Objective To isolate and purify the melanoma stem cells (MSC) in choroidal melanoma OCM-1 cells. Methods OCM-1 cells were resuscitated, and after cultured in standard Dubecco's modifided Eagle's medium (DMEM)/F12, they were cultured in serum-free medium (SFM). The cultured MSC were isolated and purified, and the positive rate of CD133, the specific markers of neurostem cells, was observed by flow cytometry (FCM). The 6th generation of the cells were stained by musashi-1 immunocytochemistry, and the rate of the positive cells was observed under the microscope. Results After the Adherent OCM-1 cells cultured in SFM, the number of the adherent number decreased obviously. The cells at the 6th generation grew as the suspended gobbets, which represented the typical grow manner of the stem cells. Positive CD133 could be found in the cells of different generations, which was 2.5％, 21.7％, and 57.8％ in the non-isolated OCM-1 cells, the 1st generation of isolated cells, and the 2nd generation cells, respectively. The positive rate of CD133 in the cells at the sixth generation was 79.8% with b positive expression of musashi-1. Conclusion MSC is in the human choroidal melanoma OCM-1 cells. The suspended stem cells may be purified by limited differentiation and serial passage in SFM. (Chin J Ocul Fundus Dis, 2007, 23: 87-90)
Objective To investigate the development and metastasis of malignant choroidal melanoma cell strain OCM-1-gfp modified with green fluorescent protein(GFP) and the factors which affected the tumor biological behaviors. Methods GFP was transfected into malignant melanoma cell strain OCM-1.Melanoma cells with high and stable expression of GFP were injected into subretinal space and the subcutaneous space of hind leg of Balb/c nude mouse respectively in order to establish orthotopic and heterotopic transplanted tumor models.The development and metastasis process of orthotopic tumor models was observed directly by fluorescence microscope，and the size of the hypodermal tumor was measured by vernier.The expressions of 13 genes in melanoma were detected by means of immunohistochemistry staining. Results Malignant choroidal melanoma cell strain OCM-1 stably expressed GFP and preserved the characteristics of parental generation，OCM-1-gfp may develop melanoma and continue to metastasize in nude mouse.Positive expression of most of the antibodies，including Rb，p53，p21，E2F，NFkappa;B，cyclin D1，proliferation cellular nuclear antigen（PCNA），bcl2、bclXL/S，bax，and epithelial growth factor（EGF）and its receptor（EGFR），was found.While the staining of inhibition gene p16 was negative. Conclusions GFP is the marker for observing the development and metastasis of malignant choroidal melanoma in vivo.The rate of tumor formation and development process in orthotopic models does not differs much from which in heterotopic models of malignant choroidal melanoma.The expressions of lots of genes in malignant choroidal melanoma developed from OCM-1-gfp including p16、p53、NFkappa;B，cyclin D，PCNA，EGF，and EGFR are abnormal. (Chin J Ocul Fundus Dis, 2006, 22: 170-173)
ObjectiveTo observe the expression and transcription of MART-1 in human uveal melanoma cell lines 92-1, 92-2, Ocm3, Me1285, as well as the possible effect of methylation on its expression.MethodsThe cell lines 92-1, 92-2, Ocm3 and Mel285 were cultured routinely and tested for MART-1 expression at protein and mRNA level by FACS analysis, Western blot and RT-PCR respectively. Methylation status of the MART-1 promoter region in all the cell lines were checked by Southern blots of DNA digested with methylation sensitive restriction enzymes.ResultsAs observed in FACS analysis and Western blot, 92-1, 92-2 and Ocm3 were MART-1 positive cell lines while Me1285 was negative cell line. Consistent with protein analysis, 92-1 and Ocm3 cell lines showed MART-1 specific PCR products and there was no product in Me1285 cell line in RT-PCR. The MART-1 positive cell lines, 92-1, 92-2, and Ocm3 show methylation at the MspI/HpaⅡ site, and the NruⅠ sites of all positive cell lines are not methylated. The MART-1 negative cell line Mel285 shows hypermethylation at the NruⅠsite and the MspⅠ/HpaⅡ site is not methylated.ConclusionsMART-1 could be expressed in human uveal melanoma cell lines 92-1, 92-2 and Ocm3. The change of methylation status of MART-1 promoter may correlate with the transcription of MART-1.
