海珠益肝加味方对免疫性肝损伤小鼠的保护作用及其免疫调节机制的研究
|
摘要
目的：
在我国慢性乙型病毒性肝炎(Chronic hepatitis B CHB)的防治虽然取得了一定的进步,但现状并不是乐观,约10%～20%慢性乙型病毒性肝炎可发展为肝硬化,1%～5%慢性乙型病毒性肝炎可演变为肝癌,CHB的防治形势依然严峻。因此慢性乙型病毒性肝炎仍然是我国突出的公共卫生问题。在慢性乙型病毒性肝炎治疗方面,干扰素和核苷类似物无疑是首要选择,但是干扰素难以适应的副作用、基本上需要终身服用的核苷类似物限制自身的使用,很显然应用干扰素和核苷类似物抗乙肝病毒不能解决慢乙肝患者的所有问题。中医药的免疫功能调节治疗无疑是积极防治慢性乙型病毒性肝炎的有效方法。近些年来,已经有许多临床治疗慢性乙型病毒性肝炎的中药单体或者复方,但其调节免疫功能机理都不是十分清楚。海珠益肝加味方是导师盛国光教授在中医理论指导下,经过多年临床验证和实验研究,在海珠益肝方的基础不断优化的纯中药复方制剂。海珠益肝加味方由叶下珠、白花蛇舌草、茯苓、海藻、白芥子、莪术、枸杞、太子参八味药组成的经验方。具有解毒、化痰、消瘀、补虚的功效。早期研究提示海珠益肝加味方可能通过某些途径、环节和靶点达到双向调节免疫功能的作用,其作用机制很有待于进行深入研究。本课题以免疫肝损伤小鼠动物模型为实验对象,对海珠益肝加味方进行研究,观察中药复方海珠益肝方加味方对实验小鼠的免疫状态、脾细胞细胞因子的mRNA基因表达、辅助性T淋巴细胞Thl/Th2、Toll样受体(TLR)及其信号转导途径的影响,并运用计算机统计学软件进行数据处理和综合分析,探讨海珠益肝加味方对免疫性肝损伤小鼠的保护作用及其免疫调节机制,进一步提高慢性乙型病毒性肝炎的临床治疗水平。
方法：实验分五个部分进行
第一部分：海珠益肝方及其扩方对Concanavalin A(刀豆蛋白A)诱导免疫性肝损伤小鼠的保护作用。
清洁级昆明小鼠100只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝方组(4)、海珠益肝方加太子参枸杞子组(5)、海珠益肝方加太子参仙灵脾组(6)、海珠益肝方加枸杞子仙灵脾组(7)、叶下珠白花蛇舌草组(8)、海藻茯苓白芥子组(9)及莪术组(10),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤动物模型。造模前12天,模型组、空白组给予蒸馏水灌胃,其余组每日分别给予醋酸泼的松、海珠益肝全方、海珠益肝方加太子参枸杞子、海珠益肝方加太子参仙灵脾、海珠益肝方加枸杞子仙灵脾、叶下珠白花蛇舌草、海藻茯苓白芥子及莪术灌胃。末次灌胃给药后1h,空白组给予生理盐水10ml/kg尾静脉注射,其余各组按照20mg/kg尾静脉注射刀豆蛋白A造模,禁食自由饮水。造模给药8h后颈椎脱臼处死动物摘取肝脏和脾脏,生理盐水中清洗后称记重量,计算肝脾指数。肝组织标本匀浆离心后取上清液检测丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、丙二醛(MDA)及超氧化物歧化酶(SOD)的值。另外取0.2g肝组织固定、脱水、石蜡包埋、切片后行HE染色,光学显微镜下观察肝脏组织形态学变化。
第二部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠血清IL-4、IL-6、INF-γ、TNF-α的影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤模型。造模前12天,模型组、空白组给予蒸馏水灌胃,其余组每日分别给予醋酸强的松、海珠益肝加味方灌胃。末次灌胃给药后1h,空白组给予生理盐水10ml/kg尾静脉注射,其余各组按照20mg/kg尾静脉注射刀豆蛋白A造模,禁食自由饮水。造模给药8h后摘眼球取血,离心后留取血清。采用ELISA方法测定小鼠血清IL-4、IL-6、IFN-γ、TNF-α的值。
第三部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠肝组织病理变化及肝组织NF-KP65的表达水平的影响。
清洁级昆明小鼠40只,随机分为正常组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用Con A尾静脉注射法制作免疫性肝损伤模型。造模给药8h后处死小鼠,取肝组织制作肝脏的超微病理切片,采用电镜透射法观察各组肝细胞内超微结构的病理变化,同时取肝组织用Western-blot法测定小鼠肝组织NF-KP65的表达水平。第四部分：海珠益肝方加味方对Con A(刀豆蛋白A)诱导免疫性肝损伤小鼠脾组织IL-6、TNF-αmRNA影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤小鼠模型。采用RT-PCR法测定小鼠脾组织IL-6、TNF-α mRNA的表达。
第五部分：海珠益肝方加味方对Concanavalin A诱导免疫性肝损伤小鼠肝组织TLR4、CD14mRNA影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤模型。采用RT-PCR法测定小鼠肝组织TLR4、CD14mRNA的表达。
结果：
第一部分：海珠益肝方及其扩方对Con A(刀豆蛋白A)诱导免疫性肝损伤小鼠的保护作用。
1成功建立小鼠免疫性肝损伤模型。与正常组比较,模型组小鼠肝脾指数、肝组织匀浆后上清液ALT、AST水平及MDA含量均明显升高(P<0.05),肝组织匀浆后上清液SOD水平明显降低。模型组肝组织内大量淋巴细胞浸润,部分汇管区淋巴细胞浸润,肝板消失、凋亡小体散在、肝细胞成脂肪样变性。血管周围的肝细胞胞浆疏松,染色明显变浅,肝窦间隙明显增宽,肝索消失,可见点状坏死、淋巴细胞浸润。
2与模型组比较,对照组和海珠益肝方加味各组可降低小鼠肝脾指数,降低肝组织ALT、AST水平及MDA含量,提高肝组织的SOD水平(P<0.05),其中以海珠益肝加太子参、枸杞子组更明显。另外与模型组比较,海珠益肝方加味组肝脏病理变化有不同程度减轻。
第二部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠血清IL-4、IL-6、INF-γ、TNF-α的影响。
1与正常组比较,模型组小鼠血清中IFN-γ、TNF-α的水平明显升高,差异有显著性(P<0.05)。模型组小鼠血清IL-4、IL-6的水平明显降低,统计学上差异有显著性(P<0.05)。
2与模型组比较,对照组和海珠益肝方加味组血清中IFN-γ、TNF-α的水平明显降低,差异有显著性(P<0.05)。对照组和海珠益肝方加味方组小鼠血清中IL-6的水平明显升高,差异有显著性(P<0.05)。对照组和海珠益肝方加味方组小鼠血清中IL-4的水平明显升高,统计学差异有显著性(P<0.05)。
第三部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠肝组织病理变化及肝组织NF-KP65的表达水平的影响。
1正常组肝细胞核呈圆形及卵圆形,核膜光滑平整,染色质均匀分布在核内。模型组肝细胞肿胀,胞质疏松,出现深浅不一的切迹,胞质中常见髓样体及空泡变性区,线粒体肿胀,嵴模糊,内质网轻度扩张。与模型组比较,对照组和海珠益肝方加味组肝细胞肿胀明显减轻,未见明显毛细胆管扩张,线粒体轻度肿胀,嵴存在,内质网无明显扩张。
2与正常组比较,模型组NF-KP65的水平明显升高,统计学有显著差异(P<0.05)；与模型组比较,对照组和海珠益肝方加味组NF-KP65的水平有明显变化,统计学有显著差异(P<0.05)。
第四部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠小鼠脾组织IL-6、TNF-α mRNA影响。
1与正常组比较,模型组IL-6mRNA的水平有明显降低,统计学有显著差异(P<0.05)；与正常组比较,模型组TNF-αmRNA的水平显著升高,统计学有显著差异(P<0.05)。
2与模型组比较,对照组和海珠益肝方加味组IL-6mRNA的水平明显升高,统计学有显著差异(P<0.05)。与模型组比较,对照组和海珠益肝方加味组TNF-α mRNA的水平明显下降,统计学有显著差异(P<0.05)。
第五部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠肝组织TLR4、CD14mRNA影响。
1与正常组比较,模型组TLR4mRNA的表达水平明显降低,具有显著性差异(P<0.05)；与模型组比较,对照组和海珠益肝方加味组TLR4mRNA的表达水平明显升高,具有显著性差异(P<0.05)。
2与正常组比较,模型组CD14mRNA的水平无明显变化,经统计学处理无显著性差异(P>0.05)；与模型组比较,对照组和海珠益肝方加味组CD14mRNA的水平无明显变化,未见明显差异(P>0.05)。
结论：
1、海珠益肝方加味对免疫性肝损伤小鼠的肝脏功能有较好的保护作用。
2、海珠益肝方加味对免疫性肝损伤小鼠的肝脏功能有较好的保护作用的机制可能是维持Th1/Th2之间的平衡；抑制过抗的脾脏的细胞免疫功能,增强调节天然免疫功能,从而达到免疫功能的双向调节。
Purpose:
Controlling Chronic hepatitis B has made certain progress in China, but the reality is not optimistic,10%-20%of CHB will develope to liver cirrhosis and1%-5%CHB will develope to primary hepatocellular carcinoma (HCC), so the prevention and controlling situation Chronic hepatitis B is still grim and the chronic hepatitis B viral hepatitis is still a prominent public health problem in China. In the treatment of Chronic hepatitis B interferon and nucleoside analogues is undoubtedly the first choice, but basically the side effects of interferon is difficult to adapt and needs of lifetime use nucleoside analogues and expensive medical costs for many patients with Chronic hepatitis B, it is clear that the application interferon and nucleoside analogues against hepatitis b virus can't solve the all problems in patients with CHB. The regulation Immune function to Traditional Chinese medicine therapy is undoubtedly positive prevention and treatment of Chronic hepatitis B of the icing on the cake. In recently years, there have been many Chinese medicine monomer in the clinical treatment of chronic hepatitis B, but its mechanism of regulating immune function is not very clear. HZYGJWF has come from the tutor professor Shengguoguang in under the guidance of TCM theory, clinical verification and experimental research for many years. HZYGJWF is on the basis of the party with the prince ginseng, medlar. HZYGJWF under bead, spreading hedyot is herb, poria cocos, seaweed, white mustard seed, rhizoma zedoariae, medlar, radix pseudostellariae experience side composed of eight herbs. With detoxification, phlegm, blood stasis, tonify deficiency effect. Early studies suggest that HZYGJWF may in some way, link and targets to achieve two-way regulating immune function, its mechanism of action is worth further study. This topic is the immune liver injury animal models in mice as experimental object, the study of HZYGJWF about mice of immune state, spleen cell cytokines gene mRNA expression, helper T lymphocytes Th1/Th2, toll-like receptor (TLR) and its signal transduction pathways, the influence of using computer and statistical software for data processing and comprehensive analysis, to explore the immunoregulatory effects of HZYGJWF and its mechanism. traditional Chinese medicine has important academic value to improving the level of Chronic hepatitis B of viral disease control and prevention has important scientific significance.
