; 61: 263.
24. Easton AJ, Eglin RP. The detection of coxsackievirus RNA in cardic tissue by in situ hybridization. Gen virol Journal, 1988:69:285～291.
25. Griffin LD, Keamey D, Ni J, et al. Analysis of formalin-fixed and frozen myocardial autopsy samples for viral genome in childhood myocarditis and dilated cardimyopathy with endocardial fibroelastosis using polymerase chain reaction(PCR). Cardiovas Pathol, 1995;4(1)3～11.
26.彭天庆,杨英珍,Kandolf,等.黄芪对柯萨奇B_3病毒核糖核酸作用的研究及其机理探讨,1994;14(11):664～666.
27.杨英珍,金佩英,等.小鼠急慢性柯萨奇B_3病毒性心肌炎的病理改变.中华医学杂志,1991,71(11):639～640.
28. Hayakawa M, Inoh T, Yokota Y, et al. A long term follow-up study of acute viral and idiopathic myocarditis.Japan Circulation Journal,1983; 47 (1): 1304～1309.
29. Simth WG. Coxsackie B myopericarditis in adults. American Heart Journal, 1970; 80:251～253.
30.王雪峰,郭津津,魏克伦,等小柴胡汤分解剂抗柯萨奇B_3病毒感染及其对心肌炎防治作用的研究.中国医科大学学报,2000;29(2):115～117.
31.王雪峰,张小梅.从三焦枢机论治小儿肠道病毒性心肌炎探要.中医函授通讯,1995;3:43。
32.王雪峰,刘芳,魏克伦,等.小柴胡汤及其分解剂对柯萨奇B_(3m)病毒感染小鼠心肌保护及细胞免疫调节作用.中国中西医结合杂志,20(8):599～602.
33.史青,聂淑琴,黄璐琦.柴胡属植物化学成分及药理研究新进展,2002;8(5):53～55.
34.杨英珍,郭棋,金佩英,等.大鼠搏动心肌细胞培养感染柯萨奇B2病毒后超微结构的研究.中华医学杂志,1985;63:524.
35. Mcmanus BM, Chow LH, Wilson JE, et al. Direct myocarditis injury by enterovirus: a central role in the evolution of murine myocarditis. Clin Immunopathol, 1993; 68(2): 159～169.
36.杨英珍,Dyke JW.人白细胞干扰素对coxsackieB-2病毒感染培养大鼠搏动心肌细胞的作用.中国药理学报,1985;6(2):102～106.
37. Yamamoto N, Shibamori M, Ogura M, et al. Effects of intranasal administration of recombinant murine interferon-gamma on murine acute myocarditis caused by encephalomyoarditis virus. Circulation, 1998; 17: 1017-1023.
38. 徐安平,崔若兰.柴胡在肾小球疾病中治疗作用的研究进展国外,医药植物药分册, 1996; 11 (2): 55-58.
39.Adams JM , Cory S.The Bcl-2 protein family :arbiters of cell surv ival.Science. 1998;281; 1322-1326.
40. Hanada M, Aime-Sempe C, Sato T , et al. Structure function analysis of Bcl-2 protein. Identification of conserved domains important for homodimerization with Bcl-2 and heterodimerization with Bax. J Biol Chem. 1995;270:1962-1969
41. Tamaru Y, Miyawaki T, Iwai K, et al. Absence of bcl-2 express by activated CD45RO+ T lymphocyte in acute infet alious mononucleosis supporting their susceptibility to programmed cell death. Blood.l993;82:521~527.
42. Chen S. In vitro mechanism of PC SPES. Urology, 2001; 58 (2suppl-1): 28-35.