灯盏花素对佐剂性关节炎大鼠血清IL-1、IL-6、TNF-α水平及滑膜MMP3/TIMP1表达的影响
|
摘要
目的：类风湿关节炎（Rheumatoid Arthritis，RA）是一病因未明的系统性疾病，特点是慢性、多关节炎症，最后导致软骨和骨的破坏。其主要病理特点是滑膜细胞增生、衬里层增厚、多种炎性细胞浸润、血管翳形成，以及软骨和骨组织的破坏。近年来的研究表明，RA的软骨破坏与细胞因子网络失调及软骨基质的降解有密切的关系。目前主要的治疗方法为早期应用免疫抑制剂来抑制关节的骨破坏,控制病情发展。临床上应用灯盏花素治疗RA取得了较好的疗效。本文旨在成功建立佐剂性关节炎（Adjuvant Arthritis，AA）模型的基础上观察灯盏花素对大鼠关节炎的治疗效果，并检测其对模型鼠血清白介素-1（interleukin-1，IL-1）、白介素-6（interleukin-6，IL-6）、肿瘤坏死因子（tumor necrosis factor-α、TNF-α）水平及滑膜基质金属蛋白酶3（ Matrix metalloproteinase 3,MMP3）和组织金属蛋白酶抑制物1(Tissue inhibitor of metalloproteinase 1，TIMP1)表达的影响，探讨作用机制。
方法：(1)选取50只Wistar雄性大鼠，随机分为五组：正常组、模型组、灯盏花素组、来氟米特组、配伍组。除正常对照组，每组大鼠右足皮内注射弗氏完全佐剂0.1ml制作关节炎模型。(2)在造模后第18天，除模型组外，各用药组分别连续给药14天，在造模及用药前后观察大鼠的一般情况，
并记录体重；根据排水法原理用容积测量仪测量大鼠后足体积，并记录关节炎指数，观察药物对继发病变的治疗作用。(3)用放射免疫法测定IL-1、IL-6、TNF-α水平。（4）采血后摘取踝关节进行固定、脱钙、石蜡包埋，HE染色，光镜下观察关节病理变化，并进行评分。（5）无菌取出踝关节处的滑膜组织应用免疫组化检测滑膜组织中的MMP3及TIMP1的表达，并采用计算机图像分析的方法对其表达量进行定量分析。（6）应用SPSS统计软件进行统积分析。
结果
1 与模型组比较，灯盏花素组、来氟米特组、联合组均能改善大鼠的一般情况；配伍组给药前后体重增加值与正常组比较，差异无显著性（P＞0.05）；与模型组比较，差异有显著性（P＜0.05）。
2 造模后18天各组大鼠之间的关节炎指数与足爪肿胀相比无明显差异（P＞0.05）；给药后，灯盏花素组、来氟米特组、配伍组均能减轻大鼠的足爪肿胀和关节炎指数，与模型组比较，差异显著（P＜0.05）；配伍组较来氟米特、灯盏花素组的作用更明显（P＜0.05)。
3 模型组的IL-1、IL-6、TNF-α水平明显高于正常对照组（P＜0.01），用药后各组的细胞因子水平较模型组降低（P＜0.05），配伍组的降低更明显（P＜0.01)。
4病理结果显示：模型组大鼠踝关节滑膜组织内有大量的炎性细胞浸润，关节面不完整，有血管翳形成；治疗后，滑膜组织内炎细胞浸润明显减轻，软骨破坏程度减弱。与正常对照组比较，模型组大鼠踝关节的病变积分显著增高（P＜0.01)；各用药组的大鼠关节病变积分与模型组比较，皆明显
下降（P＜0.05），三个用药组之间比较，配伍组的关节病变积分最低。
5 免疫组化定量分析，模型组大鼠滑膜组织MMP3 表达阳性密度显著高于正常对照组（P＜0.01）；与模型组比较，各用药组的MMP3表达阳性密度均降低（P＜0.01)；配伍组的表达阳性密度与单一用药组比较，差异显著（P＜0.05)。
与正常对照组比较，模型组大鼠滑膜组织TIMP-1的表达阳性密度明显降低（P＜0.05）。各用药组的TIMP-1表达阳性密度均明显高于模型组。配伍组的表达密度与单一用药组比较有差异（P＜0.05）。
6 IL-1、病理积分与MMP3的相关性分析
本实验得出结论：（1）IL-1与MMP3水平之间成正相关（r=0.9051，p=0.0001）；（2）病理积分与MMP3水平之间成正相关（r=0.8961，p=0.0001）。这说明MMP3在RA的软骨破坏中起着重要的作用。IL-1是MMP3的重要调节因素。灯盏花素通过降低血小板水平，影响IL-1的分泌，减少了IL-1对滑膜和软骨的刺激，使MMP3对细胞外基质的降解减轻，从而减弱了RA的软骨破坏。
结论
1灯盏花素治疗佐剂性关节炎具有显著疗效，能够明显减轻大鼠的足爪肿胀，降低血清IL-1、IL-6、TNF-α水平；减轻关节的病变损伤积分，减少滑膜组织中MMP3的表达，增加TIMP1的表达。配伍组的效果优于灯盏花素、来氟米特组，证实了中药有效成分与西药配伍应用在实验性关节炎中的增效作用。
2灯盏花素注射液治疗类风湿关节炎的可能机制是（1）
灯盏花素通过改善血循环，降低血小板水平，减少血清炎性细胞因子的分泌。（2）通过减少细胞因子IL-1、IL-6、TNF-α的分泌，减轻其对滑膜组织和软骨组织的刺激，抑制AA大鼠滑膜组织MMP3的过度表达，调整MMP3/TIMP1的失衡，从而减轻关节炎的症状和软骨破坏。
Objective: Rheumatoid arthritis（RA）is a systemic disease of unknown etiology that is characterized by chronic polyarticular joint inflammation which lead to destruction of cartilage and bone. Histopathologic features of RA encompass infiltration by macrophages and T cells synovial lining hyperplasia，neoangiogenesis，pannus formation and destruction of cartilage and bone. Study recently show that there is a correlation between cytokings IL-1、IL-6、TNF-α、 MMP3/TIMP1 and bone damage. Update，inhibiting joint destruction of cartilage is the major theapy. We found Breviscapine had a good effect in treating RA .The aim of this study is to investigate the therapeutic mechanism of Bre on cytokine IL-1、IL-6、TNF-αand MMP3/TIMP1 in the animal model of rheumatoid arthritis .
