| Background and objectiveSkin transplantation including skin autograft and skin allograft is widely used in severe burns,wounds,and cosmetic surgery.Skin autograft is the most superior graft for repairing the wound and restoring its physiological function.However,it must repeat harvesting from the same limited donor site,so that the source of autografts is often inadequate.In this situation,human skin allograft has been used for decades in clinic,and has save tens of thousands of lives.The major source of skin allografts is fresh cadaver skin,whose main advantage includes the effective physiological barrier that not only decreases the risk of infection,but also conducives to prevent loss of protein,water,heat,and electrolyte.It is also known to relieve the pain of the wound and reduce the frequency of dressing changes.Allogeneic skin transplantation has made great progress,but far failed to achieve the expected effect,which is still exist many problems in clinic application,including the possibility of disease transmission,not permanent use and limited supply.Tissue engineering skin as the new skin substitutes was invented to solve the problem of skin source.They can temporarily override the wound to prevent dehydration and keep the wound moist,but can't be used in extensive and depth burn or trauma.Heterogeneous skin mainly comes from pigskin.As the demand of the allograft skin has increased,the responsibility for looking for new source of skin has become very necessary.Teratoma used to appear in the ovary and testis,the gonads,near the axis or the axial part of the body,may also occur in the stomach,prostate,retroperitoneal,intracranial and sacral parts etc.Ovarian mature cystic teratoma,the most common benign tumor of ovarian germ cell tumor,contains multiple elements derived from ectoderm,mesoderm and entoderm.Ovarian mature cystic teratoma always contains the skin tissue,within which stratified squamous epithelium,sebaceous glands,hair and hair follicle tissue structure are present under electron microscopy.It is similar to normal skin tissues.However,up to now,it still remains poorly understood about this kind of skin.Our primary aim of this reseach was to:(1)analyze the immunological function of teratomatous skin;(2)detect the expression of P53,and P16 gene in teratomatous skin,squamous cell carcinoma of skin and squamous cell carcinoma of teratoma,and analyze the malignant tranformation rate of teratomatous skin of ovarian mature cystic teratoma;(3)observate and analyse that whether teratomatous skin can be used to repair skin defect,by transplantation experiments of teratomatous skin in nude mouse;(4)detect the expression of CyclinDl,P53,P16,P21 and c-Myc in teratomatous skin after transplantation,in order to further analyse whether it turns malignant.MethodsChapter I Comparison of immune function between the teratomatous and normal skin1.Teratomatous skins from 20 patients with ovarian mature cystic teratoma admitted to Nanfang Hospital for operation were selected.Normal skins from 10 patients with breast cancer received radical mastectomy were served as control.Part of tissue samples were fixed with 10%formalin.The remainder were stored in RPMI1640 medium at 4?.2.The expression of CD1a,CD83,CD68,CD4,CD8,and DR of the teratomatous and normal skin was examined by immunohistochemistry,in order to observe the number and distribution situation of langerhans cells,macrophages and T lymphocytes in these two kinds of skin tissues.3.The samples were made to observe the ultrastructure of immune cells of teratomatous and normal skin by transmission electron microscopy.Chapter ? Malignant transformation rate and P53,and P16 expression in ovarian mature teratoma1.The clinical specimen data from patients with ovarian mature teratoma and squamous cell carcinoma originated from the teratomatous skin in Nanfang Hospital in recent 10 years were collected and statistically analized.The expression of P53,and P16 was detected by immunohistochemistry.2.The expression of P53,and P16 was detected in 25 cases teratomatous skin after grafting and 20 cases of squamous cell carcinoma was detected by immunohistochemistry.Quantitative methods were used to analyse the differential expression through Image-Pro Plus 6.0 Image analysis software.Chapter ? Teratomatous skin grafted on nude mouse1.The teratomatous skins from 20 specimens of patients were grafted on 30 nude mice.The grafting progress is as follows.The subcutaneous adipose tissue of teratomatous skins was cut off as far as possible,but the intact epithelial tissue must be kept.Then,animals were anesthetized using an intraperitoneal injection of pentobarbital sodium(40 mg/kg of body weight)and were placed in a prone position on a standard surgical platform with the dorsum exposed.Skin was disinfected and a square graft bed was created by excising approximately 1cmx1cm of full-thickness skin and subcutaneous tissue from the subscapular region of the mouse.The graft was placed in the graft bed with a good fit.Interrupted suture was performed with 7-0 nylon line,and then the wound was covered with adhesive bandage.General condition and appearance was observed.2.The source of healing skin is identificated by HE staining,melanin staining and immunohistochemistry with antibodies such as Vimentin and CD34.Chapter IV The detection of CyclinDl,P53,and P16,P21 and C-Myc in teratomatous skin after successfully grafting and