【作者】 刘江凯；

【导师】 刘友章；

【作者基本信息】 广州中医药大学 ， 中医内科学， 2009， 博士

【摘要】 我国是病毒性肝炎的高发区,尤其是在农村及经济不发达地区,其发病率高达10-15%,由病毒性肝炎导致的重型肝炎或肝衰竭,其死亡率高达50-70%或更高,由慢性肝炎引起的肝纤维化或肝硬化,严重影响了患者的生存质量。肝纤维化进一步可发展至肝硬化,甚或终末期肝病阶段,发展至终末期肝病则缺乏广泛可行的有效治疗手段,尽管肝移植可作为治疗肝衰竭和肝硬化失代偿期的有效手段,但肝移植存在费用昂贵,手术复杂,术后可能发生严重的排斥反应,生存率不高等缺点,尤其是供体肝缺乏,使得这一治疗措施难以广泛推广。骨髓间充质干细胞(MSCs)是一群具有多向分化潜能的干细胞,在特定的诱导条件下,可以分化为多种造血以外的组织,如成骨细胞、神经元细胞、脂肪细胞、成纤维细胞、心肌细胞、肝细胞等。MSCs来源广泛,取材容易,便于自体移植,且在体外长期培养过程中,MSCs始终保持其多向分化的潜能,遗传背景相当稳定,体外基因转染率高,并能稳定高效地表达外源基因,故体内植入反应弱,是一种理想的组织工程种子细胞,具有治疗急慢性肝衰竭的潜能。因此,MSCs向肝系细胞的定向分化已受到学者们的广泛关注。1理论研究现代医学对MSCs了诱导分化为肝细胞的研究取得了一定的进展,但是单纯的MSCs进行在体移植,进入体内很容易被扩散或蛋白酶水解,能达到坏死肝细胞的MSCs不多,其肝细胞的分化率不仅很低,实际应用前景有限;而体外诱导分化则是在化学制剂的条件下实现,其肝细胞的分化率也很低,而且其临床安全性和长期有效性还难以实现。因此,如何促进MSCs向肝细胞完全意义上的转化,以便更有效改善病变肝组织的结构和功能将成为未来相关研究的重中之重。中药对MSCs诱导作用的研究起步较晚,近年来,不少研究机构证明中药对MSCs的定向分化有调控作用,并且有比传统的西医学方法高的诱导率。已有学者成功运用中药有效成分诱导MSCs分化为神经细胞、心肌细胞、脂肪细胞、成骨细胞等,目前国内外还没有关于应用中药单体有效成分诱导MSCs分化为肝细胞的研究报道,我们在众多的中药中,选用临床上较常用的治疗肝病的名贵中药冬虫夏草的有效成分虫草多糖来进行研究。现代中药药理研究表明,虫草多糖对肝细胞的损害及肝纤维化有较好的保护作用。而MSCs具有多种多分化潜能,根据既往利用中药有效成分诱导MSCs定向分化的研究成果,理论上相信虫草多糖在适当的条件下可以诱导MSCs分化为肝细胞,研究探索中药诱导MSCs分化为肝细胞,不仅将为应用中医药治疗急性肝衰竭提供新的理论依据,而且由于中药固有的安全性和长期有效性,可以克服目前现代医学研究中存在的某些困境,因此有可能与细胞移植治疗急性肝衰竭、肝硬化失代偿期的方法结合,带来广阔的应用前景,所以积极开展中药诱导MSCs分化为肝细胞的研究有重要意义。随着中医药的临床实践的不断深入,人们从中药中发现了可以治疗肝纤维的有效药物及其组分或成分,经过临床及实验得到了验证,显示中医药在抗肝纤维化治疗中的优势及潜力。冬虫夏草是我国传统滋补中药,有“保肺,益肾,补精髓,止血化痰,已劳咳,治膈症皆良”之功效,现代研究发现了其具有多种药理作用,拓展了其应用范围,已有体外实验表明虫草制剂可减轻实验型肝纤维化大鼠模型的肝纤维化指标,保护肝细胞,本研究使用虫草菌丝制剂(百令胶囊)来治疗乙肝后肝纤维化患者,观察其临床疗效,并初步探讨虫草制剂治疗肝纤维化的机制,为临床应用提供更多的科学依据。2实验研究2.1实验一虫草多糖体外诱导大鼠MSCs分化为肝细胞的研究目的:探讨虫草多糖能否体外诱导大鼠MSCs分化为肝细胞及其方法条件。方法:取成年Wister大鼠骨髓,采用贴壁法进行培养,流式细胞仪法检测细胞表面抗原CD34和CD44。对MSCs分组进行诱导,虫草组:用虫草多糖进行诱导,终末浓度为0.15mg/ml;阳性对照组:肝细胞生长因子(HGF)和表皮生长因子(EGF)联合诱导,浓度分别为20ng/ml和10ng/ml;空白对照组仅用基本培养基。倒置显微镜下连续观察细胞形态变化及增殖情况。于培养的7天、14天、21天、28天以免疫组化法检测肝细胞特异性标记AFP、CK18、ALB的表达,PAS法进行糖原染色。结果:原代培养的MSCs 24h小时后部分细胞贴壁,48h时大部分细胞贴壁,一周后细胞体积增大,多见长梭形、多角形细胞。细胞增殖明显增快,9天左右铺满瓶壁,胰酶消化传代后细胞形态多为梭形,较前均匀,部分多角形,增殖更快,成集落式生长,一般7天可铺满瓶壁90%。连续传代,P4代后,细胞形态变化不大。分离纯化的P3代MSCs检测CD34为阴性表达,CD44为阳性。对诱导分化后的细胞行免疫组化鉴定:虫草组及阳性对照组7天时出现AFP表达,14天时表达增强,28天时表达减弱;CK18阳性对照组7天时出现表达,14天时表达增强。虫草组则14天时出现表达,而后持续。ALB 7天时两组表达阴性,14天时出现阳性;对照组的AFP阳性率高于虫草组。糖原染色两组14天时出现阳性结果,28天时表达减弱。空白组结果皆为阴性。结论:虫草多糖可以体外诱导大鼠MSCs分化为类肝细胞。2.2实验二:百令胶囊治疗肝炎后肝纤维化临床疗效观察目的:探索治疗肝纤维化的有效药物。观察虫草菌丝制剂(百令胶囊)对慢性乙肝、早期肝硬化患者肝纤维化的疗效。方法:选择广州中医药大学及广东省中医院2008年1月-2008年12月份住院及门诊治疗后长期随访的慢性活动性肝炎及早期肝硬化患者36例。随机分为对照组和治疗组,两组各18例,全部组别给予肝泰乐0.2g,3次/日、维生素C2.0,口服,3次/日,甘利欣150mg+GS 250ml静滴2周,后改为甘草酸二胺胶囊,2粒,口服,3次/日等基础护肝药物。治疗组在对照组基础上给予百令胶囊(虫草菌丝体干粉,中美浙江华东制药有限公司生产)0.2g/粒,4粒/次,3次/天,口服。观察患者症状改善及血清生化学指标情况,3个月为一疗程。一个疗程后,评定疗效。结果:从临床疗效看,治疗组显效5例,有效11例,无效2例,总有效率为89%;对照组显效2例,有效10例,无效6例,总有效率为67%,两组总有效率比较有统计学意义(P＜0.05),治疗组的疗效优于对照组。两组治疗后,患者症状皆有改善,但治疗组的改善更为显著,与治疗前对比,差异有显著性(P＜0.05);对照组与治疗前相比差异无统计学意义。在肝功能检测方面两组自身治疗前后比较都有显著性差异(P＜0.05),治疗后两组肝功指标比较差异无统计学意义(P＞0.05)。两组治疗后,肝纤维化指标HA、PCⅢ、LN、Ⅳ-C皆有所改善,组内前后对比有显著差异(P＜0.05);治疗组的效果更为优异,与对照组比较差异有统计学意义(P＜0.05)。结论:百令胶囊(虫草菌丝体干粉)可有效治疗肝纤维化及早期肝硬化,保护肝细胞,改善患者的肝功能及HA、LN、PCⅢ、Ⅳ-C血清学指标。更多还原

