| Chapter one The research about the original cell of pericytes in mice wound repairResearch backgroud and objective:Pericytes,also known as adventitial cells or Rouget cell,were discovered by Eberth and Rouget in the 1870s.In recent years,with the discovery and application of more markers of pericytes,the effects of pericytes on angiogenesis caught the attention of scientists.Even more,some studies suggested that pericytes play an and important leading role in formation oof neovascular buds.Therefore,the role and mechanisms of pericyte in angiogenesis process are very important.In the past,endothelial cells(ECs)have been considered the most active cellular components in the process of angiogenesis.But in recent years,studies of the classical theory have been amended.Angiogenesis is not only dependent on invasion and proliferation of endothelial cells,but also need pericytes to cover vascular sprouts and maintain the vascular stability.When pericytes wraps on endothelial cell disorderly,angiogenesis and vascular remodeling are abnormal.Many studies have demonstrated that wound healing is closely related to peripheral cells,and hypertrophic scars,keloids,diabetes and other vascular diseases all have abnormal changes of pericytes.The pericytes have not merely the ability in stabilizing the blood vessel,maintaining local vascular and tissue homeostasis,regulating extracellular matrix protein synthesis,but also have the ability to working like macrophage,immune defense,intervention coagulation,and a series of functions.The most important is involved in the regulation of blood vessel formation.The pericytes have multipotential differentiation,sauch as fibroblasts/myofibroblasts,preadipocytes,chondrocytes,osteoblasts,dentin cells,vascular smooth muscle cells and a series of cells.Although pericytes in morphology and functional performance are polymorphic and their shape,density and cell markers in different tissues are different,it is well known that the pericytes are derived from adult pluripotent stem cell.The clinical significance of pericytes research lies in controlling angiogenesis and stabilizing status of blood vessel.however,its exact origin and differentiation mechanisms are still unclear.Pericytes derived from embryonic blood vessel formation(mesenchymal stem cells,neural crest cells and other possible sources)and postnatal angiogenesis(pre-existing pericytes,fibroblasts/myofibroblasts and circulating progenitor cell).In short,ifs thought that pericytes are multicellular origin,its precursor cells may be derived from bone marrow hematopoietic stem cells(HSCs)or bone marrow mesenchymal stem cells(MSCs).Abe and his colleagues used male mice bone marrow transplant to female mice,then made wounds model,they found the pericytes of wound healing process containing Y chromosome.It indicated that pericytes in skin wound neovascularization derived form bone marrow cells.Rajantie and other studies have shown that pericytes expressing markers of hematopoietic stem cells(CDllb and CD45RO).It may propose derived from pericytes hematopoietic stem cells.Further study found that the pericytes in woud healing expressed EGFP marker after EGFP-labeled mice hematopoietic stem cell transplantion.Now it is well known that the pericytes derive from MSCs.The latest studies suggest that mesenchymal stem eells reside in various perivascular tissues,and these cells are identified as microvascular pericytes.If like this,it may explain the current ubiquity of stem cells and their role in a variety of repairation.It offers new prospects of tissue repair.Despite there are controversy,there is growing acceptance about the relation ship between Pericytes and mesenchymal stem cells.Mesenchymal stem cells are pluripotent adult stem cells that may be present in all tissues and as a special class of cells(mural cells/pericytes)located in the vascular vessel.They originally come from bone marrow stromal microenvironment that support for growth of hematopoietic stem cell,and can differentiate into mesodermal cell lines.In short,the origin and differentiation of pericytes still have a lot of controversy.Although bone marrow is the exact source of pericytes in the angiogenesis process of wound healing.The pluripotent stem cells originated in the bone marrow have hematopoietic stem cells and marrow mesenchymal stem cells.It's unclear what