TSINGHUA UNIVERSITY GENERAL HOSPITAL OF PLA LIVER CELLS. Xu Ying Xin. Institute of Surgical Research Chinese PLA General Hospital, Beijing
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1 Harvard-MIT Division of Health Sciences and Technology HST.535: Principles and Practice of Tissue Engineering Instructors: Yingxin Xu and Qingling Feng TSINGHUA UNIVERSITY GENERAL HOSPITAL OF PLA LIVER CELLS Xu Ying Xin Institute of Surgical Research Chinese PLA General Hospital, Beijing Yingxin Xu M.D. & Qingling Feng Ph.D.
2 Liver Cells Structure and function of Liver Regenration of liver cells Liver cells research relevant to liver tissue engineering
3 Liver: the largest compound gland and chief metabolic organ Courtesy of US Dept. of Health and Human Services.
4 Different Types of Liver Cells Hepatocytes (parenchymal cells,pc) Liver endothelial cells (LEC) Kupffer cells (KC) Stellate cells(sc) Other cells: ƽ epithelial cells of bile duct ƽ endothelial cells of blood and lymphatic vessels ƽ smooth muscle cells of arteries and veins ƽ nerve cells ƽ fibroblasts ƽ inflammatory cells
5 Arrangement of liver cells Two diagrams of liver structure removed for copyright reasons. Source: Cormack, Clinically Integrated Histology.
6 Histological structure of liver Photos removed for copyright reasons. Fig.1: The direction of blood flow (arrow) from the branch of the portal vein (V) toward sinusoids (S) in the liver, (D) bile duct, (A) branch of the hepatic artery. u344 Fig.2: The direction of blood flow (arrow) from sinusoids (S) to the central vein (V) of the liver. u 140 Fig.3: A sinusoid (arrow) emptying into the central vein (V) of the liver. u 344 Irwin Berman, Color Atlas of Basic Histology
7 Functions of liver cells ƽ Intricately involved in carbohydrate, fat, and protein metabolism. ƽ Store vitamins and minerals; form specific compounds such as coagulation factors and somatomedins or growth factors. ƽ Filter the blood, removing organic by-products, cellular debris, and many other particles. ƽ Produce and secrete bile. ƽ Detoxifie or excrete cholesterol, steroid hormones, drugs, pesticides, and other toxic compounds
8 Liver Cells Structure and function of Liver cells Regenration of liver cells Liver cells research in relevant to liver tissue engineering
9 Liver Regeneration Prometheus alleged phenomenal powers of liver regeneration are enshrined in Greek mythology The most widely studied model of liver regeneration is the rat liver after two-thirds partial hepatectomy (PH), involving removal of the median (M) and left lateral (LL) lobes. regeneration in the residual lobes restores preoperative liver mass within a few days. Malcolm R. Alison CELL & DEVELOPMENTAL BIOLOGY, vol13, 2002,
10 Factors related to Liver regeneration Cell sources Hepatocytes, hepatic stem cells (oval cells) and bone marrow diliverstem cells Growth and regulating factors HGF, EGF, TGF-E,TNF TNF-D,, IL-6, IL-1, VEGF Influences of non-parenchymal cells Stellate cells, Kupfer cells, endothelial cells
11 Liver regeneration during injury Hepatoblasts Ductal Plate NORMAL Mature Hepatocytes Canal of Hering Bile Ducts DISEASE & REGENERATION Small Hepatocytes Oval Cells Atypical Ductules Bone marrow stem cells The blue boxes: bone marrow haemopoitic stem cells have been incorporated On the top: normal fetal liver development On the bottom: disease and regeneration Figure by MIT OCW. After Crosby et al., Cell and Developmental Biology. H.A. Crosby et al. CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp
12 Interactions between cells during regeneration IL-10 (-ve) TNF- (+ve) BILIARY EPITHELIAL CELL TNF TNF- (+ve) Apoptosis inhibited TNFRI NFK TGF- (-ve) KUPFFER CELL IL-6 (+ve) TGF- (-ve) IL-1 (-ve) gp 80/IL-6R gp 130 cmet gp 80/IL-6R gp 130 Activated STAT 3 HGF HGF (+ve) IL-6 (+ve) SINUSOIDAL ENDOTHELIAL CELL HEPATOCYTE TGF- (-ve) HGF (+ve) STELLATE CELL Figure by MIT OCW. R. Malik et al. CELL & DEVELOPMENTAL BIOLOGY, Vol. 13, 2002: pp
