Experrimental Study On Hepatocyte Like Cells Induced By Cordyceps Polysaccharide In Vitro From Bone Marrow Mesenchyma Stem Cells

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.