Study On The Protective Effects Of HO-1 Transfected BMMSCs On Reduced-size Liver Transplantation In Rats

Objectives: Through the establishment of the model of 50% reduced-size liver transplantation, this study was designed to investigate the protective effects of HO-1/MSCs in rats after reduced-size liver transplantation, and explore the possible mechanisms.Methods:(1) The way of isolating BMMSCs was density gradient centrifugation combined to adherent culture. Microscope was used to observe the cellular morphology. Flow cytometry analyse was used to detect the surface markers of cells. Also, staining technique was used to detect the differentiation ability of BMMSCs to lipocytes and osteoblasts. BMMSCs were infected by recombinant adenovirus bearing HO-1 to form HO-1/MSCs, and fluorescence microscope was used to observe the infection efficiency.(2) Orthotopic group、50% group and 30% group reduced size liver transplantation rejection models were developed, survival rates were observed on 1、3、7 and 14 d. Besides, the levels of ALT, AST and TBIL in the peripheral blood plasma were detected. The liver tissues were gathered, HE staining was used to detected the pathological alteration and rejection levels of specimen of the liver.(3) Frozen section technology was used to detect whether BMMSCs were over distributed in the liver tissue. 50% reduced size liver transplantation rejection model was chose and divided into 4 groups: NS control、HO-1 、BMMSCs 、HO-1/MSCs. After transplantation, the clinical situation and survival rates in each group were observed. Besides, the liver was weighed before transplantation and when the recipients died,then the liver regeneration rate was calculated. The expression of proliferating cell nuclear antigen were detected with immunohistochemical staining method and the levels of HGF were detected by ELISA.(4) Besides, expression of ALT, AST and TBil in the peripheral blood plasma were detected. The liver tissues were gathered, HE staining was used to detected the pathological alteration and rejection levels of specimen of the liver. The ultrastructure of liver tissue was observed under TEM. ELISA was used to detected the expression of IL-10、TGF-β、IL-2、IL-17、IL-23 and TNF-α. And the percent of Treg cells was determined by flow cytometry technology.Results:(1) Cells were attachment cultured with fusiform somata, which fit for the morphological characteristics of typical MSCs. The expression of CD29、CD90 and RT1 A on the cell surface were positive, rates were 97.50%、93.30% and 96.70%, but the expression of CD34、CD45 and RT1 B were negative, rates were more than 99%. Staining results showed that red lipid droplet within cytoplasm by Oil Red O staining, black calcium salt deposit within cytoplasm by Von Kossa staining. At 48 h after itransfected the cells with HO-1 gene recombinant adenovirus vectors, the positive rates of GFP(+) cells were about 85%.(2) The survival rates in each group were as follows: orthotopic group: 1d, 91.20%; 3d, 86.50%; 7d, 81.10%; 14 d, 75.70%. 50% group: 1d, 85.00%; 3d, 80.00%; 7d 62.50%; 14 d, 45.00%. 30% group: 1d, 65.00%; 3d, 50.00%; 7d, 35.00%; 14 d, 17.50%. The survival rates at 1、3、7 and 14 d in 30% group were clearly different with other groups. HE-staining results suggested that the liver tissue at 7d after transplantation in each group all showed immoderate to advanced rejection.(3) Frozen section technology showed that BMMSCs were over dispersed in the liver. The survival time in group NS、 group HO-1、 group BMMSCs and group HO-1/MSCs were separately 13、15、25 and 38 d. The survival time in group HO-1/MSCs had a significant difference than other groups(P<0.05), and group BMMSCs also had a significant difference than group NS and HO-1(P<0.05). Liver regeneration rate, PCNA index and peripheral blood levels of HGF were also the same trend.(4)The results of survival rates and general condition of the recipients in this part were the same as the third part. In the four groups, ALT level raised after the transplantation surgery, reached peak at 3h, then declined to lowest at 3d, and raised to 5d, then declined; the levels of AST were the same as ALT, except for reached to peak after 6h; the expression of TBIL level raised slowly after surgery until 5 days, then raised rapidly until 14 days. The expression of above three index of liver function in group HO-1/MSCs was lower than other three groups(P<0.05),and group BMMSCs also had a significant difference compared to group NS and HO-1(P<0.05). The pathology in group NS and HO-1 was almost the same: the acute rejection after surgery was progressing and HE-staining suggested immoderate to advanced rejection. The situation in group HO-1 is lighter than group NS. The situation in group BMMSCs and HO-1/MSCs was almost the same: the rejectiondegree was light in 7days, but after 7 days, the degree of rejection showed a sharp increase, and there was no significant difference compared to control group at 14 days. The situation in group HO-1/MSCs showed lighter than group BMMSCs. The degree of rejection was classified in each group according to standard of Banff, and the results showed that group HO-1/MSCs was lower than other three groups(P<0.05), and group BMMSCs was lower than group NS and HO-1(P<0.05).The expression of cytokines, IL-10 and TGF-β, were raised in group HO-1/MSCs, while the expression of IL-2、IL-17、IL-23 and TNF-α were lower. The percent of Treg cells were also the same trend.Conclusions:(1) The wall with density gradient centrifugation combined method of separation, purification and amplification was a simple and feasible way to obtain BMMSCs with high cell density, good dynamic and stable growth characteristics. This method was easy to operate, low cost and can meet the basic research and clinical application in large quantities. On this basis, using Adv as the carrier to build HO-1, and transfected into BMMSCs to form HO-1/MSCs laid a foundation for the follow-up study.(2) After continuous training, summary and improvement, 50% reduced size liver transplantation model was stable and conform to the requirements of the follow-up study.(3) Compared with BMMSCs, HO-1/MSCs could promote liver regeneration better after liver transplantation, and may affect the level of HGF to promote liver regeneration. This conclusion provided a new clue and theoretical basis in promoting the clinical application of liver regeneration with HO-1/MSCs.(4) In addition, HO-1/MSCs could play a more effective role to suppress the immune process and had better protective effect in reduced-size liver transplant rejection model. This conclusion provided a new clue and theoretical basis in the application of the HO-1/MSCs to control immune rejection after liver transplantation.