Methods:the experiment is divided into five parts
The first part:HZYGJWF and its expanding party to Con A induction of protective immunity liver injury of mice.
Clean level100kun ming mice were randomly divided into ten group. normal group (1), model group (2), prednisone group (3), HZYGF group (4), HZYGF and radix pseudostellariae medlar group (5), HZYGF and radix pseudostellariae fairy spleen group (6), HZYGF and medlar fairy spleen group (7), leaf under bead spreading hedyotis herb group (8), tuckahoe white mustard group (9) and rhizoma zedoariae group(10), each group of10, only by Con A tail vein injections made immune hepatic injury animal model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily spilt, HZYGF, HZYGF add radix pseudostellariae medlar, HZYGF add radix pseudostellariae fairy spleen, HZYGF add medlar fairy spleen, hepatic lobe under bead spreading hedyotis herb, kelp, tuckahoe white mustard and rhizoma zedoariae lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Make mould for cervical dislocation executed after8h animal liver, spleen, liver and spleen index is calculated. Specimens of liver tissue homogenate after testing ALT, AST, SOD and MDA. In addition in liver tissue paraffin embedding sectioning will been done to HE staining.
The second part:the influence of HZYGJWF to Con A-induced immunological liver injury in mice serum IL-4, IL-6, INF-γ, TNF-α.
Clean level40kun ming mice were randomly divided into normal group (1), model group(2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily over the loose, HZYGF lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Building after8h pick eyeball blood, centrifugal file.usd10.00serum. Using ELISA method for determination of mice serum IL-4, IL-6, IFN-γ, TNF-α value.
The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression of NF-KP65level.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model. Building to put to death in mice after8h, take make ultrastructural slices of liver, the liver tissue by tem observation group the pathological changes of the ultrastructure of liver cell, at the same time take liver tissue in mice liver tissue was determined by Western blot method-the expression of NF-KP65level.
The fourth part:HZYGJWF to Con A-induced immunological liver injury in mice spleen in mice group, IL-6and TNF-α mRNA.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury in mice model. The mice spleen tissue was evaluated by RT-PCR method, IL-6, TNF-a mRNA expression.
The fifth part:HZYGF sides to con A induced immunological liver injury mice liver tissue TLR4, and CD14mRNA in mice.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injection made immune liver injury model. The liver tissue of mice was evaluated by RT-PCR method the expression of TLR4and CD14mRNA.
Results:
The first part:hai zhu yi liver and its expanding party to Con A induction of protective immunity liver injury of mice. Success immune liver injury in mice model was established.
1Compared with normal group, model group mice liver and spleen index, liver tissue homogenate supernatant after ALT, AST andMDA content has significantly increased (P<0.05), liver tissue homogenate supernatant after the SOD level is significantly reduced. Model group liver tissue in a large number of lymphocyte infiltration, part of the portal area lymphocyte infiltration, erythrocyte sedimentation within liver sinus, a large number of liver cell nucleues is broken, scattered apoptotic body, liver steatosis in cell plasma. Liver cells around vascular cell pack loose, color becomes shallow, all loose cytoplasm of liver cells, staining obviously becomes shallow, hepatic sinus gap widened significantly, liver disappear, sometimes visible point necrosis, lymphocyte, neutrophil infiltration.
2Compared with model group, control group and haizhu yi liver flavored group can decrease the mice liver and spleen index, lower ALT, AST and MDA content in liver tissue, improve the level of liver tissue SOD (P<0.05), among them with HZYGF with radix pseudostellariae, medlar more obvious.
Other party HZYGF flavored group obviously reduce degree of liver pathological changes than model group.
The second part:the influence of HZYGJWF to Con A induced immunological liver injury in mice serum IL-4, IL-6, INF-γ and TNF- a.
1Compared with normal group, the level of serum IFN-y and TNF-a of model group mice has increased significantly with significant difference (P<0.05). the level of serum IL-4and IL-6Model group mice has decreased with significant difference (P<0.05).
2Compared with model group, the level of serum IFN-γ and TNF a of control group and HZYGJWF group has lower significantly, there is significant difference (P<0.05). the level of mice serum IL-6in Control group and HZYGJWF has increased significantly with significant difference (P<0.05). the level of mice serum IL-4Control group of mice and HZYGJWF has increased a lot, there is significant difference (P<0.05).
The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression level of NF-KP65.
1set of normal liver cells for polygon, neatly, nuclear assumes the circular or ovoid, nuclear membrane smooth level off, the chromatin evenly distributed within the box. Model group liver cell swelling, cytoplasm osteoporosis, appear incisure, two-tone cytoplasm common in myeloid body and vacuoles degeneration area, the capillary bile duct expansion, some microvilli medullary degeneration, mitochondria swelling and cristae vague, mild expansion of endoplasmic reticulum. Compared with model group, control group and HZYGJWF group liver cell swelling significantly reduce, did not see clearly the capillary bile duct expansion, mild swelling of mitochondria crest, no obvious expansion of endoplasmic reticulum.
2Compared with normal group, the NF-KP65level of model group was increased significantly, statistically significant difference(P<0.05); Compared with model group, the NF-KP65level of control group and HZYGJWF group has obvious changes, statistically significant difference(P<0.05). HZYGJWF party on immunological liver injury in mice liver tissue ultra structure has obvious protective effect and reduced immune liver injury in mice liver tissue of NF-KP65level, so as HZYGJWF can protect the immunological liver injury of con A-induced.
The fourth part:HZYGJWF to A sword bean protein induced immunological liver injury in mice spleen tissues in mice, IL-6, TNF-a mRNA.
1Compared with normal group, model group, the level of IL-6mRNA has significantly reduced with statistically significant difference(P<0.05); Compared with normal group, the level of TNF-a mRNA of model group has significantly increased, statistically significant differences (P<0.05).
2Compared with model group, the level of IL-6mRNA of control group and HZYGJWF group has increased significantly, statistically significant differences (P<0.05). Compared with model group, the level of TNF-a mRNA of control group and HZYGJWF group has dropped significantly, statistically significant difference (P<0.05).
The fifth part:HZYGJWF to Con A-induced mice immune liver injury in mice liver tissue TLR4, CD14mRNA.
1Compared with normal group, model group of TLR4mRNA expression level increased obviously, statistically significant difference (P<0.05); Compared with model group, control group and HZYGJWF group TLR4mRNA expression level decreased obviously, statistically significant difference (P<0.05).
2Compared with normal group, model group of CD14mRNA level no obvious changes, statistics has no obvious difference (P>0.05)。 Compared with model group, control group and HZYGJWF group CD14mRNA level no obvious changes, statistics has no obvious difference.
Conclusion:
HZYGJWF has obvious protective effect to immune liver injury in mice by Con A induced. the mechanism may inhibit hepatoly cellular lipid per oxidation; inhibit cell inflammatory factor the release of TNF-α and IFN-γ, improve cell suppression of inflammatory factor, IL-6and IL-4release, so as it can adjust the balance between Th1/Th2; improve the activity of TLR4and Inhibiting the activity NF-KP65, reduce the happening of natural immune.
在我国慢性乙型病毒性肝炎(Chronic hepatitis B CHB)的防治虽然取得了一定的进步,但现状并不是乐观,约10%～20%慢性乙型病毒性肝炎可发展为肝硬化,1%～5%慢性乙型病毒性肝炎可演变为肝癌,CHB的防治形势依然严峻。因此慢性乙型病毒性肝炎仍然是我国突出的公共卫生问题。在慢性乙型病毒性肝炎治疗方面,干扰素和核苷类似物无疑是首要选择,但是干扰素难以适应的副作用、基本上需要终身服用的核苷类似物限制自身的使用,很显然应用干扰素和核苷类似物抗乙肝病毒不能解决慢乙肝患者的所有问题。中医药的免疫功能调节治疗无疑是积极防治慢性乙型病毒性肝炎的有效方法。近些年来,已经有许多临床治疗慢性乙型病毒性肝炎的中药单体或者复方,但其调节免疫功能机理都不是十分清楚。海珠益肝加味方是导师盛国光教授在中医理论指导下,经过多年临床验证和实验研究,在海珠益肝方的基础不断优化的纯中药复方制剂。海珠益肝加味方由叶下珠、白花蛇舌草、茯苓、海藻、白芥子、莪术、枸杞、太子参八味药组成的经验方。具有解毒、化痰、消瘀、补虚的功效。早期研究提示海珠益肝加味方可能通过某些途径、环节和靶点达到双向调节免疫功能的作用,其作用机制很有待于进行深入研究。本课题以免疫肝损伤小鼠动物模型为实验对象,对海珠益肝加味方进行研究,观察中药复方海珠益肝方加味方对实验小鼠的免疫状态、脾细胞细胞因子的mRNA基因表达、辅助性T淋巴细胞Thl/Th2、Toll样受体(TLR)及其信号转导途径的影响,并运用计算机统计学软件进行数据处理和综合分析,探讨海珠益肝加味方对免疫性肝损伤小鼠的保护作用及其免疫调节机制,进一步提高慢性乙型病毒性肝炎的临床治疗水平。
方法：实验分五个部分进行
第一部分：海珠益肝方及其扩方对Concanavalin A(刀豆蛋白A)诱导免疫性肝损伤小鼠的保护作用。
清洁级昆明小鼠100只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝方组(4)、海珠益肝方加太子参枸杞子组(5)、海珠益肝方加太子参仙灵脾组(6)、海珠益肝方加枸杞子仙灵脾组(7)、叶下珠白花蛇舌草组(8)、海藻茯苓白芥子组(9)及莪术组(10),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤动物模型。造模前12天,模型组、空白组给予蒸馏水灌胃,其余组每日分别给予醋酸泼的松、海珠益肝全方、海珠益肝方加太子参枸杞子、海珠益肝方加太子参仙灵脾、海珠益肝方加枸杞子仙灵脾、叶下珠白花蛇舌草、海藻茯苓白芥子及莪术灌胃。末次灌胃给药后1h,空白组给予生理盐水10ml/kg尾静脉注射,其余各组按照20mg/kg尾静脉注射刀豆蛋白A造模,禁食自由饮水。造模给药8h后颈椎脱臼处死动物摘取肝脏和脾脏,生理盐水中清洗后称记重量,计算肝脾指数。肝组织标本匀浆离心后取上清液检测丙氨酸氨基转移酶(ALT)、门冬氨酸氨基转移酶(AST)、丙二醛(MDA)及超氧化物歧化酶(SOD)的值。另外取0.2g肝组织固定、脱水、石蜡包埋、切片后行HE染色,光学显微镜下观察肝脏组织形态学变化。