type of stem cells is the source of pericytes?It will be the basis of this study research topics.Research methods:1.Isolate and culture mice HSC and MSC,then identify the phenotype by flow cytometry.2.In vitro,clonal culture was carred out to demonstrate that the isolated HSCs met the standard of stem cells.MSCs were cultured to be induced and differentiated into adipocyte and osteocyte,then they were carried oil red o staining and alizarin red staining.3.Mice were irradiated by 7Gy,8Gy and 9Gy dose,then HSCs and MSCs were transplanted to mice by tail intravenous injection to test the differentiation of HSCs and MSCs in vivo.4.Mice were irradiated(8Gy dose)to induced bone marrow suppression.After that,RFP labeled HSCs and EGFP-labeled MSCs were transplanted to mice.The mice were carried out wound model.The whole-body fluorescence imaging technique and iununohistochemical staining were used to monitor the location and differentiation of stem cells.Research result:1·The suspension HSCs had grown colony formation.Flow cytometry showed HSCs expressed Seal and didn't express lin,CD48 and CD34.MSCs grew adherent culture dish like fusiform.MSCs expressed CD29 and CD44,and didn't express CD45 and CD34.2.The colony culture showed HSC differentiated into erythroid(BFU-E),granulocyte macrophage cell line(CFU-GM)and granulocyte macrophage red line(CFU-GEMM)colonies.MSC can be induced to differentiate into adipocyte and osteocyte.3.When the mice were transplanted with PBS,70%mice died with irradiated by 7Gy,all mice died in 8Gy and 9Gy group.When the mice were transplanted with HSCs,only one mouse in 9GY group died,the rest mice all lived.Therefore,we use the 8Gy dose as the radiological dose.14 days after the mice were irradiated and transplanted with EGFP-labeled HSCs,there was no green fluorescent cells in the skin,heart,liver,kidney and other tissues.The mice,which were irradiated and transplanted with PBS,had few bone marrow cells and the cells can't cultured.Mice in HSC transplantation group had many bone marrow cells and the cells can be clony and common cultured.4·The whole-body fluorescence imaging showed there were much red fluoreseence around the wound and a very small amount of green fluorescence in the wound area of irradiated mice.Further,immunohistochemicai staining showed much RFP expression and no EGFP expression around wound.Meanwhile,desmin expressed at the same area with RFP.The results demonstrated the pericytes originated from HSCs.Research conclusion:We successfully isolated and cultured mouse HSCs and MSCs and the two cells have the potential of stem cells.When the two cells simultaneously were transplanted in mice with wound,HSCs participated in wound healing and MSC didn't.The pericytes in wound healing originated from HSCs.Chapter two The research about regulation of sympathetic nerve on stem cells in bone marrowResearch backgroud and objective:In the previous chapter,we found the stem cells in the bone marrow participate in wound healing.The bone marrow is surrounded by sympathetic nerve.The previous study showed that the HSCs can,t be mobilized into the blood in sympathectomized mice.The studies demonstrated the sympathetic nerve regulates the stem cells in bone marrow in vivo.However,how the sympathetic nerve regulates the stem cells in bone marrow in vitro was unclear.Diabetes mellitus is a common disease with increasing incidence throughout the world and may cause significant health care burden especially in the working-age population.In the clinic,it has been shown that diabetes lead to disorders in Glucose metabolism,resulting in peripheral neuropathy,microangiopathy,then bring a series of diseases,such as retinopathy'diabetie nephropathy and diabetic foot(chronie ulcers)and osteoporosis.It has been focused by scholars.In China,as a developing country,the incidence of diabetes increased quickly,coupled yith rapid economic growthand a negative change(such as increased dietary fat intake and reducing physical activity)lifestyle.In the clinic,it has been shown that diabetes leads to osteoporosis and chronic wound.It's thought the impaired ability of stem cells in bone marrow was the cause.The previous studies demonstrated the abilities of bone stem cells were impaired in diabetic mice.MSCs have the potential to differentiate to lineages of mesenchymal tissues,including