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Effect Of TLR5 Agonist CBLB502 On Proliferation Of Hematopoietic Stem Cells Induced By PTN

Posted on:2020-04-25Degree:MasterType:Thesis
Country:ChinaCandidate:W M SunFull Text:PDF
GTID:2404330575976650Subject:Immunology
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As the earliest researched stem cells,hematopoietic stem cells have achieved many results in clinical application and cell transplantation research,but there are still many problems to be solved in basic research,such as how to make hematopoietic stem cells expand in vitro,how to maintain its long-term hematopoietic reconstitution ability in the body and so on.The recombinant protein CBLB502,which is a derivative containing the main functional domain of flagellin,has the same TLR-5 activating activity and anti-radiation effect as flagellin,can effectively prevent lethality due to irradiation.The midkine factor PTN can greatly stimulate the proliferation of hematopoietic stem cells in vivo and in vitro,making it one of the most promising extracellular regulatory factors in the stem cell field.However,the regulation mechanism of PTN on HSC in the hematopoietic microenvironment in vivo,and the information on the biological effects of family member MK on HSC,are still not completely clear.In order to maintain the hematopoietic stem cell sternness and improve hematopoietic reconstitution,new ideas can be provided.This study aims to analyze each protein or ion by using C57BL/6J female mice as separate subjects,using separation and purification,r-ray irradiation,RTPCR technology and flow cytometry.Content change and expression,To explore the biological mechanism of PTN-induced hematopoietic stem cell expansion in vitro,to clarify the promoting effect of Toll-like receptor 5 agonist CBLB502 on PTN-induced hematopoietic stem cell proliferation,to establish a platform for in vitro expansion of hematopoietic stem cells,and to fight bone marrow failure and stem cell regeneration medicine.The research provides a theoretical basis and has certain clinical guiding value and potential social benefits.MethodsIn this experiment,SPF grade C57BL/6J mice were used as experimental subjects 25 g female mice of 8 weeks old were selected,Cage in cages,5 per cage,and they were given free access to drinking water.1.The mice were anesthetized and then sacrificed by decapitation.The femurs were taken,bone marrow cells were collected,flow cytometry sorting target cellsand.The mRNA was extracted according to the kit,and then realtime-PCR was performed to observe the protein expression.2.Production of purified CBLB502 protein.3.Irradiation with an irradiator,the experimental group is 7Gy+CBLB502 group,the 9Gy+CBLB502 group,the control group was 7Gy+saline group,9Gy+ saline group,intraperitoneal injection of normal saline 30min before irradiation,and the survival rate was observed for 28 days.4.Mouse bone marrow cells were extracted and CD34-Lin-Scal+c-kit+ ells were sorted by flow cytometry.The experimental groups were:PTN+CBLB502 group,PTN group,PBS group.Cell culture for 7 days,and the ratio of hematopoietic stem cells was observed by flow cytometryResultsFor the expression of TLR family molecules in mouse bone marrow blood cells,only TLR4 and TLR5 were significantly expressed in hematopoietic stem cells(P<0.05);Whether it is 7.5Gy experimental group or 9.5Gy experimental group,compared with the control group,the percentage of survival of mice injected with CBLB502 increased significantly after radiation exposure,CBLB502+PTN experimental group has lower levels of active oxygenthe and the ratio of hematopoietic stem cells expanded in vitro was significantly increased(P<0.05).Conclusions1.TLR-5 agonist CBLB502 can effectively inhibit the apoptosis of hematopoietic stem cells caused by radiation irradiation,increase the regeneration ratio of hematopoietic stem cells,and thus protect the individual mice from radiation,and effectively prolong the survival rate after irradiation.2.CBLB502 can promote the PTN-induced hematopoietic stem cell expansion in vitro by inhibiting the oxidative stress level of hematopoietic stem cells cultured in vitro.
Keywords/Search Tags:TOLL-like receptor 5 agonist, CBLB502, HSC, PTN
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