Methods: (1)Fifty male Wistar rats were randomly divided into 5 groups：the normal group、the model group、the leflunomide group、the Bre group、the lef and bre group.Except for the rats of normal control group，the others were intracutaneously injected with 0.1ml Freund，s complete adjuvant in the right hind limb. (2) The medicines were given from the
18th day to the 32th day once a day to observe its effect on multiple arthritis. AA was assessed ：by measuring arthritic index、paw volume、general status and body weight. (3)The levels of IL-1、IL-6、TNF-αwere measured with radioimmunoassay. (4) Under high microscope，we observe the pathological change of ankle joint and record the remark，in order to study the effect on cartilage destruction in AA in rat. (5) Immunohistochemistry was used to detected the expression of MMP3/TIMP1in synovial tissue. The intensity of MMP3/TIMP1 in synovial tissue was analyzed by computerized image system. (6)There was positive correlation between IL-1 and MMP3 、between pathological change and MMP3.
Results
1 bre、 lef and combined group could markedly reduce the degree of rat pedal swelling，improve general status，increment the body weight as compared to the model group（P＜0.05）.There was difference between model group and combined group（P＜0.05）.The increment body weight of combined group had no difference as compared to the control group.
2 There was no significantly difference of arthtitis indexs and paw swelling in every group after modeled group .After treatment,every group could reduce the arthritis indexs and paw swelling.there was difference as model group.The effect of combined group was superior to the lef group or bre group.
3 The levels of IL-1、IL-6、TNF-αin model group was higher than control group.After treatment,the serum IL-1、IL-6、
TNF-αdecreased significantly in the bre group as compared to that in the model group. there was significantly difference（P＜0.05）.the levels of IL-1、IL-6、TNF-αof combined group were lower than bre and lef groups（P＜0.05）.
4 The pathologicals results showed that there was a lot of macrophages and T cells in synovial tissue. There was pannus formatted in synovial tissue. The remarks of model group was higher than that in control group.
