【作者】 刘亚梅；

【导师】 郭兴伯；

【作者基本信息】 广州中医药大学 ， 中西医结合基础， 2007， 博士

【摘要】 1研究设想乙肝慢性化机制与T淋巴细胞亚群的低反应性、NK活性降低、Th1类细胞因子水平下降等免疫功能不全、体内高滴度病毒含量及对感染细胞的免疫耐受状态有关。目前尚未发现根除HBV的药物，尚未明确HBV免疫耐受发生在哪一个应答环节或关键步骤上。中药复方对慢性乙型肝炎机体免疫功能表现出良好的调节作用，并能有效地促进HBV的清除。从CHB病毒水平与机体免疫状念二者的相关性入手，运用中药复方（疏肝健脾补肾方）对CHB患者、HBV转基因小鼠进行抗病毒和免疫调节作用研究目前尚未见报道。2研究目的本课题观察疏肝健脾补肾方联合ADV治疗CHB患者前后病毒DNA水平与免疫功能的相关性，进行抗病毒、免疫调节和中西医结合疗效研究，为防治CHB提供新思路和实验依据。以体外细胞模型来探讨中药抑制HBV复制的机理和意义。并在以往研究基础上观察疏肝健脾补肾方对HBV转基因小鼠的抗病毒和免疫调节作用，探讨中医药治疗HBV的疗效及作用机制。3研究方法3．1疏肝健脾补肾方对慢性乙型肝炎患者的免疫学调节机制51例肝郁脾虚兼脾肾阳虚型CHB患者按随机数字分成A、B、C三组。A为疏肝健脾补肾方组；B为疏肝健脾补肾方+ADV组；C为ADV对照组，每组各17例。A组患者在对症护肝的基础上，服用疏肝健脾补肾方，6个月为1用药疗程；B组患者在对症护肝的基础上，服用疏肝健脾补肾方和ADV；C组患者在对症护肝的基础上，服用ADV。治疗前、治疗后6个月均检测肝功能、免疫功能和病毒学指标。观察CHB患者的临床症状和体征；在自动生化仪上用常规的生化方法检测血清ALT、AST、STB、A／G等指标；采用荧光定量PCR法检测血清HBV DNA，乙肝两对半ELASA法检测HBsAg、HBeAg、抗-HBe；流式细胞仪免疫荧光法检测外周血T淋巴细胞亚群百分率、自然杀伤细胞；放免法检测血清IL-2。3．2疏肝健脾补肾方体外抗乙肝病毒的实验用2.2.15细胞作为体外模型，将疏肝健脾补肾方药物原液倍比稀释成7个浓度上药，同时设无药物细胞组进行对照。上药8天后收集上清用ELISA法测定HBsAg、HBeAg。MTT法检测药物细胞毒性，并计算药物对抗原的半数抑制浓度(IC₅₀)及治疗指数（TI）。3．3疏肝健脾补肾方对小鼠的急性毒性实验实验前把BALB／C小鼠随机分为6组，疏肝健脾补肾方160g／kg／d组、80g／kg／d组、40g／kg／d组、20g／kg／d组、10g／kg／d组和正常对照组（等量生理盐水），每组10只。以确定不同浓度的疏肝健脾补肾方安全剂量范围。动物禁食16h后一次性灌胃给予受试药物，观察5d，逐日记录中毒症状、死亡情况，死亡动物应及时尸解，记录病变情况，肉眼观察其病变组织。给药后同时观察动物死亡数、耸毛、蜷卧、耳廓苍白或充血、突眼、步履蹒跚、大小便、瘫痪、昏迷、抽搐、步态、呼吸困难等情况。最后断头处死所有动物。3．4疏肝健脾补肾方对HBV转基因小鼠免疫病理的干预作用实验动物共分为6组。正常对照组为6-8周龄非HBV转基因雄性BALB／C小鼠10只，灌以等量灭菌等渗生理盐水；SPF级HBV转基因雄性小鼠50只，HbsAg均为阳性，随机分为以下5组：模型组（灌以等量灭菌等渗生理盐水）、疏肝健脾补肾方低剂量组（按10g克生药／kg体重／天灌胃）、疏肝健脾补肾方中剂量组（按20g克生药／kg体重／天灌胃）、疏肝健脾补肾方高剂量组（按40g克生药／kg体重／天灌胃）、ADV组（按50mg／kg体重／天灌胃ADV），每组10只。每天按时喂药，连续21d。采血前禁食12h，最后1次采血后处死动物。分别于给药前1天（T0）、给药第10天（T10）、给药第21天（T21）、停药后3天（P3）取血，离心取血清，-80℃冻存，用ELASA法检测血清HBsAg，荧光定量PCR法测定血清中HBV DNA。取脾，按常规制备脾单细胞悬液，上流式细胞仪用荧光标记抗体法测定脾T淋巴细胞亚群，用放免法测定脾培养上清中的IL-2、ELISA法测定脾培养上清中的IFNγ。取病变明显处的肝脏作病理学切片及HbcAg免疫组化。4研究结果4．1疏肝健脾补肾方对慢性乙型肝炎患者的免疫学调节机制B组在胁肋胀痛、腹胀便溏、畏寒肢冷、抑郁烦闷、身倦乏力、少腹腰膝冷痛等症状改善方面，优于其它二组。临床疗效方面，三组总有效率之间比较差异均无显著性意义。三组在ALT、AST、TSB、A／G等肝功能恢复方面效果均好，其中B组（疏肝健脾补肾方+ADV）ALT复常率高于单一中药、西药组。三组HBV DNA转阴率总体比较差异有显著性意义，以B组（疏肝健脾补肾方+ADV）转阴率最高。A组、B组、C组治疗前后HBV DNA含量差异有显著性意义，尤以B组为甚。上述结果提示，中西医联合用药比单一中药和单一西药抑制病毒复制的效果更好。三种治法均可增加CD3⁺、CD4⁺百分比，减少CD8⁺细胞百分比，尤以A组、B组增加CD3⁺细胞、A组增加CD4⁺细胞、B组减少CD8⁺细胞的效果更为显著。而ADV单用时增加CD3⁺、CD4⁺和减少CD8⁺的作用则较弱。B组可同时非常显著性提高NK细胞活性和IL-2分泌；C组仅对NK细胞有正向激活作用，对IL-2则无显著影响；相反，A组对NK细胞活性影响不显著，对IL-2水平则有显著提升作用。B组治疗前后CD3⁺百分比的增加、NK细胞的增加、CD8⁺百分比的减少与HBV DNA载量的减少呈显著正相关。A组治疗前后CD4⁺百分比的增加与HBV DNA载量减少呈显著正相关。B组、C组患者各1例偶见药物不良反应。4．2疏肝健脾补肾方体外抗乙肝病毒的实验疏肝健脾补肾方对2．2．15细胞的半数毒性浓度TC₅₀为30.28mg／ml，对2．2．15细胞分泌的HBsAg、HBeAg半数抑制浓度为25.96 mg／ml、36.46 mg／ml，治疗指数分别为1.17、0.83。说明疏肝健脾补肾方能低效抑制细胞表达HBsAg，且有毒性作用，而无抑制HBeAg抗原的作用。4．3疏肝健脾补肾方对小鼠的急性毒性实验疏肝健脾补肾方160g／kg／d组显示出一定的不良反应。从第2天开始均表现出纳差、饮水量少、乏力、行动迟缓、蜷卧、大便溏泄等情况。在第4天死亡1只，在第5天死亡2只。经解剖发现肝脏呈暗红色，胃肠肿胀并扭曲。可能由于药物的浓度太大，引起小鼠肝脏水肿、出血、坏死所致。而疏肝健脾补肾方80g／kg／d组、40g／kg／d组、20g／kg／d组、10g／kg／d组给药后动物一般状况良好，未发现明显的毒性反应，未见动物死亡。因此，本课题实验所用剂量均在80g／kg／d以下范围进行。4．4疏肝健脾补肾方对HBV转基因小鼠免疫病理的干预作用疏肝健脾补肾方中、大剂量及ADV组HBV Tg鼠血清HBsAg转阴率分别在给药后第10天、第21天、停药后第3天进行比较，差异均无显著性意义。ADV具有较好的减少HBV Tg鼠血清HBV DNA载量的作用，但停药后3天有反跳。疏肝健脾补肾方大、中剂量组均具有降低HBV Tg血清HBV DNA载量的作用，尤其以大剂量效果明显，虽然没有减少到ADV组的水平，但治疗前后差异有显著性意义，且停药后无明显反跳，说明中药作用持久、平稳。模型组Tg鼠肝脏病理改变类似临床轻度肝炎表现，随着中药疏肝健脾补肾方浓度的增大，炎症得以缓解，大剂量组的肝板排列整齐，淋巴细胞浸润程度减轻。而ADV治疗小鼠肝组织汇管内见散在的淋巴细胞浸润，无纤维组织增生，但中央静脉周围肝细胞轻度脂肪变性。疏肝健脾补肾方中、大剂量和ADV均能减少肝组织中HbcAg表达，尤其以大剂量明显。疏肝健脾补肾中药能够上调HBV转基因小鼠CD3⁺、CD4⁺、CD4⁺／CD8⁺，下调CD8⁺T细胞比值水平，具有正向T淋巴细胞免疫调节作用，尤其以大剂量组效果更为显著。ADV也具有提高HBV转基因小鼠细胞免疫的功能（如CD3⁺百分比），但作用相对中药较弱。运用大、中、小剂量疏肝健脾补肾方和ADV均能提高HBV转基因小鼠血清IL-2、IFN-γ含量，与模型组相比差异有显著性意义。且运用大剂量和中剂量的疏肝健脾补肾方治疗后IL-2、IFN-γ水平已高于正常对照组。疏肝健脾补肾方中剂量组CD3⁺、IFN-γ与给药前后HBV DNA载量的减少呈显著正相关，疏肝健脾补肾方大剂量组CD4⁺、CD8⁺、CD4⁺／CD8⁺与给药前后HBV DNA载量减少呈显著正相关。5结论1、疏肝健脾补肾方是较好的免疫调节剂，能明显地增强Th1类细胞因子的分泌，使低下的免疫功能得到改善。2、疏肝健脾补肾方通过对机体免疫功能的调节，进而抑制HBV复制，这符合中医的“扶正”以“祛邪”理论。3、ADV具有较好的减少体内血清HBV DNA载量的作用，而疏肝健脾补肾方调节机体免疫功能强于ADV。中西医结合用药能明显改善CHB患者临床症状，具有抗病毒、免疫调节等整体功能。更多还原