第二部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠血清IL-4、IL-6、INF-γ、TNF-α的影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤模型。造模前12天,模型组、空白组给予蒸馏水灌胃,其余组每日分别给予醋酸强的松、海珠益肝加味方灌胃。末次灌胃给药后1h,空白组给予生理盐水10ml/kg尾静脉注射,其余各组按照20mg/kg尾静脉注射刀豆蛋白A造模,禁食自由饮水。造模给药8h后摘眼球取血,离心后留取血清。采用ELISA方法测定小鼠血清IL-4、IL-6、IFN-γ、TNF-α的值。
第三部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠肝组织病理变化及肝组织NF-KP65的表达水平的影响。
清洁级昆明小鼠40只,随机分为正常组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用Con A尾静脉注射法制作免疫性肝损伤模型。造模给药8h后处死小鼠,取肝组织制作肝脏的超微病理切片,采用电镜透射法观察各组肝细胞内超微结构的病理变化,同时取肝组织用Western-blot法测定小鼠肝组织NF-KP65的表达水平。第四部分：海珠益肝方加味方对Con A(刀豆蛋白A)诱导免疫性肝损伤小鼠脾组织IL-6、TNF-αmRNA影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤小鼠模型。采用RT-PCR法测定小鼠脾组织IL-6、TNF-α mRNA的表达。
第五部分：海珠益肝方加味方对Concanavalin A诱导免疫性肝损伤小鼠肝组织TLR4、CD14mRNA影响。
清洁级昆明小鼠40只,随机分为空白组(1)、模型组(2)、醋酸强的松组(3)、海珠益肝加味方组(4),每组10只,采用刀豆蛋白A尾静脉注射法制作免疫性肝损伤模型。采用RT-PCR法测定小鼠肝组织TLR4、CD14mRNA的表达。
结果：
第一部分：海珠益肝方及其扩方对Con A(刀豆蛋白A)诱导免疫性肝损伤小鼠的保护作用。
1成功建立小鼠免疫性肝损伤模型。与正常组比较,模型组小鼠肝脾指数、肝组织匀浆后上清液ALT、AST水平及MDA含量均明显升高(P<0.05),肝组织匀浆后上清液SOD水平明显降低。模型组肝组织内大量淋巴细胞浸润,部分汇管区淋巴细胞浸润,肝板消失、凋亡小体散在、肝细胞成脂肪样变性。血管周围的肝细胞胞浆疏松,染色明显变浅,肝窦间隙明显增宽,肝索消失,可见点状坏死、淋巴细胞浸润。
2与模型组比较,对照组和海珠益肝方加味各组可降低小鼠肝脾指数,降低肝组织ALT、AST水平及MDA含量,提高肝组织的SOD水平(P<0.05),其中以海珠益肝加太子参、枸杞子组更明显。另外与模型组比较,海珠益肝方加味组肝脏病理变化有不同程度减轻。
第二部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠血清IL-4、IL-6、INF-γ、TNF-α的影响。
1与正常组比较,模型组小鼠血清中IFN-γ、TNF-α的水平明显升高,差异有显著性(P<0.05)。模型组小鼠血清IL-4、IL-6的水平明显降低,统计学上差异有显著性(P<0.05)。
2与模型组比较,对照组和海珠益肝方加味组血清中IFN-γ、TNF-α的水平明显降低,差异有显著性(P<0.05)。对照组和海珠益肝方加味方组小鼠血清中IL-6的水平明显升高,差异有显著性(P<0.05)。对照组和海珠益肝方加味方组小鼠血清中IL-4的水平明显升高,统计学差异有显著性(P<0.05)。
第三部分：海珠益肝方加味方对Con A诱导免疫性肝损伤小鼠肝组织病理变化及肝组织NF-KP65的表达水平的影响。
1正常组肝细胞核呈圆形及卵圆形,核膜光滑平整,染色质均匀分布在核内。模型组肝细胞肿胀,胞质疏松,出现深浅不一的切迹,胞质中常见髓样体及空泡变性区,线粒体肿胀,嵴模糊,内质网轻度扩张。与模型组比较,对照组和海珠益肝方加味组肝细胞肿胀明显减轻,未见明显毛细胆管扩张,线粒体轻度肿胀,嵴存在,内质网无明显扩张。
2与正常组比较,模型组NF-KP65的水平明显升高,统计学有显著差异(P<0.05)；与模型组比较,对照组和海珠益肝方加味组NF-KP65的水平有明显变化,统计学有显著差异(P<0.05)。
第四部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠小鼠脾组织IL-6、TNF-α mRNA影响。
1与正常组比较,模型组IL-6mRNA的水平有明显降低,统计学有显著差异(P<0.05)；与正常组比较,模型组TNF-αmRNA的水平显著升高,统计学有显著差异(P<0.05)。
2与模型组比较,对照组和海珠益肝方加味组IL-6mRNA的水平明显升高,统计学有显著差异(P<0.05)。与模型组比较,对照组和海珠益肝方加味组TNF-α mRNA的水平明显下降,统计学有显著差异(P<0.05)。
第五部分：海珠益肝方加味方对刀豆蛋白A诱导免疫性肝损伤小鼠肝组织TLR4、CD14mRNA影响。
1与正常组比较,模型组TLR4mRNA的表达水平明显降低,具有显著性差异(P<0.05)；与模型组比较,对照组和海珠益肝方加味组TLR4mRNA的表达水平明显升高,具有显著性差异(P<0.05)。
2与正常组比较,模型组CD14mRNA的水平无明显变化,经统计学处理无显著性差异(P>0.05)；与模型组比较,对照组和海珠益肝方加味组CD14mRNA的水平无明显变化,未见明显差异(P>0.05)。
结论：
1、海珠益肝方加味对免疫性肝损伤小鼠的肝脏功能有较好的保护作用。
2、海珠益肝方加味对免疫性肝损伤小鼠的肝脏功能有较好的保护作用的机制可能是维持Th1/Th2之间的平衡；抑制过抗的脾脏的细胞免疫功能,增强调节天然免疫功能,从而达到免疫功能的双向调节。
Purpose:
Controlling Chronic hepatitis B has made certain progress in China, but the reality is not optimistic,10%-20%of CHB will develope to liver cirrhosis and1%-5%CHB will develope to primary hepatocellular carcinoma (HCC), so the prevention and controlling situation Chronic hepatitis B is still grim and the chronic hepatitis B viral hepatitis is still a prominent public health problem in China. In the treatment of Chronic hepatitis B interferon and nucleoside analogues is undoubtedly the first choice, but basically the side effects of interferon is difficult to adapt and needs of lifetime use nucleoside analogues and expensive medical costs for many patients with Chronic hepatitis B, it is clear that the application interferon and nucleoside analogues against hepatitis b virus can't solve the all problems in patients with CHB. The regulation Immune function to Traditional Chinese medicine therapy is undoubtedly positive prevention and treatment of Chronic hepatitis B of the icing on the cake. In recently years, there have been many Chinese medicine monomer in the clinical treatment of chronic hepatitis B, but its mechanism of regulating immune function is not very clear. HZYGJWF has come from the tutor professor Shengguoguang in under the guidance of TCM theory, clinical verification and experimental research for many years. HZYGJWF is on the basis of the party with the prince ginseng, medlar. HZYGJWF under bead, spreading hedyot is herb, poria cocos, seaweed, white mustard seed, rhizoma zedoariae, medlar, radix pseudostellariae experience side composed of eight herbs. With detoxification, phlegm, blood stasis, tonify deficiency effect. Early studies suggest that HZYGJWF may in some way, link and targets to achieve two-way regulating immune function, its mechanism of action is worth further study. This topic is the immune liver injury animal models in mice as experimental object, the study of HZYGJWF about mice of immune state, spleen cell cytokines gene mRNA expression, helper T lymphocytes Th1/Th2, toll-like receptor (TLR) and its signal transduction pathways, the influence of using computer and statistical software for data processing and comprehensive analysis, to explore the immunoregulatory effects of HZYGJWF and its mechanism. traditional Chinese medicine has important academic value to improving the level of Chronic hepatitis B of viral disease control and prevention has important scientific significance.
Methods:the experiment is divided into five parts
The first part:HZYGJWF and its expanding party to Con A induction of protective immunity liver injury of mice.
Clean level100kun ming mice were randomly divided into ten group. normal group (1), model group (2), prednisone group (3), HZYGF group (4), HZYGF and radix pseudostellariae medlar group (5), HZYGF and radix pseudostellariae fairy spleen group (6), HZYGF and medlar fairy spleen group (7), leaf under bead spreading hedyotis herb group (8), tuckahoe white mustard group (9) and rhizoma zedoariae group(10), each group of10, only by Con A tail vein injections made immune hepatic injury animal model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily spilt, HZYGF, HZYGF add radix pseudostellariae medlar, HZYGF add radix pseudostellariae fairy spleen, HZYGF add medlar fairy spleen, hepatic lobe under bead spreading hedyotis herb, kelp, tuckahoe white mustard and rhizoma zedoariae lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Make mould for cervical dislocation executed after8h animal liver, spleen, liver and spleen index is calculated. Specimens of liver tissue homogenate after testing ALT, AST, SOD and MDA. In addition in liver tissue paraffin embedding sectioning will been done to HE staining.
The second part:the influence of HZYGJWF to Con A-induced immunological liver injury in mice serum IL-4, IL-6, INF-γ, TNF-α.
Clean level40kun ming mice were randomly divided into normal group (1), model group(2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model.12days before the date of the mold, model group and blank group was given distilled water to fill the stomach, the rest of the group were given daily over the loose, HZYGF lavage. Time to fill stomach1h after the treatment, at the end of the blank group was given saline10ml/kg tail intravenous injection, the rest of the group according to20mg/kg tail vein injection of Con A building, fasting, free drinking water. Building after8h pick eyeball blood, centrifugal file.usd10.00serum. Using ELISA method for determination of mice serum IL-4, IL-6, IFN-γ, TNF-α value.
The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression of NF-KP65level.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury model. Building to put to death in mice after8h, take make ultrastructural slices of liver, the liver tissue by tem observation group the pathological changes of the ultrastructure of liver cell, at the same time take liver tissue in mice liver tissue was determined by Western blot method-the expression of NF-KP65level.
The fourth part:HZYGJWF to Con A-induced immunological liver injury in mice spleen in mice group, IL-6and TNF-α mRNA.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injections made immune liver injury in mice model. The mice spleen tissue was evaluated by RT-PCR method, IL-6, TNF-a mRNA expression.
The fifth part:HZYGF sides to con A induced immunological liver injury mice liver tissue TLR4, and CD14mRNA in mice.
Clean level40kun ming mice were randomly divided into normal group (1), model group (2), prednisone group (3), HZYGJWF group (4), each group of10, only by Con A tail vein injection made immune liver injury model. The liver tissue of mice was evaluated by RT-PCR method the expression of TLR4and CD14mRNA.
Results:
The first part:hai zhu yi liver and its expanding party to Con A induction of protective immunity liver injury of mice. Success immune liver injury in mice model was established.
1Compared with normal group, model group mice liver and spleen index, liver tissue homogenate supernatant after ALT, AST andMDA content has significantly increased (P<0.05), liver tissue homogenate supernatant after the SOD level is significantly reduced. Model group liver tissue in a large number of lymphocyte infiltration, part of the portal area lymphocyte infiltration, erythrocyte sedimentation within liver sinus, a large number of liver cell nucleues is broken, scattered apoptotic body, liver steatosis in cell plasma. Liver cells around vascular cell pack loose, color becomes shallow, all loose cytoplasm of liver cells, staining obviously becomes shallow, hepatic sinus gap widened significantly, liver disappear, sometimes visible point necrosis, lymphocyte, neutrophil infiltration.
