| Backgrounds: The mission of military preventive medicine is to research and service the army members,and study the effect of the health of military members and danger caused by military environment,weaponry,military operations and other factors,such as,lifestyle,health services,such as biological inheritance and the protection,prevention and control the occurrence of injury and popularity through micro and macro research,and to promote physical and mental health and improve the battle effectiveness.As the largest organ detoxification in body,the liver can transform many non-nutrients such as poison,all kinds of drugs,and metabolites in the body that both come from in vivo and in vitro with its biotransformation.Liver metabolism can eject them out of the body in prototype or complete decomposition.But liver is easily affected by the special way of life,psychological factors and chemical warfare agents such as N-dimethyl nitrosamine,biological warfare agents brucella bacillus,toxin from botulinum,etc,under the unique condition,members gather in military would finally suffer from influenza a or hepatitis b,viral hepatitis,liver cancer or cirrhosis of the liver,this affect the army personnel health a lot,cause fatal damage,and finally impact the operational capability.There are too many people suffer from liver disease in our country,because of the long duration and the high fatality rate,it will greatly affect the quality of our national people and influence the construction of our national defense.Liver failure remains a dramatic,unpredictable disease with high mortality rates ranging from 60 to 90 percent.Many studies have shown that liver failure is caused by a large number of liver necrosis resulting in a widespread toxic substances in the blood,causing the liver to function as an acute decline.Hepatic encephalopathy,an acute and chronic liver failure of severe neuropsychiatric complications,is closely related to the high concentration of ammonia.The current treatment of liver failure is mainly based on reducing ammonia production and increasing the absorption of ammonia in the gut,and it works through rifaximin and lactulose.Orthotopic liver transplantation is the only treatment for liver failure,but the amount of liver that can be used now is limited,artificial liver can provide some nutrient requirement for the patient and eliminate harmful substance in the body,it is has been the effective reserve treatment method of liver transplantation.The newly isolated human liver cells are the most desirable cell source for drug development,clinical research,and biological artificial liver,however,due to the severe shortage of liver donor and the difficulty to keep the cells functional in vitro,etc,making it impossible to extend the application scope.And the analysis model of other cells,such as the original liver cells from the animal source,or the cell lines of human liver cancer,they all can't compare to normal human liver cells for their functions are exponentially different from those of the original liver cells;stem cells are cells that are self-replicating and of differentiation potential to be induced into a variety of cells and also of low immunogenicity,there were no obvious side effects or adverse reactions in clinical use,so the medical community called it "universal cell." It is thought to be the seed cell that is ideal for biological artificial liver.Based on the research of many scientists from all over the world in recent years,liver cells differentiation from stem cells is the cells that most close to normal liver cells,but the present bottleneck problem is that this kind of differentiated liver cells in vitro,may not show the perfect function,for the presence of transcription factors or proteins that do not adequately match the specific expression in mature liver cells,or there are very few mature cells,and it is very difficult to apply them on a large scale.The liver in body is a huge "chemical plant",taking many functions such as sugar,protein,and lipid metabolism,synthesis of bile,albumin,detoxification,immune,production of blood coagulation factor,keeping electrolyte balance.For patients with hepatic disease are often accompanied by hepatic encephalopathy with hyper-ammonia,so we focus on the speed limit enzyme beginning in ammonia metabolism cycle,aiming at get enough cells that could be used to clear ammonia and be able to operate in vitro,to evaluate the differentiated liver cells' mature situation through direct observation.Carbamyl phosphate synthetase ?,it exists in hepatic mitochondria,promote the transformation of toxic ammonia into non-toxic product urea and it is vital to maintain the function of liver cells used in the artificial liver for the treatment of liver disease.This kind of transformation ability of carbamyl phosphate synthetase ?can be a mature index of liver cell function,telling us that small molecule compounds induced CPS1 expression could also increase the other functions of liver cells,or,which reduced function and expression of CPS1 may could change the cell morphology,reverse cell fate.For the cell fate may be reversed,somatic cells that terminal differentiated can be reprogrammed to pluripotent stem cells and express some specific transcription factor,shows that the fate of cells is reversible.The combination of different lineages specific transcription factor could also directly transfer human and mouse cells to differentiate into myocardial cells,liver cells,or neurons,and provides alternative way for model building and regenerative medicine.Compared with the genetic recombination factors,small molecules compounds used in chemical methods with cell permeability,and easy to synthesise,protect and standardize,and also have the plasticity effect at the same