【Abstract】 1 Investigative presumptionThe mechanism of chronic hepatitis B is concerned with immunologic inadequacy, forexample, the hyporesponsiveness of T lymphocyte subset, the lower of NK activity and thelower level of Th1 cytokine, virus content of high titer in vivo and the immune tolerance ofinfected cell. Now antiviral drug to eradicate HBV has not been found yet, and the immunetolerance of HBV exactly existing on which reaction link or committed step has not beenidentified. Chinese crude drug complex prescription had favorable regulation on immunefunction of CHB organism, and had effectively facilitated the clearance of HBV. From theassociativity of CHB virus levels and immune state of the organism, now there are fewreports about the studies on Chinese crude drug complex prescription (SHU GAN JIAN PIBU SHENG FANG, SGJPBSF) on the antiviral therapy and immunological regulation forCHB patients and HBV transgenic mice.2 AimsWe observed the associativity of virus DNA levels and immune function of the organismon CHB patients using SGJPBSF combination with ADV pre-treatment and post-treatmentin this topic, studied antiviral therapy, immunological regulation and the curative effect ofcombination of TCM with Western medicine, offered the new thought and experimentalevidence for prevention and therapy of CHB. We used the vitro cell model to explore themechanism and significance of the traditional Chinese medicine inhibiting the HBVreplication too. On the basis of formerly study and research, we observed theimmunological regulation and antiviral therapy of SGJPBSF on HBV transgenic mice toexplore the curative effect of traditional Chinese medicine for HBV and functionarymechanism.3 Methods 3.1 the immunologic regulatory mechanism of SHU GAN JIAN PI BU SHENG FANGon CHB patients51 CHB patients with stagnation of liver-QI with deficiency of the spleen andasdthenic splenonephro-yang were divided into Group A, B, C according to randomnumber. A was SGJPBSF group, B was SGJPBSF plus ADV group, and C was ADV group.Each group had 17 patients. Group A was administrated SGJPBSF on the basis of theaccording to the sign and the protecting from the liver, 6 months each medication. Group Bwas administrated SGJPBSF plus ADV on the basis of the according to the sign and theprotecting from the liver. Group C was administrated ADV on the basis of the according tothe sign and the protecting from the liver. The liver function, immune function and virologyindex of all patients were detected in per-treatment and in post-treatment 6 months. Weobserved the clinic sings and symptoms of patients. The serum levels of ALT, AST, STBand A/G was detected in automatic biochemistry meter by routine biochemistry method.The serum HBV DNA titers were detected by fluorescent quantitation PCR. The serum ofHBsAg, HBeAg and anti-HBe were detected by HBV two half ELASA. The T lymphocytesubset and