| The change of lifestyle,such as diet,exercise,stress and sleep,is a major independent risk factor for overweight and obesity in the world.China has become the largest population of obesity in the globe as the economy increasing and large population base.The morbidity of obesity,obesity related diseases is increasing,which also called metabolic syndrome,for example including dyslipidemia,hypertriglyceridemia,insulin resistance(IR),type 2 diabetes mellitus(T2DM),cardiovascular disease(CVD)and non-alcoholic fatty liver disease(NAFID).All of them bring great economic burden and pains to the whole society and the family itself.So it is urgent to find the reason and take the action to prevent and treat it,which could be for the betterment of the society and whole family.Increasing evidence show that obesity and type 2 diabetes belongs to a chronic low-grade inflammation,some main cytokines have been found in both rodent models and humans,such as tumor necrosis factor-a(TNF-a),interleukin-6(IL-6)and C-reactive protein,then later substantiated by further research that this low-grade inflammation in T2DM has pushed an impetus to the field of impaired the insulin resistance,insulin secretion disorders,even the complications of type 2 diabetes,such as diabetic nephropathy(DN).Ferrante and Chen in 2003 showed that adipose tissue of obese mice was infiltrated with a lot of macrophages,the same phenomenon can been seen in human pancreatic tissue from individuals with type 2 diabetes,therefore,Inflammation is key involved in the pathogenesis of obesity and T2DM,obesity and type 2 diabetes could be considered as an inflammatory disease.However,it is not the classical definition of acute or chronic inflammation,which always accompanied with infection,but there is no massive tissue injury and the dimension of the inflammatory activation is also not so large.Therefore it is often called "low grade" chronic inflammation or "meta-inflammation',meaning metabolically-triggered inflammation or even "para-inflammation",whatever the name used the inflammatory process that characterizes the metabolic syndrome has its own unique features but its causes are far from being fully understood.Macrophage is original from bone marrow hematopoietic stem and progenitor cells(HSPCs),belongs to the mainly member of body's innate immune system.They differentiate into monocytes in the bone marrow and come into the peripheral blood.if there is infectious in the body,monocytes can get into the infectious tissue,transfer to macrophages.Later,they can phagocytize or produce some cytokine to kill and clear the hurt cells to protect our body,besides this,they can secrete chemokines to recruit other immune cells to the inflammatory tissue.This is the classical inflammation role.Of course,macrophages also take part in the process of obesity and type 2 diabetic mellitus.Just as what said above,the adipose tissue of obesity and type 2 diabetic mellitus is infiltrated with a lot of macrophages,and the number of macrophage has a positive relationship with the proliferation of adipose cells.Further research showed that macrophages of adipose tissue is the mainly original of pro-inflammatory cytokine,TNF-?.In the 3T3-L1 adipose cell,if silence the TNF-a expression using some methods,the normal insulin signal transduction can be damaged.On the contrary,using the molecular biological technique to knock out the TNF-a or its receptor,which can't easy to occur insulin resistance when giving the high fat diet.subsequently,IL-6,IL-? and chemokines CCL-2 secreted from adipose cells were expression increasing in the obesity model.That means,adipose tissue macrophages(ATMs)connect the obesity,insulin resistance and type 2 diabetic mellitus.Besides,an increase in peripheral leukocytes of myeloid lineage(monocytes and neutrophils)is a common feature of obesity and type 2 diabetic mellitus,and the change of monocytes have a great relationship with the development and progression of atherosclerosis,coronary heart disease and stroke.In the ob/ob model,other research shows that myelopoiesis in the bone marrow may induce the increasing circulation monocytes in high fat diet induced obesity.When talk to the original of adipose tissue macrophage in obesity and type 2 diabetic mellitus,there are two different opinion,some research think circulating monocytes can differentiate into inflammation macrophages,but other articles show it may be because of local resident macrophage proliferation.In a word,it is still not clear about the relation between tissue macrophage and peripheral monocytes,so we design this