5 The Immunohistochemistry showed that the expression of MMP3 in model group was higher than control group.Bre could decrease the expression of MMP3.there was difference between bre group and model group. The expression of MMP3 in combined group was most lowest in test group. The result of TIMP1 was contrast to the expression of MMP3.
6 The correlation between serum IL-1、pathological remark and MMP3 was positive significantly（P＜0.01）.The result showed that IL-1 played an important role in joint destruction. In the other hand, the result also showed that MMP3 was an important factor in cartilage and bone.
Conclusion: 1 The result showed that bre had a defined effect in treating animal model of rheumatoid arthritis. It could reduced the paw swelling and arthritis indexs; It could decrease the levels of serum IL-1、IL-6、TNF-α; Moreover, Bre could decrease effectively the expression of MMP3 in synovial tissue. To sum up, Bre could relieve AA symptoms and suppressed cartilage destruction.
方法：(1)选取50只Wistar雄性大鼠，随机分为五组：正常组、模型组、灯盏花素组、来氟米特组、配伍组。除正常对照组，每组大鼠右足皮内注射弗氏完全佐剂0.1ml制作关节炎模型。(2)在造模后第18天，除模型组外，各用药组分别连续给药14天，在造模及用药前后观察大鼠的一般情况，
并记录体重；根据排水法原理用容积测量仪测量大鼠后足体积，并记录关节炎指数，观察药物对继发病变的治疗作用。(3)用放射免疫法测定IL-1、IL-6、TNF-α水平。（4）采血后摘取踝关节进行固定、脱钙、石蜡包埋，HE染色，光镜下观察关节病理变化，并进行评分。（5）无菌取出踝关节处的滑膜组织应用免疫组化检测滑膜组织中的MMP3及TIMP1的表达，并采用计算机图像分析的方法对其表达量进行定量分析。（6）应用SPSS统计软件进行统积分析。
结果
1 与模型组比较，灯盏花素组、来氟米特组、联合组均能改善大鼠的一般情况；配伍组给药前后体重增加值与正常组比较，差异无显著性（P＞0.05）；与模型组比较，差异有显著性（P＜0.05）。
2 造模后18天各组大鼠之间的关节炎指数与足爪肿胀相比无明显差异（P＞0.05）；给药后，灯盏花素组、来氟米特组、配伍组均能减轻大鼠的足爪肿胀和关节炎指数，与模型组比较，差异显著（P＜0.05）；配伍组较来氟米特、灯盏花素组的作用更明显（P＜0.05)。
3 模型组的IL-1、IL-6、TNF-α水平明显高于正常对照组（P＜0.01），用药后各组的细胞因子水平较模型组降低（P＜0.05），配伍组的降低更明显（P＜0.01)。
4病理结果显示：模型组大鼠踝关节滑膜组织内有大量的炎性细胞浸润，关节面不完整，有血管翳形成；治疗后，滑膜组织内炎细胞浸润明显减轻，软骨破坏程度减弱。与正常对照组比较，模型组大鼠踝关节的病变积分显著增高（P＜0.01)；各用药组的大鼠关节病变积分与模型组比较，皆明显
下降（P＜0.05），三个用药组之间比较，配伍组的关节病变积分最低。
5 免疫组化定量分析，模型组大鼠滑膜组织MMP3 表达阳性密度显著高于正常对照组（P＜0.01）；与模型组比较，各用药组的MMP3表达阳性密度均降低（P＜0.01)；配伍组的表达阳性密度与单一用药组比较，差异显著（P＜0.05)。
与正常对照组比较，模型组大鼠滑膜组织TIMP-1的表达阳性密度明显降低（P＜0.05）。各用药组的TIMP-1表达阳性密度均明显高于模型组。配伍组的表达密度与单一用药组比较有差异（P＜0.05）。
6 IL-1、病理积分与MMP3的相关性分析
本实验得出结论：（1）IL-1与MMP3水平之间成正相关（r=0.9051，p=0.0001）；（2）病理积分与MMP3水平之间成正相关（r=0.8961，p=0.0001）。这说明MMP3在RA的软骨破坏中起着重要的作用。IL-1是MMP3的重要调节因素。灯盏花素通过降低血小板水平，影响IL-1的分泌，减少了IL-1对滑膜和软骨的刺激，使MMP3对细胞外基质的降解减轻，从而减弱了RA的软骨破坏。
结论
1灯盏花素治疗佐剂性关节炎具有显著疗效，能够明显减轻大鼠的足爪肿胀，降低血清IL-1、IL-6、TNF-α水平；减轻关节的病变损伤积分，减少滑膜组织中MMP3的表达，增加TIMP1的表达。配伍组的效果优于灯盏花素、来氟米特组，证实了中药有效成分与西药配伍应用在实验性关节炎中的增效作用。
2灯盏花素注射液治疗类风湿关节炎的可能机制是（1）
灯盏花素通过改善血循环，降低血小板水平，减少血清炎性细胞因子的分泌。（2）通过减少细胞因子IL-1、IL-6、TNF-α的分泌，减轻其对滑膜组织和软骨组织的刺激，抑制AA大鼠滑膜组织MMP3的过度表达，调整MMP3/TIMP1的失衡，从而减轻关节炎的症状和软骨破坏。
Objective: Rheumatoid arthritis（RA）is a systemic disease of unknown etiology that is characterized by chronic polyarticular joint inflammation which lead to destruction of cartilage and bone. Histopathologic features of RA encompass infiltration by macrophages and T cells synovial lining hyperplasia，neoangiogenesis，pannus formation and destruction of cartilage and bone. Study recently show that there is a correlation between cytokings IL-1、IL-6、TNF-α、 MMP3/TIMP1 and bone damage. Update，inhibiting joint destruction of cartilage is the major theapy. We found Breviscapine had a good effect in treating RA .The aim of this study is to investigate the therapeutic mechanism of Bre on cytokine IL-1、IL-6、TNF-αand MMP3/TIMP1 in the animal model of rheumatoid arthritis .