bone,cartilage,fat,tendon,muscle,and marrow stroma.It showed the osteoporosis and chronic wound were related with impaired ability of stem cells in bone marrow.However,the exact mechanisms that cause diabetic bone loss have not been clearly identified.The MSCs isolated from diabetic rats showed impaired proliferation and differentiation potential,which may lead to a net loss of trabecular bone.The onset and progression of osteoporotic complications are delayed in diabetic patients with good glycaemic control.It is therefore hypothesized that hyperglycaemia play an important role in regulating MSCs biological activity to modulate the development of osteoporosis.The sympathetic nervous system(SNS)is prominently involved in bone marrow(BM)niche function and bone formation.Diabetic patients often experience autononic neuropathy,which is characterized by pauperization of SNS fibers in multiple organs.Mattia Albiero,et al found diabetie miee are characterized by a profound depletion of BM SNS fibers and impaired stem cells mobilization.It has been shown that NE,a major mediator of the sympathetic nervous system,influenced rat MSC migration both in culture and in vivo.However,it remains unclear whether NE regulates MSC fate in diabetic mice.To provide new insight into the mechanisms of diabetic bone loss,the aim of this present study is to investigate whether NE can protect high glucose-induced apoptotic in MSCs and primarily to investigate its mechanism of anti-apoptosis.A balance between the expression of anti-apoptotic and pro-apoptotic factors underlies apoptosis and this balance may be altered by certain extracellular signals.The phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)/Akt pathway has been reported to play an important role in MSC growth,mobilization and survival.In the present study,the expression levels of key molecules associated with the PI3K/Akt signaling pathway,including PI3K and Akt,were evaluated by western blotting at the corresponding time-points.The results of the present study may aid the elucidation of the involvement of dysregulation of the PI3K/Akt signaling pathway in apoptosis of MSCs in diabetes.Research methods:1.The HSCs and MSCs were stimulated by 10-6M,10-SM and 10-4M NE,then the proliferation of stem cells were detected by CCK8 assay.2.The effect of NE on HSC migration was detected by transwell and CCK8 assays.The MSCs migration was detected by cell scratch wound model.3.The effect of NE on CD31,Desmin,a-SMA and K5 expression of HSC and MSC were detected by immunohistochemistry staining.4.We explored the effects of glucose on the apoptosis of MSCs following exposure to 33 mM glucose.To investigate the protective effects of NE,the cells were cultured with combination of high glucose and NE.The CCK8 cell proliferation assay,Elisa,immunofluorescence,and flow cytometry were used to assess cell proliferation and apoptosis,respectively.5.After that we analyzed the expression of PI3K,p-Akt,Bcl-2,Bax and cleaved-Caspase3 by western blots.Research result:1.The CCK8 assay showed 10'6M and 10-5M NE promoted the proliferation of HSC,and 10-5M NE promoted most obviously.10-4M NE showed inhibiting effect on proliferation of HSC.We also found 10-6M and 10-5M NE promoted the proliferation of MSC,10-4M NE showed inhibiting effect on proliferation of MSC.The concentration of 10-5M was used at the next experiments.2.10-5M NE promted migration of HSC and inhibited migration of MSC.3.The immunohistochemistry staining showed that CD31,Desmin,a?SMA and K5 expression of HSC and MSC had no obvious differences between NE group and control group.It meaned NE had no effect on CD31,Desmin,a-SMA and K5 expression of HSC and MSC.4.The Elisa,Tunel and flow cytometry assays showed the apoptosis rate was higher in 33mM D-glucose group than in 4.5mM D-glucose group,suggesting that high D-glucose can induce apoptosis of MSC.When 10-5M NE was added in the high D-glucose culture medium,the apoptosis rate reduced and had no significant difference with 4.5mM D-glucose group.The results suggested that NE can protect MSC apoptosis induced by high glucose.To confirm the protective effect of NE,the MSCs were pretreated with PH and PR.After pretreatment with PH,the protective effect of NE disappeared.However,pretreatment with PR didn't affect the protective effect of NE.It suggested that PH can block the protective effect of