Establishment And Application Of Two Types Of Zebrafish Model Of Human Multiple Myeloma

Multiple Myeloma is the second highly malignant haematopoietic tumor at present.The overall survival of MM patients are prolonged obviously with the development of new dugs these years like Bortezomib and Lenalidomide.Also lots of people can survive more than 10 years if he can get the better treatment effect.DNA instability is the basis for the development of cancer,even MM.In the process of DNA damage repair,Endo/Exo-nucleases play an important role.Maybe the overexpression of APEs will cause DNA damage and it will induce chromosomal translocation,even lead to the generation of tumor.A lot of studies confirm that APEs enzymes plays the important role in the process of DNA damage repair.we also observed the expression is abnormal in multiple myeloma,so we hope to build the animal model with the overexpression of APEs Maybe it can cause chromosome translocation until develop the tumor,And we can verify the relationship with overpression of APEs and the development of MM,Also we want to establish a very simple and effective MM animal model,which we can evaluate the clinical efficacy and do new drug research work.Now people alreasy developed a lot of mature mouse tumor models of MM,such as application of severe combined immunodeficiency(SCID-hu)mice model,although It can basically imitate the tumor microenvironment is similar to the human body to allow the MM cell growth,these models have significant limitations including they need the insertion of Fetal bones,the number of the generation of mice is limited and it needs about 2 months for human MM growth and,highlighting the need for a model that allows growth of human MM cells rapidly and in larger number of animals.So we want to develop two zebrofish models.First we want to study the overexpression of APEs gene in vitro and in vivo and the relationship between APEs and the development of tumor,thus proving APEs gene may be a potential marker of MM.First we mainly detected the expression of the APEs in DNA and protein expression level,We transfected the fiberblast cell lines with the plasmid contains APE1 to observe the change of cell growth and chromosomal changes.At the same time,we designed the plamid including APEs based on the Cre-Loxp system.Then we injected them into the zebra fish eggs to established the transgenic zebrafish with Endo/Exo-nucleases,like APEs.In the vitro experiment,gene array shows that the expression of APEs in patients with multiple myeloma and cell lines were significantly higher than that from normal plasma cells.In the protein expression level,it is 3.6 fold than the normal level.APE1gene is mainly expressed in the cytoplasm of MM tumor cells.After transfected the fibroblasts cell,we can clearly observed cell grows fast,The doubling time shortened from 4.8 days to 1 day,The chromosome karyotype analysis shows:From the transfection cells,we observed the chromosome translocation 11CR29t(6;17)(q13;q25)in the cells transfected with APEX2 gene and the chromosome translocation 11CR30t(1;19)(p13;p13)in the cells transfected APEX1 and APEX2genes.Next we established the transgenic zebrafish express the APEs gene through injected the embryos of zebrafish with the plamid carry Loxp sequence.After 6 weeks,8 weeks and 10 weeks we made the pathological section.Unfortunately we did not find the tumor from the transgenic zebrafish.only found a mild inflammatory reaction on the surface of zebrafish.Next,we utilized Casper fish,which are transparent and hence ideal for in vivo observation of tumor,Moreover,their 48 hours post fertilization(hpf)stage embryos are immune deficient allowing growth of xenogeneic human MM cells.We have tested growth of both MM cell lines as well as primary patient cells.About 50-200 cells from MM cell lines or CD138~+plasma cells from MM patient bone marrow samples labeled by CM-Dil were injected into the abdominal perivitelline space of 48 hpf stage Casper fish larvae and observed tumor progression,including tumor size and cell invasion by fluorescence microscopy at 24to 96 hrs post injection.MM cells were confirmed by immunofluorescence histochemistry.We observe over 80%larvae demonstrating MM cell survival for up to 9 days.As we require very small number of cells we were able to inject and observe growth of primary cells from all the patients producing over 50 larvae,We next treated the MM cells in vivo by adding first common anti-MM agents into fish water through 24 hrs to 72 hrs post injection(hpi)and observed change in tumor cell survival by fluorescence microscopy again at 24,48 and 72 hours post treatment(hpt).We have here evaluated response of MM cell lines and MM primary patient cells to drug treatment,and defined response.We defined the response into three groups:0-30%means no response,30-60%means relative sensitive and>60%means sensitive.Here we have observed response of the traditional and novel agents as predicted.we have confirmed the response of dexamethasone to MM1.S and MM1.R cell lines which are sensitive or resistant to dexemathasone in this model.We observed that 100 nM dexemathasone treatment led to observation of MM cell survival,and found response ratio of MM1.S and MM1.R xenograft tumors to were37.5%vs 87.5%fish at 24 hpt and 26.7%vs 87.5%at 48 hpt respectively.We have confirmed efficacy of bortezomib as well as lenalidomide in this model using MM cell lines(MM1S,MM1R,OPM1 and RPMI8226)as well as observed efficacy against primary MM cells.Importantly,we have been able to confirm similar drug resistance profile as patients;for example cells from a patient with bortezomib resistance survived bortezomib treatment in this model.We also compared the result between this xenograft model and the cell proliferation in vitro use MTT.It showed similar indicating that MM xenograft zebrafish model can predict drug response accurately.Conclusion:We developed two zebrafish model successfully.The MM xenograft zebrafish model can predict drug response accurately.And maybe can be used for the prediction of drug screening in vivo.Though the transgenic zebrafish is successfully established,but we did not find the development of tumor.We confirmed that the traditional transgenic technology can not be useful to induce gene mutation in zebrafish,Maybe only with the new technology like TALEN or CRISPR-Cas can improve the phenomenon;And we also confirmed that only rely on a single APEs gene abnormalities can not result in the tumor.