| Objective:(1) BMSCs were cultured and detected identificated in vitro.(2) Rat model with ALF was established by injecting D-galactosamine and lipopolysaccharide (LPS). BMSCs were injected via the tail vein and portal vein to study the effect and possible mechanism of BMSCs in treatment of ALF rats, to compare the efficiency of transplantation route of BMSCs transplantation.(3) By measuringthe the expression of cytokine in serum and liver tissue to explore the mechanism of BMSCs transplantation for ALF, to understand BMSCs homing factors, cytokine expression in liver tissue and liver cell apoptosis, to conduct clinical BMSCs transplantation therapy、to provide reliable clinical prognostic evaluation system, to evalute immune status, condition and prognosis of ALF in real-time accurately. Methods:(1) BMSCs were separated from rat bone marrow, labeled by DAPI, cultured and identified by flow cytometry, immunofluorescence.(2)10%D-galactosamine1.4g/kg/second,12hours and0.005%lipopolysaccharide20μg/kg were prepared by intraperitoneal injection to establish rat ALF model and check the levels of serum ALT, AST. The pathological changes of liver tissues were observed by microscope after stained with HE.(3) Through the tail vein and the portal vein injection of the way to transplant BMSCs.66rats were randomly divided into three groups. the control group:no transplantation, given an equal volume of normal saline. Tail vein graft group: to give1.4×107cells/kg for intravenous injection of BMSCs. Portal vein transplantation group:rats were anesthetized, exposing the abdominal cavity, via the portal vein injection of1.4×l07cells/kg of BMSCs for transplantation. At24h,72h,120h and168h after BMSCs transplantation, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were measured by the automatic biochemical analyzer.(4) Pathological changes of the liver were observed by hematoxylin-eosin (HE) staining. Apoptosis was analyzed by TUNEL assay. ELISA was used to dectect the serum levels of CD163, IL-10and the expression of CD163, IL-10proteins in the rat liver tissue were detected by RT-PCR. In situ hybridization and Western blot were measured to determine the expression of SDF-la protein. The expression of VEGF proteins were detected by immunofluorescence and Western blot. Caspase-1and IL-18proteins were detected by immunohistochemistry, Western blot. Results:(1) primary cultured rat BMSCs after passage, expressed CD29and CD90, no expression of CD11b and CD45. We used DAPI to label BMSCs and observed all BMSCs were labeled blue fluorescence under the fluorescence microscope.(2) By using D-galactosamine (D-Gal)/lipopolysaccharide (LPS) to induce ALF model, Model was established, HE staining showed typical manifestations of acute liver injury. Compared with BMSCs transplantation group, the serum ALT, AST levels and liver cell apoptosis of ALF control were significantly increased, and were time-dependent. These results support the modeling success.(3) The serum ALT, AST level of ALF control group were gradually increased with the course of the disease. Compared with control group, a significant improvement of liver functional test were observed in the tail vein group and portal vein group after transplantation120h and168h (P<0.01). But there was no difference between the tail vein and portal vein group (P>0.05).(4) After BMSCs transplantation168h, the tail vein group and the portal vein group liver tissue were seen a lot DAPI-positive labeled cells. ALF model control group had no DAPI-positive labeled cells.(5) The inflammatory cell infiltration was significantly reduced, lobular architecture gradually restored, periportal visibled bile duct hyperplasia in the tail vein group amd the portal vein transplantation group after BMSCs transplantation120h,168h. Compared with the control group, the situation of liver inflammation in the tail vein group transplantation group and the portal vein group were statistically significant (P<0.05). And the difference improved situation of liver inflammation between the tail vein and portal vein group had no significant (P>0.05).(6) Hepatocyte apoptosis in the transplantation group were significantly lower at120h and168h after BMSCs transplantation compared with the control group (P<0.05). The apoptosis index of the tail vein group and the portal vein group was22.00±16.84%,20.60±7.60%respectively after BMSCs transplantation group120h. The apoptosis index of the tail vein group and the portal vein group was18.33±8.78%,12.67±8.78%respectively after BMSCs transplantation group168h. Compared with the control group, the difference was statistically significant (P<0.05). The apoptosis index between the tail vein and the portal vein group had no significant difference (P>0.05).