Heat Shock Protein 70-peptice Complex Loading With Dendritic Cells As An Potential Immunotherapeutic Candidate For Osteosarcoma: An Experiment Study

Osteosacoma is one of the commonest malignant bone tumors, which takes20% of the bone tumor cases. It happens frequently among the adolescentsbetween 10 to 30 years old. At present surgery operation and adjuvantchemotherapy are used to treat osteosacoma in clinical practice with 60% of thepatients cured. However, there is still 40% of the patients dead of recurrence andmetastasis. Nowadays with the advocacy and development of osteosacoma limbprotection technique, not only curing tumor but enhancing life quality as well hasbecome an eager demand of the patients. Therefore, it is significantly importantfor orthopaedists to find a new adjunctive therapy to raise the cure rate ofosteosacoma, especially that of the chemotherapeutics medicine tolerant tumorsand reduce chemotherapeutics doses and side effect. Heat shock proteins (HSP) are highly conservative in biological evolutionand widely distributed in prokaryotes and eukaryotes. Heat shock protein 70 is akey member of HSPs family who is generally considered as molecular chaperonewho plays an important role in the antigen presentation pathway and can aid thefolding and transport of protein. Dendritic cells are a kind of professional antigen presenting cells (APC) andhave very important function in innate and adaptive immune response. Above all,it is the only kind of APC, which can elicit innate immune-response. However theresearch of DC's functions inside and outside of the body is restricted in a longperiod of time because of its low quantity and the difficulty in abstraction.Comparatively speaking the collection of human cord blood is convenientand painless. Take cord blood as the material to induce the multiplication of DC,via joint cultivation of GM-CSF, IL-4 and TNF-α, plenty of DC, high-expressedCdla and HLA-DR may generate from the precursor cell of cord blood. Then takethe tumor antigen as the carrier to induce specific T-cell to kill the activity oftumor cell.The interaction of HSP-peptide complexes with high affinity receptor CD91in dendritic cells (DC) helps antigenic peptides present on DC cells by MHC Imethod and then activates antigen specific cytotoxicity T lymph cell to kill tumorcell. Research shows that HSP-peptide complexes can induce the maturity of DC.The DC secretion of inflammatory cytokines and chemotatic factors like TNF-α,IL-2, IL-6 and IL-12 can promote the expression of MHC and costimulatorymolecules. Matured DC can stimulate T cells to proliferate more effectively andthen initiate immune response.Therefore in this study human osteosarcoma HSP70 gene was used toreconstruct procaryon expression carrier and HSP70 was expressed in bacteriumcoli and collected by purification, which set up a base for the study of HSP70 andits function in osteosarcoma immunotherapy. Taking cord blood as the material toinduce the multiplication of DC, we approached the immunologic mechanism ofHeat Shock Protein 70-peptide Complex from osteosarcoma cell loading withDendritic Cells.Ⅰ. Cloning of HSP70 Gene from Osteosarcoma Cell and its Expressionand Purification in E. coliObjective: To clone HSP 70 gene from and express it in E.Coli in order toget antigen for the functional experiment of HSP 70. Method: After heat shockedthe osteosarcoma cell, HSP70 gene was amplified by One-Step and RT-PCRmethod from the total RNA of osteosarcoma cell. The PCR product was insertedinto PBS and sequenced. Then the HSP 70 gene was subcloned into expressionvector pET28α, and successfully constructed the recombinant vectorpET28α-HSP70 which was transformed into E.coli: BL21 (DE3) competent cell.After inducing the protein expression by IPTG, the expressed product wasanalyzed by SDS-PAGE and Westernblot. Results revealed that the PCR productof HSP70 gene from osteosarcoma cell is about 1.9 kb. The DNA sequencingresult shows that the HSP70 sequence of the PCR product coincides withpublished sequence, which means the result is correct. The recombinantexpression plasmid pET28α-HSP70 can express in E.coli and after being purifiedHSP70 can be got. Conclusion: The successful abstraction of HSP70 gene fromosteosarcoma and cloning and expression of it in E.coli provides basis forstudying the structure, functions and clinical applications of HSP70 inosteosarcoma.