The Role Of Pancreatic Stellate Cells On Neovascularization Of Human Pancreatic Carcinoma

Pancreatic stellate cells are important effect cells on the occurrence and development of chronic pancreatitis. Recent studies had showed that PSCs played an important role in the growth, invasion and metastasis process of pancreatic cancer too. Our previous study had confirmed that there were a lot of activated PSCs around human pancreatic cancer cells, together with an obvious neovascularization and a large mean density of blood vessels in tissues of human pancreatic cancer, which suggested that PSCs may play a role in the early invasion and metastasis of pancreatic cancer. We supposed that there were interactions between PSCs and pancreatic cancer cells. Pancreatic cancer cells may secrete a lot of growth factors to stimulate the activation of PSCs, and activated PSCs may secrete MMPs to degrade extracellular matrix and stimulate the neovascularization process, which lead to the early hematogenous metastasis of pancreatic cancer. This study will investigate the role of PSCs in the early hematogenous metastasis of pancreatic cancer from the point of angiogenesis.This study was divided into four parts:PartⅠ: The expression of pro-antigenic growth factors VEGF, IL-8 and bFGF in cultured PSCs.To detect the expression and activity of pro-antigenic growth factors VEGF, IL-8 and bFGF in primer cultured PSCs. Human PSCs were isolated from pancreatic cancer and normal pancreatic tissues firstly, with being detected the expression of VEGF, IL-8 and bFGF in cultured PSCs by ELISA, RT-PCR and Western Blot. Human PSCs expressed VEGF, IL-8 and bFGF in genetic and proteinic level. The supernatants of cultured PSCs contained large amount of VEGF, IL-8 and bFGF cytokine. This suggested that human PSCs could secret of VEGF, IL-8 and bFGF by themselves.PartⅡ: The effects of PDGF and its monoclonal antibody on secretion of VEGF, IL-8 and bFGF by PSCs.Different concentrations of PDGF or its monoclonal antibody were added into media during the culturing of PSCs, and their effects on the expression of VEGF, IL-8 and bFGF were observed. PDGF-BB (10, 25 and 50ng/ml) and PDGF monoclonal antibody (5 and 10 ng/ml) were added into culturing media. Human PSCs were cultured with PDGF-BB or its antibody for 36 hours before harvested. RT-PCR and Western Blot were used to detect the expression of VEGF, IL-8 and bFGE The effects of PDGF and its monoclonal antibody on the expression of VEGF, I1-8 and bFGF were semi-quantitative analyzed. PDGF-BB could up-regulate the expression of VEGF and IL-8, and 50ng/ml PDGF-BB had the most obvious up-regulation effects. The expression of bFGF was not affected by PDGF-BB. The monoclonal antibody of PDGF-BB could inhibit the expression of VEGF and IL-8 to a certain extent. PDGF-BB may up-regulate the expression of VEGF and IL-8 in cultured PSCs.PartⅢ: The effects of PSCs on the angiogenesis process of pancreatic cancer observed by studies in vitro.To observe the effects of PSCs on HUVECs' proliferation and formation of tubular structure by studies in vitro. The effects of supernatants of PSCs on the proliferation of HUVECs were evaluated by MTT assay and flowcytometry. Tubular formation assay were used to evaluate the effects of PSCs' supernatants on the tubular formation ability of HUVECs. Supernatants of PSCs can significantly stimulate the proliferation of HUVECs, without significant differences between different concentrations of PSCs' supernatants. Flowcytometry had showed PSCs supernatants could raise the total ratio of HUVECs in S phase and G2/M phase. In tubular formation study, after the stimulation of PSCs supernatants, HUVECs connected each other and formed hollow tubular structure. PSCs supernatants could significantly stimulate the proliferation of HUVECs in vitro.PartⅣ: The effects of PSCs on the angiogenesis of SW1990 pancreatic cancer cells observed by studies in vivo.To show the effects of PSCs' supernatants on angiogenesis of pancreatic carcinoma in vivo by examine the mean vascular density (MVD) in nude mice model and blood vessel growth in chorioallantoic membrane model (CAM). Human PSCs and SW1990 cells were cultured in vitro. Nude mice were divided into three groups, and were differently hypodermically injected into SW1990 cells, PSCs and SW1990 cells with PSCs. Tumor size and weight was evaluated by 21days after injection. The MVD was evaluated by detecting the expression of CD34 in tumor tissues. The proliferation of SW1990 cells in tumor tissues were evaluated by detecting the expression of PCNA. In chorioallantoic membrane model, SW1990 cells suspended with PBS or PSCs' supernatants were dropped onto the surface of chorioallantoic membrane and cultured for 72 hours. The number and diameter of blood vessels around the tumor was evaluated. PSCs and their supernatants could stimulate the proliferation of human pancreatic cancer cells in nude mice model, and PSCs had a stronger stimulation effects than their supernatants (P<0.05). The MVD of these groups were 35.12±12.89, 27.28±10.14 and 9.06±3.12, and the PCNA index of these groups were 87.32±31.51, 86.21±28.9 and 64.32±11.28. PSCs groups had no tumors formed in CAM. SW1990 cells with PSCs supernatants groups had larger number and diameter of blood vessels than control groups. PSCs could stimulate neovascularization process around caner tissues, and this may be related to the early hematogenous metastasis of pancreatic cancer.