2Compared with model group, control group and haizhu yi liver flavored group can decrease the mice liver and spleen index, lower ALT, AST and MDA content in liver tissue, improve the level of liver tissue SOD (P<0.05), among them with HZYGF with radix pseudostellariae, medlar more obvious.
Other party HZYGF flavored group obviously reduce degree of liver pathological changes than model group.
The second part:the influence of HZYGJWF to Con A induced immunological liver injury in mice serum IL-4, IL-6, INF-γ and TNF- a.
1Compared with normal group, the level of serum IFN-y and TNF-a of model group mice has increased significantly with significant difference (P<0.05). the level of serum IL-4and IL-6Model group mice has decreased with significant difference (P<0.05).
2Compared with model group, the level of serum IFN-γ and TNF a of control group and HZYGJWF group has lower significantly, there is significant difference (P<0.05). the level of mice serum IL-6in Control group and HZYGJWF has increased significantly with significant difference (P<0.05). the level of mice serum IL-4Control group of mice and HZYGJWF has increased a lot, there is significant difference (P<0.05).
The third part:HZYGJWF to Con A-induced immunological liver injury in mice liver tissue pathological changes and the expression level of NF-KP65.
1set of normal liver cells for polygon, neatly, nuclear assumes the circular or ovoid, nuclear membrane smooth level off, the chromatin evenly distributed within the box. Model group liver cell swelling, cytoplasm osteoporosis, appear incisure, two-tone cytoplasm common in myeloid body and vacuoles degeneration area, the capillary bile duct expansion, some microvilli medullary degeneration, mitochondria swelling and cristae vague, mild expansion of endoplasmic reticulum. Compared with model group, control group and HZYGJWF group liver cell swelling significantly reduce, did not see clearly the capillary bile duct expansion, mild swelling of mitochondria crest, no obvious expansion of endoplasmic reticulum.
2Compared with normal group, the NF-KP65level of model group was increased significantly, statistically significant difference(P<0.05); Compared with model group, the NF-KP65level of control group and HZYGJWF group has obvious changes, statistically significant difference(P<0.05). HZYGJWF party on immunological liver injury in mice liver tissue ultra structure has obvious protective effect and reduced immune liver injury in mice liver tissue of NF-KP65level, so as HZYGJWF can protect the immunological liver injury of con A-induced.
The fourth part:HZYGJWF to A sword bean protein induced immunological liver injury in mice spleen tissues in mice, IL-6, TNF-a mRNA.
1Compared with normal group, model group, the level of IL-6mRNA has significantly reduced with statistically significant difference(P<0.05); Compared with normal group, the level of TNF-a mRNA of model group has significantly increased, statistically significant differences (P<0.05).
2Compared with model group, the level of IL-6mRNA of control group and HZYGJWF group has increased significantly, statistically significant differences (P<0.05). Compared with model group, the level of TNF-a mRNA of control group and HZYGJWF group has dropped significantly, statistically significant difference (P<0.05).
The fifth part:HZYGJWF to Con A-induced mice immune liver injury in mice liver tissue TLR4, CD14mRNA.
1Compared with normal group, model group of TLR4mRNA expression level increased obviously, statistically significant difference (P<0.05); Compared with model group, control group and HZYGJWF group TLR4mRNA expression level decreased obviously, statistically significant difference (P<0.05).
2Compared with normal group, model group of CD14mRNA level no obvious changes, statistics has no obvious difference (P>0.05)。 Compared with model group, control group and HZYGJWF group CD14mRNA level no obvious changes, statistics has no obvious difference.
Conclusion:
HZYGJWF has obvious protective effect to immune liver injury in mice by Con A induced. the mechanism may inhibit hepatoly cellular lipid per oxidation; inhibit cell inflammatory factor the release of TNF-α and IFN-γ, improve cell suppression of inflammatory factor, IL-6and IL-4release, so as it can adjust the balance between Th1/Th2; improve the activity of TLR4and Inhibiting the activity NF-KP65, reduce the happening of natural immune.
引文
[1]中华医学会传染病与寄生虫病学分会、肝脏病学分会.病毒性肝炎的诊断标准[C].中西医结合肝病杂志,2001,11(1)：56-60.
[2]王伯祥主编.中医肝胆病学[M],第一版,北京,中国医药科技出版社,1993： 2.
[3]盛国光,黄育华,张建军,等.海珠益肝胶囊治疗慢性乙型肝炎近期疗效观察[J].中国中西医结合杂志,2002,22(2)：136-137.
[4]谭大琦,李秋华,竺稽能,等.海珠益肝胶囊对免疫性肝损伤的影响[J].中西医结合肝病杂志,2002,12(2)：36-38.
[5]谭大琦,李秋华,竺稽能,等.海珠益肝胶囊对免疫低下小鼠免疫作用的实验研究[J].中药药理与临床,2002,18(12)：24-27.
[6]黄育华,晏雪生,彭亚琴,等.海珠益肝胶囊对免疫肝损伤小鼠神经肽SPVIP及辅助性T细胞亚群的影响[J].中华中医药学刊,2007,25(11)：2267-2269.
[7]王福根,孙静霞.肝损伤动物模型的建立和应用[J].中国药房,2006,17(9)：702-703.
[8]宋雨鸿,刘强,陈育尧,等.红背叶根对小鼠免疫性肝损伤的保护作用[J].南方医科大学学报,2008,28(3)：494-496.
[9]曲建慧,张修礼.ConA性小鼠肝损伤模型的研究及应用.肝脏[J],2001,6(3)：180-181.
[10]TiegsG,Hentschel J,WendelA. A T Cell-dependent experimental liver injury in mice inducible by concanvalin A[J].J Clin Invest,1992,90 (1):196-203.
[11]李跃华,朱丽,哈团柱,等.刀豆素A诱导小鼠肝损伤增加肝脏NFKB活性[J].中华肝脏病杂志,2003,11(2)：115.
[12]Filinonov PN, Sukhenko TG, Pulitonopulo AN, et al Level of liver fibrosis and immune status of mice of different age after heroii treatment and long abstinence[J]. Bull Exp BiolMed, 2005,140(6):723-725.
[13]Imose M, Nagaki M, Kimura K, et al.Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB[J], Hepatology.2004,40(5):1160-1169.
[14]Ksontini R, Colagiovnnai DB, Joseqhs MD, et al. DisParate roles of TNF-alpha and Fas ligand in Concanavalin A induced hepatitis. JImmunol.1998; 160(8):4082-4089.
[15]刘巧丽,高月求.刀豆球蛋白A诱导小鼠免疫性肝损伤的凋亡机制研究.中国免疫学杂志[J].2008；24(10)：876-878.
[16]Kajiya M, Sato K, Silva M, et al. Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis [J].Biochem Biophys Res Commun,2009,386(2):316-321.
[17]Trautwein CR, Akemann T Bernner DA, et al. Concanavalin A induced liver cell damage:activation of intracellular Pathways tirggered by tumor necrosis factor in mice, Gasrtoenterology,1998,114 (4):1035-1045.
[18]李小刚,王天才,李绍白,等.抗TNF-a单抗对刀豆蛋白A诱导急性肝损伤的保护作用.同济医科大学学报2000,29：513-514.
[19]Shin J, Wang J, Kim H, et al. CGX, a traditional Korean medicine ameliorates concanavalin A-induced acute liver injury[J]. Food Chem Toxicol,2010,48(12):3308-3315.
[20]张引强,唐旭东,王凤云,等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-455.
[21]Adams D, Ju C, Ramaiah SK, et al. Mechanisms of immune-mediated liver injury [J]. Toxicology Sci,2010,115 (2):307-321.
[22]辛永宁,宣世英,孙樱.慢性乙型肝炎Th1/Th2细胞的免疫功能研究进展[J].临床肝胆病杂志,2005,21(6)：182-184.
[23]Diehl S, Anguita J, Hoffmeyer A, et al. Inhibition of Thl differentiation by IL-6 is mediated by SOCS1 [J]. Immunity,2000, 13(6):805-815.
[24]张晓园,杭永付,王健,等.重组鲨肝刺激物质类似物对刀豆蛋白A致小鼠急性肝损伤的保护作用.中国药科大学学报[J].2011,42(4)：375-379.
[25]Hong F, Radaeva S, Pan H N, et al. Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease. Hepatology,2004,40(4):933-941.
[26]Sun R, Tian Z G, Kulkarni S, et al. IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+T cell- and STAT3-dependent manners. J Immunol,2004,172(9): 5648-5655.
[27]ANNETTE E,MARKUS B R, THOMAS P,et al. IL-10, Regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice[J].Hepatology,2007,45 (2):475-485.
[28]Sun WY, Wei W, Gui SY, et al. Protective effect of extract from paeonia lactiflora and Astragalus membranaceus against liver injury induced by bacillus calmette-Gu tin and lipopolysaccharide in mice[J]. Basic Clin Pharmacol Toxicol,2008,103 (2):143-149.
[29]Lu, JW, wang H, Yan-Li J, et al. Differential effects of pyrrolidiue dithiocarbamate on TNF-alpha-mediated liver injury in two different models of fulminant hepatitis [J].J Hepatoloy,2008,48 (3):442-452.
[30]Yu HB,Zhang HF,Zhang X,,et al. Resveratrol inhibits VEGF expression of human hepatocellular carcinoma cells throughaNF-kappaB-mediatedmechanism[J]. Hepatogastroenterolo gy,2010,57(102):1241-1246.
[31]李常青,刘丽丽,莫传伟,等.苦参碱对刀豆蛋白A所致小鼠肝损伤的保护作用.现代消化及介人诊疗[J].2005,10(增刊)：154-156.
[32]Dao-fang LIU, Wei Wei, Li-hua SONG. Upregulation of TNF-a and IL-6 mRNA in mouse liver induced by bacille Calmette-Guerin plus lipopolysaccharide. Acta Pharmacologica Sinica[J].2006, 27 (4):460-468.
[33]Akira S, Uematsu S, Takeuchi 0. Pathogen recognition and innate immunity. Cell,2006,124:783-801.
[34]Chuang T, Ulevitch R J. 工dentification of TLR10:a novel human Toll-like receptor preferentially expressed in immune cells [J]. Biochim Biophys Acta,2001,1518:157-161.
[35]JunWu, MengjiLu, ZhongjiMeng, etal. Toll-like-Receptor-Mediated Control of HBV Replication by Nonparenchymal Liver Cells in Mice. [J]. Hepatology,2007,46 (6):1769-1778.
[36]Zhi Ao Chen a,bYuming Cheng a,b, Yongfen Xu, etal. Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients [J]. Clinical Immunology,2008,128:400 408.
[37]Kumar Visvanathan, Narelle A. Skinner, Alex J. V. Thompson, etal. Regulation of Toll-Like Receptor-2 Expression in Chronic Hepatitis B by the Procure Protein [J]. Hepatology,2007,45 (1):102-110.
[38]Saito T, Gale M. Principles of intracellular viral recognition [J]. Current Opinion in Immunology,2007,19 (1):17-23.
