| Objective:1.To investigate the clinicopathological characteristics of cutaneous melanoma and the mutation analysis of susceptibility genes.2.Immunodeficient animals were used to complete the melanoma formation experiment and simulate the development process of melanoma,providing a preclinical experimental basis for the diagnosis and treatment of tumors.Methods:1.Data of melanoma patients diagnosed in People’s Hospital of Xinjiang Uygur Autonomous Region from January 2009 to December 2019 were collected to retrospectively analyze the etiology and clinicopathological characteristics.The mutations of BRAF,NRAS,c-kit genes and human telomerase reverse transcriptase(h TERT)gene promoter region in melanoma tissues were detected by polymerase chain reaction(PCR)and direct DNA sequencing,and the relationship between gene mutations and clinicopathological characteristics was analyzed.2.Twenty BALB/c immunodeficient nude mice were purchased and randomly divided into four groups:A,B,C and D,with 5 mice in each group.Human malignant melanoma A375 cells were cultured and counted in vitro,and the cell suspension concentrations were adjusted to 1×10~5,1×10~6and 1×10~7cells/m L.0.2ml of each was subcutaneously inoculated into the back of nude mice in group A,B and C,respectively,and D was the healthy control group.The growth,tumor growth time,tumor volume and body weight of nude mice in all experimental groups were observed.Tumor growth curves were drawn.The mice were sacrificed on the 42nd day after observation,and the tumor bodies were removed for HE staining and immunohistochemical staining of Melan-A,SOX-10 and Ki-67.RESULTS:1.Among the 94 patients,there were 46 males(48.9%)and 48 females(51.1%).There was no statistical difference in gender(P>0.05),and the mean age was(58.5±16.0)years old.There were 41 cases of Han nationality(43.6%)and 53 cases of ethnic minorities(56.4%).The most common site of disease was the extremities,with 50 cases(53.2%),27 cases(28.7%)in the head,face and neck,10 cases(10.6%)in the trunk,and 7 cases(7.4%)in the extremities.27.7%(26/94)of the patients had a clear history of trauma before onset.The positive expressions of S-100,Melan-A,HMB45 and Vimentin were93.8%-100%in melanoma patients.43.6%(41/94)of the patients were classified asⅣand normal.At the time of consultation,the clinical stages of AJCC(8 edition)of the patients were:Ⅰstage 9 cases(9.6%),Ⅱstage 26 cases(27.7%),Ⅲstage 15 cases(16.0%),Ⅳstage 21 cases(22.3%),and the remaining 23 cases(24.5%)could not be judged.Among the 94 patients,48 cases of wax block were well preserved,including11 cases of BRAF mutation with a mutation rate of 23.1%(11/48),5 cases of NRAS mutation with a mutation rate of 10.4%(5/48),6 cases of c-kit gene mutation with a mutation rate of 12.5%(6/48),and 7 cases of h TERT gene promoter mutation.The mutation rate was 14.6%(7/48).There were statistically significant differences between BRAF gene mutation and melanoma patients’age and site of disease(P<0.05);NRAS,c-kit gene mutation,h TERT gene promoter mutation and lymph node metastasis were statistically significant(P<0.05).2.Of the 15 nude mice,13 developed melanoma successfully,1 nude mouse in group A showed no tumor formation and 1died of unknown cause.The tumor formation rate of human malignant melanoma cell line A375 was 86.7%when subcutaneously injected into BALB/c nude mice.The tumor tissue was confirmed to be melanin-derived tumor by immunohistochemical staining.Tumors of nude mice in group A,B and C were all formed by subcutaneous nodules.The mean time of tumor formation was(13±1.73)days in group A,(7.8±1.64)days in group B,and(4.2±1.64)days in group C.There were significant differences in tumor formation time and tumor volume between group A and group C(P<0.05).There was no statistical significance in tumor formation time and tumor volume between group A and B,or between group B and C.Mice in the three groups were well fed with water,their body weight increased steadily,and no obvious cachexia was observed.There was no significant difference between the body weight of mice and the concentration of melanoma cells injected on day 42(P>0.05). Conclusion:1.Cutaneous melanoma in this group was mainly middle-aged and elderly patients,and the most common site was the extremities,and most patients had a late stage when seeking treatment.The incidence of melanoma is related to ethnic differences,and the formation of melanoma ulceration is related to the history of trauma.2.BRAF gene mutation is closely related to the age and site of melanoma.NRAS,c-kit gene mutation and h TERT gene promoter mutation were closely related to lymph node metastasis.3.The tumor formation rate of human malignant melanoma A375 cell model in nude mice was higher.The time of tumor growth and tumor volume were related to the concentration of subcutaneous injection cells.The tumor formation effect of subcutaneous injection of at least 10~6cells was better.The body weight of mice was not related to subcutaneous injection cell concentration. |
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