| The research on anti-tumor immunity has a long history. For several decades, immune mechanism of tumor's incidence,development and metastasis have been explored and illuminated. People have gradually recognized the correlation of tumor disease and immunology,and actively applied theory on anti-tumor practice. Especially recent years,anti-tumor researches have achieved a new phase with the finding and applying of tumor specific antigen and tumor non-specific antigen. Using different tumor antigens as anti-tumor vaccine,people can cure tumor disease more directly and obtain anti-tumor effect aiming at tumor cells,which can overcome the disadvantage of present anti-tumor measures have more side effects and poor specific. However,the targets of today's anti-tumor vaccine are simplex,which can't induce powerful enough and unlimited anti-tumor effect. To induce more strengthen and complete anti-tumor immunity,we need to combine several kinds of tumor antigens or directly produce the immunogen containing many tumor antigens.To include more tumor antigens,the normal method is irradiating tumor cells with X-ray and then immunizing the tumor cells as an immunogen into human body. But some researches have found that this therapy method has some risks,because the irradiated tumor cells still have the possibility of tumorigenesis. The research of Donna H.D and his colleges had found that the tumor cells irradiated by X-ray couldn't be completely killed,and some parts of the tumor cells had the ability of division and proliferation. Another strategy is using tumor cell lysate as immunogen (TCL). TCL is tumor antigens mixture prepared by lysis tumor cells with physical or chemical methods. Theoretically,TCL contains all the tumor antigens in tumor cells. Except using as an anti-tumor vaccine to applying on tumor therapy,TCL can solve the problem that present anti-tumor vaccine's antigenicity is simplex.Besides prepareing effective immunogen,there is a problem that tumor antigen immunized exogenously can't effectively activate cellular immunity should be solved. That is because tumor antigens immunized exogenously usually are presented by antigen present cell (APC) in MHC II pathway priming humoral immunity producing specific antibody. But antigen specific cytotoxicity T lymphocyte (CTL) will play a key role in anti-tumor immunity rather than antigen specific antibody. Based on this fact,the key point of tumor antigen immunized exogenously priming effective anti-tumor immunity is activating CTL effectively. To achieve this goal,the problem how tumor antigen immunized exogenously can be presented by APC in MHC I pathway should be solved at first,because basic immunology has illuminated that CTL's activation must depend on the combination of tumor antigen peptides and MHC I cell surface molecular. Some researches have found that some immune cells,like dendritic cell (DC),can cross-present exogenous tumor antigen in MHC I pathway priming activation of antigen specific CTL. In this process,some immune adjuvant is needed helping the tumor antigens to be presented. Many evidences have proved that heat shock protein (HSP) is a kind of protein can assist tumor antigens to be cross-presented. Thus,combining HSP and tumor antigen is a feasible anti-tumor strategy. Tumor-derived chaperone-rich cell lysate (CRCL) prepared via a free solution-isoelectric focusing (FS-IEF) technique were demonstrated therapeutic and prophylactic in mice against B-16 melonoma , leukemia (12B1) and B-cell leukemia/lymphoma (A20). A cellular vaccine prepared by combining the CRCL preparations with dendritic cells could eradicate pre-existing tumors in a high percentage of cases. In addition,recombinant HSP fusion proteins between tumor antigens and HSPs were developed as tumor vaccines. A HSP65-MUC I fusion protein between mycobacterial HSP65 (MHSP65) and MUC1 derived peptide was demonstrated capable of inducing MUC1 specific CTL that contributed to the growth inhibition of B-16 melonomas in mice. Another fusion protein between MHSP65 and human papillomavirus (HPV) E7 derived peptide was tested in clinical trials to treat HPV associated diseases. Alternatively , tumor cell lysate-pulsed human monocyte-derived DCs heat-shocked were found increased in their capacity to generate CTL responses against medullary thyroid carcinoma (MTC) tumor cells in vitro,showing that heat shocking DCs,due to the expression of HSPs,may be a kind of efficient tumor vaccines. In our research,we combined MHSP65 and TCL from Lewis lung cancer cells producing MHSP65-TCL anti-tumor vaccine and investigated its anti-tumor immunity.1. The expression and purification of MHSP65 and the preparation of TCL or MHSP65-TCLTo obtain MHSP65 used in the development of anti-tumor vaccine and the following experiments in vivo and