NE on MSC apoptosis induced by high glucose.5·To further determine the underlying mechanism of the effects of HG and NE on MSCs,we tested the p-Akt expression,molecules involved in the transduction of anti-apoptotic signals.The level of p-Akt was increased 1 day,3 days,5 days after NE treatment and returned to baseline on seventh day(date not shown).So we tested the p-Akt,Bcl-2,Bax,cleaved-caspase3 of treatment with NE or HG for 5 days.As shown in Fig.5,after exposure of MSCs to HG for 5 days,p-Akt significantly fell,and the HG induction of p-Akt was markedly reversed by inclusion of NE.As expected,PH blocked enhancement effect of NE on p-Akt,and PR had no such effect.Meanwhile,HG treatment resulted in a decrease in Bcl-2 expression and an increase in expression of Bax and cleaved-caspase-3 compared with the control group,a tendency that was reversed by NE treatment.And PH blocked the effect of NE on expression of Bcl-2,Bax and cleaved-caspase-3,PR didn,t have this function.Research conclusion:NE promoted HSC and MSC proliferation.NE also promoted HSC migration,but inh1bitd migration of MSC.NE had no effect on CD31,Desmin,a-SMA and K5 expression of HSC and MSG-NE had protective effect on mesenchymal stem cells apoptosis induced by high glucose and the Akt/Bcl-2 pathway was involved in.Chapter three Regulation of sympathetic nerve on bone marrow-deriyed stem cells involved in wound healingResearch backgroud and objective:The pericytes in wound healing originated from stem cells in bone marrow.It's known that there is space between bone marrow and wound.So,how do the stem cells move to the wound?Some scholars have found that the sympathetic nervous system signals regulate hematopoietic stem and progenitor cells release from the bone marrow to blood.And the stem cells migrate with the flow of blood.When the adrenergic neurotransmission were blocked by specific drugs,the G-CSF(granulocyte colony-stimulating factor)controlled by NE inhibited the activity of osteoblasts,and CXCL12 in bone(is a protein of bone marrow specific niches to attract hematopoietic stem cells and progenitor cells)expression increased induced the HSPC stayed in bone.Therefore,the article published in Cell commented that the nervous system may be a new axis which controls the relationship between stem cells and microenvironment.The previous studies have shown that hematopoietic stem cells and progenitor cells is one of sources of pericytes.Whether this niches(including bone marrow mesenchymal stem cells)are affected by this neuromodulation Axis,it has not been clarified.We want to learn more about the various niches of bone marrow and the repair mechanism which the autonomic nervous system regulate the endogenous bone marrow cells moving into the blood,homing,and transforming into wound.Research methods:1.The mice were randomly divided into 2 groups(60HDA group,CTR group).Then each group were randomly divided into 2 groups again,one for the transplant group and other for non-transplantation group.Therefor,the four groups were named 60HDA+transplantation group,6OHDA non-transplantation group,CTR+transplantation group,CTR non-transplantation group.The mice were carried by sympathetic denervation or not.After 6 days,the mice were transplanted by stem cells or not before wound modeling.The stem cells contained 106 green fluorescence labeled HSCs and 106 red fluorescence labeled MSCs.We immediately make wound model after the completion of transplanting stem cells.Afterthat,the wounds were immediately photographed to measure the wound area.The wounds were recorder on day 0,day 1,day 3,day 5,day 7and day 9.Wound healing rate=(original wound area-did not heal the wound area)/original wound area2.We isolated and cultured mouse dermal fibroblasts.We used cell scratch to detect the regulation of NE on migration of fibroblasts.Research result:1.In sympathectomized mice group,the stem cells can accelerate the rate of wound healing.However,in normal sympathetic nerve mice group,there was no significant difference in the rate of wound healing between stem cells transplanted group and non-transplanted group.In non-transplanted mice,there was also no significant difference in the rate of wound healing between normal mice group and sympathectomized mice group.2.The cell scratche assy show NE inhibited migration of fibroblasts,and PH can block the effect