| BackgroundThe shortage of donors for kidneys, liver, heart and other organ transplants is a global problem with one patient dying every16minutes. According to China Red Cross, there are five patients waiting for a suitable donor organ in the US. In the UK, more than8,000people need organ transplantation and every day three people die while waiting for a suitable donor organ. Statistics from the Chinese Ministry of Health show that each yeare nearly1.5million Chinese patients require organ transplants, but only10,000operations are made due to a severe shortage of donors. Making full use of so-called expanded-criteria organs or organs that used to be considered unsuitable for transplant could allevitate the donor shortage to some extent. For liver transplantation, it is helpful to increase the utilization of marginal donor (especially moderate/severe fatty liver graft).Fatty liver graft is easy to develope primary nonfunction (PNF) in the process of liver transplantation (LT) since it is extraordinarily sensitive to ischemia-reperfusion injury (IRI) and immune injury. How to reduce these injures is one of the focuses in the field of liver surgery and transplantation. Mesenchymal stem cells (MSCs) are widely used in a variety of fields in anti-inflammatory injury and tissue repairment for they have the abilities of chemotactic migration, tissue repairment and regeneration. This aim of the present study is to investigate the protective effects and possible mechanism of human umbilical cord mesenchymal stem cells (huc-MSC) on the fatty liver graft after rat LT, and to find a new strategy to improve the utilization of marginal donor. Chapter1Establishment of a rat fatty liver modelObjectiveTo establish a rat model of moderate/severe fatty liver by fat-rich diet feeding.MethodsTwenty male Sprague Dawley (SD) rats, weighting from40to50g, were randomly divided into high-fat diet group and regular control group (ten rats in each group). The rats in high-fat group were fed with a fat-rich diet (48.32%of energy from fat) and the rats in regular control group were fed with standard diet (18.23%of energy from fat). All animals were fed rat chow and water ad libitum. The weight of rats was monitored every week and the hairs, general condition, behavior of the rats were observed every day. All animals were killed by exsanguination (under chloral hydrate anesthesia) after8weeks continued feeding. The wet liver weight and liver index were calculated. Blood and liver speciments were harvested to detect serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC) and triglycerides (TG). The degree of fatty liver was assessed by hepatic pathology.Results1. Body weight and liver index of rats in the high-fat diet group was significantly higher compared with the rats in the regular diet group.2. The levels of serum ALT, AST, and TC in high-fat diet group were significantly higher than those in the regular diet group, which indicated that high-fat diet feeding induced liver cell impairment and cause lipid metabolism disorder.3. Histological examination showed that the liver structure of the regular diet group had no abnormal changes. However, the rats in high-fat diet group developed diffused macrovesicular steatosis, derangement, vacuolar, ballooning change of liver cells, and lobular inflammatory.ConclusionsFatty liver model was successfully established in rats through high-fat diet for continued8weeks, which had a mimic pathological and metabolic change of human fatty liver. Chapter2Protective effects of human umbilical cord mesenchymal stem cells on ischemia-reperfusion injury of fatty liver inratsObjectiveTo investigate the protective effects of human umbilical cord mesenchymal stemcells (hus-MSCs) on ischemia-reperfusion injury of fatty liver in rats.MethodsThe isolation and expansion of hus-MSCs were successfully established in our group. The Wharton's Jelly were isolated and digested by type I collagenase combine with hyaluronidase, and further cultured with the mixture of LG-DMEM and MesenPro medium at a ratio of1:1for further expansion. The rat fatty liver model was established by fat-rich diet feeding. Sixty female SD rats were randomly divided into MSC-treated and control groups (n=30). The rats in MSC-treated group were infused with MSC (2×106cells resuspended in1.5ml of sterile phosphate-buffered saline solution) by intra-venous injection through the tail vein. The first administration