| Objective:A new model organism zebrafish as a model was used to study the protective effect and mechanism of pig-hide gelatin on immune injury and hematopoietic inhibition induced by chemotherapy.Provide the theoretical foundation and experimental basis for further study of pig-hide gelatin in anti-cancer clinical application.Methods:The wild-type AB strain zebrafish embryos?2 hours post fertilization,2 hpf?were selected as experimental materials,and treated with different concentrations of pig-hide gelatin for a certain time.The effects of pig-hide gelatin on the mortality,hatching and developmental malformation of zebrafish embryos were observed,and the tolerance dose range of zebrafish was investigated.The transgenic zebrafish?48 hpf?with green fluorescence-labeled neutrophils was used as experimental animals.The imaging was observed using a fluorescence microscope,and the number of its neutrophils was analyzed by the Image Pro Plus software.This was used to investigate the response of the positive drugs such as vinorelbine,rapamycin and chloramphenicol in a zebrafish model,as well we establish an evaluation model of immune function;Moreover,the transgenic zebrafish?48 hpf?with green fluorescence-labeled blood platelets and the wild-type AB strain zebrafish?24hpf?were selected as experimental animals.Fluorescence microscopy imaging and o-dianisidine staining were used to investigate the response of the positive drugs such as methotrexate,doxorubicin,and nitrogen mustard hydrochloride in the zebrafish model.And a evaluation model of hematopoietic function was established.Using the above zebrafish model,after adding pig-hide gelatin for a certain time,the number of neutrophils,blood platelets,macrophages engulfing ink particles and erythrocytes in the zebrafish were used to examine the protective effect of pig-hide gelatin on the damage models of immune and hematopoietic.Then,the quantitative real-time PCR?qPCR?method was used to detect mRNA expression levels of zebrafish related factors,including immune-related factors?il-6,il-8,il-10,tnf-?,ifn-??and hematopoietic expression-related factors?scl1 c-myb,gata1,pu.1,runx1 genes?.Results:In the study of zebrafish tolerance,when concentration of pig-hide gelatin was in range of 0-100?g·mL-1,the mortality,hatching rate and malformation rate of zebrafish embryos had no significant effect.However,when its concentration was more than 200?g·mL-1,the certain effect on development of zebrafish embryos had been shown,such as reduced hatching rate,increased mortality,etc.Under the experimental conditions,the known immunosuppressive agents such as vinorelbine,rapamycin and chloramphenicol could significantly inhibit the number of neutrophils in zebrafish juvenile?72 hpf?,and it showed a certain dose-effect relationship.And the known hematopoietic inhibitors such as methotrexate,doxorubicin and nitrogen mustard hydrochloride could also significantly inhibit the formation of blood platelets and erythrocytes in zebrafish juvenile,indicating that the zebrafish model could sensitively characterize immune damage and hematopoietic disorders caused by chemotherapy and other.In the range of experimental doses,pig-hide gelatin significantly reversed the decrease in neutrophils quantity induced by vinorelbine,rapamycin and chloramphenicol in zebrafish,it also significantly enhanced the ability of zebrafish macrophages to engulfe ink particles,showing that pig-hide gelatin had some protective effect on the immune injury caused by chemotherapy and other drugs.The results of qPCR showed that,after vinorelbine treatment,the mRNA expression levels of il-6,il-8,il-10 and ifn-?were down-regulated in different degrees,while the expression level of tnf-?was significantly increased.In contrast,after pig-hide gelatin treatment,the mRNA expression levels of il-6,il-8,il-10 and ifn-?were increased while the expression of tnf-?was decreased,suggesting that the protective effect of pig-hide gelatin on immune injury might be related to the above factors.In the range of experimental doses,pig-hide gelatin significantly reversed the decrease in blood platelets quantity induced by vinorelbine and chloramphenicol in zebrafish,it also significantly enhanced the reductions in erythrocytes quantity caused by methotrexate,doxorubicin,and nitrogen mustard hydrochloride,indicating that pig-hide gelatin had a protective effect on hematopoietic disorders induced by chemotherapy drugs.The results of qPCR showed that,methotrexate significantly down-regulated the expression of related genes and transcription factors that promoted hematopoietic stem cell production,such as scl1,gata1 and c-myb.But,the expression of pu.1,which affected the differentiation of hematopoietic stem cells into myeloid cells during hematopoiesis,significantly increased.Similar to immune factor results,the expression levels of hematopoietic factors,such as scl1,gata1 and c-myb,increased with increasing concentrations of pig-hide gelatin.This indicated that its protective effect on hematopoietic disorders might be related to the above factors.Conclusion:Zebrafish model could better characterize the chemotherapy-induced damage of immune and hematopoietic,use fewer samples,and have short test cycles,which provided a new experimental method for the further study on the activity of modified drugs.This model was used to study the protective effect of pig-hide gelatin on immunologic and hematopoietic function after chemotherapy for the first time,which provided a theoretical foundation and experimental basis for the treatment of malignant tumors using pig-hide gelatin and chemotherapeutic drugs. |
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