vitro,we firstly constructed a pET28a-HSP65 recombinant plasmid and expressed MHSP65 protein in BL21 escherichia coli. Then we purified the MHSP65 protein using Ni+ affinity chromatography method. The level of LPS in purified MHSP65 protein should be detected. Only when the detected results accorded with the standard LPS level,the MHSP65 could be used in immunization in vivo and cell experiment in vitro. The results of SDS-PAGE detection,protein quantitation and western blot detection showed that we successfully expressed and purified the MHSP65. The purity of this protein was over 90%,and the concentration of this protein was 330μg/ml. The level of LPS in MHSP65 was lower than. This purified protein corresponded with the requirement of following experiments.To preparing TCL,we froze and thawed between -70℃and 37℃for 5 times. Our results exhibited that all the Lewis lung caner cells were destroyed after the freezing and thawing and stained into blue by trypan blue under microscope. Protein quantitation and SDS-PAGE revealed that all the protein in tumor cells had released out of the cells,and the concentration of this protein solution is 3.15mg/ml,which achieved our expect preparing a more safer tumor antigen peptides mixture.To preparing MHSP65-TCL,we directly mixed MHSP65 and TCL in vitro. Some researched have proved that direct mixing antigen and MHSP65 can effectively induce anti-tumor effect. So in our research,we directly mixed TCL from 1×10~6 Lewis lung cancer cells and MHSP65 in 0.85% NaCl producing MHSP65-TCL used in following experiments.2. Investigation of TCL or MHSP65-TCL's componentTo explore the component of TCL and MHSP65-TCL,we carried on degenerated SDS-PAGE electrophoresis analysis with different components in TCL or MHSP65-TCL.Besides that , we carried on non-degenerated SDS-PAGE electrophoresis analysis and Western blot detection with different components in MHSP65-TCL. The results showed that TCL contained various proteins with different molecular weight,which equably distributed in cell lysate and cell precipitation. In MHSP65-TCL,MHSP65 couldn't be digested by the hydrolytic enzyme probably existed in TCL,and maintaining the integrity of its structures. Moreover we detected a kind of MHSP65-tumor antigen complex in MHSP65-TCL by non-degenerate SDS-PAGE and Western blot analysis,and the formation of this complex didn't correlated with the mixing time of MHSP65 and TCL. Some researches have proved that this complex may be the structure basis on which MHSP65 helps exogenous antigens to be cross-presented. These results suggested that TCL we prepared contained a large number of intra-cellular peptides. Besides that,there were MHSP65-tumor antigen complexes existed in MHSP65-TCL,which play a crucial role in inducing specific anti-tumor immunity.3. MHSP65-TCL induced anti-Lewis lung cancer effect in miceTo test the anti-tumor effect of MHSP65-TCL , we firstly carried on tumor-suppressive experiment in mice. In the experiment,we immunized mice on day -7,1,8,15,and inoculated Lewis lung cancer tissue on day 0,then observing tumor incidence,mice living condition and measuring tumor size to draw tumor growth curve,tumor incidence curve and mice survival curve (P<0.05). The results showed that immunization of MHS65-TCL could significantly inhibit the growth of Lewis lung cancer in mice (P<0.05),whereas MHSP65 or TCL immunization didn't exhibit such anti-tumor effect. By the results of tumor incidence,we found that tumor incidence of NaCl injected,TCL immunized,MHSP65 immunized mice had arrived 66.67%,50% and 83.33% separately,whereas that of MHS65-TCL immunized mice was only 16.67% on day 11 after tumor challenge. By the results of mice survival,we found that 100% MHSP65-TCL immunized mice survived,whereas 83.33% NaCl injected mice had dead on day 50. These results suggested that MHSP65-TCL immunization could significantly delay Lewis lung cancer incidence,prevented tumor's growth and prolong the survival of mice. Following that,we investigated the immunizing dose of MHSP65 in MHSP65-TCL and the immunizing schedule in vivo. Compared with control groups,TCL or MHSP65 immunization couldn't inhibit the growth of tumor (P>0.05),whereas all 1μg MHSP65,10μg MHSP65,100μg MHSP65 plus 1×10~6 TCL immunization had anti-Lewis lung cancer effect (P<0.05). Among them,1μg MHSP65 and 10μg MHSP65 plus 1×10~6 TCL immunization induced more powerful anti-tumor immunity (p<0.001) than 100μg MHSP65 plus 1×10~6 TCL immunization (P=0.004). Compared with the schedule immunizing after tumor challenge,immunizing before and after tumor challenge obtained better anti-tumor effect (P=0.014).4. MHSP65-TCL induced specific anti-lung cancer immunityTo investigate the role of specific immunity in anti-tumor process,we detected activation status of CD8+ T cells