【Abstract】 China is a region of high incidence of viral hepatitis its incidence rate is as high as 10-15%,especially in rural and underdeveloped areas of the economy.Serious hepatitis or liver failure caused by viral hepatitis,its mortality rate as high as 50-70%or higher.Hepatic cirrhosis and hepatic fibrosis caused by chronic hepatitis,seriously affected patients in the quality of life.Hepatic fibrosis can further develope cirrhosis of liver,even final stage liver disease. Lack of effective therapeutic tool in the stage,although liver transplantation can be used as the effective means of treating liver failure and cirrhosis,but it has several drawbacks, Expensive,complex surgery,may occur severe rejection after surgery and the survival rate is not high.the lack of donor liver is a big problem.Those restrictions make it difficult to promote this therapeutic tool.Mesenchymal stem cells(MSCs)is a group of multi-differentiation potential of stem cells.It can differentiate into a variety of out the hemopoietic tissue in specific conditions. for example,osteoblast,neuron cell,adipocyte,fibroblast,cardiac muscle cell,hepatocyte etc.MSCs can be obtained from different organizations of the body,and can be autografted conveniently.In the course of long-term culture in vitro,MSCS has always been to maintain its multi-directional differentiation potential,its genetic background is relatively stable in vitro,it has a high rate of gene transfection and can express foreign genes stable and efficient.Therefore,when MSCs is implanted in the body,the weak response to implan in vivo is one of the features.So MSCS is an ideal seed cells for tissue engineering,wih the potential of treating acute and chronic liver failure.MSCs can directed to differentiate into liver cells,which has been widespread concerned by scholars.1.Theoretical researchModern medicine has made some progress in MSCs differentiate into liver cells.But MSCs can easily diffused or hydrolysed by enzymatic for pure MSCs transplantation in vivo,the MSCs that can achieve hepatocyte is not much,and it differentiation rate is low,so its prospects is lmited for practical application.The differentiation in vitro is realized under conditions of chemical praeparatum,the differentiation rate of it is low too.And its clinical safety and long-term effectiveness is also difficult to achieve,therefore it’s the most important how to promote the MSCS into hepatocytes fully and more effectively to improve the structure and functions of liver organizations.The study was started relatively late that make use of chinese medicine induce MSCs differentiate.Many research institutions have proved chinese medicine the differentiation regulation to MSCs in recent years,and wih higher induce rate than Western medicine method.It were reported that scholars applied active ingredients of Chinese medicine had been successful induced MSCs differentiate into nerve cells、adipocyte,cadiocyte, fibroblast and hepatocyte etl.There are no reports that someone apply monomer composition of traditional Chinese medicine to induce MSCs differentiate into liver cells at home and abroad.Chinese caterpillar fungus is a commonly Chinese medicine used to treat liver disease in clinical,We chose its active ingredients Chinese caterpillar fungus polysaccharide to study.Pharmacological studies show that Cordyceps polysaccharide:it has protective effect to liver damage and liver fibrosis.MSCs has a wide range of differentiate potential,According to oriented research results of applying chinese medicine active ingredients to induce differentiation of MSCs,we can believe theoretically that cordyceps polysaccharide can induce MSCs differentiate into liver cells in the right conditions.Research on