Ⅱ. Investigating the Way of Precursor in Human Cord Blood to Induceand Amplify Dendritic CellsObjective: To investigate the way of precursor in human cord blood toinduce and amplify dendritic cells (DCs). Methods: Cord blood was collectedunder sterile condition and the cord blood mononuclear cells (CBMCs) wereseparated by extraorgan (methyl cellulose method). Then the CBMCs werecultured with GM-CSF, IL-4, TNF-α and immunohistochemical technique wasused to determine its surface marker. Results: The precursor cells in cord bloodcan induce considerable dendritic cells (DCs) and highly express CD1a, HLA-DR.Cytokines, like GM-CSF, IL-4, TNF-α, can induce CBMCs to generate DCs invitro. Conclusion: Cord blood is ease to collect and has wide sources and lowimmunogenicity as well. GM-CSF, IL-4 and TNF-α are all essential in generatingDC. The generated DCs have all the features of DC either in the shape or in thephenotype, which pave the way for further study of DC.Ⅲ . The Lethal Effect of HSP 70-peptice Complex Loading withDendritic Cells on Osteosarcoma Cell Line Saos-2Objective: To take Heat Shock Protein 70-peptice Complex fromosteosarcoma cell as the tumor antigen, and load it with Dendritic Cells from cordblood to induce the specific T cell to kill the activity and observe its effect onSaos-2 cell, for the purpose of providing a base for the osteosarcomaimmunotherapy applying DCs loaded by HSP 70-peptice Complex. Method:Combined the tumor antigen peptice and HSP70 in vitro and loaded it with theDendritic Cells extracted from cord blood. Observed its specific lethal effect onSaos-2 cell. Results: Loading with antigen, the DCs could activate the CTLs tobecome tumor specialized CTLs, which had shown significantly inhibition ongrowth of Saos-2 tumor cell. After antigen stimulation the anti-tumor function ofkaryocyte in cord blood could be enforced effectively. Conclusion: This researchprovides theoretical basis for osteosarcoma immunotherapy.Up to now, although the research of osteosarcoma immunotherapy has beencarried on for many years, its clinical curative effect results are very tiny. One ofthe most important reasons is that osteosarcoma is a kind of weak antigen tumor,the accurate tumor antigen can't be found. HSP 70-peptice Complex appears to bea good molecular chaperone and causes an effect of internal immunologicadjuvant. Heat Shock Protein 70-peptice contains all tumor antigens and canactivate multiple CTL clone. Thus the difficult problems of tumor heterogeneityand tumor escaping can be surmounted. Without identifying the composition ofantigen peptide it can induce specific anti-tumor immunization.The research of Heat Shock Protein 70-peptice Complex Loading withDendritic Cells shows significant clinical application value:(1) This new tumor immunotherapy can induce body anti-tumor immunityrespond more forcefully and eliminate immune suppression caused bychemotherapy medicines. Thereby the cure rate of osteosarcoma can be enhanced.(2) The combination of this new osteosarcoma immunotherapy withchemotherapy medicine can correspondingly reduce the dosage of thechemotherapy medicine, thus diminish the side effect of the medicine, which leadsto the improvement of patients' living quality. Immunotherapy becomes one of theeffective adjuvant treatment methods for osteosarcoma.(3) Treatment of drug fast osteosarcoma is not a tough problem for theclinical doctors any more. This new immunotherapy for osteosarcoma functionsby inducing body's anti-tumor immunity. There is a method now to deal with thedrug fast tumor.This research provides a theoretical basis for enhancing the survival rate ofosteosarcoma patients, improving their living quality, and making immunotherapyan effective adjunctive therapy for osteosarcoma after surgical operation, andchemotherapy. Immunotherapy will play an important role in exterminating micrometastasis and make it possible to cure osteosarcoma. Relevant research in thisaspect hasn't been found in China or abroad. Therefore, the expected results ofthis research will have great value in clinical application and economicperformance.