Methods: (1)Fifty male Wistar rats were randomly divided into 5 groups：the normal group、the model group、the leflunomide group、the Bre group、the lef and bre group.Except for the rats of normal control group，the others were intracutaneously injected with 0.1ml Freund，s complete adjuvant in the right hind limb. (2) The medicines were given from the
18th day to the 32th day once a day to observe its effect on multiple arthritis. AA was assessed ：by measuring arthritic index、paw volume、general status and body weight. (3)The levels of IL-1、IL-6、TNF-αwere measured with radioimmunoassay. (4) Under high microscope，we observe the pathological change of ankle joint and record the remark，in order to study the effect on cartilage destruction in AA in rat. (5) Immunohistochemistry was used to detected the expression of MMP3/TIMP1in synovial tissue. The intensity of MMP3/TIMP1 in synovial tissue was analyzed by computerized image system. (6)There was positive correlation between IL-1 and MMP3 、between pathological change and MMP3.
Results
1 bre、 lef and combined group could markedly reduce the degree of rat pedal swelling，improve general status，increment the body weight as compared to the model group（P＜0.05）.There was difference between model group and combined group（P＜0.05）.The increment body weight of combined group had no difference as compared to the control group.
2 There was no significantly difference of arthtitis indexs and paw swelling in every group after modeled group .After treatment,every group could reduce the arthritis indexs and paw swelling.there was difference as model group.The effect of combined group was superior to the lef group or bre group.
3 The levels of IL-1、IL-6、TNF-αin model group was higher than control group.After treatment,the serum IL-1、IL-6、
TNF-αdecreased significantly in the bre group as compared to that in the model group. there was significantly difference（P＜0.05）.the levels of IL-1、IL-6、TNF-αof combined group were lower than bre and lef groups（P＜0.05）.
4 The pathologicals results showed that there was a lot of macrophages and T cells in synovial tissue. There was pannus formatted in synovial tissue. The remarks of model group was higher than that in control group.
5 The Immunohistochemistry showed that the expression of MMP3 in model group was higher than control group.Bre could decrease the expression of MMP3.there was difference between bre group and model group. The expression of MMP3 in combined group was most lowest in test group. The result of TIMP1 was contrast to the expression of MMP3.
6 The correlation between serum IL-1、pathological remark and MMP3 was positive significantly（P＜0.01）.The result showed that IL-1 played an important role in joint destruction. In the other hand, the result also showed that MMP3 was an important factor in cartilage and bone.
Conclusion: 1 The result showed that bre had a defined effect in treating animal model of rheumatoid arthritis. It could reduced the paw swelling and arthritis indexs; It could decrease the levels of serum IL-1、IL-6、TNF-α; Moreover, Bre could decrease effectively the expression of MMP3 in synovial tissue. To sum up, Bre could relieve AA symptoms and suppressed cartilage destruction.
