Establishment Of An Animal Model Of Choroidal Melanoma And The Expression Of Tissue Factor In It

BACKGROUND:Choroidal melanoma(choroidal melanoma,CM) is the most common malignant uveal,the most common primary malignant tumors of the adults eye.They are characterized by a high degree of malignancy,early metastasis and widespread,with the vision and life pose a serious threat.Choroidal melanoma in the study encountered by the main difficulty is compared with other tumors such tumor incidence rate is very low,so in order to obtain a significant number of samples is very difficult.In the past few decades,scientists study choroidal melanoma faced by the main challenge is to establish a suitable animal model.OBJECTIVE:To establish choroidal melanoma rabbit model with human choroidal melanoma cell lines OCM-1.This study was conducted for two purposes: First,to compare the tumor growth and the survival rate of different doses of cyclosporine A(CsA) in the rabbit model of choroidal melanoma;second,to illustrate tissue factor expression and localization in CM using immunohistochemical, and count the number of MVD,in order to investigate the relationship between TF and MVD.METHODS:The human choroidal melanoma cell OCM-1 was used to establish rabbit model of choroidal melanoma.Disregarding sex,35 matured rabbits were medially divided into 4 groups randomly:Group A(non-injection cyclosporine A),Group B(cyclosporine A 15mg/kg/day),Group C(cyclosporine A 20mg/kg/day), Group D(cyclosporine A 20mg/kg/day,after two weeks 15mg/kg/day,after four weeks 10mg/kg/day,after six weeks 5mg/kg/day,and continued until last).5 animals in group A;group B,C and D were 10 animals each.All experimental animals left eye for surgery.B-mode ultrasonography,MRI scans and fundus photography were used after operation.After natural death,the eyeball,lung tissue and liver tissue were obtained for biopsy HE pathological and immunohistochemical staining.RESULTS:1.Survival time:only one animal in Group A died in the fourth week,the remaining 4 animals were sacrificed in the eighth weeks.The mean survival times were 5.74±0.64 weeks in Group B,4.33±0.41 weeks in Group C,5.71±0.81 weeks in group D.The difference in survival between Group B and Group C was significant(P=0.01＜0.05).The difference in survival between Group D and Group C was significant(P=0.012＜0.05).The difference in survival between Group B and Group D was no significant(P=0.878＞0.05).Kaplan-Meier cumulative survival rates are shown that group B and group D display a better survival.2.B-mode ultrasonography:With B-mode ultrasonography we measured the largest basal tumor diameter and height and calculated volume estimate based on the assumption of a half-rotation ellipsoid with the formula 4/3 p a~2H,then divided by 2. The formula is:2/3 p a~2H(H=tumor maximum height,a= half of tumor basal diameter,p=3.14).In the first week we were not able to measure tumor height,just found partially thickening of sclera wall,since the second week we recorded basal tumor diameter and height.All from the same period in each experimental animal eyes,the average tumor volume rendering tumor growth curve.In the first four weeks Group C and Group D intraocular tumor of laboratory animals grew faster, there was no significant difference.After four weeks intraocular tumors average volume of Group C continued to increase,increasing at a faster speed.After four weeks intraocular tumors average volume growth of Group D laboratory animals had slowed,after five weeks the volume became less,in which one tumor in the eye in the eighth week was unable to detect with B-mode ultrasound,whose volume was recorded as 0.Intraocular tumors average volume of Group B experimental animals increased gradually,the growth rate was slower than the group C,but in the end there was no significant difference between the average size of group B and group C.3.HE staining:Histopathological detection found intraocular tumor incidence rates were 60%in group B(6),80%in group C(8),40%in group D(4).Among these three groups there was no significant difference.Group A found no tumor growth.In all groups tumors were confined to the choroid,without any case of iris and ciliary body metastasis.Lung metastasis:two in group B(33.3%),one in group C(12.5%),one in group D(25%).The difference of incidence of lung metastasis in these three groups was not significant.Liver metastasis were not found in all three groups.4.Tissue factor(TF) expression in tumor tissue:Positive rate of TF staining in intraocular tumor was 88.9%.TF staining intensity of intraocular tumor in Group A, B,and C increase with tumor size,moderately positively stained in the majority.In group D tumor volume decreased gradually after five weeks,the TF staining intensity does not increase with time,the intensity of staining were mainly suspicious positive.TF staining intensity of intraocular tumor in those animals that lung metastasis occurred were significantly higher than those that lung metastasis didn't happen.In Lung lesions of metastasis TF positive rate was 100%.TF staining intensity in pulmonary metastasis were significantly higher than the original intraocular tumor expression.Tissue factor(TF) expression was positively correlated with the MVD.The number of MVD in metastasis is more than in the eyes.CONCLUSION:With the use of human choroidal melanoma cell lines OCM-1 cells,with the application of different doses of cyclosporine A for rabbit immunosuppression,we founded rabbits choroidal melanoma animal models through a surgical procedure and conducted following research,and come to the following conclusions:1.we successfully establishing a rabbit model of choroidal melanoma.This model is close to the characteristics of human diseases,providing experimental basis for the further study.2.Evaluating the role of different doses of cyclosporine A in animal model foundation,as well as the complication and survival rates of experimental animals at different doses.3.It is easy and convenient to measure intraocular tumors with B-mode ultrasonography.It was applied in the animal model building process of choroidal melanoma,in the measurement of intraocular tumors diameter and height precisely, and that the tumor volume,to get the effects of different doses of cyclosporine A on the tumor growth rate. 4.Tissue factor(TF) expression was positively correlated with the MVD,which was correlated with occurrence of choroidal melanoma and the incidence of metastasis and prognosis,to provide a theoretical basis for tissue factor targeted therapy of choroidal melanoma.