| The systematical researches concentrating on normal stem cells (NSCs) andcancer stem cells (CSCs) are very important for the diagnosis and therapy ofkinds of diseases. The NSCs are not only essential for the maintaining normalfunction of organism, but also necessary for treating many typed diseases. Thestem cell therapy is one of the most promising strategies to treat acute andchronic end-stage liver diseases. At present, there are many kinds of stem cellsthat can be used for therapying liver diseases, which can be divided into two maincategories, intra-hepatic stem cells and extra-hepatic stem cells. Among thesestem cells, fetal liver stem/progenitor cells (FLSPCs) is a typical representative ofintra-hepatic stem cells. Because FLSPCs have many advantages for cell therapy,such as easy survival, rapid proliferation, small size and high security, they aresupposed to be one of the promising stem cells fro treating liver diseases.Applying a three-step method previously established by our group, in this projectwe isolated FLSPCs from embryonic day14rat fetal livers. The efficiency of ourmethod is compatible with fluoresence activated cell sorting (FACS). Howver, tothoroughly investigate the therapeutic potential of FLSPCs, there are two questions need to be solved urgently. First, how to guarantee FLSPCs inundifferentiated state when they are amplied? Second, how to induce FLSPCs tosteadily differentiate into functional liver cells, such as hepatocytes and bile ductcells. All the above researches are planning to investigate the therapeuticpotential of FLSPCs at least in animal models first.Hepatocellular carcinoma (HCC) is one of the most frequently occurred cancertypes and one of the most deathly cancer types. Although there are someimprovements in diagnosis and therapy of HCC, it still remains big problembecause the clarification of molecular mechanisms responsible for HCC genesisand metastasis is unclear. HCC is speculated to oringinate from a small set ofcells, containing many stem cell properties., considered as hepatic cancer stemcells (HCSCs). Meanwhile, some of these HCSCs are suppsosed to come frommalignant transformation of hepatic normal stem cells (HNSCs), which gainmultiple genetic changes under the stimulation of cancer inductive factors. Inorder to explore similarities and differences between these two kinds of stem cells,we isolated both HCSCs and HNSCs using our established methods. Bycomparing the miRNA and genetic expression profiles, we found many distinctlyexpressed miRNAs and genes between HNSCs and HCSCs.Although HCSCs are speculated from the malignant transformation of HNSCs,there is no sounding evidence to prove it and the detailed mechanism is far fromclear. Among a great number of dys-regulated miRNAs and genes, we selectedthe most down-regulated miRNA, miR-200a (almost lack of expressionin inHCSCs) for thorough exploration. We found that the inhibition of miR-200acould push HNSCs to proliferate at much fast speed, gain the ability to migrate,and in the mean time maintain a status of differentiation block. As a result,through the above mechanisms, the inhibition of miR-200a could finally induceHNSCs to go through malignant transformation. Although the above mentionedfindings have been sequentially published in several high influence journalsincluding Stem Cell Rev, the detailed molecular mechanism responsible for themalignant transformation of HNSCs induced by the miR-200a inhibition is still unclear. We hypothesized some genes, which highly expressed in HCSCs, may beregulated by miR-200a, such as ZEB. To analyze the possibility of dys-regulatedmiRNAs as early diagnostic markers for HCC, we detected the expression ofmiRNA-200a in both HCC tissues and sera during every stage of HCC genesis.We found that the change of miR-200a is much earlier than AFP. That is to say, itmay serve as a potential marker for HCC.[Aims]The aim of this project is to compare the differential biological characteristicbetween HNSCs and HCSCs, and further to investiage the molecular mechanismsresponsible for malignant transformation from HNSCs to HCSCs. The main aimconsists of three detailed purposes. First, address several key issues in the area ofFLSPCs treating liver failure. That is to say, find an optimal culture conditiaon toamplify FLSPCs in large-scale and maintain them in undifferentiated state; obtaina proper inductive strategy to induce FLSPCs efficiently differentiate intofunctional liver cells, such as hepatocytes. Second, systematically compare thebiological characteristic between HNSCs and HCSCs, including self-renewal,directed differentiation, in vivo transplantation. Meanwhile, it is better toestablish the differential miRNA profiles and gene expressions between HNSCsand HCSCs. Third, select some representative dys-regulated miRNA or gene,investigate its roles in the process of malignant transformation.