Metabolism Of Cholesterol Effects On Hematopoietic Stem Cell Self-renewal And Short-term Hyperthermia Effectes On Red Blood Cells Injury

There are two sections in this thesis.In section one,the relationship between metabolism of cholesterol and proliferation and differentiation of EML cells was discovered.In section two,the changes of erythrocyte structure and function induced by short-term hyperthermia was observed,and the mechanism of erythrocyte injury induced by heat stroke was also explored.Section one: As known to everyone,hematopoietic stem cells(HSCs)were a group of cells which had the tissue specificity.In order to maintain normal hematopoiesis,HSCs would adjust itself in number stable by asymmetric division and generate more single hematopoietic progenitor cells.In the general physiological conditions,the content of HSCs and the number of hematopoietic progenitor cells maintained in a state of dynamic balance.When the balance was broken,a series of diseases may cause.A lot of researches about the mechanism were carried out mainly on immune system.While,except for immune system,cellular cholesterol metabolism was also closely related to hematologic malignancies.It had been revealed that cellular cholesterol metabolism took part in the occurrence of hematologic malignancies through regulating the balance of hematopoietic stem cells(HSCs)proliferation and differentiation.But all of the researches above were just focused on the cholesterol metabolism-related gene abca1,abcg1,abcg4 and apoe,except for which there were NPC1 and NPC2 genes that also can regulate the metabolism of cellular cholesterol.Defection of NPC1 protein will cause Niemann-Pick disease,in which there were a large number of cholesterol accumulation in neuron of patients.And at the same time,hematopoietic function obstacle was also existed in patients,but the mechanisms were not clear.In order to reveal the relationship between NPC1,NPC2 genes and balance of HSCs proliferation and differentiation,we constructed RNA interference(RNAi)lentiviral vector targeting NPC1 and NPC2 of mouse and to investigate its effect on the proliferation and differentiation of erythroid myeloid lymphoid(EML)cells.Research methods:Firstly,we tested the correlation between sca-1 and NPC1,NPC2 through FCM.After that,DNA plasmids containing NPC1 shRNA or NPC2 shRNA were transfected into 293 FT cells to produce lentiviral particles.Then,the EML cells were transfected by the lentiviral particles.After that,the mRNA and protein expression of NPC1 and NPC2 in the transfected EML cells was detected by real-time PCR and Western Blot,respectively.And cholesterol distribution changing in EML cells was analyzed by filipin staining and laser confocal.The proliferation of transfected EML cells was tested by cell count kit-8(CCK-8)and Trypan blue staining respectively.And the expression of CD34,sca-1,c-kit,TER-119,CD11 b and B220 among EML cells were analyzed by FCM(flow cytometry).Then,phosphorylation of c-kit affected by NPC1 and NPC2 was detected by Western Blot after SCF stimulation.At last,phosphorylation of c-kit affected by U18666 A,?-CD and cholesterol was detected by Western Blot.Research results:Data of flow cytometry and Real-time PCR showed that expression of NPC1 and NPC2 had reduced in sca-1-low/EML cells,which indicated that the expression of NPC1 and NPC2 was positively correlated with sca-1.Data of Western blotting and Q-PCR revealed that the lentiviral vector pLKO.1/NPC1,NPC2 shRNA effectively down-regulated the expression of NPC1 and NPC2,and filipin staining showed that there was a lot of cholesterol in shNPC1/EML and shNPC2/EML cells and there was more cholesterol in shNPC1/EML cells than in the shNPC2/EML cells,which indicated that the NPC1 and NPC2 knock down EML cell modle had been created successfully.The results of CCK-8 and Trypan blue staining showed that the growth of EML cells were slower after NPC1,NPC2 gene kock-down,which showed that NPC1 and NPC2 genes would regulate the growth of EML cells.And data of flow cytometry showed that the percents of CD34 and sca-1were decreased in shNPC1/EML cells,which demonstrated that differentiation of EML cells was inhibited after NPC1 gene knock down.According to the data of flow cytometry,the expression of CD11 b was constant after NPC1 and NPC2 genes were knocked down,which showed that NPC1 and NPC2 genes would not affect EML cells to differentiate into granulocyte.But in shNPC1/EML cells the expression of TER-119 was decreased,which indicated that knock down of NPC1 gene would inhibit EML cells from differentiating into erythroid.While,in both of shNPC1/EML and shNPC2/EML cells the expression of B220 was decreased significantly,which showed that after knocking down of NPC1 and NPC2 genes the differentiation into lymphocytes of EML cells would be inhibited.Data of western blotting showed that phosphorylation of c-kit was inhibited in EML/shNPC1 cells after the stimulation of SCF,which indicated that NPC1 gene may regulate the phosphorylation of c-kit in EML cells.After treated by U18666 A and beta-CD the phosphorylation of c-kit was inhibited in EML cells,which was as the same as silence of NPC1 gene in EML cells.But the inhibition was rescued after using beta-CD and cholesterol at the same time,which revealed that NPC1 gene may regulate the phosphorylation of c-kit in EML cells through regulating the cholesterol on cell membrane.Section two: Red blood cells were the largest number of blood cells in mammals blood system.Except for as the main media of the transportation of oxygen,RBCs were important immune cells.RBCs were generated in the bone marrow,and new born RBCs would die after 120 days.Before apoptosis,RBCs would experience of young,middle and old three stages,finally,the aging RBCs could be deleted by immune system or Kupffer Cells in liver.While,when it was on the pathological state,RBCs death would intensify.According to cell biology researches,high temperature can cause the cytoplasm membrane deformation.And the formation of the bubble is adaptive,which could increase the cell survival rate.But the stronger cells was heated,the higher cell death rate would be.In order to observe the changes of erythrocyte structure and function induced by short-term hyperthermia,and to explore the mechanism of erythrocyte injury induced by heat stroke,venous blood collected from healthy volunteers was incubated for 10 minute at 37? and 42?.The erythrocytes from different age groups were separated by Percoll density gradient centrifugation,and the zeta potential was measured.The cell size,apoptotic cell marker molecule Annexin V and reactive oxygen species(ROS)in erythrocytes was analyzed by flow cytometry.Research results: Compared with 37 ?,erythrocytes was incubated at 42 ? for 10 minutes,the surface charge decreased and aging accelerated,further found that erythrocytes volume reduced,apoptosis and intracellular ROS increased.At last,we can get that short-term high hyperthermia can cause changes of physicochemical properties of erythrocytes,then leading erythrocytes senescence and apoptosis.Those may be the mechanism of blood system pathologically change induced by heat stroke.