The Yiqijiedu On Rat Liver Stem Cell Proliferation And Liver Regeneration Mechanism

1. ObjectiveAmerican ginseng, the Bezoar have the Yiqiyangyin Qingrejiedu function, to conform Yiqijiedu "basic principles. Compatibility combination of the active ingredients of the two drugs to observe the impact and effect of liver regeneration "Yiqijiedu Law on rat liver stem cell lines induced differentiation, proliferation, to explore the role of principle Yiqijiedu treatment of chronic liver disease, in order to further study therapeutic principle to provide new ideas, and also provide a theoretical and experimental basis for clinical application.2Methods2.1GroupingNormal group, operation group (orally2-AAF), model group, Chinese medicine decoction group5%group,10%of the group,20%of the group, Chinese medicines group group5%,10%group,20%of the group.2.2modelingSelected the frequently used the solt-Farbar modeling method to make some improvements. Randomized housed separately one week after the gastric tube fed AAF (10mg/kg mouse weight), once a day, four consecutive days, on the5th of sodium pentobarbital (50mg/kg) by intraperitoneal injection anesthesia for liver three two-thirds (left lobe and middle) resection (the day stopping irrigation AAF) hepatectomy method, supine fixed conventional skin preparation disinfection, to take abdominal incision Rufu, free left and middle lobes artery ligation, to be left mid-liver-yellowing, resection of the left and middle lobes, ligation of the artery, vein and bile duct, no bleeding, biliary fistula in the abdomen was closed. The day of surgery is not administered. The6th consecutive irrigation serving AAF1weeks, respectively Chinese medicine orally (dose, the effective dose of the overall model animal), and the normal control group, the control group received saline intragastrically.13,16,20,25days after surgery were taken three rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg), the abdominal cavity was opened, exposing the liver, sterile abdominal aortic blood, separation of the serum set stored at-30℃for inspection. Quickly clipping liver10-20g quick frozen in liquid nitrogen overnight at-80℃cold kept examination and take the regeneration of liver tissue and the right lobe of the liver, a fixed concentration of10%formaldehyde, embedded in paraffin,5um serial sections alternate.2.3OUTCOME MEASURES detectionLiver stem cells (oval cell) proliferation:hepatic oval nucleus use computer image analysis system of oval cells, liver cells count quantitative analysis and comparison. In situ hybridization detection of liver tissue TGF-a, TGF-β1mRNA expression:Preparation of TGF-aCDNA and the TGF-β1RNA probe, TGF-a RNA-RNA hybridization using DNA-RNA in situ hybridization, TGF-β1, press The kit instructions.Quantitative detection of TGF-β1in liver tissue:Take0.1g liquid nitrogen frozen liver tissue homogenates, extraction of total RNA, detected using RT-PCR the United DotBlot statutory amount of TGF-β1.Regenerating liver cell division index (MI):take regeneration in liver tissue fixed in10%formalin, embedded in paraffin, hematoxylin-eosin staining, oil microscope to count the total number of liver cell nuclei and hepatocyte nuclear division of the following formula:MI%=hepatocyte nuclear mitotic (a)/(a) x100%the total number of hepatocyte nuclear.Liver tissue EGFR, of TGF-βR2, and HGFR receptor protein:liver tissue5um consecutive slices by SABC immunohistochemical method to detect the receptor, known EGFR of TGF-βR1, R2, and HGFR positive rat liver tissue as a positive control for blank negative control antibody was replaced with PBS.General observation of indicators:body weight of rats, hair, feces, secretions, activity status, blood liver function biochemical detection, liver tissue biopsy tissue observations.3Results3.1component group decoction group, PCNA, vimentin, liver cells CK8, CK18expression is greater than the surgical group and the normal group (P<0.05), indicating a successful hepatic oval cell model.3.2component group decoction group, the expression of TGF-p is greater than the model group and the surgery group (P<0.05), and through TGF-β intervention liver stem cells induced to differentiate that Yiqijiedu law. 3.3component group decoction group the TGF-a, TGF-β1mRNA expression are better than the model group and the surgery group (P<0.05), through the TGF-a that Yiqijiedu law, TGF-β1mRNA intervention liver stem cells induced to differentiate.3.4component group with the of water decoction group of EGFR of TGF-βR1, R2, HGFR receptor protein are better than the model group and the surgery group (P<0.05), indicating the Yiqijiedu law through EGFR the TGF-βR2, HGFR, The receptor proteins intervention liver stem cells induced to differentiate.3.5component group decoction group are better than in the improvement of the general situation of the rat model with operation group (P<0.05), that Yiqijiedu law can regulate liver stem cells induced to differentiate.4Conclusion4.1.2-AAF combined liver injury than a simple modeling of liver injury, the the rat mortality rate is low.4.2Yiqijiedu by regulating TGF-β intervention induced liver stem cells, proliferation and differentiation.4.3Yiqijiedu through regulation of TGF-a, TGF-β1mRNA intervention liver stem cells induced proliferation and differentiation.4.4Yiqijiedu through regulation of EGFR, the TGF-βR2, HGFR the intervention liver stem cells induced proliferation and differentiation.4.5Yiqijiedu to improve the general situation of the rat, to improve their quality of life, improve liver function.