| Pancreatic cancer is a common malignant tumor with difficult early diagnosis, patients in advanced stage post-surgery showed poor 5-year survival rate less than 5%. Incidence of pancreatic cancer in recent years has rise rapidly and presented obvious trend of getting younger over the disease. Less specific symptoms in early stage, rapid growth, early distant metastasis and poor prognosis were all Pancreatic cancer characteristics.Regulatory T cells(Tregs) was a specific T lymphocyte subgroup present negative control on immunity. Plenty of archives have demonstrated that patients with multiple malignancies including pancreatic cancer gain an increased CD4+CD25+Treg than normal control, which was associated with poor prognosis. Chemokine CCL22 showed chemotaxis on CD4+CD25+Treg because the latter can express ligand of CCL22--CCR4. It has also been verified that CCL22 can be secreted by a myriad of pancreatic cancer cell lines.CCL22 Expression can be regulated at transcription level by genes such as NF-kappaB and AP-1. The proto-oncogene Pim-1 with serine/threonine kinase activity can up-regulate NF-kappaB transcription. The Pim family has three members:Pim-1,Pim-2 and Pim-3. Among them, Pim-3 showed homology with Pim-1, so it is reasonable to assume that:Pim-3 can also be capable to up-regulate NF-kappaB transcription, then up-regulate CCL22 secretion, due to its high homology with Pim-1. It has been known that Pim-3 was overexpressed in pancreatic cancer and colon cancer tissue and absence in normal tissue. Also Pim-3 can up-regulate pSTAT signaling pathway activity, while the latter is an important medium of various tumorigenesis. However, the pathophysiological mechanism Pim-3 is not fully understanded. Our research will try to explore relation between Pim-3 and CCL22, and determine whether Treg in different location can be used to predict recurrence/metastasis and prognosis of pancreatic cancer patients.PART 1 Correlation between Pim-3 and CCL22 expression and Treg distribution in pancreatic ductal adencarcinomaObjective:To Explore whether Pim-3 and CCL22 was linked to Treg distribution in pancreatic ductal adencarcinoma tissue, and whether the three markers above can predict recurrence, metastasis and prognosis of PDAC patients.Method:Flow cytometry was used to detect Treg in peripheral blood, tumor draining lymph nodes(TDLN), and immunohistochemistry(IHC) for Pim-3 and CCL22 expression and Treg amount in tumor tissue.61 patients’ clinical and pathological characteristics were retrospectively reviewed to analyse the association with Pim-3,CCL22 and Treg.Results1. Flow cytometry test:TDLN Treg ratio is higher than in the peripheral blood and normal control peripheral blood, the difference was statistically significant.2. IHC testing:tumor cell Pim-3, CCL22 expression and tumor infiltrating lymphocytes FOXP3+ Treg Numbers were significantly higher than that of normal tissue adjacent to carcinoma.3. Survival analysis1) A total of 54 in 61 PDAC patients got complete follow-up information and 7 patients lost. The disease-free survival (PFS) rate is 44.4%(24/54),while the overall survival (OS) rate 53.7% (29/54);recurrence/metastasis rate 55.6%(30/24), the morality 46.3% (25/54)2) Kaplan-Meier univariate analysis showed:higher Pim-3 and CCL22 expression and higher TDLN Treg group gained shorter PFS and OS than that of lower Pim-3, CCL22 expression and lower TDLN Treg group,respectively. Cox multivariate survival analysis showed that TDLN Treg was independent prognosis factor of PFS and OS for PDAC patients.ConclusionPim-3 and CCL22 expression was up-regulated in PDAC tissue. Treg ratio in TDLN was higher than that in peripheral blood. TDLN Treg ratio was independent risk factor to predict recurrence/metastasis and prognosis for PDAC patients.PART 2 Correlation between Pim-3 and CCL22 transcription and expression in pancreatic cancer cell lines and ductal adencarcinoma tissueObjective Explore Pim-3 and CCL22 expression in human pancreatic cancer cell line PCI-55 and PKD, the latter of which means knocking down Pim-3 gene stably. Also the expression of the two above were detected in human pancreatic ductal adencarcinoma tissue.MethodsREAL-TIME RT-PCR was used to detect Pim-3 and CCL22 transcription in human pancreatic cancer cell line PCI-55 and PKD. Western Blot was used to detect Pim-3 expression in these cell lines. ELISA was used to test CCL22 secretion in culture supernatant of these cell lines. Immunohistochemistry was used to detect Pim-3 and CCL22 expression in pancreatic ductal adencarcinoma tissue from 61 patients.Result1. The real-time rt-pcr results showed that PCI-55 showed an obviously higher Pim-3 and CCL22 mRNA level than PKD, differences were significant, and the two were positively correlated, difference was statistically significant.2. Western Blot results show that the PKD cell lines’Pim-3 expression is lower than the original PCI-55 cell lines, the difference was statistically significant.3. The ELISA results show that the PKD cell lines showed a lower CCL22 secretion in the culture supernatant than the original PCI-55 cell lines, the difference was statistically significant.4. The IHC results show that The Tumor Infiltrating Lymphocytes FOXP3+Treg amount increased significantly, and CCL22 expression was associated with TIL FOXP3+Treg amount.ConclusionPim-3 was associated with CCL22 at both transcription and expression level in pancreatic cancer cell lines and ductal adencarcinoma tissue, respectively. Also the two marker showed a positive correlation. |
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