(7) In situ hybridization, immunofluorescence, Western blot results showed that:The expression levels of SDF-la, VEGF protein in BMSCs transplantation group were gradually increased with the improvement of liver function. Compared with the control group, there were significant difference after transplantation120h and168h And the difference of SDF-la, VEGF protein between the tail vein and portal vein group had no significant (P>0.05). Correlation analysis showed that the expression levels of SDF-la was positive correlation with VEGF (P<0.05). The expression of SDF-la, VEGF protein in liver tissue were negative correlation with hepatocyte, the correlation coefficients were-0.293,-0.274respectively (P<0.05).(8) ELISA and RT-PCR results showed that:The expression levels of CD163and IL-10in serum and in the control group were increased with with time prolonged and liver function deterioration. The expression levels of CD163and IL-10in BMSCs transplantation group were gradually decreased with the improvement of liver function. Compared with the control group, there were significant difference after transplantation120h and168h in BMSCs transplantation (P<0.05). And the difference of CD163and IL-10between the tail vein and portal vein group had no significant (P>0.05).There was a positive correlation between the expression levels of CD163and IL-10(P<0.05); And there were significant correlation among the serum level of CD163, IL-10ALT and AST (P<0.01).(9) The expression of Caspase-1and IL-18protein had the same trend in rat liver tissue. The expression of Caspase-land IL-18protein in liver tissue were significantly decreased after transplantation120h and168h compared with the control group (P<0.05). The expression of Caspase-land IL-18protein had no significant difference between the tail vein group and the portal vein group (F<0.05). Correlation analysis showed:The expression of Caspase-1protein was positively correlated with the expression of IL-18protein in liver tissue (P<0.05). The expression levels of Caspase-1and IL-18protein were significant correlation with hepatocyte apoptosis (P<0.05). Conclusion:(1) Using bone marrow adherent screening method can be successfully isolated and cultured, amplified high purity rats BMSCs, and to maintain its original biological characteristics. DAPI can be successfully labeled BMSCs.(2) ALF model can be successfully established by using10%D-Gal1.4g/kg/times, every12hours, two times;0.005%LPS (10mg/support)20μg/kg, intraperitoneal injection of rat. This method was reliable, reproducible, model the formation of stable and had longer survival in rats, which can meet the research needs.(3) BMSCs can homing to ALF rat liver after BMSCs transplantationthrough tail vein and portal vein.(4) BMSCs transplantation ALF can promote the recovery of liver function, reduce inflammation of the liver tissue necrosis. BMSCs had a therapeutic effect on ALF rats.(5) Both tail vein and portal vein migration path can improve liver function, inhibition of hepatocyte apoptosis and reduce the extent of damage liver pathology, but two kinds of ways had no statistically significant difference.(6) BMSCs transplantation can reduce immune ALF liver inflammation, inhibition of hepatic apoptosis.(7) BMSCs can damage the liver-specific homing to promote the secretion of VEGF and promote hepatocyte proliferation and liver vascular regeneration, promote liver tissue repair.(8) The levels of CD163and IL-10were decreased with the degree of improvement of liver inflammation, which reflected the sharp deterioration in liver function and disease severity. BMSCs can reduce CD163and IL-10levels, regulate proinflammatory and anti-inflammatory cytokines to reach a new equilibrium. CD163, IL-10can be used as an early assessment of prognosis of liver transplantation in patients sensitive serum marker proteins indicators.(9) Caspase-1and IL-18played an important role in hepatocyte apoptosis and the pathogenesis. BMSCs can reduce Caspase-1, IL-18levels. Caspase-1and IL-18were expected to be a predictor of ALF and future therapeutic targets. |
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