NK of the peripheral blood were detected by fluorescent-labeled antibody inflow cytometry. IL-2 was detected by radioimmunity.3.2 the anti-effect experiment of SGJPBSF in vitroThe 2.2.15 cell line was used as cell model. SGJPBSF was diluted into 7 dosage groups.At the same time cell control group was set. After 8 days drug administration, the HBsAgand HBeAg in the supernatant were tested with ELISA assay. The cytotoxicity of drug wasevaluated with the MTT assay, and the IC50 (50%inhibiting concentration) and TI(therapeutic index) of SGJPBSF to antigen were calculated.3.3 acute toxicity experiment of SGJPBSF to miceBefore the experiment, BALB/C mice had been randomly divided into six groups,SGJPBSF 160g/kg/d, 80g/kg/d, 40g/kg/d, 20g/kg/d, 10g/kg/d and normal control (isodosenormal sodium) to definite the safety dose range of different density SGJPBSF. After allanimals were fasted about 16 hours they were intragastricly administrated one time. Weobserved them for 5 days, recorded daily toxic symptom and death information. The deadmice must be autopsied in time and the pathological changes must be noted. The tissuemust be checked if these pathological changes were observed by naked eye. The number ofdeath, piloerection, tired to lie down, pale auricle or congest, exorbitism, walking haltingly,urination and defecation, plegia, cataphora, convulsion, carriage and anhelation at the sametime were observed. At last, all animals were sacrificed by decapitation.3.4 the immunopathogenesis intervention of SGJPBSF to HBV transgenic mice The experimental animals were divided into six groups. The normal control group were6-8 weeks age, 10 non-HBV transgenic male mice, those were intragastric administratedisodose, sterilizing, iso-osmia normal sodium. 50 HBV transgenic male mice of SPF grade,those HbsAg were positive, were randomly divided into 5 groups, model group(wereintragastric administrated isodose, sterilizing, isooosmia normal sodium), low-dose groupof SGJPBSF (were intragastric administrated 10g crude drug /kg body weight /d),middle-dose group of SGJPBSF (were intragastricly administrated 20g crude drug/kgbody weight/d), high-dose group of SGJPBSF (were intragastric administrated 40g crudedrug/kg body weight/d), and ADV group (were intragastric administrated 50g ADV/kgbody weight/d), 10 mice each group. Everyday they were administrated on time, for 21days consecutively. They were fasting for 12 hours before their blood samples were taken.Their blood samples were taken in the time points of T0, T10, T21 and P3 in accordancewith the time point of drug intragastric administration and they were sacrificed after at lastsampling. The serums were collected by centrifugalization, and were keeped under 80℃.The serum HBsAg was detected by ELASA. The serum HBV DNA titers were detected byfluorescent quantitation PCR. The spleens were taken and unicell suspension of the spleenswas prepared by secundum artem. The T lymphocyte subset of the spleen was detected byfluorescent-labeled antibody in flow cytometry. The cytokine was detected in the spleencultivation supematant, for example, IL-2 was detected by radioimmunity, and IFNγwasdetected by ELISA. The liver tissues of obviously pathological changes were taken to dopathology slice and HbcAg immunohistochemistry.4 Results4.1 the