Expression And Significance Of TGF-β/Smad Signaling Pathways And Related Genes In Actinic(Solar) Keratosis

The first part:Analysis of clinical and pathological features of actinic(solar) keratosisObjective:The clinical skin lesion of actinic(solar) keratosis(AK) are variegated and atypism,resulting in high rates of clinical misdiagnosis.The different clinical lesion need to indentify with different clinical diseases,and histopathologic diagnosis with high diagnostic rate.To improve the standards of diagnosis,precaution and treatment of AK for reducing the incidence of disease and skin carcinogenesis,a retrospective review had been conducted.A integrated analysis about the clinical,pathological,diagnostic characteristics have been done.Methods:Descriptive epidcmiological study was applied to analyze the cases from the dermatology outpatient clinic of the first affiliate hospital of Kunming Medical College between 1997 and 2008.The paraffin sections of this disease were checked and some samples were sliced again.Ninty cases of AK by pathological diagnosis were collected.The subjects were all Yunnan population.Their clinical features were analyzed with pathological aspects observed.Excel and SPSS 10.0 were used for data analysis.Results:A total of 790851 person-time of outpatient,14550 biopsy examples and 90 cases of AK were being cpidcmiologic survey during a 12 year study period from 1997 to 2008.The average constituent ratio of AK was 1.13/10 000,and the annual constituent ratio was 9.4/100 000.The onset of AK increases with advancing age.There was a distinct characteristic of seasonal difference.The ratio of male/female was 1:1.5.The patients'ages ranged from 33 to 85 years and averaged 65.94±10.46 years.92.2%patients occurs the macules after 50 years old and over.The average of courses of AK was 2.6 years.The macules were located on face in 83 cases(93.3%),on the cheek in 44 cases(48.9%),2 cases(2.2%) have developed to squamous cell carcinoma.Among these 90 cases,46(51.1%) cases were reddish brown macules,23(25.6%) were blackish brown macules,8(8.9%) cases were black papules,7(7.8%) cases were comu cutaneum,4 cases were anabrosis,2 cases were combined with ulcer.Six histopathology types of AK were included:hypertrophic(40%),atrophic(24.4%), bowenoid(16.7%),acantholytic(5.6%),pigmented(6.7%) and lichenoid(6.7%).2 cases which combined with ulcer developed to invasive squamous cell carcinoma.Some lesions(20%) show spread into the infundibular and isthmic segments of follicles or less commonly along eccrine ducts(12.2%).Dermal changes include solar elastosis,an infiltrate of lymphocytes and plasma cells and increased vascularity.The diagnose accordance rate of clinic and pathological was only 42.2%.cases were previously diagnosed as seborrheic keratosis, melanocytic nevus,basal cell carcinoma,lupus vulgaris or other diseases.Conclusions:Actinic(Solar) keratosis was most common in the fifth and over decades of life and involved sun-exposed areas.Actinic(Solar) keratosis and showed a marked preponderance in females in Yunnan people.Under the situation of patient's senior age, slow progression of the disease course and the non-typical clinical presentations.The misdiagnosis often happen in clinic works.Pathological examination is important to improve accuracy of diagnosis and to differential diagnosis from other diseases.The second part:In vitro culture and biological characteristics of keratinocytes isolated from actinic(solar) keratosisObjective:A variety of culture methods about keratinocyte under diseases have been established and developed.However,few studies reported in vitro culture of keratinocytes isolated from AK.This experiment aims are to culture keratinocytes isolated from AK and to identify its biological characteristics in vitro,to establish a cell model of human AK for mechanism analysis.Methods:Primary dysplastic keratinocytes were isolated from patients with AK(AKKc), nomal keratinocytes from face skin of old people(O-NKc) using serum-free culture technology.A431 cell line of skin squamous