[Methods]Part1: The isolation and identification of HNSCs and HCSCs1. The isolation of FLSPCs by three-step method (in master’s degree thesis)Percoll discontinuous gradient centrifugation (PDGC), differentialtrypsinization and differntial adherence (DTDA) and Percoll continuous gradientcentrifugation (PCGC) were sequentially used to isolate FLSPCs.2. The isolation of HCSCs by a three-step method (in Wang Xing’s degree thesis)After HCC model was completed by diethylinitrosamine (DEN) induction inFisher344rats, the HCSCs were isolated by the three-step method.3. The isolation of side population (SP) from hepatic normal cells (HNCs) After the livers were injured by carbon tetrachloride in Fisher344rats, theendoneous HNSCs were greatly motivated. The HNCs suspensions were gainedby trypsinization from liver tissues. Because SP containing stem cells coulddiscard the Hoechst33342dye and exhibited low fluorescence, the non-stainedSP cells from HNCs (SP-HNCs) were isolated by fluorescence activated cellsorting (FACS). The stem cell properties were identified for SP-HNCs.4. The isolation of SP cells from HTCsAfter HTCs were isolated from DEN induced HCC model, they wereincubated with Hoechst33342dye for isolation of SP cells. Afther the thresholdwas set as the area with double low fluorescence, SP cells from HTCs (SP-HTCs)were isolated by FACS. The CSCs properties were identified for SP-HTCs.Part2: The self-renewal and differentiation of FLSPCs1. In vitro amplification of FLSPCs in undifferentiated stateTo keep FLSPCs undifferentiated, they were maintained in half-suspensionstate by soft agar culture. Actually, FLSPCs were cultured in the middle interfaceof double soft agar mediums containing leukaemia inhibitory factor (LIF).2. In vitro differentiation of FLSPCs by chemical inductionWhen FLSPCs were cultured in adherent state, several kinds of inductivestrategies were used to promote FLSPCs properly differentiate into functionalliver cells. These strategies included induction by adding single dimethylsulfoxide (DMSO) at different concentrations, single hepatocellular growth factor(HGF) at different concentrations, or combination of HGF and DMSO.Part3: The differential nucleic acid profiles between HNSCs and HCSCs1. The differential miRNA profilesThe total RNAs were isolated from SP-HNCs and SP-HTCs. The miRNAarray was used to detect the expression of miRNAs. The upregulation by2foldsand downregulation by one second were identified as dys-regulated, and thedifferential expressed miRNAs were clarified by real-time PCR.2. The differential gene profilesThe inhibitory cut hybrid technology was used to detect the expression of genes in both SP-HNCs and SP-HTCs. The upregulation by2folds anddownregulation by one second were identified as dys-regulated, and thedifferential expressed genes were clarified by real-time PCR.Part4: The malignant transformation from HNSCs to HCSCs1. The cell model of malignant transformationAmong the numerous dys-regualted miRNAs, we selected the mostdown-regulated miRNA, miR-200a as the target molecules. Using RNAitechnique, the miR-200a was inhibited in FLSPCs.2. The outcomes of malignant transformationThe proliferative capacity was checked by flow cytometry. The migrativeability was evaluated by scratch and transwell tests. The in vivo tumorigenesiswas detected by transplanting FLSPCs into subcutaneous of NOD/SCID mice.3. The mechanisms of malignant transformationUsing several widely used miRNA-target gene predicting systems, thepotential regulated genes by miR-200a were forecasted. Comparing with thedifferential gene profiles between HNSCs and HCSCs, the possible targets wereselected. By double fluorescent element enzyme report system, the exact targetgene was identified.Part5: The relationship between the miR-200a level and HCC genesis1. The developing stages of HCC modelThe HCC model was produced by DEN induction. According to thepathological results, the process of HCC genesis was divided into six main stages,listed as below: normal stage, degeneration stage, fibrosis stage, cirrhosis stage,early HCC stage, metastatic HCC stage.2. The expression of miR-200aThe expression of miR-200a in HCC tissues was examined by northernblotting. The miR-200a expression in HCC sera were detected by real-time PCR.3. The analysis of relevanceUsing SPSS13.0statistical software, the relationship between the miR-200aexpression and the pathological scores of HCC genesis was analyzed. [Results]Part1: The isolation and identification of HNSCs and HCSCs1. The isolation of FLSPCs by a three-step method (in master’s degree thesis)FLSPCs were supposed to be located in the first and second layers of PCGC.These cells were with small size, larger nuclear/plasma, highly express stem cellsCD49f and CD133, low expression of mature liver markers. In addition, theisolated FLSPCs could generate mature ALB expressing hepatocytes.2. The isolation of HCSCs by a three-step method (in Wang Xing’s degree thesis)The HCSCs were enriched in the third layer of cells fractions by a three-stepmethod. These cells had larger nuclear/plasma, high expressions of stem cellmarks EpCAM and CD133. In