Study Of Hair Follicle Stem Cells' Differentiation Into Epithelium And Related Regulating Signal

PART ONE The localization and characteristics of mousevibrissa follicle stem cells and human frontal hair follicle stemcellsObjective: To investigate the localization and characteristic of mouse vibrissafollicle stem cells and human frontal hair follicle stem cells, and compare thenumber difference between normal adult's and adult alopecia's frontal hair folliclestem cells.Methods: 8 weeks old BALB/C mouse vibrissa pad samples and adult frontal skinsamples were harvested and fixed overnight in 10% neutral-buffered formalin.Formalin-fixed tissue was fixed by self-made sample fixator and dehydrationfollowed. Samples was embedded in paraffin and sectioned at 5 um thickness. Partof paraffin skin sections were stained with Haematoxylin and Eosin(HE), the otherwere processed and CK15 immunohistochemistry was done. Then examinedmicroscopically. CK15 positive cell counts was conducted to compare thenumber difference between normal adult's and adult alopecia's frontal hair follicle.Results: HE and immunohistochemistry both indicated the hair follicle iscomposed of concentric rings of an external outer root sheath(ORS) attached to thebasal lamina and contiguous with epidermis,a channel(IRS), and finally the hairshaft. CK15 is highly expressed in the bulge region, a specialized portion of theouter root sheath epithelium defined as the insertion site of the arrector pili musclein mouse vibrissa. However, in human hair follicles the bulge is an inconspicuousswelling. And normal adult's frontal hair follicle's CK15 expression is higher thanthat of adult alopecia's (P＜0.05).Conclusions: In mouse vibrissa hair follicle stem cells is localized in the bulge region. However, in human hair follicles the bulge is an inconspicuous swelling.And normal adult's frontal hair follicle's CK15 expression is higher than that ofadult alopecia's.PART TWO Study of mouse hair follicle stem cellsdifferentiating into TA cellsObjective: To investigate the accelerating process of mouse hair follicle stemcells'differentiating into TA cells after the intervention of exogenous EGF andwound stimulation..Methods: 8 weeks old BALB/C mice were divided 4 groups at random, all mice'svibrissa pad were shaved the epidermis. 1 group is blank group, the others wereinjected EGF daily through buccal mucosa 1000IU, 5000IU, 10000IU respectively.Oberserved the wound healing and compared the difference, decided the best EGFdose.8 weeks old BALB/C mice were divided 3 groups at random. That is A(EGFinterference group), B (positve control group,wounding group) andC(blank control group). Daily EGF(5000IU) buccal mucosa injection was appliedin A group, and shaving epidermis of vibrissa pads was applied in group B. Vibrissapads were harvested at d0, d1,d3,d5,d7,d9 respectively.①CK15/CK14immunohistochemistry of every sample was done and examinedmicroscopically.Cell counting was applied, the data was analysed by ANOVA.Samples were processed and ORS cells were harvested. FACS analyzed theCK15 and CK14 positive cell of every sample,and compared the changingtendency.Results: All three dose EGF can promote the healing of the vibrissa wound, andthe 5000IU is best dosage.Immunohistochemistry:①From dl,CK15 positive cells decreased but CK14positive cells increased obviously in A and B group(P＜0.05), and A group wasmanifester than B group(P＜0.05).However, C group had no obviouschange(P＞0.05).And at the same time from dl, CK15 positive cells countingresults: A＜B＜C(P＜0.05),CK14 positive cell counting results: A＞B＞C(P＜0.05).②FACS results indicated that CK15 positive cells'ratio decreased but CK14 positive cells' increased in A group and B group from dl, and A group was manifesterthan B group. C group had no obvious change.Conclusions:①Three dose of EGF(1000IU,5000IU,10000IU) all could promotethe wound healing.②EGF and wound stimulation both can prompt mouse hair follicle stem cells'sdifferentiation into TA cells, and the former is more obvious.PART THREE Notch signal's change of mouse vibrissafollicle ORS after EGF's intervention and wound stimulaitonObjective: To investigate Notch signal's change of mouse vibrissa follicle ORSafter EGF's intervention and wound stimulation.Methods: 8 weeks old BALB/C mice were divided three groups at random. That isA(EGF interfering group), B (wotmding group) and C(blank group). Daily EGFbuccal mucosa injection was applied in A group, and shaving epidermis of vibrissapad was applied in group B. Vibrissa pads were harvested atd0,d1,d3,d5respectively. Samples were processed and ORS cell were harvested.The expression of Notchl, Delta, ICN and EGFR gene were assessed by RT-PCR,and expression difference was evaluated.Results: Notchl, Delta, ICN and EGFR gene were all detected and there were nosignificant difference in groups and different time. The key component of activatedNotch signaling(ICN) only can be detected in EGF group at the first day afterEGF's inetervention.Conclusions:①Exogenous EGF can activate the Notch signaling, and the effectonly restricted at the early stage of the intervention.②Wotmd stimulation can not activate the Notch signaling.PART FOUR Treatment of large chronic wound usingautogenous human hair follicleObjective: To investigate the possiblity of treatment of large chronic wound usingautogenous human hair follicle.Methods: With the patient's consent, effective anti-infection and regular changing dressings were applied until the healthy granulation covered the wound. A2cm×8cm split-thickness skin grafting was harvested from occiput, and trimmed to 0.5cm×0.5cm stamps. The beneath occipital scalp was excised and 400 singlefollicles were dissected out as described by Philpott. The hair follicles dissectedfree from the subcutaneous fat and epidermis were implanted into the lateral side ofwound at intervals of 0.5cm.The stamp like split-thickness graftings weretransplanted onto the inside of wound. Gentamicin hydropathic compress wasapplied. Regular changing dressing and observating the newborn skin followed.Little samples were harvested to to dectect CK pan and vimentin to evaluate thenewborn skin.Results:①General observation: At 2 weeks after operation, a pale epitheliumextending out from the transplanted hair follicles looked like neoepidermis. Thehistology analysis confirmed neoepidermis generating from the transplanted hairfollicles. However, the neoepidermal layer was cobble-like and not confluent.Thesplit-thickness grafting surviveded but grew slowly; 8 weeks later, the newbornepidermis merged and confluent,which expressed a good appearance with somelanugo-like hair growing ;The split-thickness grafting area indicated obviousscarfing and was shaggy. 4 months later, the hair follicle transplantation zone wascompletely epithelialized with some lanugo-like hair growing. The new skin wasmature, which can resist some kind of scrape. On the other hand, the grafting zonewas scarfing and shaggy, with obvious scar and no hair growing.②Histology and immunohistology: 2 weeks after operation, neoepitheliumoriginated from implanted hair follicles with high level of ck pan and obvious reteridges, which is a important structure connecting the epidermis and dermis so thatskin can resistant scrape. In spit-thickness grafting zone rete ridges also weredectected.The new epithelium grew slowly.8 weeks later, a large amout of reteridges were observed in the hair follicle transplantation area. The new skinexpressed strong CK pan and vimentin. No glands were dectected; so was thesplit-thickness grafting area.Conclusions: Transplanting autogenous hair follicle can generate new skincovering the wound. It would be a good alterative in treating the large chronicwound.