13 Liver Cells Structure and function of Liver cells Regenration of liver cells Liver cells research relevant to liver tissue engineering
14 Liver Tissue Engineering Cell sources Compatibility of materials to hepatocytes Cytological research related to tissue vascularization
15 Hepatocyte sources Primary hepatocytes Tumour-derived cell lines HepG2 and C3A Embryonic stem cells Adult stem cells ƽ small hepatocyte ƽ oval cell ƽ bone marrow derived stem cell
16 bone marrow Stem cells Hapatocytes Bile ductular cells Endothelial cells Stem cells Functional cells Tissue and Organ
17 From BMSCs to Hepatocytes Culture medium: DMEM, IMDM, Growth Factors: HGF, EGF and extraction of regenerative liver tissue ECM: Collagen coating, Poly-Lysine coating Identify methods: Morphology observation Immunofluorescence(Albumin, CK8, CK18), RT-PCR (Albumin) Radioimmunon analysis (AFP )
18 From Bone Marrow Cells to Hepatocytes Photos removed for copyright reasons. BMSCs in IMDM BMSCs+HGF+EGF Photos removed for copyright reasons. CK18 Albumin
19 From BMSCs to Hepatocytes Effect of Partial Hepatectomy Experiment I: Animal : Kunming mouse Group A: sham operation (n=20) Group B: partial hepatectomy (2/3)( (n=20) BMSCs isolation At 12h, 24h, 36h 48h, 72h after operation respectively BMSCs culture BMSCs were cultured in IMDM +HGF + EGF Immunoflurescence stain Counting the ALB positive cells and calculating the differentiation rate ALB positive rate: At 24h following operation: Group A: %, Group B: 9.83 % (P<0.05 <0.05)
20 From Bone Marrow Cells to Hepatocytes Albumin staining Photos removed for copyright reasons. CK 18 staining sham operation (24h) partial hepatectomy (24h)
21 From BMSCs to Hepatocytes Effect of Partial Hepatectomy Experiment II: Animal : Kunming mouse, partial hepatectomy (PH 2/3) Liver tissue lixivium (LTL) Regenerative liver tissue were extracted at 36h after PH BMSCs isolation and culture BMSCs + IMDM BMSCs + LTL BMSCs + IMDM +HGF + EGF BMSCs + IMDM +HGF + EGF+ LTL Immunoflurescence stain Counting the ALB positive cells and calculating the differentiation rate
22 From Bone Marrow Cells to Hepatocytes BMSC labelled by BrdU Photos removed for copyright reasons. Photos removed for copyright reasons. Induced cells labeled with BrdU(CLSM) Distribution of induced cells labeled by Brud in liver fibrosis tissue (liver tissue section) Red: albumin positive Green: BrdU positive Orange: albumin+brdu positive
23 Endothelial Cells Source Primary endothelial cells Endothelial progenitor cells(epcs) Embryonic stem cells Bone marrow derived stem cell
24 From Bone Marrow Cells to Endothelial cells Photos removed for copyright reasons. Rat BMSCs At 14 day after induced Photos removed for copyright reasons. vwf-fitc(vegf ) 7day FLK1(VEGFR-2)-TRITC 14 day
25 Liver Tissue Engineering Cell sources Compatibility of materials to hepatocytes Cytological research related to tissue vascularization
26 Evaluating biocompatibility of scaffold materials Liver cells isolation and culture Contrast microscopy Scan electronic microscopy SEM SEM Laser confocal microscan system (LSCM) Biochemical analysis of culture medium
27 Several slides removed for copyright reasons.
28 Liver Tissue Engineering Cell sources Compatibility of materials to hepatocytes Cytological research related to tissue vascularization
29 Several slides removed for copyright reasons.
30 Research Group Institute of General Surgery General Hospital of PLA Xu Yingxin, M.D Wu Shihe, M.D An Weide,M.D Cao Liang,M.D Song Xuhua, Wang Jingjing, Biomaterial lab, MSE Dept Tsinghua University Feng Qingling, Ph.D Wang WenJie, M.S Wang Xiaohong, Ph.D Li Dapeng, M.S Zhang Yong, Ph.D Lv Qiang, Ph.D
31 Thank you!
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