[39]刘悦晖,范学工,李宁,等.刀豆球蛋白A所致实验性肝损伤模型的构建[J].中国感染控制杂志,2008,7(5)：297-301.
[40]F. V. Chisari, M. Isogawa, S. F. Wieland. Pathogenesis of hepatitis B virus infection Pathogene sede 1'infection par levirus de' hepatits B[J]. Pathologie Biologie,2010,58 (2):258 266.
[41]叶维法,钟振义主编.肝病免疫学[M],第一版,天津科技出版社,天津,1997.
[42]Zhang Y, Lian JQ, Huang CX, et al. Overexpression of Toll-like receptor2/4 on monoeytes modulates the activities of CD4 (+) CD25 (+) regulatory T cells in chronic hepatitis B virus infection. Vimlogy,2010,397(1)):34-42.
[43]郭建巍.Th细胞活化与树突状细胞.国外医学免疫学分册,2002；25(1)：13-17.
[44]0'Garra A. T-cell subsets:Chemokine receptors guide the way[J]. Immunity,1998,8 (18):275-283.
[45]Viney JL. Dendritic cell subsets:the ultimate T cell differentiation decision makers [J]. Gut,1999,45 (5):640-641.
[46]Steinman RM, Cohn ZA. Identification of a novel cell type in periph 2 eral lymphoid organs of mice.I. Morphology, quantitation, tissue dis2 tribution [J].J Exp Med,1973,137 (5):1142-1162.
[47]黄亚东,程树军.树突状细胞提取与鉴定方法的研究进展.华南国防医学杂志,2008,22(4)：74-77.
[48]杨璞,徐安平.树突状细胞分化与信号转导.国际内科学杂志,2009,36(5)：278-281.
[49]杨莉,刘玉珍,侯军良,等.慢性乙型肝炎患者外周血树突状细胞免疫功能的研究.临床荟萃,2010,25(21)：1871-1877.
[50]张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用.国际免疫学杂志,2010,33(2)：121-123.
[51]Sun J, Walh M, AV, et al. TLR ligands can activate dendritic cells to p rovide a MyD88-dependent negative signal for Th2 cell development. JImmunol,2005,174 (2):742-751.
[52]钱毅,章廉,陈仁馨,等.慢性乙肝患者IFN-a治疗后Th1/Th2细胞因子表达与HLA-DRB107的相关性.上海免疫学杂志,2002,22(5)：44-45.
[53]盛国光.慢性乙型肝炎的中医病因病机探讨.中西结合肝病杂志,1997,(2)：126-127.
[54]Wei H S, Lu H M, Li D G, etal. The regulatory role of AT1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CC14 [J]. World J Gastroenterol,2000,6 (6):824-828.
[55]Liu H L, Li X H, Wang D Y, etal. Matrix metal loproteinase-2 and tissue inhibitor of metalloproteinase-lexpression in fibrotic rat liver [J].WorldJ Gastroentero,2000,6 (6):881-884.
[56]Wetzel C C, Leonis M A, Dent A, et al. Short-form Ron re-ceptor is required for normal IFN-gamma production in con-A-induced acute liver injury[J]. Am J Physiol Gas-trointest Liver Physiol,2007,292 (1):253-261.
[57]张引强,唐旭东,王凤云,等.刀豆蛋白A (Con A)肝损伤模型的研究应用[J].世界中医药学会联合会消化病专业委员会首届消化病国际学术大会,2010,1(1)：518-519.
[58]黄凯,杨新波,陈红艳,等.两种急性免疫性肝损伤小鼠模型的对比研究[J].中国比较医学杂志,2009,19(11)：32-34.
[59]何晓静,邱枫,肇丽梅.鱼腥草对免疫性肝损伤的保护作用[J].中国现代医学杂志,2011,21(28)：3475-3477.
[60]Imose M, Nagaki M, Kimura K,etal. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappa B [J]. Hepatology,2004,40(5):1160-1169.
[61]李敏,金晓琨,李卫东.双氢青蒿素保护刀豆蛋白A诱导的小鼠肝损伤及机制探讨[J].中国药理学通报,2009,25(5)：630-633.
[62]Tan Y W, Wu J C. Establishment of rats model with actue hepatic injury induced by injection of concanavalin A [J]. Jiangsu Med J,2009,35 (6):702-704.
[63]宋军,王晓东,赵军宁,等.芍药甘草汤提取物(芍甘多苷)对实验性肝损伤的影响[J].中药药理与临床,2010,26(2)：40.
[64]周明眉,范自全,赵爱华,等.山豆根非生物碱部位对免疫性肝损伤模型小鼠的影响[J].时珍国医国药,2011,22(11)：2709-2710.
[65]李兆杰,薛湖,陈磊,等.低分子量海带岩藻聚糖硫酸酯清除活性氧自由基和体内抗氧化作用[J].水产学报,2001,25(1)：64-68.
[66]Bockhamn J, Lutgetmann M. Intrahepatic analysis of cytokine and Toll like receptors signaling expression in chronic hepatitis B patients with different HBV replication activity[J]. Hepatology,2008,48(14):680.
[67]Cai H, Song YH, Xia WJ, Jin MW. Aqueous extract of Yin-Chen-Hao decoction, a traditional Chinese prescription, exerts protective effects on concanavalin A — induced hepatitis in mice through inhibition of NF-kappaB[J]. J Pharm Pharmacol,2006,58:677-684.
[68]邓利娟,李湛军,罗楹,等.Caspase抑制剂F1013对急性肝衰竭大鼠肝细胞凋亡的影响[J].中国临床药理学与治疗学,2010,15(11)：1229-1233.
[69]Yin H Z, Zhao C Y, Liu Y H, et al. Role of two kinds of adipo-kines on hepatocellular apop tosis in mice with acute liver injury [J]. Basic & Clin Med,2009,29 (10):1070-1074.
[70]Kusters S, F Gantner, G Kunstle. Interferon-y plays a critical role in T cell-dependent liver injury in mice Initiated by concanavalin A [J]. Gastroenterology,1996, 111:462-471.
[71]郑加嘉,黄清松,吴蔓.茵栀黄注射液对小鼠免疫性肝损伤炎症因子含量的影响[J].成都中医药大学学报,2012,35(4)：8-10.
[72]黑常春,马宁芳,王燕蓉.Th1/Th2类细胞因子的变化对急性免疫性肝损伤的影响研究[J].中国组织化学与细胞化学杂志,2011,20(6)：635-636.
[1]殷小兰.慢性乙型病毒性肝炎免疫耐受的机理与治疗现状[J].湖北中医药大学学报,2011,13(4)：73-78.
[2]辛永宁,宣世英,孙樱.慢性乙型肝炎Th1/Th2细胞的免疫功能研究进展[J].临床肝胆病杂志,2005,21(6)：182-184.
[3]Adams D, Ju C, Ramaiah SK, et al. Mechanisms of immune-medi-ated liver injury [J].Toxicol Sci,2010,115 (2):307-321.
[4]O'Garra A. T-cell subsets:Chemokine receptors guide the way[J]. Immunity,1998,8 (18):275-283.
[5]Viney JL. Dendritic cell subsets:the ultimate T cell differentiation decision makers[J]. Gut,1999,45 (5):640-641.
[6]Steinman RM, Cohn ZA. Identification of a novel cell type in periph 2 eral lymphoid organs ofmice. I. Morphology,quantitation,tissue dis2 t ribution[J]. J Exp Med,1973,137 (5):1142-1162.
[7]黄亚东,程树军.树突状细胞提取与鉴定方法的研究进展[J].华南国防医学杂志,2008,22(4)：74-77.
[8]李树龙,姜颖,贺福初.乙型肝炎免疫发病机制研究进展[J].军事医学科学院院刊,2006,30(6)：580-583.
[9]杨璞,徐安平.树突状细胞分化与信号转导[J].国际内科学杂志,2009,36(5)：278-281.
[10]杨莉,刘玉珍,侯军良等.慢性乙型肝炎患者外周血树突状细胞免疫功能的研究[J].临床荟萃,2010,25(21)：1871-1877.
[11]张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用[J].国际免疫学杂志,2010,33(2)：121-123.
[12]Sun J, WalhM, AV, et al. TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development [J]. J Immunol,2005,174 (2):742-751.
[13]钱毅,章廉,陈仁馨,等.慢性乙肝患者IFN-a治疗后Th1/Th2细胞因子表达与HLA-DRB1* 07的相关性[J].上海免疫学杂志,2002,22(5)：44-45.
[14]王伯祥主编.中医肝胆病学[M],第一版,中国医药科技出版社,北京,1993：2
[15]田红剑,陈斌.当归对大鼠免疫性肝损伤的保护作用[J].中南药学,2003,1(5)：271.
[16]邹丽宜,吴铁,崔燎.甘草酸对四氯化碳致小鼠肝纤维化及骨丢失的防治作用[J].中国药理学通报,2002,18(4)：437-438.
[17]周明眉,范自全,赵爱华,等.山豆根非生物碱部位对免疫性肝损伤模型小鼠的影响[J].时珍国医国药,2011,22(11)：2709-2710.
[18]陈象青,王钦茂,方华武,等.白芍总苷和当归提取物对小鼠免疫性肝损伤的保护作用.承德医学院学报[J],2004,21(1)：8-9.
[19]薛冬英,刘光伟,陈一奕,等.干扰素-a联合柴胡皂苷对免疫性肝损伤小鼠血清T淋巴细胞亚群和肝组织细胞因子的影响[J].中国中西医结合杂志,2012,32(1)：69-71.
[20]甘乐文,王国俊,李玉莉.苦参素对大鼠慢性肝损伤的防护作用[J].中草药,2002,33(4)：339-340.
[21]林兴,黄权芳,张士军,等.山芝麻水提取物对小鼠免疫性肝损伤的保 护作用[J].中国现代应用药学,2012,29(1)：1-4.
[22]郭昱,吴梧桐,巫冠中.鲨肝肽对小鼠免疫性肝损伤的保护作用及免疫调节作用[J].中国新药杂志,2001,10(1)：29.
[23]Wen juan Cao, Wei Zhang, Jingjing Liu, et, al. Paeo niflorin improves survival in LPS-challenged mice through the suppression of TNF-α and IL-1β release and augmentation of IL-10 production[J]. International Immunol pharmacology, 2011,20(11):162-167.
[24]刘铁铮,郭爱洁,王桂春,等.多棘海盘车多糖对正常小鼠免疫功能的影响[J],中国药学杂志,2011,46(24)：1894-1896.
[25]张庭廷,聂刘旺,刘爱民,等.金樱子多糖的免疫活性研究[J].中国实验方剂学杂志,2005,11(2)：55-56.
[26]邹丽宜,吴铁,崔燎.黄芪多糖对肝纤维化小鼠及骨丢失的防治作用[J].中西医结合肝病杂志,2002,12(2)：95-96.
[27]桂蜀华,王贵均,熊艺花,等.狭基线纹香茶菜对免疫性肝损伤小鼠TNF-amRNA和蛋白表达水平的影响[J].中药药理与临床,2011,27(4)：43-45.
[28]丁虹,彭仁王秀,于皆平.当归多糖对免疫性肝损伤小鼠肝中一氧化氮合酶及凋亡相关基因的影响[J].中华肝脏病杂志,2001,9(增刊)：50-51.