studies to focus on the macrophages and monocytes in the high fat diet mice,and hope to know more mechanism about obesity and type 2 diabetic mellitus.Part I The characteristic of peripheral blood monocytes in the high fat diet induced obesity in C57BL/6J male miceObjectiveThe increasing macrophages infiltrating into adipose tissue,especially visceral fat tissue,is the key to the development of obesity,insulin resistance and type 2 diabetic mellitus,but the activation and original of macrophage is not known.In the state,peripheral blood monocytes can develop into the tissue and differentiate into the macrophage,it is reported that there is an increase in circulating monocytes in obesity and type 2 diabetic mellitus,but when and how the characteristic of monocytes change in the process of obesity under the triggers of high fat diet,hyperglycemia,exercise or drinking,it is still not known,too.And the first part of our study is to observe the dynamic features about circulating monocytes in the different time under the high fat diet,the aim is to help find the role of blood monocytes in this process.Methods1.C57BL/6J male mice were purchased from Jackson laboratory of USA,the age is 5-6 weeks,after adapting feed for one weeks,the mice were divided into two groups randomly,high fat diet(HFD)and low fat diet(LFD),the former is given 60%of calories from fat,and later is 10%of calories.Each group is 5 mice unless special instruction,at the 3days,7days,15days,4weels,12weeks,15weeks and 19 weeks,sampled blood from orbital venous plexus and put into EDTA-treated sampling tubes,then take about 100?l for staining,first adding 2?l CD 16/32 antibody for each sample to block the Fcr receptor,and following adding the different antibody,PE-CD115,APC-CD11b,Alexa Flour 430-Ly6C,put the tubes on the ice for 30mintus,then 1ml ACK Lysis buffer for each tube was added,centrifuged in the 4? for 15minutes in 1500 rpm,300-500?l FACS was adding to suspend the cells,and 15?l count Beads(15000/?l)was adding at last for each tube.After above operation,the sample was measured with flow cytometry,the image was collected by Flow Jo 7.0 software,the data was analyzed with Prism 7.0,the results are presented as meanħSEM.Statistical analyses were conducted using a 2-sample Student's t-test,with significance set at p-value<0.05.2.At the 3days,4weeks,8weeks and 15weeks,after blood sampling,euthanized the mice by cervical dislocation,and made a small incision at the lower abdomen,peered the skin down to knees,dissected the femur and tibia,cut at the both ends of femur and tibia,flushed the BM cells using lml FACS(0.2mM)into a 5ml tube,dispersed the bone marrow cells by fleshing up and down several times with 1ml syringe until cells are visually dispersed,and added 1ml ACK lysing Buffer,mixed,set at room temperature less than 1 minute,then added 300?l FACS to centrifuged in the 4? for 15minutes in 1500 rpm,discarded supernatant,and re-suspend the cell pellet in 500?l FACS.Took 100 ?l for staining,first adding 2?l CD 16/32 antibody for each sample to block the Fcr receptor,and following adding the different antibody,PE-CD115,APC-CD11b,Alexa Flour 430-Ly6C,put the tubes on the ice for 30mintus,then 1ml ACK Lysis buffer for each tube was added,centrifuged in the 4?for 15minutes in 1500 rpm,300-500?l FACS was adding to suspend the cells,and 15 ?l count Beads(15000/?l)was adding at last for each tube.After above operation,the sample was measured with flow cytometry,the image was collected by Flow Jo 7.0 software,the data was analyzed with Prism 7.0,the results are presented as meanħSEM.Statistical analyses were conducted using a 2-sample Student's t-test,with significance set at p-value<0.05.the last operation was the same as step 1.3.using the 3days and 15 weeks to represent the early and late stage in our experiment,Tissue(adipose tissue and pancreas)were took and fixed overnight in 4%paraformaldehyde at room temperature,part was dehydrated by 30%sucrose,all then embedded in paraffin,tissue blocks were sectioned at from 5 ?m to 8 ?m for immunofluorescence.The operation was carried out following the standard protocols.4.Isolated the perigonadal adipose depot and removed lymph nodes,washed it with PBS to remove any contaminats,then transfered minced tissues into 10ml round-bottom tube containing 7ml of ice-cold digestion buffer,10 X collagenase buffer was added and adjusted the final concentration of collagenase to lmg/ml.then incubated at 37? for 30-45min,EDTA should be added to stop digestion,centrifuged in the 4? for 15minutes in 1500 rpm to get the SVF,the other step was the