Identification Of Anti-Pro-gastrin Releasing Peptide McAb And The Effect Of Anti-Pro-gastrin Releasing Peptide McAb On The Growth Of Lung Cancer Cell Line

Objectives1.To identify the group and subgroup,specificity,valence and immune response of anti-Pro-gastrin releasing peptide McAb.2.To investigate the effect of anti-Pro-gastrin releasing peptide McAb on human lung cance cell line A549 growth,which supports experimental base for the early diagnosis and therapy of lung cancer based on anti-Pro-gastrin releasing peptide McAb.Methods1.The group and subgroup of McAbs to Pro-GRP secreted by 2A₆,2D₁₂two strains of hybridoma cell were examined by double antibody sandwich method.2.Apply low salt precipitation,McAbs to Pro-GRP were depurated by Protein A-Sepharose-4B collumn(antibody affinity laminar analysis method).3.Though ELISA plank invested by Pro-GRP and independence proteinum,the specificity of anti-Pro-gastrin releasing peptide McAb was identified with McAbs to Pro-GRP secreted by 2A₆,2D₁₂two strains of hybridoma cell and the valence of anti-Pro-gastrin releasing peptide McAb was identified with depurant McAbs to Pro-GRP by ELISA indirect method.4.The expression of Pro-GRP on human lung cancer cell line A549 was determined by immunocytochemistry with depurant anti-Pro-gastrin releasing peptide McAb,which was to identify the immune response of McAbs to Pro-GRP.5.In a series of experiments,the proliferation of A549 cells was evaluated by MTT assay on cells grown in serum-free medium and treated with Pro-GRP and depurant anti-Pro-gastrin releasing peptide McAb.Results1.The subgroups of McAbs to Pro-GRP secreted by 2A₆,2D₁₂two strains of hybridoma cell were IgG2a and IgG2b.2.Apply low salt precipitation,McAbs to Pro-GRP were depurated by Protein A-Sepharose-4B column to obtain depurant McAbs to Pro-GRP.3.ELISA result displayed McAbs to Pro-GRP had better specificity and the valence of McAbs to Pro-GRP was 1:800.4.Immunocytochemical examination showed Pro-GRP was expressed in endochylema and nucelus of A549 cells. 5.After a period when A549 cells were treated with Pro-gastrin releasing peptide and depurant anti-Pro-gastrin releasing peptide McAb,Pro-gastrin releasing peptide stimulated A549 cells proliferation at the concentrations of 1×10^-6mol/L～1×10^-3mol/L in a dose-dependent manner.The proliferation of A549 cells treated with anti-Pro-gastrin releasing peptide McAb diluted at the level of 1:80～1:10,eithor alone or in combination with Pro-gastrin releasing peptide was inhibited,which demonstrated a level dependent relationship.Conclusions1.Depurant anti-Pro-gastrin releasing peptide McAb has better specificity;the expression information of Pro-GRP on human lung cancer cell line A549 is determined with depurant anti-Pro-gastrin releasing peptide McAb,which hints Pro-GRP stimulates the growth of cancer cell by autocrine or paracrine pattern and plays a role in genesis and development of lung cancer.2.Pro-GRP is able to promote human lung cancer cell line A549 proliferation,anti-Pro-gastrin releasing peptide McAb not only inhibits A549 cells proliferation but also intercepts the promoting effect,which hints anti-Pro-gastrin releasing peptide McAb might bind to Pro-GRP secreted by cancer cell in order to inhibit the effect of promoting proliferation.