| 【Background】Recently, liver failure caused by viruses, alcohol, toxicity and metabolism has increased. Especially, acute liver failure, sub-acute liver failure and acute-on-chronic liver failure had become serious problems due to their high morbidity, mortality, complex pathogeny and complications. Liver transplantation is considered as the most effective way to treat liver failure. However, the clinical application was restricted by scarce of organs, expensive fees and immuno-rejection after surgery.Artificial liver support system could partially relieve the toxic materials and was helpful for patients’ liver to regenerate and recover. However, the shortage of synthetic and metabolic ability of mature hepatocytes restricted the treatment effect. Thus, it is necessary to develop bio-artificial liver support system(BLASS) with liver function. Recently, BLASS is becoming one of the effective and promising methods for acute liver failure. BLASS consists of functional cells, bioreactor and assistant device. Among them, the functional cells were the key of BLASS. At current, cell source of BLASS was limited by the technology of culture and preservation of primary hepatocytes and immuno-rejection of heterogeneous liver cells. So, we have to find an ideal cell which can bring into wide production. The development of stem cells provides new hope for the cell source.In our previous study, we compared the expression of mi RNA between human umbilical cord derived MSCs(h MSC) and HGF-induced hepatocytes by chip analysis. Six overexpression mi RNAs(mi R-1246, mi R-1290, mi R-148 a, mi R-30 a, mi R-424 and mi R-542-5p) which were also confirmed by quantitative reverse transcription polymerase chain reaction were identified during the hepatic differentiation. Then, we combined liver-enriched mi R-122 and elucidated six specific micro RNA profiles of hepatic differentiation and transfected into h MSCs. We found that HLC induced by seven mi RNAs(HLC-7) not only exhibited the liver function such as LDL uptake, glycogen storage and urea production, but also illustrated the exciting role in CCl4-injuried mice model in vivo. Later, we continued to screen out and found that mi R-30 a and mi R-1290 were dispensable for hepatic differentiation.In this study, we aimed to ensure the hepatic function of hepatocyte-like cell induced from five mi RNAs(HLC-5) and get the optimal mi RNA combination furtherly based on five mi RNAs pool. We tested the hepatic gene expression and explore their liver function after transfection with less mi RNAs. According to the optimal mi RNA transfection, we triggered acute liver injury and acute liver failure by injection of CCl4. We examined the improvement of liver injury or liver failure by serum parameters, histological analysis and survival condition after HLC transplantation. 【Objectives】1. To investigate the optimal mi RNA combination for the transdifferentiation of MSCs into HLCs.2. To clarify the role of HLCs in the nude mice model with liver injury and liver failure.【Methods】 1. The morphology of adherent MSCs was observed by microscope when cultured for several days. Flow cytometry was applied to test surface markers. The standard condition kits were used to induce the osteogenic differentiation and adipogenic differentiation and illustrate multi-potential ability of MSCs.2. Screening out the mi RNA combination: illustrating the efficiency of transfection and the differentiation ability of MSCs into HLCs by five mi RNAs again(mi R-122,148 a,424,542-5p and 1246). Then, remove one from the pool and test the effect of four mi RNA combination transfection, including liver specific genes by RT-PCR, liver function by LDL uptake, PAS staining and urea production analysis.3. Nude mice were triggered acute liver injury by CCl4 injection. They were divided into three groups, consisting of saline control, HLC-7 positive control and HLC-N induced by the optimal mi RNA combination. 1x106 cells and saline were transplanted into the mice via tail vein. The liver condition was evaluated by serum parameters like AST, ALT and ALB, H&E staining and Sirius Red staining.4. High concentration of CCl4 was used to trigger acute liver failure of nude mice. These mice were treated with saline, HLC-7 and HLC-N randomly through the tail vein. The survival conditions of mice were observed within one week.【Results】 1. The adherent MSCs exhibited fibroblast-like shape when cultured. The result of Flow Cytometry showed that MSCs were positive for marker CD105(99.8%) and negative for CD34(0.2%) and CD31(0.2%), which were the marker of hematopoietic stem cells and endothelial cells respectively. The alizarin red staining and the oil red staining showed that MSCs could differentiate into osteocyte and adipocyte, indicating the multi-potential ability of MSCs.2. The result of five-mi RNA combination transfection into MSCs demonstrated that HLC-5 could increase the expression of hepatic gene ALB, HNF4 A and AFP significantly compared with the negative control, which were similar with HLC-7. While, HLC-5 could not increase the expression of PDX1,Ep CAM and CK7. Most of HLC-5 could uptake LDL and 40% could uptake ICG. Meanwhile, PAS staining illustrated that more than 60% HLC-5 store glycogen. HLC-5 could also produce urea by BUN analysis. The result had a big difference compared with the negative control.3. The result of four mi RNA transfection found that there was no big difference of morphology after mi RNA transfection. There was no big difference about the m RNA level of liver marker gene ALB,HNF4 A,CYP3A4 and TF among different groups. The result of LDL uptake showed that the induced cells from four mi RNAs could not intake LDL.4. After CCl4 treatment, the level of serum ALB decreased a lot, while the level of AST and ALT increased obviously. H&E analysis showed the damage of liver architecture and infiltration of inflammatory cells. Compared with saline treatment, the transplantation of HLC-5 and HLC-7 could improve the serum parameters such as ALB, AST and ALT, and the result of histology improved as well.5. High concentration of CCl4 induced acute liver failure for one day, with the detriment of architecture and inflammatory cells infiltration. According to the observation of survival time within one week, HLC-5 and HLC-7 transplantation significantly improved the survival condition of nude mice suffered from liver failure. Interestingly, there was no big difference between HLC-5 and HLC-7 treatment, indicating the HLC-5 could mimic HLC-7 in vivo.【Conclusion】 This study found that five mi RNA combination, including mi R-122,mi R-148 a,mi R-424,mi R-542-5p and mi R-1246, were the optimal group for the transdifferentiation of human umbilical cord derived MSCs into HLCs. The HLC-5 not only exhibited liver function in vitro, but also showed inspiring results in animal model with liver injury and liver failure, which mimicked HLC-7 in vivo. It provided experimental base for the application of HLC in bio-artificial liver support system. However, the regulation mechanism of mi RNA during this differentiation process and characterization of HLC-5 need further exploration. |
PDF Full Text Request