[29]李艳秋,赵德化,潘伯荣,等.龙胆苦甙抗鼠肝损伤的作用[J].第四军医大学学报,2001,22(18)：1645-646.
[30]朱逸宽,吴洪海,王立明,等.黄芩苷对卡介苗加脂多糖诱导免疫性肝损伤小鼠的作用[J].中国现代应用药学杂志,2005,22(1)：14-16.
[31]黄华,哈木拉提,杨丽丽,等.苦豆子生物碱保肝降酶和免疫调节作用研究[J].中药药理与临床,2005,21(2)：16-18.
[32]王晓燕,庞建新.胡黄连总甙对小鼠免疫性肝损伤的保护作用[J].广西医科大学学报,2002,19(4)：524-525.
[33]李颖,彭仁王秀.阿魏酸钠和当归醇沉物对免疫性肝损伤的干预作用[J].中草药,2000,31(4)：274-275.
[34]陈明,王英锋.肝毒清复方对BCG/LPS所致小鼠免疫性肝损伤免疫功能的影响[J].中国医药学报,2004,19(5)：273-274.
[35]郑加嘉,林英,黄清松,等.茵桅黄注射液防治Con A诱导小鼠免疫性肝损伤的药效学观察[J].中国药师,2011,14(6)：831-834.
[36]王文杰,王济.小柴胡汤对实验性肝损伤小鼠Th1/Th2免疫平衡的影响[J].山西中医,2004,20(1)：49-50.
[37]盛国光,黄育华,张建军等.海珠益肝胶囊治疗慢性乙型肝炎近期疗效观察[J].中国中西医结合杂志,2002,22(2)136-137.
[38]李卫星,谭大琦,李秋华,等.海珠益肝胶囊对免疫性肝损伤的影响[J],中西医结合肝病杂志,2002,12(2)36-38.
[39]谭大琦,李秋华,竺稽能等.海珠益肝胶囊对免疫低下小鼠免疫作用的实验研究[J].中药药理与临床,2002,18(6)：24-27.
[40]黄育华,晏雪生,彭亚琴等.海珠益肝胶囊对免疫肝损伤小鼠神经肽SPVIP及辅助性T细胞亚群的影响,中华中医药学刊[J],2007,25(11)：2267-2269.
[41]赵艳玲,肖晓河,袁海龙,等.肝得宁片对小鼠免疫功能的影响[J].中草药,2002,33(2)：147-148.
[42]陈斌,孙克伟,谢凤瑛,等.莪术治疗慢性肝损伤时的肝毒作用研究[J].中西医结合肝病杂志,2003,13(1)：20-22.
[43]熊益群,周大桥,姚小萍,等.青芪散对小鼠淋巴细胞亚群及白细胞介素2的影响[J].中国中西医结合消化杂志,2005,13(13)：152-154.
[44]李常青,刘丽丽,朱余同,等.清肝排毒饮对刀豆蛋白A所致小鼠免疫性肝损伤的保护作用[J].中华现代中西医杂志,2005,3(8)：69-73.
[45]江艳艳,王胜春,刘明义,等.五灵丸与五灵胶囊对免疫性肝损伤小鼠作用的研究[J].中成药,2001,23(7)：508-510.
[46]高普均,王小丛,朴云峰,等.五杞胶囊对酒精/CC14所致大鼠慢性肝 损伤的影[J]响.中成药,2003,25(7)：585-587.
[47]XU L, ZHAO Y, QIN Y, et al. A novel model of acute liver injury in mice induced by T cell-mediated immune response to lactosylated bovine serum albumin [J].Clin Exp Immumol,2006,144 (2):125-133.
[48]MCFARLANE I G, WOJCICKA B M, ZUCKER G M, et al. Puri-f ication and characterization of human liver-specific membrane lipoprotein (LSP) [J]. Clin Exp Immunol,1977,27 (3):381-390.
[49]陈莉萍,陈立峰,徐琳本,等.肝复乐对小鼠异种免疫性肝损伤的保护作用[J].中药药理与临床,2000,16(1)：25-27.
[50]徐焕宾,龚燕萍,蒋正刚,等.CXCL16在小鼠免疫性肝损伤中的作用及意义[J].中华肝脏病杂志,2005,13(4)：282-285.
[51]王继光.夏枯草合剂的保肝作用[J].中草药,2001,32(7)：625.
[52]邱英锋,缪晓辉,王俊学,等.卡介苗加脂多糖建立大鼠急性免疫性肝损伤模型的研究[J],实验动物科学与管理,2004,21(3)：15-18.
[53]王福根,孙静霞.肝损伤动物模型的建立和应用[J],中国药房,2006,17(9)：702-703.
[54]宋雨鸿,刘强,陈育尧等.红背叶根对小鼠免疫性肝损伤的保护作用,南方医科大学学报[J],2008,28(3)：494-496.
[55]LUCA S, STEFANO F, FRANCESCO C, et al. Galectin-1 exerts immunomodulatory and protective effects on concanavalin a-induced hepatitis in mice [J]. Hepatology,2000, 31(2):399-407.
[56]陈丽,谭友文,叶云,等.刀豆蛋白A诱导小鼠肝纤维化动物模型的建立及机制探讨[J].肝脏,2012,17(4)：258-259.
[57]姚敏,李宏伟,曲均革,等.刀豆蛋自A引起小鼠免疫性肝损伤机制研究[J].蚌埠医学院学报,2011,36(5)：445-447.
[58]张引强,唐旭东,王凤云等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝 损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-455.
[59]张引强,唐旭东,王凤云,等.荣肝合剂对ConA诱导慢性免疫性肝损伤小鼠的影响[J].胃肠病学和肝病学杂志,2008,17(6)：492-495.
[60]ANDREW F G, SLATER C, STEF. AN I, etal. The role of inlra-cellular oxidants in apoptosis[J]. Biochim. Biophysics. Acta,1995,12 (1):59-62.
[61]WANG J, SUN R, WEI H M, et al. Pre-activation of T lymphocytes by low dose of concanavalin A aggravates toll-like receptor-3 ligand-induced NK cell-mediated liver injury[J]. Irate Immuno pharm,2006,6(5):800-807.
[62]潘红芳、赵凯、张炜.不同剂量刀豆蛋白A腹腔注射诱导昆明小鼠肝纤维化的效果观察[J].中国保健营养,2012,6：1244-1245.
[63]Dao-fang liu, Wei wei, Li-hua song. Upregulation of TNF-a and IL-6 mRNA in mouse liver induced by bacilli Calmette-Guerin pluslipopolysaccharide[J]. ActaPharmacologicaSinica,2006,27( 4):460-468.
[64]刘巧丽,高月求.刀豆球蛋白A诱导小鼠免疫性肝损伤的凋亡机制研究.中国免疫学杂志[J].2008；24(10)：876-878.
[65]TAGAWA Y, SEKIKAWA K, IWAKURA Y. Suppression of concanaval in A-induced hepatitis in IFN-gamma(-/-)mice, but not in TNF-alpha (-/-)mice; male for IFN-gamma in act ivating apoptosis hepatocytes[J]. J Immunol,1997,159 (3):1418-1428.
[66]ANNETTE E,MARKUS B R, THOMAS P, et al. IL-10, Regulatory T cells, and Kupfter cells mediate tolerance in concanavalin A-induced liver injury in mice[J].Hepatology,2007,45 (2):475-485.
[67]Kozie 1 MJ. Cytokines in viral hepatitis [J]. J Semin Liver Dis, 1999,19:157-169.
[2]王伯祥主编.中医肝胆病学[M],第一版,北京,中国医药科技出版社,1993： 2.
[3]盛国光,黄育华,张建军,等.海珠益肝胶囊治疗慢性乙型肝炎近期疗效观察[J].中国中西医结合杂志,2002,22(2)：136-137.
[4]谭大琦,李秋华,竺稽能,等.海珠益肝胶囊对免疫性肝损伤的影响[J].中西医结合肝病杂志,2002,12(2)：36-38.
[5]谭大琦,李秋华,竺稽能,等.海珠益肝胶囊对免疫低下小鼠免疫作用的实验研究[J].中药药理与临床,2002,18(12)：24-27.
[6]黄育华,晏雪生,彭亚琴,等.海珠益肝胶囊对免疫肝损伤小鼠神经肽SPVIP及辅助性T细胞亚群的影响[J].中华中医药学刊,2007,25(11)：2267-2269.
[7]王福根,孙静霞.肝损伤动物模型的建立和应用[J].中国药房,2006,17(9)：702-703.
[8]宋雨鸿,刘强,陈育尧,等.红背叶根对小鼠免疫性肝损伤的保护作用[J].南方医科大学学报,2008,28(3)：494-496.
[9]曲建慧,张修礼.ConA性小鼠肝损伤模型的研究及应用.肝脏[J],2001,6(3)：180-181.
[10]TiegsG,Hentschel J,WendelA. A T Cell-dependent experimental liver injury in mice inducible by concanvalin A[J].J Clin Invest,1992,90 (1):196-203.
[11]李跃华,朱丽,哈团柱,等.刀豆素A诱导小鼠肝损伤增加肝脏NFKB活性[J].中华肝脏病杂志,2003,11(2)：115.
[12]Filinonov PN, Sukhenko TG, Pulitonopulo AN, et al Level of liver fibrosis and immune status of mice of different age after heroii treatment and long abstinence[J]. Bull Exp BiolMed, 2005,140(6):723-725.
[13]Imose M, Nagaki M, Kimura K, et al.Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB[J], Hepatology.2004,40(5):1160-1169.
[14]Ksontini R, Colagiovnnai DB, Joseqhs MD, et al. DisParate roles of TNF-alpha and Fas ligand in Concanavalin A induced hepatitis. JImmunol.1998; 160(8):4082-4089.
[15]刘巧丽,高月求.刀豆球蛋白A诱导小鼠免疫性肝损伤的凋亡机制研究.中国免疫学杂志[J].2008；24(10)：876-878.
[16]Kajiya M, Sato K, Silva M, et al. Hydrogen from intestinal bacteria is protective for Concanavalin A-induced hepatitis [J].Biochem Biophys Res Commun,2009,386(2):316-321.
[17]Trautwein CR, Akemann T Bernner DA, et al. Concanavalin A induced liver cell damage:activation of intracellular Pathways tirggered by tumor necrosis factor in mice, Gasrtoenterology,1998,114 (4):1035-1045.
[18]李小刚,王天才,李绍白,等.抗TNF-a单抗对刀豆蛋白A诱导急性肝损伤的保护作用.同济医科大学学报2000,29：513-514.
[19]Shin J, Wang J, Kim H, et al. CGX, a traditional Korean medicine ameliorates concanavalin A-induced acute liver injury[J]. Food Chem Toxicol,2010,48(12):3308-3315.
[20]张引强,唐旭东,王凤云,等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-455.
[21]Adams D, Ju C, Ramaiah SK, et al. Mechanisms of immune-mediated liver injury [J]. Toxicology Sci,2010,115 (2):307-321.
[22]辛永宁,宣世英,孙樱.慢性乙型肝炎Th1/Th2细胞的免疫功能研究进展[J].临床肝胆病杂志,2005,21(6)：182-184.