time.In addition,we can adjust the whole process through the control concentration and duration of small molecule compounds.Via chemical methods to control several signaling pathways and regulate special gene expression of transcription factors of cells,so as to change cell morphology and functions.This way bypass the technology and safety problems that brought up by other cells reprogramming method,bringing hope to "incurable" disease that defined by the traditional treatment method.Objectives: The main challenge is how to obtain enough cells that can be used in biological artificial liver cell in vitro detoxification,especially aiming at scavenging free ammonia in the body and improving the effect of the biological artificial liver in clinical use,and could be able to operate in vitro,to evaluate the differentiated liver cells' mature situation in vitro through direct observation.Then we can optimize the conditions that the existing drugs act on liver cells on this basis,and to improve the efficiency as well as to depress the loss from the drugs.Methods: Making use of the CRISPR/Cas9 technology to built the sg RNA and the donor carrier that target CPS1,and the two plasmids were transferred to the hepatic hepatic cell line Hep G2,the permanent biochemical cell line LO2 and human embryonic stem cells by electric instrument and reagents respectively.Using PCR amplification techniques to inspect DNA fragments from cells.The groups of cells that have been edited sorted by flow cytometry.Using immunofluorescence to target,screen,and identify the target proteins.The expression of genes related to liver function from different fluorescence intensity cells were analyzed by real-time fluorescent quantitative PCR.The albumin secretion,CYP3A4 enzyme activity,ammonia removal ability and urea formation ability of cells with different fluorescence intensity are tested and analysed by the specifical kits.Statistical analysis reports of the impact from different drugs and different drug concentration to the fluorescence intensity of the cells were worked out by the high throughput screening system.The toxicity influence of different drug concentrations on cells were tested with CCK8 kits.Examined the changes of carbamyl phosphate synthetase ? in protein level after resveratrol and si CPS1 treatment by Western Blotting.We also evaluated the impact of these drugs on cells by observed the form of LO2 cell clone,and preliminarily guess that these results may related to reprogramming.Results: Gene sequencing shows that we built the donor plasmids correctly;G418-screened Hep G2,LO2 cells and human embryonic stem cells are labeled by the enhanced red fluorescent protein(td Tomato)which from donor plasmid and express red fluorescent protein spontaneously.Results show that the size of PCR amplification of the DNA fragment from cells were correct.Then divided these cells with different fluorescence intensity into groups by flow cytometry,and the target protein was identified by immunofluorescence staining.We finally get cell clones that express mature CPS1 in mitochondria.There are five Hep G2-CPS1-2atd Tomato cell clones,and four LO2-CPS1-2atd Tomato cell clones.The results indicates that Hep G2 cell clones and LO2 cell clones with different fluorescence intensity shows different genes expression levels including CPS1?AAT?ALB?CYP450 and TF,different albumin secretion,CYP3A4 enzyme activity,ammonia removal ability and urea formation ability.These is a positive correlation between the fluorescence intensity in Hep G2 cells and LO2 cells with all these functions,therefore,we screen the small molecule compounds intuitively through the increase and decrease of fluorescence intensity on this basis.And sodium phenylbutyrate and resveratrol can significantly improve cell fluorescence intensity,CPS1 expression level and a mmonia removal and urea formation ability among these compounds,in addition,they also improve the gene expression effectively of AAT,ALB,TF,CYP1A2,CYP2E1 and CYP3A4,C/EBP?,HNF4?,FOXO3 a.And resveratrol and si RNA,respectively enhance and inhibit the red fluorescence intensity also enhance and inhibit the of CPS1 protein levels.When stem cells which the CPS1 gene are marked with red fluorescent protein induced into mature liver stage,their red fluorescent protein in mitochondria began to express,and this once again proved that expressed in mitochondria CPS1 is truly mature.Small molecule compounds weakened the fluorescent intensity of the Hep G2 and LO2 cells can also change the cell morphology and characters and improve the expression levels of genes related to stem cells in the long-term cells culturing process.Conclusions: We established CPS1-2atd Tomato liver cell line Hep G2 and LO2,embryonic stem cells report system based on the gene targeting technology of CRISPR/Cas9,combined with cell image screening to choose the small molecule compound on regulation of cell fate and the directional differentiation of human embryonic stem cells into hepatic cells system,can make up a reliable model of people liver cells that have stable metabolism in vitro,thus having extremely vital significance providing the donor cells for later use,establishing humanized animal model in vivo and using the differentiated stem cell to drug screening in vitro.What's more,if we can get liver cells that have similar protein expression and m RNA transcription level to human adult liver cells by using this evaluation system,then the cells from this evaluation system is likely to be used in the dectection of drug liver toxicity,and the assessment of drug interactions.More importantly,if the small molecular compounds screened can promote cell reprogramming,then we can open cell reprogramming gate through chemical methods. |
PDF Full Text Request