Research On Possible Damage Of Murine Pancreas By HSP65-MUC1 Via Molecular Mimicry

People expected the treatment with better target and lower toxicity all the times, for traditional radiative treatment and chemical treatment killed normal cell as killing tumor cell. Tumor vaccine belong to one kind of independent tumor biology treatment, yet, it usually combined with standard treatment.The ideal tumor vaccine scarcely harmed the normal cells when it got rid of tumor cells.By contraries,Some tumor vaccine prolong patients'life without reduce tumor size compared with former chemical drug,which become one of standards for evaluating tumor vaccine treatment was curative and effective or not.it is neccssary to carry safety and curative rearch on animals for developing tumor vaccine,then,enter stage III of clinical test.HSP-peptide, tumor vaccine, has some charatrers as follows: (1) HSP-peptide was not restricted to recognize special antigen epitope,(2) HSP-peptide induced special CTL that killed tumor cell specially,(3) HSP-peptide can be immunized without adjuvant.As vaccine anti-lung cancerHSP65-MUC1 involved two parts:heat BCG shock protein 65 and MUC1 CTL epitope.The mechnism of fusion protein was HSP65 transfer MUC1 CTL epitope to DCs where CTL epitope was processed and presented by MHCI pathway, then, CTL was activated by epitope.HSP kept conservation during evolution. great similarity existed between HSP from other kinds of pathogens and human HSP,when bacteria invade host, T lymphocytes were primed by HSP60 as foreign antigen through MHCI molecular, since highly homology existed between mammal HSP60 and bacteria HSP60,autoimmunity reaction occured when primed T lymphocytes recongnized self antigen,HSP60, by molecular mimicry.Autoimmune disease was aroused further.So,HSP65-MUC1 fusion protein acted as vaccine to treat lung cancer, researching on curative and safety as HSP65-MUC1vaccine was applied, we concentrate on three parts.The first part made research whether absorption and distribution of tumor vaccine was localized selectively or not, the second part made research whether cross reaction happened under different immunity mode, and cross reaction will give rise to insulin dependent diabetes mellitus. The third part made research on antitumor effective of tumor vaccine on mice which were inoculated with tumor. It basically proved that tumor vaccine developed by our lab was able to induce T cell mediated immune response to inhibit MUC1 positive tumor cell growing, and well done in treatment. MUC1 express highly on ovary cancer, pancrea cancer, liver cancer, stomach cancer, lung cancer, prostate cancer, so, HSP65-MUC1, gene engineering vaccine, was may valuable to therapy and prevention of these cancer. Our work involved several parts as following:1.absorption and distribution in miceTo be sure of HSP65-MUC1 destroying tissue by settling, fluorescence quantitative test detect distribution of HSP65-MUC1 in mice.1.1 Acquirements of HSP65-MUC1, HSP65 and BSA labeled by FITCUnder alkaline condition, FITC labeled HSP65-MUC1, HSP65 and BSA by blending, at low temperature, overnight. The dissociative FITC was filtrated by G25. Protein labeled with FITC was qualified in purity and speciality.1.2 Establishment of fluorescence protein tracer methodeFirst,Total protein concentration in homo-plasm was measured by standard specimen of BSA.Three kinds of fluorescence protein in homo-plasm was determined measured by standard specimen ofthree fluorescence protein . Calculating mass of three fluorescence protein in unit tissue from mice that immunized with three fluorescence protein.Calculational formula as follow :Mass of fluorescence protein (sample-blank)/ Mass of total protein=Mass of fluorescence protein in unit tissue1.3 Research on HSP65-MUC1 distribution in miceWhen mice were immunized with protein labeled with FITC in 40μg dose and for 70 min. Three kinds of protein all distribute a largest amount of drug in intestine, the next one was kidney and liver, the distribute in spleen, lung, pancrear were found less and the distribute in pancrear was the least one among three tissue. The least one was found in heart.1.4 Analysis of HSP65-MUC1 on drug metabolize dynamicsWhen mice were immunized with protein labeled with FITC in 40μg dose and for different time, Three kinds of protein all distribute a largest amount of drug in intestine, The least one was found in heart. Except 240min of immunization, distribution in kidney and liver was inferior to distribution in intestine; there is increasing amount of protein in spleen, on the contrary, it is decreasing in distribution in lung and pancrear which is slightly much more than quantity of heart. 