and their cytotoxicity. To detect CD8+ T cells in mice,we immunized mice on day 0,7,14,20 and sacrificed the mice on day 15,19,21. Then spleen cells were taken and stained with FITC anti-CD8 and PE anti-CD69 mAb to analysis by flow cytometer. The results showed that activated CD8+ T cells in splenocytes from MHSP65-TCL immunized mice significantly decreased on day 19 (P<0.01) and didn't show any difference on day 15 or 21 ( P>0.05). We considered this result might correlate with the migration of CTL and memory CTL weren't activated effectively. When recalled in vitro with TCL,activated CD8+ T cells in splenocytes from MHSP65-TCL immunized mice significantly increased compared with that in splenocytes from NaCl injected,TCL immunized or MHSP65 immunized mice (P=0.001). Following that,we detected the cytotoxicity of CD8+ CTL in mouse splenocytes. The results showed that splenocytes from MHSP65-TCL immunized mice couldn't significantly kill Lewis lung cancer cells compared with that from NaCl injected,TCL immunized and MHSP65 immunized mice in E:T ratio of 5:1 or 15:1 (P>0.05). When E:T radio is 45:1,splenocytes from MHSP65-TCL immunized mice could significantly kill Lewis lung cancer cells,and the killing rate of splenocytes from MHSP65-TCL immunized mice is 38.23 %,which is higher than that of splenocytes from NaCl injected,TCL immunized and MHSP65 immunized mice (P=0.02). These results suggested that MHSP65-TCL immunization could induce TCL specific anti-tumor effect in mice. 5. MHSP65-TCL induced non-specific anti-lung cancer immunityTo investigate non-specific anti-lung cancer immunity induced by MHSP65-TCL immunization. We detected expression of CD69 and TNF secretion in splenocytes from MHSP65-TCL immunized mice,and their cytotoxicity. Besides that,we detected the activation of NK cells in vivo and vitro. To CD69 detection,splenocytes were taken from na?ve mice,and stimulated the splenocytes with MHSP65-TCL,MHSP65,TCL or NaCl to analysis CD69's expression. The results showed that CD69 expression of splenocytes stimulated with MHSP65-TCL was significantly higher than NaCl group and MHSP65 group (P<0.001). To TNF detection,we obtained culture supernatant of activated splenocytes to evaluate its TNF level. The results showed that there was high level TNF could kill L929 cells in vitro existed in the culture supernatant. To cytotoxicity assay of activated splenocytes , we took splenocytes as effect cells and Lewis lung cancer cells as target cells. When E:T radio is 45:1 or 15:1,splenocytes from MHSP65-TCL immunized mice could significantly kill Lewis lung cancer cells,and the killing rate of splenocytes from MHSP65-TCL immunized mice is significantly higher than that of splenocytes from NaCl injected,TCL immunized and MHSP65 immunized mice (P<0.05). To cytotoxicity assay of activated NK,we took splenocytes as effect cells and YAC-1 cells as target cells. When E:T radio is 100:1 or 50:1,splenocytes from MHSP65-TCL immunized mice could significantly kill Lewis lung cancer cells,and the killing rate of splenocytes from MHSP65-TCL immunized mice is significantly higher than that of splenocytes from NaCl injected,TCL immunized and MHSP65 immunized mice (P<0.05). To detect activated NK in vivo,we immunized mice on day 0,7,14,20 and sacrificed mice on day 6,15,19,21. Then spleen cells were taken and stained with FITC anti-CD8 and PE anti-CD69 mAb to analysis by flow cytometer. The results showed that activated CD8+ T cells in splenocytes from MHSP65-TCL immunized mice significantly increased on day 6 or 15 (P<0.01) and didn't show any difference on day 19 or 21 ( P>0.05). 6. MHSP65-TCL produced with B16 melanoma cells induced anti-melanoma effect in mice.Besides the anti-lung cancer effect mentioned above, we investigated the anti-tumor effect induced by MHSP65-TCL produced with B16 melanoma. Our experiments proved that MHSP65-TCL produced by combining B16 TCL and MHSP65 could induce anti-melanoma effect in mice and significantly prolong the survival of immunized mice. These initiatory results provided a new direction of MHSP65-TCL research.Collectively,to develop effective anti-lung cancer vaccines,we directly mixed MHSP65 and TCL from Lewis lung cancer cells in vitro and tested its efficacy on stimulating anti-tumor immunity. Our results show that MHSP65-TCL immunization significantly inhibits the growth of lung cancer in mice and prolongs the survival of lung cancer bearing mice. In vivo and vitro data suggest that MHSP65-TCL could induce specific CTL responses as well as non-specific immunity,both of which contribute to the effective tumor elimination. Thus,this report provides a more easy method to prepare MHSP65-TCL anti-tumor vaccine,which might be further developed to treat lung cancer.
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