| Immunosuppressive cell located in cancer tissue microenvironment(FoxP3+Treg,neutrophils)and HIF-la mediated glycolysis effect induced by hypoxia play the important role of promoting cancer cell proliferation,invasion and,the specific mechanism is still not clear.Our previous study confirmed that there was a negative correlation between the tumor differentiation and the prognosis with regulatory T cells in patients with HCC,and also found that sirolimus(SRL)combined Huaier granule(Huaier)and thymalfasin(Thy)(SRL+H+Thy)helps prevent tumor recurrence after liver transplantation.In the rat model of hepatocellular carcinoma,confirmed that high expression of FoxP3 +Treg located in tissue and peripheral blood inhibit T cells by secreting IL-10 and TGF-to induce tumor growth,and SRL+H+Thy therapy can reduce the expression of FoxP3+Treg,inhibit tumor growth,but the molecular mechanism remains need to be further studied,and whether it regulate neutrophil and HIF-1 a pathway induced by hypoxia in promoting tumor growth is still unknown.Therefore,this thesis was based on the prior research to further explore the correlation between FoxP3+Treg,neutrophils and HIF-1 α mediated glycolytic effect with prognosis and differentiation,analysis of risk factors for the prognosis of HCC.At the same time;to explore the regulation effect on the proliferation in HepG2 of hypoxia inducible HIF-1 alpha activation pathway,the anticancer molecular mechanism of the SRL+H+Thy therapy and role in the regulation of HIF-1 a signaling effect,in order to provide the basis for clinical treatment,meanwhile,to evaluate the progonisis value of long-term survival with the sirolimus-based schemes for the liver transplant patients.Based on this,this research is divided into two major parts.The main purpose and content are as follows:Part Ⅰ:Correlation analysis of neutrophils,FoxP3+Treg and glycolytic effect with tumor immunity and prognosis of HCCObjective:1,phenotypic analysis of neutrophil in differentiation hepatocellular carcinoma;2,analyze the correlation of CD274+NEUT and FoxP3+Treg,NLR with the prognosis of HCC;3,correlation analysis of glycolysis mediated by hypoxia inducible HIF-1 a signaling pathway and HCC;4,Cox regression model analysis of the risk factors affecting clinical prognosis of HCC.Methods:To enrolled 136 cases of HCC which diagnosed by clinical and pathological results with the standard of NLR cutoff value,and divided into high-mid differentiated and low differentiated groups,neutrophils phenotype(CD62L and CD274),FoxP3+Treg,absolute count of lymphocytes subgroup in peripheral blood and tumor tissue(NEUT)expression detected with flow cytometry,the relationship between the expression of FoxP3+ Treg with tumor differentiation and its effects on CD8+T cells and IL-10,TGF-β,discusses the roles in carcinogenesis of hepatocellular carcinoma;and immunohistochemical staining was used to detect CD274(PDL-1),FoxP3,and p-Akt p-mTOR,PTEN and p27,HIF-1 alpha and glycolysis in LDHA,GLUT-1,VEGF protein expression in cancer and paracancerous tissues,and analyze their correlation and degree of differentiation of the tumor,and to explore the mechanism of HIF-1 alpha in hepatocellular carcinoma;Cox regression model analysis of the risk factors affecting clinical prognosis of HCC for the two different groups.Results:(1)the expression of FoxP3+Treg in peripheral blood and tumor tissue of HCC was significantly increased,and the low differentiated HCC expression level was significantly increased(P<0.001),CD274+CD62L+CD15+NEUT(CD274+NEUT)levels in poorly differentiated HCC patients had significantly higher expression(P<0.001),the MFI was difference(P=0.021),and was positively correlated with FoxP3+Treg expression(r = 0.58,P<0.0001);at the same time,NLR expression were significantly different in patients with differentiated HCC(P<0.01),and the expression is a positive correlation between FoxP3+Treg and CD274+ NEUT(P<0.001),also found that CD8+T cells in patients low differentiation group was significantly lower than that of high differentiation group,and inhibition of cytokine IL-10 and TGF-beta increased significantly(P<0.01);(2)the MIOD of CD274(PDL-1),FoxP3,p-Akt,p-mTOR,LDHA,GLUT-1,HIF-1 α,VEGF,PTEN,p27 protein were higher in cancer tissues than in the adjacent tissues(P<0.01),and for the different differentiation of HCC,the expression of CD274(PDL-1),FoxP3,p-Akt,p-mTOR,HIF-1α,LDHA,GLUT-1,VEGF in low differentiation in patients and high-mid differentiation patients had significant difference(P<0.01),while the expression of p27 and PTEN was significantly lower than that in high-mid differentiation patients(P<0.01);(3),univariate survival analysis indicated that In different degree of differentiation,survival are significantly different,s relatively short in low differentiation group;multi factor COX regression model analysis showed that NLR,CD274+NEUT and FoxP3+Treg are the independent risk factors for the prognosis of hepatocellular carcinoma(P = 0.024;0;0.006);the relative risk of RR and 95%CI 1.362-.(1.085-1.709);2.631-(2.091-3.310);1.409-(1.105-2.798),and the NLR and Ki67%analysis shows that the two are independent prognostic factors(P= 0),RR value of 1.225 and 1.396;95%CI(1.027-1.041)-(1.277-1.446).Conclusion:(1)FoxP3+Treg and CD274+NEUT are negative associated with the differentiation of hepatocellular carcinoma;and by secreting IL-10 and TGF-β to play the inhibitory on CD8+T cell killing efficiency;(2)CD274(PDL-1),FoxP3 and HIF-1 αinfiltrating in carcinoma,closely related to the degree of differentiation of HCC;and to increase expression of LDHA,GLUT-1 in glycolysis effect and decrease the expression of PTEN to promote tumor growth;(3)Cox regression analysis confirmed that NLR,CD274+NEUT,FoxP3+Treg and Ki-67%are independent risk factors for the prognosis of hepatocellular carcinoma.Part Ⅱ:Mechanism antitumor effect research of SRL and fungi of huai er(PS-T)and its regulation of HIF-1 α on the proliferation and invasion of hepatocellular carcinoma cells.Objective:1 analysis mechanism of sirolimus,PS-T inhibited HepG2 growth and proliferation in cell level;2,analysis of the promoted role of hypoxia inducible HIF-1 αsignaling pathway mediated glycolytic effect on proliferation of HepG2;3,analysis of the molecular mechanism of sirolimus,PS-T anticancer and regulation of HIF-1 a;4,the interaction between cells to prove the effects of SRL+H+Thy treatment on antitumor function effect of T cells.Methods:the HepG2 human HCC cell line divided into normoxia group and hypoxia group,with sirolimus,PS-T as intervention,regulating effect of hypoxia on cell proliferation,at the cellular level by analyzing its inhibitory effect on the proliferation and invasion of HepG2 with cell proliferation assay and cell scratch in the experiment,and flow cytometry analysis of the effect of drug intervention on apoptosis and cell cycle of HepG2;and to analyze the impact of drugs on HepG2 cell proliferation,apoptosis and cell cycle under hypoxic conditions;on this basis,combined with LY294002 and KC7F2 for the intervention measures to RT-PCR detection of tumor growth,proliferation signaling pathway related genes the expression and regulation of hypoxia inducible HIF-1 α signaling pathway mediated glycolytic effect,to explore the molecular mechanism SRL+H inhibited HCC growth and proliferation,then,on the basis of the above results,magnetic beads were used to separate CD8+T and CD4+T cells,and Treg was induced and stimulated.The effects of drug intervention on T cell tumor immune response were analyzed by co-culture with HepG2 under drug intervention.Results:(1)PS-T 8mg/ml(H8)and sirolimus(S50),50 nM LY294002 25 ⒚M(Ly-25)can inhibit the proliferation of HepG2 cells(P<0.05),KCF72 20 M(KC7)intervention under hypoxia has the effect of inhibiting the proliferation of HepG2(P<0.05);(2)scratch experiments