ObjectiveTo observe the OCT angiography (OCTA) imaging features of isolated choroidal hemangioma (CCH).MethodsA retrospective case study. From January 2017 to February 2019, 18 CCH patients (18 eyes) diagnosed in the Affiliated Eye Hospital of Nanjing Medical University were included in the study. There were 13 males (13 eyes) and 5 females (5 eyes), with the mean age of 44.5 years. All the tumors were orange-red, with clear boundaries, located at the posterior pole or around the optic disc. OCTA was used to scan the 6 mm×6 mm of macular area or in the range of 6 mm×6 mm. After automatic image processing, the system provided the blood flow map of shallow capillary plexus, deep capillary plexus, outer retina and choroidal capillary plexus, as well as the corresponding structure en-face image and B-scan image.ResultsOCTA examination found that when the stratification line was adjusted to the periphery of the choroidal capillary layer, the blood flow map showed clear boundary of the tumor, and the blood vessels on the surface of the tumor presented a network crisscross with different thickness. B-scan image showed that the whole layer of retinal choroid at the tumor presented a dome-shaped uplift, and the neurocortical layer could be accompanied by thickening, subretinal effusion, exudation and splitting. En-face image showed that the boundary of the tumor was clear, the surrounding exudation was strong reflection in spots or patches, local pigmentation showed weak reflection, and the signal reflection was uneven.ConclusionOCTA can clearly show the vascular morphology on the surface of CCH.
Objective To evaluate the effect of transpupillary thermo therapy (TTT) on the treatment of intraocular tumors. Methods A total of 50 patients with intraocular tumors, including 37 choroidal hemangioma, 2 retinal capillary hemangioma, 5 choroidal osteoma, 4 choroidal melanoma, and 2 retinoblastoma (RB) underwent TTT and were followed up for 1～20 months. Results In 30 patients with choroidal hemangioma (average follow-up was 5.1 months), 29 (96.7%) had pigment scarring in different levels and the retinal detachemnts were partly or completely recovered; 1 had no obvious improvement. The visual acuity was unchanged in 24 (80.0%) patients, improved in 41 (13.3%) and declined in 2 (6.7%). In 2 patients with retinal capillary hemangioma, no effect was found. In 5 eyes (4 patients) with choroidal osteoma (average follow-up was 6 months), no change of the tumor was found in 1 and the atrophic spots were seen in 4; the visual acuity was unchanged in 3, improved in 1 and declined in 1. In 4 patients with choroidal melanoma (average follow-up was 8 months), the tumor was shrunken in 1, unchanged in 2, and enlarged in 1; the visual acuity was unchanged in 2 and declined in 2. In 2 patients with RB, RB was totally shrunken in 1 and partly shrunken in 1. Visual acuity of one child patient who was followed up for 20 months could not be examined, and was unchanged in another one who was followed up for 3 months. No severe complications were found in the patients during the treatment and the follow-up. Conclusions TTT is effective for the treatment of some intraocular tumors except retinal capillary hemangioma. It is a kind of potential treatment for intraocular tumors with few side-effect. (Chin J Ocul Fundus Dis，2003，19：144-148)
Objective To investigate the influence of vascular endothelial growth factor (VEGF) antagonist bevacizumab on the growth of human choroidal melanoma (CM) OCM-1 cell xenografts in nude mice, and to explore the probable mechanism.Methods OCM-1 cells were subcutaneously implanted on 18 nude mice to establish ectopic model of human CM. The nude mice with the tumor of 5 mm in diameter were randomly divided into three groups: untreated group (group A), normal saline （NS） group (group B), drug treated group (group C). Bevacizumab was intraperitoneally injected for 14 consecutive days in group C, and the same volume of NS was used at a same way in group B. The volume and weight of implanted tumor as well as inhibitory rates of drug on tumor were calculated, ki67 and survivin proteins were measured with immunohistochemistry, and the mRNA expression of VEGF and survivin were assessed by RT-PCR.Results The volume and weight of tumor was (598.86plusmn;321.81) mm3, (0.66plusmn;0.15) g; (1 715.15plusmn;278.16) mm3, (1.54plusmn;0.39) g and (1 750.23plusmn;206.36) mm3, (1.54plusmn;0.31) g in groups C, A and B, respectively. There were significant differences between group C and A (F=34.53, P=0.00) and group C and group B (F=8.69, P=0.01). The inhibitory rate of these three groups were 57.14%, 5.31%, 6.25%, respectively, and the proliferation index (PI) of ki67 in these three groups were (51.85plusmn;1.32)%, (46.30plusmn;1.39)%, (27.90plusmn;0.90)%, respectively, there were significant differences in ki67 PI between C group and A or B group (H=15.17, P=0.00). The expression of survivin mRNA was (0.49plusmn;0.02), (0.82plusmn;0.05) and (0.61plusmn;0.05) in groupss C, A and B, respectively, there were significant differences between C group and A or B group (F=15.17, P＜0.05) . The expression of VEGF mRNA was (0.32plusmn;0.08), (0.73plusmn;0.07), (0.80plusmn;0.04) in groups C, A and B, significant difference was found between group C and A or B group (F=12.05，P＜0.05). Conclusion Bevacizumab can inhibit the growth of human CM in nude mice probably by inhibiting the activity of VEGF and downregulating survivin expression of the tumor as well as inhibiting the growth of the tumor.