same as step1,except the Abs were changed.Besides,adipose tissue RNA was extracted by the classical trizol methods,CCL2 mRNA was measured by RT-PCR,and 2-AACT to calculate the relative expression,statistical analyses were conducted using a 2-sample Student's t-test,with significance set at p-value<0.05.Results1.The body weight of high fat diet groups was growing faster than the low fat diet groups from the 6th weeks,and there was significantly difference between two groups(P<0.05);2.We measured the randomly blood sugar at the same time(about from 3pm to 4pm)once every two weeks during the experiment,if the level of blood glucose is more than 300mg/dl for more than 2times,diabetic mellitus can be diagnosed.3.The characteristic of peripheral blood monocytes:the percent of blood monocytes of high fat diet in the whole blood(mainly refer to white blood cells)was reduced in the 3th days,reached to the lowest at 15days,compared with the low fat diet.and there was significantly difference between two groups(P<0.05).from the 4th weeks,the difference of two groups seems to reduce,and there was no significantly difference between two groups until 19th weeks.For more accurate to compare the monocytes,Count Beads was used to calculate the absolute numbers,and the results showed the numbers of blood monocytes of high fat diet was reduced in the 3th days,reached to the lowest at 15days,when compared with the low fat diet,and there was significantly difference between them(P<0.05),too.the numbers of blood monocytes of high fat diet seemed to begin increasing compared with the low fat diet in 19th weeks,but there was no significantly difference(P>0.05);4.The feature of peripheral Ly6c-hi monocytes:the percent of blood Ly6c-hi monocytes of high fat diet in the whole blood was decreased in the third days,if compared with the low fat diet.and there was significantly difference between two groups(P<0.05).from the 15th weeks,the percent of blood Ly6c-hi monocytes of high fat diet began to increase,however,and there was no significantly difference between two groups(P>0.05).Count Beads was used to calculate the absolute numbers for more accurate,the numbers of blood monocytes of high fat diet was reduced in the 3th days,reached to the lowest at 15days,when compared with the low fat diet,and there was significantly difference between them(P<0.05),the numbers of blood Ly6c-hi monocytes of high fat diet seemed to begin increasing at 15th weeks when compared with the low fat diet,and there was significantly difference at 19th weeks(P<0.05);5.The characteristic of peripheral Ly6c-low monocytes:the percent of blood Ly6c-low monocytes of high fat was increased in the third days,compared with the low fat diet,there was significantly diflference(P<0.05),but the percent began to decrease at the 15th weeks,and there was significantly difference(P<0.05);the absolute number of Ly6c-low monocytes was reduced at third days,7th days and 15th days,and there was significantly difference(P<0.05);the number of ly6c-low monocytes began to increase at the 19th weeks compared with the low fat diet,but there was no significantly difference(P>0.05).6.As to bone marrow monocytes and its subsets,our study found that there was no significantly difference about the monocytes,Ly6c-low monocyte and Ly6c-hi monocytes at third days,4th weeks and 8th weeks(P>0.05).but at 15th weeks,all these three population began to increase,and there was significantly difference.(P<0.05)7.the expression of macrophages in the adipose tissue:at third days,immunofluorescence histochemistry detection of the macrophage specific antigen F4/80 showed that F4/80-expressing cells were uniformly small,dispersed in the low fat diet,on the contrary,there are more macrophages in the high fat diet,some are aggregated;at the 15th weeks,the number of macrophages was increasing both high fat diet and low fat diet groups,but the high fat diet seems to have more macrophages,and some macrophages aggregated together like classical crownlike structures(CLS),which was a sign of inflammation.8.the expression of macrophages in the pancreas:there was no significantly difference between two groups at third days,but as the prolong of high fat diet,the infiltrating macrophages in the islet was increasing,and the size of islet in the high fat diet was larger than that in low fat diet.9.the expression of CDllc in blood monocytes and adipose tissue macrophages after 1 week of HFD:the percent of ly6c-lowCD11c+ in blood was increasing compared to LFD,CD11b+F4/80+CD1c+cells percent was increasing in adipose tissue,too,at the same time,the