immunologic regulatory mechanism of SHU GAN JIAN PI BU SHENG FANGon CHB patientsThe symptoms of hypochondrium distending pain, abdominal distention, loose stool,feeling chill, cold limbs, depression, discomfort, tired body, debilitation, psychroalgia inthe lower abdomen, lumbar and knee in Group B were superior to other groups. Inclinically therapeutic effect, there was no significant difference in total effective rate ofthree groups. The liver functional recoveries of alanine aminotransferase, aspartateaminotransferase, total bilirubin, A/G were good in three groups, but the ALTnormalization rate in Group B (SGJPBSF plus ADV) was higher than that of in Group A(SGJPBSF) and Group C (ADV). There was significant difference in three groups about therate of negative transfer of HBV DNA, and the rate in Group B was highest. There weresignificant differences of the serum titers of HBV DNA in three groups between the pre-treatment and post-treatment, especially in Group B. Those results suggest that theeffect of inhibiting viral replication in combination medication of TCM with Westernmedicine was superior to single Chinese crude drug and single western medicine. Threetherapeutic methods all could increase the ratio of CD3~+, CD4~+ cell and decrease the ratioof CD8~+ cell, especially increasing the ratio of CD3~+ in Group A and Group B, increasingthe ratio of CD4~+ in Group A, decreasing the ratio of CD8~+ in Group B were verysignificant. When using ADV treatment, the effect of increasing the CD3~+, CD4~+ cell ratioand decreasing the CD8~+ cell ratio in single was mild. The raising of NK cytoactive andIL-2 secretion in Group B was very significantly. Group C had the norientation activationon NK, but had no significant effect on IL-2. Opposite, Group A had no significantnorientation activation on NK, but had significant upgrading effect on IL-2. The increasingof CD4~+ and NK and the decreasing of CD8~+ T cell ratio were significantly positivecorrelation to the decreasing of HBV DNA titers between the pre-treatment andpost-treatment in Group B. The increasing of CD4~+ cell ratio was significantly positivecorrelation to the decreasing of HBV DNA titers between the prior treatment andpost-treatment in Group A. There was one example drug adverse reaction in Group B andGroup C respectively.4.2 the anti-effect experiment of SGJPBSF in vitroThe TC50 of SGJPBSF to 2.2.15 cell line was 30.28mg/ml. The IC50 of SGJPBSF toHBsAg, HBeAg was 25.96mg/ml and 36.46mg/ml respectively, and the TI of SGJPBSFwas 1.17 and 0.83 respectively. It suggested SGJPBSF could inhibit the HBsAg express of2.2.15 cell line, but it could have toxic effect and could not inhibit the HBeAg express.4.3 acute toxicity experiment of SGJPBSF to miceThe mice of SGJPBSF 160g/kg/d showed some adverse effect. From the second day, themice showed seldom eating, seldom drinking water, debilitation, moving slowly, tiring tolie down, loose stool, and so on. One mouse died in 4th day and two mice died in the 5thday. Under the anatomy, the livers of three died mice were madder red and their stomachintestines were swelling and twist, possibly because too high pharmacal density could causedropsy, hemorrhage and necrosis of murine liver. The general condition was good afteradministration in the mice of SGJPBSF 80g/kg/d, 