traditional Chinese medicine induced MSCs to differentiate into liver cells,not only provide a new theoretical basis for applying chinese medicine to treat acute liver failure,but also can overcome some dilemma in modern medical research due to the inherent safety and long-term effectiveness of chinese medicine.It may be related to the methods that treat acute liver failure and cirrhosis by cell transplantation,Bring about broad application prospects.So it is important to carry out chinese medicine induce MSCS to differentiate into liver cells.Along with the deepening of clinical practice of chinese medicine,people had been found some effective drugs and compositions from Chinese medicine that can treat hepatic fibrosis.Clinical and Experimental have been verificated those effects,this showed advantages and potential of chinese medicine in the treatment of hepatic fibrosis.Chinese caterpillar fungus is a traditional chinese tonic medicine,it can protect the lungs and beneficial to the kidneys,enrich the essence,relieve cough and reduce sputum,treat chronic cough and swallowing disorders.Modern research has found a variety of pharmacological effects of it,expanded its scope of application.Experiments have been showed that cordyceps can reduce indicators of experimental liver fibrosis rat model about liver fibrosis, protect liver cells.In this study,we made use of the preparations of Cordyceps sinensis(Bailing Capsule)to treat liver fibrosis with hepatitis B,observe the clinical efficacy and explore mechanisms for the treatment of liver fibrosis.Provide more scientific basis for the clinical application of it.2.Experimental StudyExperimentⅠ:Cordyceps polysaccharide induces differentiation of adult rat MSCs into a hepatocyte lineage in vitroObjective:To research the possibility of the rat mesenchymal stem cells(MSCs)to differentiate into hepatocyte-like with Cordyceps polysaccharide in vitro.Methods:Bone marrow in the femurs of Wistar rat was collected by flushing under sterile condition.MSCs were separated and cultured according to the direct anchoring method,identified corpuscular surface antigen of CD34 and CD44 by using flow cytometric analys.MSCs were divided into 3 groups to induce differentiate.Cordyceps group:induced with Cordyceps polysaccharide.The final concentration of it was 0.15mg/ml.Masccline Control group:Induced by HGF and EGF,the concentration of each was 20ng/ml and 10ng/ml. Blank group:only cultivated with basis medium.The morphological changes and proliferation of MSCSs were observed by phase-contrast microscope.On day 7,14,21 and 28,AFP,CK18 and ALB of MSCSs of each group were examined,the expressions of them were detected with immunohistochemistry technique,staining for glycogen with PAS method.Results:After 24h in primary culture,MSCs adhered to the plastic surface,most cells adhered surface,cell volume increase after a week.The cells spindle-shaped and polygonal,cell proliferation significantly faster,the cells covered culture flask about 9 days. Subcultured after trypsin digestion the cells morphology are most of the spindle patterns more homogeneous than before,proliferated even faster,the cells into a colony-type growth.7 days can covered with 90%surface of culture flask.Continuous passage.Little change in cell morphology after p4.MSCs collected from the