引文
徐叔云主编.药理实验方法学，北京：人民卫生出版社，1991：723
张钧田.现代药理实验方法，[M].北京：北京医科大学-中国协和医科大学联合出版社,1998,1383
Kenneth W. Adolph：human genome methods .New York CRC Press，1998:224
申维玺，孙燕.论中医证的化学本质是蛋白质和肽及证本质的分子标准，中国中西医结合杂志.1999,11:696-698
中华人民共和国药典[M].1997年版第1部,245
PETTIPHER ER ，HIGGS GA，HENDERSON B. IL- induces leukocyte infiltration and cartilage proteoglecan degradation in the synovial joint [J]. Proc Natl Acad Sci USA .1986,83:8749-8753
Chin JE,LinYA. Effects of recombine anthumaninter leukin-1 beta on rabbit articular chondrocytes. Stimulation of prostanoid release and inhibition of cell growth. Arthritis Rheum,1988,31(10):1290 -1296
Dibattista JA, Martel Pelletier J, WosuLOet al. Glucocorticoidre ceptormediated in hibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes. J Clin Endocriin normal human chondrocytes. J Clin Endocrinol Metab , 1991,72(2)：316 -326
Tewari A，Buhles WC，Starnes HF，et al. Preliminary report：effects of interleukin-1 on platelet counts. Lancet,
1990, 336：712
粱虹，张育.RA患者血小板参数的变化与IL-1β关系的研究.中华风湿病学杂志，1998，9：177-178
张水源.灯盏花注射液治疗脑梗死临床研究.中国新药杂志，2002，11（1）：87-89
Saxne T，Palladino MA Jr，Heinegard D，et al .Detection of tumor necrosis factor-α but not tumor necrosis factor -βin rheumatoid arthritis synovial fluid and serum. Arthritis Rheum，1988，31：1041-1045
Popp PT，Pape H，peddinghaus. Interleukin-6 in adjuvant arthritis of rats amd its pharmacol ，1992，14（4）：5657
Thomas CV，Coker ML，Zellner JL，et al. Increased matrix metalloproteinases activity andselective upregulation in LV myocardium from patients with end staged ilated cardiomyopathy Circulation，1998,97：1708 -1715
蒋明，朱立平，林孝义.风湿病学.科学出版社1995，314
Clio R, Beatrice A, Jean MJ, et al. Matrix metalloproteinase -3 serum levels are correlated with disease activity and predict clinical response in rheumatoid arthritis [J]. Rheum , 2000, 27(4)：888
李兴勇，朱任之.类风湿性关节炎与基质金属蛋白酶的研究进展.中国医学理论与实践.2003，2：141-142
季颖，张明发.米诺环素治疗类风湿关节炎.中国新药与临床.2000，3：220-223
Conway JG，Wakefield JA，Brown RH，Marron BE，Sekut L, Stimpson SA, et al. Inhibition of cartilage and bone
destruction in adjuvant arthritis in the rat by a matrix metalloproteinase inhibi-tor. J Exp Med 1995；182：449-571
霍丹群，张伟，刘佳.中药方剂配伍研究近况.浙江中医杂 志.2003，5：214-215
Chong AS，Huang WY，Liu W，et al，In vivo activity of leflunomide，pharmacokinetic analyses and mechanism of immunosuppression[J]. Transplantation，1999，68（1）：100
Li WD，Ran GX, Teng HL，et. Dynamic effect s of leflunomide on IL-1、IL-6 andTNF-aalpha activity produced from peritoneal macrophages in adjuvant arthritis rats [J]. Acta Pharmacoi Sin ，2002；23(8)：752-756
孟中，吕照江，张之南.几种中药有效成分对中性粒细胞呼吸爆发的影响.中国药理学通报，1994，10（6）：439-441
王丽娟，陈咏梅，林宇，等.灯盏花素对吲哚美辛所致胃溃疡的保护作用［J］齐齐哈尔医学院学报，1999，20（4）：317-318
林宇，王勇，孟文芬，等.灯盏花素对乙酸所致胃溃疡的保护作用［J］齐齐哈尔医学院学报，2001，22（1）：1
王荪.灯盏花注射液治疗中风后瘫痪469例报告.中草药， 1983，14（1）：33
张钧田.现代药理实验方法，[M].北京：北京医科大学-中国协和医科大学联合出版社,1998,1383
