The Pilot Study Of Immune Escape In Pancreatic Carcinoma Induced By Hypoxia And Its Correlative Signal Access

Objective Pancreatic carcinoma is a frequent digestation system malignant tumor and the best treatment is operation.But because of the low rate of the excision,the multidrug resistance and the high relapse rate,the prognosis of the pancreatic carcinoma is very poor,to find the new treatment approach is now becoming a hot spot.We first examined the hypoxia microenviroment and its associativity with MIC,then we constructed the hypoxia model in vitro and investigated the mechanism of immune escape of the pancreatic carcinoma cell induced by hypoxia. Meanwhile we activated the NO-cGMP-PKG signal access in vitro or in animal model to investigate if it can reverse the lethal effect of the NK cells to the pancreatic carcinoma cells when the lethal effect was weaken by hypoxia.It can provide the new evidence that the activation of the NO-cGMP-PKG signal access can promote the innate immunity system and inhibit the growth of pancreatic carcinoma.Method Part one:Gathered 42 pancreatic carcinoma samples and 9 chronic pancreatitis samples from Jan,2002 to May,2008 in xiangya hospital,all of the samples were obtained by operation.8 normal pancreatic tissue were offered by transplant centre or autopsy.IHC examined the expression of the HIF-lαand the MIC in pancreatic carcinoma and chronic pancreatitis and normal pancreatic tissue,then analysed the correlation between them.Part two:Constructed the hypoxia cultivate model,the PANC-1 cells were cultivated in normal or low O2 concentration.IHC and IFC examined the expression of MIC on the PANC-1 cell membrane.RT-PCR examined the expression of mRNA of MIC A or MIC B.Separated,sorted and amplified the NK cells in peripheral blood,ELISA examined the sMIC A and sMIC B in the PANC-1 cell culture in different O2 concentration or when the NO-cGMP-PKG signal access was restrained or activated. Meanwhile FACS examined the expression of the MIC on PANC-1 cells and NKG2D on NK cells contacted with medium.MTT examined the lethal activity of the NK cells to the PANC-1 cells when the PANC-1 cells were in different O2 concentration or the NO-cGMP-PKG signal access was restrained or activated.Part three:Nude mice were inoculated s.c. with PANC-1 cells to form tumor then divided into two random group:treat group or placebo group.The GTN acted as a treat drug.IHC examines the HIF-1αand MIC in two group samples.RT-PCR and Western blotting examined the MIC A and MIC B mRNA and MIC protein in two groups.ELISA examined the content of sMIC A and sMIC B in serum of two nude mice groups with tumor and normal nude mice.FACS examined the expression of the NKG2D on NK cells in the peripheral blood of two nude mice groups with tumor and normal nude mice.Result 1.IHC showed that the positive rate of HIF-1αand MIC in pancreatic carcinoma are 76.2% and 90.5%,higher than chronic pancreatitis(22.2%,22.2%) or nomal pancreatic tissues(0%,12.5%).The positive rate of HIF-1αin samples(Ⅰ-Ⅱstage) was 57.1%,less than in samples (Ⅲ-Ⅳstage) that was 92.9%.The positive rate of HIF-1αin pancreatic carcinoma with lymphnode metastasis was 91.3%,higher than samples without.In samples of different pathology and clinical stages,the expression level of MIC is significant different(P<0.05).There is inverse correlation between MIC and HIF-1α(Υ=-0.522, P<0.001).2.The purity of NK cells elevated from 13.8%±2.6% to 92.1±3.7% by the MACS immunomagnetic beads system and its amount was enlarged by adding IL-2.The three gas incubation successfully constructed the hypoxia cultivate environment.RT-PCR showed that the mRNA of MIC A of pancreatic cells in normal condition or hypoxia condition were 0.514±0.104,0.540±0.125;the mRNA of MIC B in normal condition or hypoxia condition were 0.663±0.189,0.697±0.176.No significant differentiation was found(P>0.05).FACS showed that NO-cGMP-PKG signal access activating agent can up-regulate MIC on PANC-1 cells and NKG2D on NK cells in hypoxia microenviroment. ELISA also showed that the activating agent may mainly decrease the sMIC A level;MTT showed it can also enhance the activity of NK cells to PANC-1 cells.The effect of inhibitor was averse.3.GTN had obviously depressant effect to the subcutaneous tumor formed by PANC-1 cells.The volume of the tumor in placebo and treat groups after 28 days course of treatment were 1550.4±148.6 mm3 and 750.6±71.2 mm3, the rate of restrain was 51.59%.IHC showed the expression of HIF-1αwas lower and the expression of MIC was higher in treat group (P＜0.05).RT-PCR and Western blotting examines the MIC mRNA and protrein in two groups.It showed that the level of MICA mRNA and MIC B mRNA were 0.748±0.112 and 0.321±0.131 in treat group.In placebo group,they were 0.702±0.157 and 0.298±0.173.No significant deviation (P>0.05).The MIC protein level was 0.688±0.214 in treat group and higher than 0.363±0.123 in placebo group (P＜0.01).Meanwhile ELISA shows that sMIC A and sMIC B level were 15.2±21.4 pg/ml和0±10.2 pg/ml in normal nude mice; sMIC A和sMIC B level were 345.1±125.2 pg/ml and 52.8±25.2 pg/ml in treat group and the sMIC A和sMIC B level are 674.2±205.4pg/ml and 144.4±61.8pg/ml in placebo group.The sMIC level in treat group was lower than placebo group but higher than normal nude mice (P<0.001).FACS showed that the expression rate of NKG2D in blood of normal nude mice was 68.51±19.42%,it was 60.52±10.52% in treat group and 39.88±10.14% in placebo group.The expression rate of NKG2D was higher in treat group or normal nude mice than that in placebo group and it was lower in treat group than that in normal nude mice (P<0.001).It was inverse correlation between expression rate of NKG2D and sMIC level (r=-0.905, P＜0.001).Conclusion The expression rate of HIF-1αin Pancreatic carcinoma was significant higher than that in chronic pancreatitis and normal pancreatic tissues which revealed that the hypoxia microenviroment existed in pancreatic carcinoma.HIF-1αinversely correlated with MIC,that hinted that hypoxia was concerned with the tumor immune escape.The research in vitro proved that the mechanism was MIC shedded from the membrane of pancreatic carcinoma cells then formed soluble MIC.sMIC blocked the NKG2D on the NK cells and attenuated the lethal effect of NK cells.The NO-cGMP-PKG signal access correlated with the immune escape induced by hypoxia,to restrain or active it can increase or inhibit the immune escape.The research of animal model indicated that GTN can attenuate the shed of the MIC from the membrane in pancreatic carcinoma and decrease the sMIC in serum and increase the expression of the NKG2D of the NK cells.It restrained the immune escape and decreased the growth of the tumor. NO-cGMP-PKG signal access activating agent provides a good strategy to aim directly at the immune escape in pancreatic carcinoma and may find a better immune therapy method.