the impact on organs of nude mouse1.25 cases of teratomatous skin after successfully grafting were selected as experimental group and 20 cases of cutaneous squamous cell carcinoma were served as control group.The expression of CyclinDl,P53,and P16,P21 and C-Myc was detected in the two groups by immunohistochemical and in situ hybridization method.2.At endpoint after grafting,mice were killed using 1%sodium pentobarbital anesthesia.All organs of mice were fixed with 10%neutral formaldehyde,dehydrated,transparent,wax dipped,embedded,sliced and hematoxylin-eosin stained to observe if there is the formation of metastases.ResultsChapter ? Comparison of immune function between the teratomatous and normal skin1.The immunohistochemical results show that the number and distribution situation of langerhans cells,macrophages and T lymphocytes in teratomatous skin are similar to normal skin.The number of langerhans cells,macrophages and T lymphocytes showed no significant difference between teratomatous skin and normal skin(all P>0.05).2.Under transmission electron microscopy,ultrastructure of langerhans cells of teratomatous skin is similar to normal skin langerhans cells.Chapter ? Malignant transformation rate and P53,and P16 expression in ovarian mature teratoma1.Of 1913 cases of ovarian mature cystic teratoma in nearly 10 years,only two cases of squamous cell carcinoma were found in teratomatous skin,with malignant transformation rate of 0.1045%.P53 expression was detected in 2 cases squamous cell carcinoma originated from teratomatous skin and P16 overexpression in one.2.There were no expressions of P53 and P16 in 25 cases of teratomatous skin of ovarian mature cystic teratoma.Of 20 cases of squamous cell carcinoma P53 overexpression(positive rate of 55%)was detected in 11 cases,P16 overexpression(positive rate of 35%)in 7 cases.The positive rates of P53 and P16 expressions in squamous cell carcinomas were significantly higher than that in the teratomatous skins(P<0.001,P=0.002).Chapter ? Teratomatous skin grafted on nude mouse1.Of 30 nude mice in the experimental group,13 were grafted successfully.The total successful rate was 43.33%.Morphological variation was found in grafts,including the length and density of the hair,color and area of the survival graft.2.HE staining showed that the teratomatous skin graft contains epidermis and dermis.There are about 5 to 6 layers in Keratinizing squamous epithelium,with obvious rete ridges.Hair follicles,sebaceous glands,and sweat glands can be seen in the dermis layer.However,there are about 2 to 3 layers in epidermis of mice.The dermis layer exhibited hair follicles and sebaceous glands but not sweat glands.Melanin staining showed there were no melanin granules in nude mice skins,while there were many melanin granules in basal cell layer and spinosum stratum of teratomatous skin.Immunohistochemical staining showed HLA-ABC expression was positive in the epidermis of teratomatous skin,while negative in the mouse skin.In dermis,Vimentin was positive in the teratomatous stroma,but negative in the mouse stroma.The result supported that the skin was origined from tetatoma.Chapter ? The detection of CyclinD1,P53,and P16,P21 and C-Myc in teratomatous skin after successfully grafting and the impact on organs of nude mouse1.Immunohistochemistry results showed that Of 25 cases of teratomatous skin,there were no expressions of P53 and P16,but Cyclin Dl,P21 and c-Myc were overexpressed in 2,4,and 3 cases,respectively.However,of 25 cases of squamous cell carcinoma,Cyclin D1 and P53 were overexpressed in 11 cases,and P16,P21,and c-Myc were overexpressed in 7,9,and 12 cases,respectively.The positive rates of Cyclin D1,P53,P16,P21,and c-Myc in squamous cell carcinoma were significantly higher than those of the teratomatous skin respectively(P=0.001,P<0.001,P=0.002,P=0.033,P=0.001).2.In situ hybridization results showed that of 25 cases of teratomatous skin,there were no expressions of c-Myc,P53 and P16,but Cyclin D1 and P21 were overexpressed in 1,and 2 cases,respectively.However,of 25 cases of squamous cell carcinoma,Cyclin D1,P53,P16,P21,and c-Myc were overexpressed in 8,7,5,7,and 6 cases,respectively.The positive rates of Cyclin D1,P53,P16,P21,and c-Myc in squamous cell carcinoma were significantly higher than those of the teratomatous skin respectively(P=0.003,P=0.002,P=0.013,P=0.015,P=0.005)3.At endpoint after transplantation,HE staining results show that there is not the formation of metastases in heart,liver,spleen,lung,kidneys,and brain.Conclusion1.The observation results of the number,distribution and ultrastructure of immune cells of the teratomatous skin were similar to those obtained in the normal skin.We hypothesized that the teratomatous skin has basic immunological function.2.There was low risk of malignant transformation in teratomatous skin of ovarian mature cystic teratoma which can be explained by lower P53 and P16 expressions in teratomas than that in squamous cell carcinoma.3.The teratomatous skin can graft on nude mice successfully.Hair,sebaceous and sweat glands were founded in the graft.So this kind of skin can be used for tissue repair.4.There were no expression of Cyclin D1,P53,P16,P21,and c-Myc in the teratomatous skin after grafting and no tumor formation was found in organs,which suggests that it does not cause cancer after teratomatous skin grafting. |
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