Kenneth W. Adolph：human genome methods .New York CRC Press，1998:224
申维玺，孙燕.论中医证的化学本质是蛋白质和肽及证本质的分子标准，中国中西医结合杂志.1999,11:696-698
中华人民共和国药典[M].1997年版第1部,245
PETTIPHER ER ，HIGGS GA，HENDERSON B. IL- induces leukocyte infiltration and cartilage proteoglecan degradation in the synovial joint [J]. Proc Natl Acad Sci USA .1986,83:8749-8753
Chin JE,LinYA. Effects of recombine anthumaninter leukin-1 beta on rabbit articular chondrocytes. Stimulation of prostanoid release and inhibition of cell growth. Arthritis Rheum,1988,31(10):1290 -1296
Dibattista JA, Martel Pelletier J, WosuLOet al. Glucocorticoidre ceptormediated in hibition of interleukin-1 stimulated neutral metalloprotease synthesis in normal human chondrocytes. J Clin Endocriin normal human chondrocytes. J Clin Endocrinol Metab , 1991,72(2)：316 -326
Tewari A，Buhles WC，Starnes HF，et al. Preliminary report：effects of interleukin-1 on platelet counts. Lancet,
1990, 336：712
粱虹，张育.RA患者血小板参数的变化与IL-1β关系的研究.中华风湿病学杂志，1998，9：177-178
张水源.灯盏花注射液治疗脑梗死临床研究.中国新药杂志，2002，11（1）：87-89
Saxne T，Palladino MA Jr，Heinegard D，et al .Detection of tumor necrosis factor-α but not tumor necrosis factor -βin rheumatoid arthritis synovial fluid and serum. Arthritis Rheum，1988，31：1041-1045
Popp PT，Pape H，peddinghaus. Interleukin-6 in adjuvant arthritis of rats amd its pharmacol ，1992，14（4）：5657
Thomas CV，Coker ML，Zellner JL，et al. Increased matrix metalloproteinases activity andselective upregulation in LV myocardium from patients with end staged ilated cardiomyopathy Circulation，1998,97：1708 -1715
蒋明，朱立平，林孝义.风湿病学.科学出版社1995，314
Clio R, Beatrice A, Jean MJ, et al. Matrix metalloproteinase -3 serum levels are correlated with disease activity and predict clinical response in rheumatoid arthritis [J]. Rheum , 2000, 27(4)：888
李兴勇，朱任之.类风湿性关节炎与基质金属蛋白酶的研究进展.中国医学理论与实践.2003，2：141-142
季颖，张明发.米诺环素治疗类风湿关节炎.中国新药与临床.2000，3：220-223
Conway JG，Wakefield JA，Brown RH，Marron BE，Sekut L, Stimpson SA, et al. Inhibition of cartilage and bone
destruction in adjuvant arthritis in the rat by a matrix metalloproteinase inhibi-tor. J Exp Med 1995；182：449-571
霍丹群，张伟，刘佳.中药方剂配伍研究近况.浙江中医杂 志.2003，5：214-215
Chong AS，Huang WY，Liu W，et al，In vivo activity of leflunomide，pharmacokinetic analyses and mechanism of immunosuppression[J]. Transplantation，1999，68（1）：100
Li WD，Ran GX, Teng HL，et. Dynamic effect s of leflunomide on IL-1、IL-6 andTNF-aalpha activity produced from peritoneal macrophages in adjuvant arthritis rats [J]. Acta Pharmacoi Sin ，2002；23(8)：752-756
孟中，吕照江，张之南.几种中药有效成分对中性粒细胞呼吸爆发的影响.中国药理学通报，1994，10（6）：439-441
王丽娟，陈咏梅，林宇，等.灯盏花素对吲哚美辛所致胃溃疡的保护作用［J］齐齐哈尔医学院学报，1999，20（4）：317-318
林宇，王勇，孟文芬，等.灯盏花素对乙酸所致胃溃疡的保护作用［J］齐齐哈尔医学院学报，2001，22（1）：1
王荪.灯盏花注射液治疗中风后瘫痪469例报告.中草药， 1983，14（1）：33
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |