The Experiment Study Of Anti-Ovary Cancer By Dendritic Cells With Heat Shock Protein Gp96-Peptide Complexes

Ovary cancer is a type of malignant tumor frequently seen in gynecology. The incidence of its occurrence ranks second in the tumors among gynecology procreation system. With the highest death rate, only 20-30% of its patients can survive in five years. Toxic reaction and drug tolerance brought by repeated radiotherapy and chemotherapy, the extreme difficulties of operation, and poor physical and economic conditions of the patients are among the main factors for the victims of ovary cancer to lose hope. It is urgent to find a more efficient adjunctive therapy to treat the main problem of drug tolerance of ovary cancer in the medical field of gynecology.Firstly, disrupt the ovary tumor cell(sSKOV3), then precipitate them by saturated MGW, after affinity chromatography and ion exchange chromatography, heat shock protein 96-peptide complex is extracted. Appraise the complex through SDS-PAGE gel electrophoresis and Western blotting; then make quantitative estimation of it with a UV spectrophotometer. Secondly, put cytokines-GM-CSF,IL-4,TNFa-into CB(cord blood) to induce DC. Observe its maturation process through an inverted microscope and appraise it from cyto-morphology through a transmission microscope. Then further appraise the phaenotype of matured DC through immunohistochemistry and flow cytometry. On the forth day of DC maturation, add the extracted heat shock protein 96-peptide complex, and compare it with the one without the complex. Thirdly, increase the density of carboplatin in the culture solution gradually. After a 12-month inducing cultivation, an anti-carboplatin multidrug tolerance cell strain of SKOV3 is established, and is appraised by immunohistochemistry and RT-PCR. Extract the karyocyte and human PB T lymphocyte from CB, after adding simple DC and the DC activated by the heat shock protein 96-peptide complex into the karyocyte and human PB T lymphocyte from CB, observe their vitro CTR effects on the TCs separately. Based on the above results of vitro experiments, athymic mice of ovary cancer drug tolerance are culturated. Then use the DC activated by the heat shock protein 96-peptide complex to induce the activation of human PB T lymphocyte and form specific CTL cells to conduct adoptive immunotherapy on athymic mice bearing cancer.The result of the experiment shows: 1: the extracted protein processed by SDS-PAGE polyacrylamide gel electrophoresis displays one stain-limpid strap of protein at molecular weight 96kD, which proves to be heat shock protein gp96. Heat shock protein gp96 80-100ug can be extracted from 1X 1010 SKOV3 cells in the protein quantitative appraisement.Secondly. Observe the maturation process of CB oriented DCs through an inverted microscope: on the third day, the cells assemble to be evenly distributed cyto-mass, with some deformed and prolonged. On the sixth day, vague ecphyma can be seen, cyto-morphous being irregular. On the ninth day, typical dendritic processes DCs can be seen in adherence growth and suspension growth sparsely. Under the TEM, the irregular morphous of cellular nucleus, located on the side of kytoplasm, can be seen. In the shape of kidney or horseshoe, the DCs are abundant in kytoplasm, its ratio being multiplied. Also, cell organs and chondriosomes are plenty. Cytolysosomes are less, and the surfaces of the cells are rough, with obvious ecptoma, which are the typical morphous of matured DCs. Examine the cell surface by APAAP, and the CD1a and HLA-DR induction expressions are higher than the ones before induction. The molecule expression of HLA-DR on the matured DC surface is up-regulated, and is significantly different in statistic terms. Flow cytometry also shows the maturity of the DCs, who can more effectively stimulate the secretion of IL-12 and TNF-αthan the immature DCs. When checked about their MLR by MIT, the cultivated cells stimulate more effectively both the proliferative response of allogene T cells and the multiplication of karyocyte of CB, compared with the ones without cultivation, in statistic terms.Thirdly. Establish drug tolerance cell strain of ovary cancer successfully. the index of drug tolerance detection indicates that SKOV3/DDP is tolerant to carboplatin, NSC-123127, Efudex as well as carboplatin. Cytomembrane and endochylema of SKOV3 is stained blue through immunohistochemistry. There are buffy colouration expressed by P-glycoprotein in cytomembrane and endochylema of SKOV3/DDP cell which indicates that cells of ovary cancer have the gene expression of multidrug tolerance. In addition, The establishment of SKOV3/DDP multidrug tolerance cell strain has been further verified by RT-RNA electrophoresis succesfully. Then two kinds of DCs have evident effects on the activation to CB karyocyte and human PB T lymphocyte, but DCs which are stimulated by heat shock protein gp96 promote more powerful activation to kill the non-drug tolerance cell and multidrug tolerance cells of ovary cancer.Finally. Successfully establish drug tolerance cell strain of bearing ovary cancer athymic mice. The tumor achievement ratio exceeds 70%. T- lymphocytes of human peripheral blood, which are vitro stimulated and induced by DC, are used conduct vivo experiment in ovary cancer bearing athymic mice. The result indicates that it can control the growth speed of the tumor, extend the life span of the mice and the competent T- lymphocytes which are stimulated by HSPgp96-PC can cure the multidrug tolerance ovary cancer effectively.On the whole, firstly, the method of extraction and appraisement of gp96 of ovary cancer cell strain is built up. Thereby, it is possible to produce ovary cancer vaccine binding gp96 heat shock protein. Secondly, the cultivation system which can vitro induce DC from monocyte presenting CB is set up. That gp96 can promote not only DC maturing but also CB karyocyte and T- lymphocyte multiplication in human PB is testified.. Thereby, the foundation of DC vaccine stimulated by gp96 is erected. Thirdly, multidrug tolerance cell strain of ovary cancer is made and the vaccine which originats from it has the function of killing the non-drug tolerance cell of ovary cancer and multidrug tolerance cell of ovary cancer, thus providing patients troubled by ovary cancer with an adjunctive therapy. Finally, by establishing the athymic mice model of multidrug tolerance, DCs which are stimulated by gp96 extracted from ovary cancer can induce CTL lethal effect. Furthermore, they can cure multidrug tolerance ovary cancer effectively when injected into bearing cancer athymic mouse. This indicates once more that it is possible to cure multidrug tolerance ovary cancer by immunotherapy.