1.5 Analysis of time- and dose-dependent on HSP65-MUC1Three kinds of protein all distribute a largest amount of drug in intestine, the next one was kidney and liver, the distribute in spleen, lung, pancrear were found less and the distribute in pancrear was the least one among three tissue. The least one was found in heart.2 Research on possible damage of normal murine pancreas via autoimmune disease induced by HSP65-MUC1Normal mice immunized with HSP-MUC1, specific lyphocytes were produced in vivo, HSP65 as foreign antigen activated T lymphocytes via MHCI molecular. For mammal HSP60 showed high homology with HSP65 from bacteria, multiple clone T lymphocytes activated by HSP65-MUC1 may cross-reactived with self-reactive antigen, HSP60 and lead to autoimmunization. So, we penetrate with possibility that HSP65-MUC1 cross-reactived with self-reactive antigen and organ specific autoimmune disease induced by HSP65-MUC1.2.1 Generation of CTL specific to HSP65 of mice induced by HSP65-MUC1Mice immunized with HSP65-MUC1 generated multiple specific CTL and HSP65 specific to CTL, except MUC1 epitope specific to CTL.To prove HSP65 as fusion partner have strong immunity, First,proliferation of HSP65-MUC1 specific to CTL were detected via CTL assay in vivo.The results showed mice inoculated HSP65-MUC1 or HSP65 not only produced HSP65 specific to CTL, but generated MUC1 epitope specific to CTL. To explor increasing immunizing treatments impact on cellular immunity effect, Mice was respectively immunized twice and four times under the same condition, then, CTL proliferation of two situation was compared. Specific CTL proliferation of mice immunized was checked by CTL assay in vivo. CTL proliferate remarkably in mice which were detected on 7 days later from twice immunization, the specific killing rate reached over 90%. CTL proliferate slightly in mice which were detected on 7 days later from four times immunization. the specific killing rate missed reaching 60%.The result presented us that mice produced specific CTL in vivo, but it also proved that immunization for multiple time fail to accelerate immune response.2.2. Lymphocytes induced by HSP65-MUC1 cross-reactive with HSP60To detect lymphocytes activated by HSP65-MUC1 or HSP65 recongnize HSP65 or HSP60, lymphocyts proliferation test in vitro was carried on. Spleen cell was separated,and stained with CFSE. The spleen lymphocytes incubated with macrophge cells that were dealed by heat shock or unsettled before dealed with colchicines according to ratio of 1:10.10μg/mL of HSP65-MUC1, HSP65 and Vp1 was added into cell supernatants for five days, cells collected was tested for FACS. The result presented: Lymphocytes from mice immunized with HSP65-MUC1 and HSP65, proliferated by stimulus, such as two forms of macrophage, HSP65-MUC1 and HSP65. The results suggested that lymphocytes generated from mice immunized with HSP65-MUC1 and HSP65 were multiclone, that is, lymphocytes activated by HSP65-MUC1 include lymphocytes specific for HSP65 epitope and lymphocytes specific for MUC1 epitope. Besides, Lymphocytes responsed better to macrophage cell dealed with heat shock, by control with two forms of macrophage as simulus, the lyphocytes proliferate in the presence of HSP60 expressed by macrophge, and the reponse was accelerated when HSP60 upregulated. 2.3 Analysis of possible damage of murine pancrear by HSP65-MUC1By using European Molecular Biology Open Software Suite, amino acid sequence of mammal HSP60 was contrast with amino acid sequence of HSP65 from Mycobacterium tuberculosis. The results proved similarity between HSP65 and HSP60 reach over 60%.3 Research on possible damage of tumor-bearing murine pancreas via autoimmune disease induced by HSP65-MUC1Based on paper, HSP60 as member of heat shock protein family was significantly influenced by temperature and pressure. Accordingly, first, macrophage cells were separated from mice ascites, then, the expression of HSP60 on macrophage cells was altered by changing temperature. when HSP65 and HSP60 polycolonal antibody was used for western-blottin test,Macrophage cells incubating under 43℃and 37℃were collected respectively, it was detected that HSP60 upregulate distinctly ,and HSP65 antibody can cross-recognize HSP60 antigen,3.1 Anti-tumor cellular immunity response of tumor-bearing mice induced by HSP65-MUC1The principle of tumor vaccine is induced specific CTL generated to kill tumor cells, tumor was fadeaway and patients lives were prolonged. Therefore, we researched that whether mice with tumor immunized with HSP65-MUC1 generated specific CTL, and led to eppitope extention as unspecific killing melanin tumor.First, we separated spleen cell of mice with tumor, the spleen cell cocultured with MUC1 positive B16 inactivated by colchicum, B16 inactivated by colchicum acted as negative control, HSP65-MUC1 acted as positive antigen. The cells were cultured for five days, and marked by CD8+ monoclone antibody labeled with PE, FACS check after gathered the cell. Compared with PBS group, proportion of CD8+ lymphocytes increased distinctively when lymphocytes of mice immunized with HSP65-MUC1 were pulsed with MUC1 positive