| Background and aims Pancreatic cancer is one of the refractory malignant tumor in clinical and the morbility rate of people was ascending year by year. Surgical intervention is the most effective way to treat this disease at present, but it is easy to recur and metastasis. Generaly speaking,70%-90% patients were lost the chance to receive radical operation when got the final diagnosis,the survival rate of those who diagnosised pancreatic cancer over 5 years is less than 5%.Thus,it was an important task to seek new therapeutic measures in order to improve the treatment of pancreatic cancer through combined therapy.As the unceasing research of the mechanisms in tumorigenesis,growth and aversion from the angle of the molecular biology and immunology,immunogene therapy which based on immunology and molecular biology brings a new hope for people who suffered from pancreatic cancer.Otherwise,it had reported that most gene therapeutic measures were all adopt single anti-tumor target gene and the therapeutic effect were usually restricted, the results weren't satisfied.Therefore, it is more rationality and superiority for combining multiple genes'therapy to treat pancreatic cancer than singal gene .Moreover, it is bound to the development direction of gene therapy for cancer. The ememging and development of a neoplasma, was a complex process, involving in multi-genes. Single anti-tumor target gene therapy had less stronger enough and limited to supress the tumor cell proliferation.For this reason, use two or more genes'therapy to treat malignant tumor can induce cooperation with anti-ongene,and reinforce the effciency predominantly.NK4 was a kind of HGF specificity antagon, founded by Date in 1997. They got NK4 from schizolysis HGF by pancreatic elastate ,finding that NK4 was composed of 447 amino acids which originated from HGF chain-αand four Kringle domains. HGF,also named Scatter Factor ,was a kind of mutifunctional cytokine which came from interstitial cells and played an important part in adhere,degradation,invasion and aversion in tumor cell. HGF activated its specificity receptor, c-Met,which encouraged tumor cells to move,invasion,degradation of stromous and induced neovascularization.As a result, the tumor happened and then caused metastasis. Researches have indecated that the HGF/c-Met signal transdution pathway had a relationship with the malignancy of pancreatic cancer cell and c-Met showed high expressions in pancreatic cancer tissues.Fibroblast in pancreatic cancer tissues can excrete HGF by paracrine way, inducing c-Met tyrosine phosphorylation which stimulated pancreatic cancer'growth,infiltration and metastasis. [2].According to activated receptor c-Met,HGF taked an important part in regulating tumor'invasion,metastasis and angiogenesis.Thus,NK4 was not only the antagon of HGF,but also the inhibitor of angiogenesis blocking HGF/c-Met signal transdution pathway and neovascularization.therefore NK4 becomes one of the strategy to treat the malignant tumor.[3] Kushibiki[4] etal founded that transfecting NK4 gene can change biological behaviour of malignant pancreatic cancer, through suppressing tumor cells to growth,invasion and metastasis. Murakami[5]'etal indicated that on nude model NK4 can inhibit pancreatic cancer cells transferring to liver;they also founded that NK4 , a inhibitor of neovascularization,can inhibit angiopoiesis induced by HGF,FGF,VEGF.Monocyte chemoattractant protein-1(MCP-1) which belongs to chemotatic factor superfamily CC subgroup, is one of the most activity factors for histoleucocyte, the antineoplastic potency of MCP-1 in intern have been proved in some animal experiments. MCP-1 could inhibit the tumor to growth by T-cell dependent or not T-cell dependent ways[7]. Monti[8]'etal founded that the blood plasma level of MCP-1 in patients who suffered from pancreatic cancer was obviously higher than normal people and patients who were detected in high MCP-1 level had a better prognosis than those detected in low level after surgery operation.the density of MCP-1 in blood plasma had a positive correlation with macrophagus,but had a negative correlation with multiplication of tumor cells.In a word,MCP-1 played a down-regulation part in the process of pancreatic cancer'growth,invasion and metastasis. The futher study had indecated that the reasons of MCP-1 inhibited the tumor to growth and invasion,because MCP-1 could induce mononuclear macrophage intra-tumoral infiltration.This topic aimed to allosterism NK4 