vitro, these cells had strong capacities toproliferate, migrate, be resistant to chemotherapy. In vivo, these cells could causetumors not only in subcutaneous, but also in the liver of NOD/SCID mice.3. The isolation of side population (SP) from hepatic normal cells (HNCs)The proportion of SP-HNCs accounts for4%of the HNCs, these cells were insmall size, with large nuclear/plasma, high expression of stem cell marks, lowexpression of mature liver marks, could differentiate into ALB positive cells byHGF induction. When SP-HNCs were introduce to Fisher344rats with liverinjury caused by carbon tetrachloride, they could repair the acute liver damage.4. The isolation of SP cells from HTCsThe isolated SP-HTCs was2.3%or so of HTCs. These SP-HTCs had largenuclear/plasma, high expression of stem cell markers, and had strong capacitiesto in vitro proliferate, migrate. In addition, by HGF induction, the SP-HTCscould generated mature HTCs with low expression of stem cell markers andsteady expression of AFP. In vivo, these cells could form tumors not only insubcutaneous, but also in the liver of NOD/SCID mice.Part2: The self-renewal and differentiation of FLSPCs1. In vitro amplification of FLSPCs in undifferentiated stateComaring with adherent cultured FLSPCs, the FLSPCs in half suspensionculture of double soft agar medium, were in smaller size, had larger nuclear/plasma, formed more obvious cell clones with strong positive alkaliphosphorus staining. During culture period, the FLSPCs in soft agar culturestably expressed stem cell markers CD49f and CD90, highly expressed immaturehepatic marker AFP, and weakly expressed late mature liver marker ALB.2. In vitro differentiation of FLSPCs by chemical inductionThe most appropriate concentration of DMSO to promote FLSPCsdifferentiation was1%; the optimal concentration of HGF to induce FLSPCsdifferentiation was20ng/ml; and comparing with single use, the combination ofHGF and DMSO could better better induce FLSPCs differentiation.Part3: The differential nucleic acid profiles between HNSCs and HCSCs1. The differential miRNA profilesComparing with SP-HNCs, there were78miRNAs dys-regulated in SP-HTCs,including68miRNAs up-regualted,10miRNAs down-regulated.2. The differential gene profilesComparing with SP-HNCs, there were585genes dys-regulated in SP-HTCs,including524genes up-regualted,61genes down-regulated.Part4: The malignant transformation from HNSCs to HCSCs1. The comfirmation of malignant transformationThe inhibition effect of miR-200a was confirmed by real-time quantitativePCR detection. Because of the miR-200a inhibition, cell proliferated faster thanbefore. In addition, the FLSPCs with miR-200a inhibition got strong migrativeability. Furthermore, afther the miR-200a inhibited FLSPCs were transplanted inNOD/SCI mice, although there was no obvious tumor formed in subcutaneous,they caused sereve damage to liver tissues and even generated tumors in the liver.2. The related mechanisms of malignant transformationAmong the dys-regulated genes, ZEB was predicted to be regulated bymiR-200a. In miR-200a inhibited FLSPCs, the expression of ZEB was increasedgreatly at both mRNA and protein levels. Double fluorescent element enzymereport system certificated that ZEB was the target gene of miR-200a.Part5: The relationship between the miR-200a level and HCC genesis 1. The expression of miR-200a in different stages of HCC genesisIn tissues, the expression of miR-200a began to decrease from the fibrosisstage. In contrast, serum miR-200a dramatically reduced from the cirrhosis phase.2. The correlation between miR-200a expression and pathological scoresFrom the cirrhosis stage, the serum miR-200a level was positively correlatedwith the tissue miR-200a expression. Moreover, the serum miR-200a level wasalso greatly compatible with the pathological scores.[Conclusions]Part1: The isolation and identification of HNSCs and HCSCsUsing our three-step method, both FLSPCs and HCSCs were effectivelyisolated from FLCs and HTCs, respectively; through SP separation, bothHNSCs and HCSCs-like cells were enriched from HNCs and HTCs, respectively.Part2: The self-renewal and differentiation of FLSPCsDouble soft agar culture was more suitable for the amplification ofundifferentiated FLSPCs than adherent culture; the combination of HGF andDMSO could better promote the FLSPCs to mature into functional HNCs.Part3: The differential nucleic acid profiles between HNSCs and HCSCsThere were multiple differential expressed miRNAs and genes betweenSP-HNCs and SP-HTCs, these dys-regulated molecules could be responsible forthe great biological difference between them.Part4: The malignant transformation from HNSCs to HCSCsThe inhibition of miR-200a could promote FLSPCs to go through malignanttransformation probably by directly inhiting the expression of ZEB.Part5: The relationship between the miR-200a level and HCC genesisThe miR-200a expression was closely related to the pathological process ofHCC genesis. Thus, miR-200a could be used as a potential marker of HCC. |