B16. It presented CD8+ lymphocytes specific proliferated. B16 fails to stimulate CD8+ cell proliferate specificly, that is, no epitope extention happened. So, mice induced by HSP65-MUC1 produced CTL specific for MUC1 epitope.3.2 Generation of CTL specific to HSP65 of mice induced by HSP65-MUC1HSP65 as foreign antigen activeted by T lymphocytes via MHC class I molecular, since HSP60 showed high homology with bacteria HSP65, lthe primed T lymphocytes recongnize self HSP60, and lead to autoimmune reaction, the autoimmune disease occured. HSP60 was self antigen on isletβcell, when the primed lymphocytes of mice immunized by HSP65-MUC1 cross- reactived with HSP60, the cross-reaction are likely to cause autoimmun response, then, insulin dependent diabetes mellitus was triggered. First, blood sugar of mice was detected by blood sugar instrument on the time of experiments earlier and each immunization later. The results showed: blood sugar of mice immunized with HSP65-MUC1 or HSP65, fluctuated below normal value, 6.00mmol/L, no abnormity was found. Second, mice was killed and its pancreas were separated which were sent to pathology and physiology lab for slice, the results of HE staining presented that pancreas of mice were clear and found no lymphocytes invading. The mice generated cross-reactive lymphocytes after immunization, yet, the primed lymphocytes failed to response to self antigen, thus, islet was not damage to change blood sugar concentration for lymphocytes invading islet. 3.3 Lymphocytes induced by HSP65-MUC1 cross-reactive with HSP60To detect lymphocytes activated by HSP65-MUC1 in tumor-bearing mice recongnize HSP65 or HSP60, lymphocyts proliferation test in vitro was carried on. Spleen cell was separated,and stained with CFSE. The spleen lymphocytes incubated with macrophge cells that were dealed by heat shock or unsettled before dealed with colchicines according to ratio of 1:10.10μg/mL of HSP65-MUC1, HSP65 and Vp1 was added into cell supernatants for five days, cells collected was tested for FACS. The result presented: Lymphocytes from mice immunized with HSP65-MUC1 and HSP65, proliferated with stimulus, such as two forms of macrophage, HSP65-MUC1 and HSP65. The results suggested that lymphocytes generated from tumor-bearing mice immunized with HSP65-MUC1 were multiclone, that is, lymphocytes activated by HSP65-MUC1 include lymphocytes specific for HSP65 epitope and lymphocytes specific for MUC1 epitope. Besides, Lymphocytes responsed better to macrophage cell dealed with heat shock, by control with two forms of macrophage as simulus, the lyphocytes proliferate in the presence of HSP60 expressed by macrophge, and the reponse was accelerated when HSP60 upregulated. In a whole, to compare with normal mice immunized, ymphocytes in tumor-bearing mice immunized with HSP65-MUC1 proliferated weakly.3.4 Analysis of possible damage of pancrear by HSP65-MUC1HSP65-MUC1 as foreign antigen activeted by T lymphocytes via MHC class I molecular, since HSP60 showed high homology with bacteria HSP65, lthe primed T lymphocytes recongnize HSP60, and lead to autoimmune reaction, the autoimmune disease occured. HSP60 was self antigen on islet　c?ell, when the primed lymphocytes of mice immunized by HSP65-MUC1 cross- reactived with HSP60, the cross-reaction are likely to cause autoimmun response, then, insulin dependent diabetes mellitus was triggered. First, blood sugar of mice was detected by blood sugar instrument on the time of experiments earlier and each immunization later. The results showed:blood sugar of tumor-bearing mice immunized with HSP65-MUC1, fluctuated below normal value, 6.00mmol/L, no abnormity was found. Second, mice was killed and its pancreas were separated which were sent to pathology and physiology lab for slice, the results of HE staining presented that pancreas of tumor-bearing mice were clear and found no lymphocytes invading. The tumor bearing mice generated cross reactive lymphocytes after immunization, yet, the primed lymphocytes failed to response to self antigen, thus, islet was not damage to change blood sugar concentration for lymphocytes invading islet.In conclusion, HSP65-MUC1 fusion protein may induced mice produced effective cellular immunity response, thus, it play an important role in prevention and tumor theropy. We design HSP65-MUC1 labeled with FITC, and made a detailed research on metaboliz.ing and distribute, in addition, HSP65 not only acted as vector of antigen, but also induced mice to produce specific lymphocytes,which could recongnize cross-reactive self HSP60. Though HSP60 was self antigen of isletβcell, yet, whatever was normal mice or tumor-bearing mice,cross-reactive immunity failed to destroy pancreas.HSP65-MUC1 , gene engineering vaccine acquaring first stage clinical warrant, these results presented HSP65-MUC1 could company with chemical drug or single used for treat tumor with MUC1 antigen safely and effectively in clinic.