cell carcinoma was used to control using DMEM medium contain with 10%FBS.Morphological changes and ultrastructure of cells were observed under the microscope and transmission electron microscope.The expressions of keratin in keratinocytes were detected by indirect immunofluorescence.In addition,the capacity of migration was observed by scratch test,the double-soft-agar colony forming ability and tumorigenecity of the cells were identified to confiem the mglignant characteristics of AKKc.Results:The AKKc possessed typical epidermal cell characteristics like normal keratinocytes (O-NKc):that is,revealed a typical "paving stone" like under an inverted microscope, showed keratin-positive,and massive bundles of tonofibrilla could be seen in the plasma under a transmission electron microscope.Compared with the normal groups:nucleolus, heterochromatin and number of mitochondria were increased,number of endoplasmic reticulum was reduced,and the characters of keratinocyte under electron microscope showed more immature in the keratinocytes from AK.The growth curve,scratch test, double-soft-agar colony formation rate experiments showed that AKKc have a stronger proliferation,migration and tumorigenic capacity than O-NKc,but lower than A431.Conclusion:Keratinocytes can be isolated from samples of AK.These researches show that AKKc is a kind of special transitional cell type exist between normal cell and SCC,which has dysplastic typical cell structure and biologic behavier.This cell can be used as a cell model to study carcinogenesis and prevention of AK.The third part:Expression of TGF-β/Smad signaling pathways and related genes in actinic(solar) keratosisObjective:To investigate the expressions of TGF-β1,receptor(TβRⅠ,TβRⅡ),Smads protein and P53,MMP-2,Fas,FasL in AK and the correlations between each other,for an insight into the mechanism of aberrant TGFβ/Smad signaling and tumorous hyperplasia in AK.Methods:First,the real-time quantitative polymerase chain reaction(RT-Real time PCR) and MaxiVision^TM IHC techniques were utilized to detect the expresions and localization of TGF-β1,TβRⅠ,TβRⅡ,Smads related proteins and P53,MMP-2,Fas,FasL mRNA and protein in AK,SCC and control normal facial skin paraffin specimens.Then the digestion method of separation and primary cultured keratinocytes and fibroblasts of AK lesions and the normal aged face skin.After the cells were identified,RT-Real time PCR and Western Blot were utilized to detect expressions of TGF-β1,TβRⅠ,TβRⅡ,Smad related proteins and P53,MMP-2,Fas,FasL mRNA and protein in keratinocytes and fibroblasts of AK and control cells.Results:In the normal control skin,AK and squamous cell carcinoma,RT-Real time PCR revealed that the expression of Smad7,MMP-2,P53,FasLmRNA showed increasing trend, while Fas mRNA expression decreased gradually,there are statistically significant't;TGF-β1, TβRⅠ,TβRⅡ,Smad2,Smad3,Smad4 mRNA to increase in the AK,to decrease in the SCC. These results were consistent with the results of immunohistochemical,that the expression of Smad7,P53,FasL protein as the disease progress is gradually,Fas protein gradually decreased, while the expression of TGF-β1,TβRⅠ,TβRⅡ,Smad2,Smad3,Smad4 and MMP-2 protein were down-regulates in the basal layer of AK lesions,but significantly up-regulated in the upper epidermis and dermis.In the primary culture of keratinocytes and fibroblasts of AK,the expression trends of TGF-β1,p53,Smad4,Fas and FasL were same(down or up regulation) compared with control cells,respectively.The expression trends of TβRⅠ,TβRⅡ, Smad2,Smad3,Smad7 of MMP-2 is opposite,that the decreased expressions of TβRⅠ, TβRⅡ,Smad2,Smad3,and MMP-2 mRNA and protein in AKKc,but significantly up-regulated in AKFb,Smad7 expression was the opposite.Conclusion:The disorders of the TGF-β/Smad signaling pathway in AK involving many important aspects.It may be not caused by one genetic disorder,but multiple gene disorders in the TGF-β/Smad signaling pathway and the interaction between keratinocytes and fibroblasts in the occurrence and development of AK.