[23]Diehl S, Anguita J, Hoffmeyer A, et al. Inhibition of Thl differentiation by IL-6 is mediated by SOCS1 [J]. Immunity,2000, 13(6):805-815.
[24]张晓园,杭永付,王健,等.重组鲨肝刺激物质类似物对刀豆蛋白A致小鼠急性肝损伤的保护作用.中国药科大学学报[J].2011,42(4)：375-379.
[25]Hong F, Radaeva S, Pan H N, et al. Interleukin 6 alleviates hepatic steatosis and ischemia/reperfusion injury in mice with fatty liver disease. Hepatology,2004,40(4):933-941.
[26]Sun R, Tian Z G, Kulkarni S, et al. IL-6 prevents T cell-mediated hepatitis via inhibition of NKT cells in CD4+T cell- and STAT3-dependent manners. J Immunol,2004,172(9): 5648-5655.
[27]ANNETTE E,MARKUS B R, THOMAS P,et al. IL-10, Regulatory T cells, and Kupffer cells mediate tolerance in concanavalin A-induced liver injury in mice[J].Hepatology,2007,45 (2):475-485.
[28]Sun WY, Wei W, Gui SY, et al. Protective effect of extract from paeonia lactiflora and Astragalus membranaceus against liver injury induced by bacillus calmette-Gu tin and lipopolysaccharide in mice[J]. Basic Clin Pharmacol Toxicol,2008,103 (2):143-149.
[29]Lu, JW, wang H, Yan-Li J, et al. Differential effects of pyrrolidiue dithiocarbamate on TNF-alpha-mediated liver injury in two different models of fulminant hepatitis [J].J Hepatoloy,2008,48 (3):442-452.
[30]Yu HB,Zhang HF,Zhang X,,et al. Resveratrol inhibits VEGF expression of human hepatocellular carcinoma cells throughaNF-kappaB-mediatedmechanism[J]. Hepatogastroenterolo gy,2010,57(102):1241-1246.
[31]李常青,刘丽丽,莫传伟,等.苦参碱对刀豆蛋白A所致小鼠肝损伤的保护作用.现代消化及介人诊疗[J].2005,10(增刊)：154-156.
[32]Dao-fang LIU, Wei Wei, Li-hua SONG. Upregulation of TNF-a and IL-6 mRNA in mouse liver induced by bacille Calmette-Guerin plus lipopolysaccharide. Acta Pharmacologica Sinica[J].2006, 27 (4):460-468.
[33]Akira S, Uematsu S, Takeuchi 0. Pathogen recognition and innate immunity. Cell,2006,124:783-801.
[34]Chuang T, Ulevitch R J. 工dentification of TLR10:a novel human Toll-like receptor preferentially expressed in immune cells [J]. Biochim Biophys Acta,2001,1518:157-161.
[35]JunWu, MengjiLu, ZhongjiMeng, etal. Toll-like-Receptor-Mediated Control of HBV Replication by Nonparenchymal Liver Cells in Mice. [J]. Hepatology,2007,46 (6):1769-1778.
[36]Zhi Ao Chen a,bYuming Cheng a,b, Yongfen Xu, etal. Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients [J]. Clinical Immunology,2008,128:400 408.
[37]Kumar Visvanathan, Narelle A. Skinner, Alex J. V. Thompson, etal. Regulation of Toll-Like Receptor-2 Expression in Chronic Hepatitis B by the Procure Protein [J]. Hepatology,2007,45 (1):102-110.
[38]Saito T, Gale M. Principles of intracellular viral recognition [J]. Current Opinion in Immunology,2007,19 (1):17-23.
[39]刘悦晖,范学工,李宁,等.刀豆球蛋白A所致实验性肝损伤模型的构建[J].中国感染控制杂志,2008,7(5)：297-301.
[40]F. V. Chisari, M. Isogawa, S. F. Wieland. Pathogenesis of hepatitis B virus infection Pathogene sede 1'infection par levirus de' hepatits B[J]. Pathologie Biologie,2010,58 (2):258 266.
[41]叶维法,钟振义主编.肝病免疫学[M],第一版,天津科技出版社,天津,1997.
[42]Zhang Y, Lian JQ, Huang CX, et al. Overexpression of Toll-like receptor2/4 on monoeytes modulates the activities of CD4 (+) CD25 (+) regulatory T cells in chronic hepatitis B virus infection. Vimlogy,2010,397(1)):34-42.
[43]郭建巍.Th细胞活化与树突状细胞.国外医学免疫学分册,2002；25(1)：13-17.
[44]0'Garra A. T-cell subsets:Chemokine receptors guide the way[J]. Immunity,1998,8 (18):275-283.
[45]Viney JL. Dendritic cell subsets:the ultimate T cell differentiation decision makers [J]. Gut,1999,45 (5):640-641.
[46]Steinman RM, Cohn ZA. Identification of a novel cell type in periph 2 eral lymphoid organs of mice.I. Morphology, quantitation, tissue dis2 tribution [J].J Exp Med,1973,137 (5):1142-1162.
[47]黄亚东,程树军.树突状细胞提取与鉴定方法的研究进展.华南国防医学杂志,2008,22(4)：74-77.
[48]杨璞,徐安平.树突状细胞分化与信号转导.国际内科学杂志,2009,36(5)：278-281.
[49]杨莉,刘玉珍,侯军良,等.慢性乙型肝炎患者外周血树突状细胞免疫功能的研究.临床荟萃,2010,25(21)：1871-1877.
[50]张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用.国际免疫学杂志,2010,33(2)：121-123.
[51]Sun J, Walh M, AV, et al. TLR ligands can activate dendritic cells to p rovide a MyD88-dependent negative signal for Th2 cell development. JImmunol,2005,174 (2):742-751.
[52]钱毅,章廉,陈仁馨,等.慢性乙肝患者IFN-a治疗后Th1/Th2细胞因子表达与HLA-DRB107的相关性.上海免疫学杂志,2002,22(5)：44-45.
[53]盛国光.慢性乙型肝炎的中医病因病机探讨.中西结合肝病杂志,1997,(2)：126-127.
[54]Wei H S, Lu H M, Li D G, etal. The regulatory role of AT1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CC14 [J]. World J Gastroenterol,2000,6 (6):824-828.
[55]Liu H L, Li X H, Wang D Y, etal. Matrix metal loproteinase-2 and tissue inhibitor of metalloproteinase-lexpression in fibrotic rat liver [J].WorldJ Gastroentero,2000,6 (6):881-884.
[56]Wetzel C C, Leonis M A, Dent A, et al. Short-form Ron re-ceptor is required for normal IFN-gamma production in con-A-induced acute liver injury[J]. Am J Physiol Gas-trointest Liver Physiol,2007,292 (1):253-261.
[57]张引强,唐旭东,王凤云,等.刀豆蛋白A (Con A)肝损伤模型的研究应用[J].世界中医药学会联合会消化病专业委员会首届消化病国际学术大会,2010,1(1)：518-519.
[58]黄凯,杨新波,陈红艳,等.两种急性免疫性肝损伤小鼠模型的对比研究[J].中国比较医学杂志,2009,19(11)：32-34.
[59]何晓静,邱枫,肇丽梅.鱼腥草对免疫性肝损伤的保护作用[J].中国现代医学杂志,2011,21(28)：3475-3477.
[60]Imose M, Nagaki M, Kimura K,etal. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappa B [J]. Hepatology,2004,40(5):1160-1169.
[61]李敏,金晓琨,李卫东.双氢青蒿素保护刀豆蛋白A诱导的小鼠肝损伤及机制探讨[J].中国药理学通报,2009,25(5)：630-633.
[62]Tan Y W, Wu J C. Establishment of rats model with actue hepatic injury induced by injection of concanavalin A [J]. Jiangsu Med J,2009,35 (6):702-704.
[63]宋军,王晓东,赵军宁,等.芍药甘草汤提取物(芍甘多苷)对实验性肝损伤的影响[J].中药药理与临床,2010,26(2)：40.
[64]周明眉,范自全,赵爱华,等.山豆根非生物碱部位对免疫性肝损伤模型小鼠的影响[J].时珍国医国药,2011,22(11)：2709-2710.
[65]李兆杰,薛湖,陈磊,等.低分子量海带岩藻聚糖硫酸酯清除活性氧自由基和体内抗氧化作用[J].水产学报,2001,25(1)：64-68.
[66]Bockhamn J, Lutgetmann M. Intrahepatic analysis of cytokine and Toll like receptors signaling expression in chronic hepatitis B patients with different HBV replication activity[J]. Hepatology,2008,48(14):680.
[67]Cai H, Song YH, Xia WJ, Jin MW. Aqueous extract of Yin-Chen-Hao decoction, a traditional Chinese prescription, exerts protective effects on concanavalin A — induced hepatitis in mice through inhibition of NF-kappaB[J]. J Pharm Pharmacol,2006,58:677-684.
[68]邓利娟,李湛军,罗楹,等.Caspase抑制剂F1013对急性肝衰竭大鼠肝细胞凋亡的影响[J].中国临床药理学与治疗学,2010,15(11)：1229-1233.
[69]Yin H Z, Zhao C Y, Liu Y H, et al. Role of two kinds of adipo-kines on hepatocellular apop tosis in mice with acute liver injury [J]. Basic & Clin Med,2009,29 (10):1070-1074.
[70]Kusters S, F Gantner, G Kunstle. Interferon-y plays a critical role in T cell-dependent liver injury in mice Initiated by concanavalin A [J]. Gastroenterology,1996, 111:462-471.
[71]郑加嘉,黄清松,吴蔓.茵栀黄注射液对小鼠免疫性肝损伤炎症因子含量的影响[J].成都中医药大学学报,2012,35(4)：8-10.
[72]黑常春,马宁芳,王燕蓉.Th1/Th2类细胞因子的变化对急性免疫性肝损伤的影响研究[J].中国组织化学与细胞化学杂志,2011,20(6)：635-636.
[1]殷小兰.慢性乙型病毒性肝炎免疫耐受的机理与治疗现状[J].湖北中医药大学学报,2011,13(4)：73-78.
[2]辛永宁,宣世英,孙樱.慢性乙型肝炎Th1/Th2细胞的免疫功能研究进展[J].临床肝胆病杂志,2005,21(6)：182-184.
[3]Adams D, Ju C, Ramaiah SK, et al. Mechanisms of immune-medi-ated liver injury [J].Toxicol Sci,2010,115 (2):307-321.
[4]O'Garra A. T-cell subsets:Chemokine receptors guide the way[J]. Immunity,1998,8 (18):275-283.
[5]Viney JL. Dendritic cell subsets:the ultimate T cell differentiation decision makers[J]. Gut,1999,45 (5):640-641.
[6]Steinman RM, Cohn ZA. Identification of a novel cell type in periph 2 eral lymphoid organs ofmice. I. Morphology,quantitation,tissue dis2 t ribution[J]. J Exp Med,1973,137 (5):1142-1162.
[7]黄亚东,程树军.树突状细胞提取与鉴定方法的研究进展[J].华南国防医学杂志,2008,22(4)：74-77.
[8]李树龙,姜颖,贺福初.乙型肝炎免疫发病机制研究进展[J].军事医学科学院院刊,2006,30(6)：580-583.