40g/kg/d, 20g/kg/d, 10g/kg/d, obviouslytoxic reaction was not found, and there was not dead animal. So all dose used in followexperiment was below 80g/kg/d.4.4 the immunopathogenesis intervention of SGJPBSF to HBV transgenic miceThe HBsAg sero-nagative rate of HBV Tg in high-dose group of SGJPBSF,middle-dose group and ADV group had no significant difference on T10, T21 after administration and P3. ADV could reduce the serum HBV DNA titers of HBV Tg, butthere was markedly rebound on 3rd after drug withdrawal. High-dose SGJPBSF andmiddle-dose both decreased the serum HBV DNA titers of HBV Tg, especially high-doseSGJPBSF, although the serum HBV DNA titers were not decreased to the level of ADVgroup, there was significant difference between the pre-treatment and post-treatment, andthere was no rebound after drug withdrawal, its effect was steady for a long time. The liverpathology change of model HBV Tg was similar to the appearance of the clinical lightlyhepatitis, and along with the increasing SGJPBSF density, the liver inflammation wasrelieved. The hepatic plate of high-dose SGJPBSF lined up in order and the degree oflymphocyte soakage was relieved. But there was still sporadic lymphocyte soakage in theconverge duct of hepatic tissue of ADV group, there was no fibroplasias, but in the centralveins surrounding there was some lightly hepatocyte adipose degeneration. Themiddle-dose, high-dose SGJPBSF and ADV all could decrease the HbcAg express in thehepatic tissue, especially high-dose SGJPBSF. SGJPBSF could up-regulate the ratio ofCD3~+, CD4~+ and CD4~+/CD8~+ cells of HBV Tg, and could down-regulate the ratio of CD8~+T cell. So it had the norientation effect of T lymphocyte immunological regulationespecially high-dose SGJPBSF. ADV had the effect of increasing cytoimmunity of HBVTg too (for example, CD3~+ cell percentage), but its function was weaker than the traditionalChinese medicine. The low-dose, middle-dose, high-dose SGJPBSF and ADV all couldincrease the serum level of IL-2 and IFN-γof HBV Tg, and there was significant differenceas compared to the model group. And the level of IL-2 and IFN-γin middle-dose, high-doseSGJPBSF had far surpassed the normal control after treatment. The ratio of CD3~+ T cell andthe level of IFN-γwere significantly positive correlation to the decreasing of HBV DNAtiters between the pre-treatment and post-treatment in middle-dose. The ratio of CD4~+,CD8~+ and CD4~+/CD8~+ T cells were significantly positive correlation to the decrease ofHBV DNA titers between the pre-treatment and post-treatment in high-dose.5 Conclusions5.1 SGJPBSF was fine immunomodulator, and it could significantly strengthen thesecretion of Th1 cytokine and could improve the lower immune function.5.2 It suggested that SGJPBSF could clear up and inhibit HBV through regulating theimmune function of organism. It was consistent with TCM theory of strengthening bodyresistance to eliminating pathogen.5.3 ADV could have the better effect of decreasing the serum HBV DNA titers thanSGJPBSF, but SGJPBSF could have the better effect of regulating immune function than ADV. United medication of SGJPBSF plus ADV is shown to have the better clinicaleffect of improving symptoms, inhibiting the HBV duplication and regulating theabnormal immune function in CHB patients.更多还原