relative expression of CCL2mRNA was elevated.Conclusion1.The characteristic of peripheral blood monocytes in the high fat diet:first,the percent of Ly6c-low was increased,while the percent of ly6c-hi monocytes was decreased,it seemed that monocytes and its subsets was increasing,especially ly6c-hi monocytes at the 19th weeks.and the infiltrated macrophage in adipose tissue at the early stage may come from the peripheral blood ly6c-low monocytes,from 4weeks on,mainly 19th weeks,it seemed that blood ly6c-mi monocytes may develop macrophage.2.The time that infiltrated macrophages occurred in the adipose tissue was earlier than pancreas,adipose tissue,especially visceral adipose tissue may be the primitive center in the development of obesity.3.the inflammatory macrophages were appeared in the adipose tissue as early as one week of HFD,and Ly6c-low CD11c+monocytes may have an important actor in the process about blood monocytes transferring into adipose tissue macrophages.Part ? High fat diet diminishes hematopoietic stem and progenitor cells in C57BL/6J male miceObjectiveThe primary site of immune cell production was bone marrow(BM),and the hematopoietic stem and progenitor cells(HSPCs)resided here,which was the most primitive precursors.From the Part I,we knew that the blood monocytes was reduced at the early stage,and seemed to increase at the late,however,the mechanism was not clear.Besides,HSPCs is the progenitor of all immune cells,what is the effect of high fat diet on it?So we observed the change of hematopoietic stem and progenitor cells(HSPCs)in the high fat diet further,our aim was to find the primitive reason and mechanism which led to the change of blood monocytes.MethodsC57BL/6J male mice were purchased from Jackson laboratory of USA,the age is 5-6 weeks,after adapting feed for one weeks,the mice were divided into two groups randomly,high fat diet(HFD)and low fat diet(LFD),the former is given 60%of calories from fat,and later is 10%of calories.Each group is 5 mice unless special instruction,at the 3days,7days,15days,15weeks and 19 weeks,euthanized the mice by cervical dislocation,and made a small incision at the lower abdomen,peered the skin down to knees,dissected the femur and tibia,cut at the both ends of femur and tibia,flushed the BM cells using lml FACS(0.2mM,27#or 25#needles)into a 5ml tube,dispersed the bone marrow cells by fleshing up and down several times with lml syringe until cells are visually dispersed,and added 1ml ACK lysing Buffer,mixed,set at room temperature less than 1 minute,then added 300?1 FACS to centrifuged in the 4? for 15minutes in 1500 rpm,discarded supernatant,and re-suspend the cell pellet in 500 ?l FACS.Took 100 ?l for staining,first adding 2?l CD16/32-FITC antibody for each sample to block the Fcr receptor,then adding lineage marker 2 ?l for each tubes for 30min on ice,washing with FACS,following adding the different antibody,Qdot-605-Lin-,APC-CD11b,PE-c-kit,Alexa Flour430-CD34,FITC-CD16/32,Percp-Cy5-5-Flt3,put the tubes on the ice for 30mintus,then centrifuged in the 4? for 15minutes in 1500 rpm,300-500?l FACS was adding to suspend the cells,and 15?l count Beads(15000/?l)was adding at last for each tube.After above operation,the sample was measured with flow cytometry,the image was collected by Flow Jo 7.0 software,the data was analyzed with Prism 7.0,the results are presented as mean ħSEM.Statistical analyses were conducted using a 2-sample Student's t-test,with significance set at p-value<0.05.ResultsThe subsets of the hematopoietic stem and progenitor cells characteristic in the high fat diet:1)the percent of Lin-cell populations:the percent of Lin-population was reduced at 15days,compared with low fat diet,there was significantly difference with two groups(p<0.05);2)the percent of scal-ckit+ population was decreased at third days and 15th weeks by contrast with low fat diet(p<0.05);3)the percent of CMP population was reduced at third days by contrast with low fat diet(p<0.05);4)the percent of LT-HSC subsets was sharply reduced at the 7th days under high fat diet(p<0.05);5)the percent of MPP subsets was obviously decreased at the 15th days(p<0.05).Conclusion1.High fat diet could hamper the differentiation function of hematopoietic stem and progenitor cells in bone marrow,the subsets of HSPCs has been changed and re-modulated.2.the function of HSPCs was inhibited at the early stage in the high fat diet induced mice,the third day was most obvious,and the changes of HSPCs occurred earlier than that of peripheral blood monocytes(15th days). |
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