and MCP-1 recombinant adenovirus vector respectivly at first.We extracted NK4 and MCP-1 adenovirus by the process of preparation,depuration and appreciation, then transfering NK4 and MCP-1genes into pancreatic cancer cells to acquire the knowledge of the effection on NK4 gene combining with MCP-1 gene suppressing the growth of tumor cell.SW1990 cells were injected into NK4 and MCP-1, Fingally, we detected the growth of tumor cells in vitro. This study aimed to approach the possiblity and possible mechanisms of the treatment with pancreatic cancer by NK4 gene combining with MCP-1 gene.Objective To investigate the effectiveness and mechanism of HGF/NK4 mediated by adenovirus -mediated in inhibiting the growth of pancreatic cancer cells. To investigate the influence and mechanism of adenovirus -mediated MCP-1in the growth of pancreatic cancer cells.To identify the differences between the single gene therapy and the combining therapy.Methods 1. Transfer the NK4 and MCP-1 adenovirus vector into HEK293 cells and utilize viral suspension to infect SW1990 pancreatic cancer cells. 2. Use different MOI's adenovirus vector to infect SW1990 cells to judge the sensitivity. 3. NK4 combining with MCP-1 infect the SW1990 cells, then detected the mRNA expressions of NK4 and MCP-1 by RT-PCR in vitro and the expressions of NK4 and MCP-1 proteins detected by Western Blot. 4. the effection that the clone products put on the growth of vascular endothelial cell was detected by MTT method.Results After different MOI Ad-GFP adenovirus infection, we found that the effciency of infection boost along with the increasing of virus titre when MOI less then 80 efu/cell.when MOI was 60 efu/cell, the effciency of infection by SW1990 was 100%.Because of the toxicity of high MOI adenovirus, this experiment chose 60efu/cell as the infecting condition of SW1990 cells.The expression of mRNA after infection by recombinant adenovirus.Use RT-PCR to detect the expression of MCP-1 and NK4 after adenovirus infection.Take the level of expression of MCP-1 and NK4 infected after 24 hours about 100%, the max expressions of MCP-1 and NK4 that we detected after 5 days were 7.46 and 7.57 times than they were infected 24 hours.As the time goes on ,the level of expression began to decrease. 12 days later, the expressions of both were about 1.6 times,a quarter of expression of infection 24 hours .Through these experiments above, we founded that we used MCP-1 and NK4 to co-infection,the expression of genes show on identical change,otherwise,the expression of each gene was decrease by 30% in contrast to singal infection.We detemined the value of luminosity absorption in 570nm for 6 days using enzyme-labeled instrument after infection, then drew the growth curve according to database.As a result, we founded that the growth curve got from three infection models had no significant deviation with control groups and the expressions of MCP-1 and NK4 genes had no influence on the growth of pancreatic cancer cells in vitro.Conclusions Adenovirus-mediated NK4 and MCP-1 genes can effectively infect SW1990 cells and express a high level of NK4 and MCP-1 genes, which also can secret into extracellular. The peak of expression mantained about 3-7 days. The expression of adenovirus-mediated NK4 and MCP-1 genes'infection had no significant influence on the growth of SW1990 in vitro. However, Ad-NK4 infected pancreatic cancer cells expressing NK4 significantly inhibited HGF stimulating the growth of endothelial cells.It suggests that in pancreatic cancer NK4 inhibiting tumor angiogenesis may be play the role of tumor suppressor. Through the anti-tumor immune mechanisms, MCP-1 plays a key role in inhibiting tumor growth.In our study, we cann't observe that in vitro MCP-1 had impact on pancreatic cancer cell growth , which was consistent with its biological function.Through adenovirus-mediated vector infected SW1990 cells in vitro, we identified NK4 and MCP-1 genes'expression after viral infection having effectiveness and timeliness. Meanwhile, we also confirmed the mechanisms that NK4 may be play the role of inhibition in pancreatic cancer.From the functions of NK4 and MCP-1genes, in order to determine the combined treatment of two genes had anti-tumor effect on pancreatic cancer and the related theories, therefore, we should observe and analyze the findings on the basis of the animal model of pancreatic cancer. The relevant experiments are in progress... |
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