was made on day1before IRI, followed by another treatment on day3post-IRI. The rats in control group were injected with sterile PBS alone at the same intervals. Blood and liver samples were collected on day1,4and7(10rats at each time point) post-IRI respectively for enzyme activities, biochemical and histological changes examination. The levels of ALT, AST in serum, and the levels of superoxide dismutase (SOD), malondialdehyde (MDA) and tumor necrosis factor a (TNF-α) in liver tissue were measured. The pathological changes of liver tissue and apoptosis of hepatocytes were also assessed.Results Compared with the control group, the levels of ALT, AST, TNF-a, and MDA were obviously declined in the MSC-treated group on day1and day4post-IRI (P<0.05), while no difference was observed on day7post-IRI (P>0.05). There was no significant difference of the level of SOD between both groups on day1,4and7post-IRI (P>0.05). Pathological examination revealed that the inflammatory injury in the MSC-treated group was clearly alleviated compared with that in the control group.Conclusions1. The hus-MSCs were successfully isolated and expanded using combined enzymes digestion.2. The huc-MSC shows an obvious protective effect on the IRI of fatty liver in rats, which is supposed to be a new strategy to alleviate fatty liver IRI in the field of liver surgery and transplantation. Chapter3Protective effects of human umbilical cord mesenchymal stem cells on the fatty liver graftObjectiveThe aim of this part was to investigate the protective effects and to find the possible mechanism of human umbilical cord mesenchymal stem cells (huc-MSCs) on the fatty liver graft after rat liver transplantation.MethodsThe rats were randomly divided into four groups:①normal liver group (n=4), Lewis rats with eight weeks regular diet feeding.②fatty liver group (n=4), Lewis rats with eight weeks high-fat diet feeding.③MSCs group(n=16), BN rats (fatty liver graft from Lewis rats)were infused with MSCs (2×106cells resuspended in1.5ml of sterile phosphate-buffered saline solution) by intra-venous injection through the tail vein after LT.④control group (n=16), BN rats (fatty liver graft from Lewis rats)were infused with sterile PBS alone by intra-venous injection through the tail vein after LT.Blood and liver samples were collected on day1and day3(4rats at each time point) post-OLT respectively to measure enzyme activities, biochemical and histological changes, and the other8rats were kept for survival analysis.The differently expressed miRNAs in liver tissue between these four groups were detected via miRNA microassay analysis.Results1. Compared to the control group, the levels of ALT, TBIL, TNF-a, and MDA were obviously declined in the MSCs group on day1and day3post-OLT (P<0.05). There was no difference of the level of SOD between MSCs group and the control group on1and day3(P>0.05).2. Pathological examination revealed that the inflammatory injury in the MSC-treated group was clearly alleviated compared with that in the control group.3. There was significant difference in1week survival rates between MSCs group and control group (75%vs12.5%, P=0.01), which indicated that the risk of PNF could be reduced by huc-MSCs treatment in rat LT with fatty liver donor grafts.4. Microarray analysis showed that, compared with the fatty liver group, the miRNAs profile in control group has undergone a drastic change Id post-OLT (29up-regulated and27down-regulated). However, the miRNA profile in MSCs group has a minor change with8up-regulated and8down-regulated genes respectively. The expression of miR-107, miR-186, miR-25, miR-16, miR-34a, miR-339-5p were up-regulated, and the expression of miR-291a-5p,miR-490, miR-874, miR-3568were down-regulated both in MSCs and control groups. Furthermore, compare to control group, miR-375in MSCs group was up-regulated on day1and3post-OLT (2.56and4.22fold change).Conclusions1. Our present study showed that the huc-MSCs protect fatty liver grafts against IRI and immune injury and reduce the morbility of PNF post-LT, which could be a new tool for increasing the utilization of marginal donor.2. The miRNAs profile of fatty liver graft underwent a drastic change post-OLT. Some miRNAs, such as miR-21, miR-375, may play an important role in the process of IRI and immune injury, which still need to be verified by further functional experiments.
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