[9]杨璞,徐安平.树突状细胞分化与信号转导[J].国际内科学杂志,2009,36(5)：278-281.
[10]杨莉,刘玉珍,侯军良等.慢性乙型肝炎患者外周血树突状细胞免疫功能的研究[J].临床荟萃,2010,25(21)：1871-1877.
[11]张艳霞,王质刚.人类Toll样受体信号转导途径及其介导的免疫作用[J].国际免疫学杂志,2010,33(2)：121-123.
[12]Sun J, WalhM, AV, et al. TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development [J]. J Immunol,2005,174 (2):742-751.
[13]钱毅,章廉,陈仁馨,等.慢性乙肝患者IFN-a治疗后Th1/Th2细胞因子表达与HLA-DRB1* 07的相关性[J].上海免疫学杂志,2002,22(5)：44-45.
[14]王伯祥主编.中医肝胆病学[M],第一版,中国医药科技出版社,北京,1993：2
[15]田红剑,陈斌.当归对大鼠免疫性肝损伤的保护作用[J].中南药学,2003,1(5)：271.
[16]邹丽宜,吴铁,崔燎.甘草酸对四氯化碳致小鼠肝纤维化及骨丢失的防治作用[J].中国药理学通报,2002,18(4)：437-438.
[17]周明眉,范自全,赵爱华,等.山豆根非生物碱部位对免疫性肝损伤模型小鼠的影响[J].时珍国医国药,2011,22(11)：2709-2710.
[18]陈象青,王钦茂,方华武,等.白芍总苷和当归提取物对小鼠免疫性肝损伤的保护作用.承德医学院学报[J],2004,21(1)：8-9.
[19]薛冬英,刘光伟,陈一奕,等.干扰素-a联合柴胡皂苷对免疫性肝损伤小鼠血清T淋巴细胞亚群和肝组织细胞因子的影响[J].中国中西医结合杂志,2012,32(1)：69-71.
[20]甘乐文,王国俊,李玉莉.苦参素对大鼠慢性肝损伤的防护作用[J].中草药,2002,33(4)：339-340.
[21]林兴,黄权芳,张士军,等.山芝麻水提取物对小鼠免疫性肝损伤的保 护作用[J].中国现代应用药学,2012,29(1)：1-4.
[22]郭昱,吴梧桐,巫冠中.鲨肝肽对小鼠免疫性肝损伤的保护作用及免疫调节作用[J].中国新药杂志,2001,10(1)：29.
[23]Wen juan Cao, Wei Zhang, Jingjing Liu, et, al. Paeo niflorin improves survival in LPS-challenged mice through the suppression of TNF-α and IL-1β release and augmentation of IL-10 production[J]. International Immunol pharmacology, 2011,20(11):162-167.
[24]刘铁铮,郭爱洁,王桂春,等.多棘海盘车多糖对正常小鼠免疫功能的影响[J],中国药学杂志,2011,46(24)：1894-1896.
[25]张庭廷,聂刘旺,刘爱民,等.金樱子多糖的免疫活性研究[J].中国实验方剂学杂志,2005,11(2)：55-56.
[26]邹丽宜,吴铁,崔燎.黄芪多糖对肝纤维化小鼠及骨丢失的防治作用[J].中西医结合肝病杂志,2002,12(2)：95-96.
[27]桂蜀华,王贵均,熊艺花,等.狭基线纹香茶菜对免疫性肝损伤小鼠TNF-amRNA和蛋白表达水平的影响[J].中药药理与临床,2011,27(4)：43-45.
[28]丁虹,彭仁王秀,于皆平.当归多糖对免疫性肝损伤小鼠肝中一氧化氮合酶及凋亡相关基因的影响[J].中华肝脏病杂志,2001,9(增刊)：50-51.
[29]李艳秋,赵德化,潘伯荣,等.龙胆苦甙抗鼠肝损伤的作用[J].第四军医大学学报,2001,22(18)：1645-646.
[30]朱逸宽,吴洪海,王立明,等.黄芩苷对卡介苗加脂多糖诱导免疫性肝损伤小鼠的作用[J].中国现代应用药学杂志,2005,22(1)：14-16.
[31]黄华,哈木拉提,杨丽丽,等.苦豆子生物碱保肝降酶和免疫调节作用研究[J].中药药理与临床,2005,21(2)：16-18.
[32]王晓燕,庞建新.胡黄连总甙对小鼠免疫性肝损伤的保护作用[J].广西医科大学学报,2002,19(4)：524-525.
[33]李颖,彭仁王秀.阿魏酸钠和当归醇沉物对免疫性肝损伤的干预作用[J].中草药,2000,31(4)：274-275.
[34]陈明,王英锋.肝毒清复方对BCG/LPS所致小鼠免疫性肝损伤免疫功能的影响[J].中国医药学报,2004,19(5)：273-274.
[35]郑加嘉,林英,黄清松,等.茵桅黄注射液防治Con A诱导小鼠免疫性肝损伤的药效学观察[J].中国药师,2011,14(6)：831-834.
[36]王文杰,王济.小柴胡汤对实验性肝损伤小鼠Th1/Th2免疫平衡的影响[J].山西中医,2004,20(1)：49-50.
[37]盛国光,黄育华,张建军等.海珠益肝胶囊治疗慢性乙型肝炎近期疗效观察[J].中国中西医结合杂志,2002,22(2)136-137.
[38]李卫星,谭大琦,李秋华,等.海珠益肝胶囊对免疫性肝损伤的影响[J],中西医结合肝病杂志,2002,12(2)36-38.
[39]谭大琦,李秋华,竺稽能等.海珠益肝胶囊对免疫低下小鼠免疫作用的实验研究[J].中药药理与临床,2002,18(6)：24-27.
[40]黄育华,晏雪生,彭亚琴等.海珠益肝胶囊对免疫肝损伤小鼠神经肽SPVIP及辅助性T细胞亚群的影响,中华中医药学刊[J],2007,25(11)：2267-2269.
[41]赵艳玲,肖晓河,袁海龙,等.肝得宁片对小鼠免疫功能的影响[J].中草药,2002,33(2)：147-148.
[42]陈斌,孙克伟,谢凤瑛,等.莪术治疗慢性肝损伤时的肝毒作用研究[J].中西医结合肝病杂志,2003,13(1)：20-22.
[43]熊益群,周大桥,姚小萍,等.青芪散对小鼠淋巴细胞亚群及白细胞介素2的影响[J].中国中西医结合消化杂志,2005,13(13)：152-154.
[44]李常青,刘丽丽,朱余同,等.清肝排毒饮对刀豆蛋白A所致小鼠免疫性肝损伤的保护作用[J].中华现代中西医杂志,2005,3(8)：69-73.
[45]江艳艳,王胜春,刘明义,等.五灵丸与五灵胶囊对免疫性肝损伤小鼠作用的研究[J].中成药,2001,23(7)：508-510.
[46]高普均,王小丛,朴云峰,等.五杞胶囊对酒精/CC14所致大鼠慢性肝 损伤的影[J]响.中成药,2003,25(7)：585-587.
[47]XU L, ZHAO Y, QIN Y, et al. A novel model of acute liver injury in mice induced by T cell-mediated immune response to lactosylated bovine serum albumin [J].Clin Exp Immumol,2006,144 (2):125-133.
[48]MCFARLANE I G, WOJCICKA B M, ZUCKER G M, et al. Puri-f ication and characterization of human liver-specific membrane lipoprotein (LSP) [J]. Clin Exp Immunol,1977,27 (3):381-390.
[49]陈莉萍,陈立峰,徐琳本,等.肝复乐对小鼠异种免疫性肝损伤的保护作用[J].中药药理与临床,2000,16(1)：25-27.
[50]徐焕宾,龚燕萍,蒋正刚,等.CXCL16在小鼠免疫性肝损伤中的作用及意义[J].中华肝脏病杂志,2005,13(4)：282-285.
[51]王继光.夏枯草合剂的保肝作用[J].中草药,2001,32(7)：625.
[52]邱英锋,缪晓辉,王俊学,等.卡介苗加脂多糖建立大鼠急性免疫性肝损伤模型的研究[J],实验动物科学与管理,2004,21(3)：15-18.
[53]王福根,孙静霞.肝损伤动物模型的建立和应用[J],中国药房,2006,17(9)：702-703.
[54]宋雨鸿,刘强,陈育尧等.红背叶根对小鼠免疫性肝损伤的保护作用,南方医科大学学报[J],2008,28(3)：494-496.
[55]LUCA S, STEFANO F, FRANCESCO C, et al. Galectin-1 exerts immunomodulatory and protective effects on concanavalin a-induced hepatitis in mice [J]. Hepatology,2000, 31(2):399-407.
[56]陈丽,谭友文,叶云,等.刀豆蛋白A诱导小鼠肝纤维化动物模型的建立及机制探讨[J].肝脏,2012,17(4)：258-259.
[57]姚敏,李宏伟,曲均革,等.刀豆蛋自A引起小鼠免疫性肝损伤机制研究[J].蚌埠医学院学报,2011,36(5)：445-447.
[58]张引强,唐旭东,王凤云等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝 损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-455.
[59]张引强,唐旭东,王凤云,等.荣肝合剂对ConA诱导慢性免疫性肝损伤小鼠的影响[J].胃肠病学和肝病学杂志,2008,17(6)：492-495.
[60]ANDREW F G, SLATER C, STEF. AN I, etal. The role of inlra-cellular oxidants in apoptosis[J]. Biochim. Biophysics. Acta,1995,12 (1):59-62.
[61]WANG J, SUN R, WEI H M, et al. Pre-activation of T lymphocytes by low dose of concanavalin A aggravates toll-like receptor-3 ligand-induced NK cell-mediated liver injury[J]. Irate Immuno pharm,2006,6(5):800-807.
[62]潘红芳、赵凯、张炜.不同剂量刀豆蛋白A腹腔注射诱导昆明小鼠肝纤维化的效果观察[J].中国保健营养,2012,6：1244-1245.
[63]Dao-fang liu, Wei wei, Li-hua song. Upregulation of TNF-a and IL-6 mRNA in mouse liver induced by bacilli Calmette-Guerin pluslipopolysaccharide[J]. ActaPharmacologicaSinica,2006,27( 4):460-468.
[64]刘巧丽,高月求.刀豆球蛋白A诱导小鼠免疫性肝损伤的凋亡机制研究.中国免疫学杂志[J].2008；24(10)：876-878.
[65]TAGAWA Y, SEKIKAWA K, IWAKURA Y. Suppression of concanaval in A-induced hepatitis in IFN-gamma(-/-)mice, but not in TNF-alpha (-/-)mice; male for IFN-gamma in act ivating apoptosis hepatocytes[J]. J Immunol,1997,159 (3):1418-1428.
[66]ANNETTE E,MARKUS B R, THOMAS P, et al. IL-10, Regulatory T cells, and Kupfter cells mediate tolerance in concanavalin A-induced liver injury in mice[J].Hepatology,2007,45 (2):475-485.
[67]Kozie 1 MJ. Cytokines in viral hepatitis [J]. J Semin Liver Dis, 1999,19:157-169.