suggested that,compared with S50 plus Ly-25,KC7 and Ly-25+KC7,S50+H8 could significantly inhibit the invasion of HepG2(P<0.001),and has time-dose dependence;(3)S50,H8 can significantly promote the apoptosis of HepG2 cells.The apoptosis rate of 70-80%(P<0.001).Oxygen environment,S50,H8 still has a significant promoting effect of HepG2 cell apoptosis,the apoptosis rate of 60-70%(P<0.001),but significantly higher than normal oxygen levels decreased(P<0.001),suggesting that hypoxia can promote the proliferation of HepG2 cells;(4)compared with S50,H8,S50+H8 significantly inhibited cell growth in S/G2 phase,and the difference was significant(P<0.05),under hypoxic conditions,the proportion of cells in S/G2 phase of S50+H8 group increased than of SRL group,there was no significant difference between the PS-T group,but are often less in oxygen group(P<0.05);(5)the RT-PCR showed that CoC12 simulation of the hypoxic environment,can activate HIF-1α lead to accumulation increased,up-expression of LDHA,GLUT-1 mRNA,down-regulation of PTEN,p27 mRNA expression,up-regulation of Akt/mTOR mRNA expression,VRGF,FoxP3 mRNA expression increased;compared with the single drug group,S50+H8 intervention could significantly decrease expression the level of Akt/mTOR mRNA and HIF-1α mRNA,and VEGF,FoxP3 at the normoxia and hypoxia conditions;moreover,at the same time,blocking PI3K/Akt/mTOR and HIF-1 α,enhancement of down-regulation effect in Akt/mTOR,HIF-1,LDHA,GLUT-1 α mRNA,and further reduced the expression of p27,PTEN,VEGF mRNA,with significant difference(P<0.001);(5)Co-culture with PBMCs,S50+H8 increased apoptosis of HepG2 cells,and co-culture with CD8+T,Thy can enhance the killing effect of CD8+T cells on HepG2;combined with Treg can reduce the killing effect of S50+H8 plus CD8+T on HepG2 cells(P<0.005).Conclusion:(1)Cocl2 hypoxia can activate PI3K/Akt/mTOR-HIF-1 aand HIF-1 α-PTEN-Akt pathway to up-regulate the HIF-1 α mediated glycolytic effect,promote the proliferation and invasion of HepG2 cells;(2)SRL plus PS-T reduced hypoxia induced HIF-1 accumulation and decreased expression of HIF-la mediated enhancement effect of glucose metabolism,and through the PI3K-Akt-mTOR-HIF a and HIF-1 a-PTEN-Akt/mTOR pathway play anti-tumor effect;(3)S50+H8 combined with Thy can reduce the inhibitory effect of Treg on CD8+T cells,play the anti-cancer proportion effect.Part Ⅲ:The Survival value of sirolimus based combined programs on long-term graft survival after liver transplantation-5 years of follow-up studiesObjective:Explore the safety and effectiveness of the scheme with sirlimus and thymalfasin and huaier granules on the long-term graft survival after liver transplantation.Methods:The present study retrospectively analyzed data from 36 non-University of California at San Francisco criteria-eligible patients with advanced HCC who underwent LT,and then treated them with sirolimus(RAPA)-based therapy with thymalfasin and huaier granules(RAPA+,n=18),or with tacrolimus-based therapy(controls;n=18).Results:The RAPA+ group had significantly longer recurrence times(P=0.008)and survival times(P<0.0001)(OS,1-year:100%,3-year:94.4%,5-year:55.6%;DFS,1-year:88.9%,3-year:77.8%,5-year:50.0%).Furthermore,compared with pre-LT values and the control group,the RAPA+ group had significantly lower serum a-fetoprotein(AFP)levels(both P<0.0001)and percentage of FoxP3+ Treg lymphocytes(P<0.001)during the first year.In the SRL+ group,FoxP3+Treg and FoxP3+Treg/CD8+ T lymphocyte percentages decreased significantly following LT(P<0.001);however,CD8+/CD3+ T-cells significantly increased(P<0.001).Levels of serum AFP and FoxP3+ Treg cells increased when tumors relapsed and decreased to near-normal when relapse foci were cured or stabilized.Conclusion:RAPA-based therapy appears to be safe and effective in preventing HCC recurrence following LT with no significant adverse events. |
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