of NE.Research conclusion:Sympathetic nerve did participate in regulating the bone marrow stem cells moving into the blood to take part in wound healing.Meanwhile sympathetic nerve participated in the regulation of skin.We found that NE inhibited migration of mouse skin fibroblasts.Chapter four Norepinephrine regulates keratinocyte proliferation to promote the growth of hair folliclesResearch backgroud and objective:In previous studies,we unexpectedly found that hair growth has changed in sympathectomized mice.Therefore,we would like to further explore the role of sympathetic nerve on regulation of mouse hair.Psychological factors have been demonstrated to cause hair loss.Data obtained from animal models support the hypothesis that stress may act as a cofactor to promote hair loss.The sympathetic-adrenal-medullary(SAM)and hypothalamic-pituitary-adrenal(HPA)axes are activated by higher brain centers in response to psychological stress.The skin is a neuroendocrine organ that is a target of hormones,neurohormones,neurotransmitters,and neural responses.The skin is also regulated by the SAM and HPA axes and expresses elements of these axes,including proopiomelanocortin(POMC),corticotropin releasing hormone(CRH),the CRH receptor-1(CRH-R1),and adrenoreceptors(ARs).Stress-induced signals induce neurons to produce and release CRH.CRH then binds CRH-R1 and induces the release of POMC-derived adrenocorticotropic hormone(ACTH),which stmulates glucocorticoid(GC)synthesis and secretion.Activation of the SAM axis increases the release of the neurotransmitters,epinephrine and norepinephrine(NE),from the adrenal medulla and stimulates the sympathetic norepinergic nerves to promote further NE secretion.Past studies focused on the regulation of the skin by the HPA axis.However,little is known about the SAM axis,and specifically,the sympathetic nerve.Therefore,we wouldlieeto investigate the effectof SAM axis onhair.As a component of the SAM axis,the sympathetic nerve can regulate wound healing by secreting neurotransmitters such as NE,a key mediator of cell proliferation,differentiation,and apoptosis.Sympathetic nerve fibers are located between the blood vessels and HFs,and insert into the HF ORS at the level of the bulge region,the probable reservoir of HF stem cells.In addition,some circumstantial evidence supports the involvement of sympathetic skin nerves in HF growth.Using C57BL/6 mice,we administered the neurotoxin,6-hydroxydopamine(6-OHDA)to selectively destroy noradrenergic nerve fiber endings as a chemical-induced model of sympathectomy.So we wound research the effect of sympathetic nerves on hair in sympathectomized mice using 60HDA.Research methods:1.Mice were randomly assigned to three groups in a 1:1:1 ratio:60HDA group,PR group,control(CTR)group(n=30/group).PR(0.5 g/l;Sigma,Buchs,Switzerland)was administered to the drinking water of mice in the PR group,whereas pure water was provided to mice in the 60HDA and CTR groups.After 2 weeks,6-week-old mice in the 60HDA group received intraperitoneal(i.p.)injections of 6-OHDA(Sigma)diluted in a sterile saline solution containing 0.01%L-ascorbic acid.Six-week-old mice in the PR and CTR groups received i.p.injections of equal amounts of solvent(sterile saline solution containing 0.01%L-ascorbic acid).Depilation was conducted on the seventh day of 6-OHDA or solvent administration in mice(7 weeks old)over 20 g,the HFs immediately and uniformly entered the anagen stage.Skin was harvested on day 1,3,5,7,9 and 14 after depilation.To detect HF epithelial proliferation,mice received i.p.injections of BrdU(0.2 mg/g body mass;Sigma)2 h before harvesting the skin.2.Ki67 expression was detected by Immunohistochemical staining.3.The tissue culture of mouse skin,NE detected its growth regulation.Research result:1.The hair grew slowlyer in sympathectomized mice than in normal mice and PR mice.Meanwhile,the skins were thicker and the hair follicles were longer in 60HDA group.2.Brdu and Ki67 staining showed proliferation of keratinocytes in 60HDA group was less than the PR group and the control group.3.Skin Culture showed NE promoted hair grow,PH can inhibit NE promotion,PR had no inhibition.Research conclusion:This study provides suggestive evidence that sympathetic nerves regulate keratinocyte behaviors to promote hair growth. |
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