femora of Wister rats expressed antigens of CD44,but not CD34.Immunocytochemical analysis for AFP showed positive staining reaction on day7,in Cordyceps group and Masccline Control group, strengthen on day 14,then weaken on day 28.Positive staining reaction on 14 day.Masceline Control group was higher than Cordyceps group in ratio of masccline for AFP.On 14 day both group show positive staining reaction in staining for glycogen of PAS,then weaken on day 28.The blank group all showed positive staining reaction. Conclusion:MSCs induced by cordyceps polysaccharide in vitro can differentiate into hepatocyte-like cells. ExperimentⅡ:Bailing capsule to treat hepatic fibrosis after hepatitis clinical observationObjective:To explore the effective drug of treating hepatic fibrosis.Observe cordyceps sinensis preparation(Bailing Capsule)for hepatic fibrosis and early liver fibrosis clinical efficacy in patients.Methods:Choose chronic active hepatitis and early liver cirrhosis 36 cases of patients,which cases come from the First Affiliated Hospital of Guangzhou University of TCM and the Guangdong Provincial TCM Hospital since January 2008 to December 2008.The 36 cases randomly divided into control group and treatment group.18 cases of each group.Two groups all gave glucurolactone 0.2g,3 times/d,po;Vitamin C 2g, 3 times/d,po;Diammonium glycyrrhizinate 150mg+GS 250ml ivgtt in 2 weeks,1 times/day,replaced by diammonium Glycyrrhizinate capsule after 2 weeks,2 pills,po,3 times/day;Treatment group gave Bailing Capsule.Treatment group gave Bailing Capsule(Cordyceps Mycelium Powder,East China Pharmaceutical Co,Ltd, Zhejiang,China and the United States)in addition than the control group,0.2g / tablets,4 tablets/times,3 times/d,po.Observed symptom improvement and serum biochemical indicators in patients,3 months for a course,after a course of treatment,assess of efficacy.Results:See from the Clinical Efficacy 5 cases of treatment group markedly,11 cases of effective,2 cases of invalid,the total effective rate was 89%;2 cases of the control group markedly,10 cases of effective,6 cases of invalid,the total effective rate was 67%,The total efficiency of the two groups was statistically significant(P＜0.05),Efficacy of the treatment group was superior to the control group.The patients had improvement in symptoms in two groups after treatment,however,the improvement of the treatment group was more significantly,Compared with before treatment,there was a significant difference (P＜0.05),The control group compared with before treatment there was no significant difference(P＞0.05).Comparison of the two groups own before and after the treatment has its significant difference in liver function testing(P＜0.05).The two groups was no significant difference in indicators of liver function after treatment(P＞0.05).Indicators of hepatic fibrosis HA、PCⅢ、LN、Ⅳ-C all were improved in two groups after treatment, Comparison before and after treatment in group were significantly different(P＜0.05),the effect of the treatment group is more superior,Compared with the control group were statistically significant differences.Conclusions:Bailing Capsule(cordyceps sinensis preparation)can treat liver fibrosis and early cirrhosis,protects liver cells,improve liver function and serum indicators of patients with hepatic fibrosis.更多还原