ObjectiveTo observe the multimodal imaging characteristics of choroidal metastasis.MethodsA retrospective clinical observation study. From January 2016 to November 2018, 28 patients with choroidal metastasis diagnosed in Department of Ophthalmology in the Second People’s Hospital of Yunnan Province were included in the study. There were 12 males and 16 females, with the mean age of 50.8±6.9 years. There were 18 unilateral patients and 10 bilateral patients. The lesion of choroidal metastasis was regressed after systemic antitumor therapy in 3 patients (4 eyes). All patients underwent ultra-wide-angle fundus photography, infrared fundus imaging, fundus autofluorescence, FFA, frequency-domain OCT, and B-ultrasound examinations.ResultsIn the ultra-wide-angle fundus photography, metastatic tumors were located in the posterior or middle part of the retina, of which 26 were isolated lesions and 12 were multifocal. A yellow-white bulge lesion with (11 eyes) or without pigmentation (27 eyes). There were 12 eyes with exudative retinal detachment. Infrared photography of the fundus showed that the tumor area showed varying degrees of mottled brightness change, and the infrared photograph of the exudative retinal detachment area was relatively low. Fundus autofluorescence showed that 14 eyes had plaque-like strong autofluorescence in the tumor, 13 eyes had a mottled autofluorescence formed by strong and weak fluorescence in the tumor; 3 eyes of old lesions showed " leopard-like” autofluorescence. Among the 38 eyes in the fluorescein angiography, 32 eyes of the early lesions showed low fluorescence, and the venous phase showed a needle-like high fluorescence point, and the post-leakage fluorescence gradually increased. Two eyes with old lesions showed a " leopard-like” change. In 38 eyes, OCT showed wavy ridges of the choroid and pigment epithelium, and a large number of fine-grained or cluster-like high-reflector accumulations were observed between the retinal neuroepithelial layer and the pigment epithelial layer. B-ultrasound showed substantial lesions in the posterior pole and uniform internal echo. There were 23 eyes with flat shape, 12 eyes with flat hemisphere, and 3 eyes with irregular shape.ConclusionsColor photography of the fundus showed the size, location, pigmentation and peripheral retinopathy of the metastatic lesions. Infrared photography showed different reflex signals in the tumor, exudation, and atrophy. The autofluorescence of the fundus showed the damage of pigment epithelium in the lesion. In the fluorescein angiography, the fresh tumor showed fluorescence leakage, while the atrophic tumor showed transmitted fluorescenc. OCT reflected the height of the lesion and the change of pigment epithelium.
Objective To observe the therapeutic effect of plaque radiotherapy (PRT) on choroidal melanoma. Methods PRT was performed on 21 patients (21 eyes) with chroidal melanoma who had been examined by ophthalmoscopy, fundus fluorescein angiography (FFA), indocyanine green angiography (ICGA), and B-scan echography. The visual acuity was le;0.05 in 3 eyes, 0.06-0.2 in 4 eyes, and ge;0.3 in 14 eyes before the treatment. Choroidal melanoma, round or oval brown solid hunch, was located at the area around macula in 7 eyes, around the optic disc in 7 eyes, at or near the vascular arcade in 5 cases, and at the periphery in 2 eyes. The maximum length、width and thickness of tumor was 13 mm, 11.6 mm, and 9.59 mm. The isotope we used was125I, and the quantum of designed radiation was 100-120 Gy. Fourteen patients with choroidal melanoma at the macular area or around the optic disc underwent plaque radiotherapy associated with transpupillary thermotherapy (TTT). The average follow-up duration was 12 months with the longest duration of 3 years. The basis and thickness (height) of tumors were measured by B-scan echography. The aggrandizement of the tumor would be regarded if the height increased 15% or the basis boundary aggrandized 250mm. Results The visual acuity after the treatment decreased in 9 eyes, remained unchanged in 10, and increased in 2. The dimension of tumo increased in 6 eyes, remained unchanged in 12, and decreased in 3. The complication was vitreous hemorrhage in 2 eys, vascular occlusion in 1, branch retinal venous occlusion in 1, macular pucker in 1, retinal hemorrhage in 3, partial optic atrophy in 3, neovascular glaucoma in 1, and extraction of eye in 3. Conclusion The domestic plaque design is effective on choroidal melanoma, and is of a sort on the thick tumor and the tumor located at macula or beside the optic disc. (Chin J Ocul Fundus Dis, 2006, 22: 157-160)
Objective To evaluate the therapeutic effects of treatments of eye-retaining and enucleation for choroidal melanomas. Methods The clinical data of 44 patients (44 eyes) with choroidal melanomas after eye-retaining treatments and enucleation surgery were retrospectively analyzed. The metastasis, retention rate of eyeball after eye-retaining treatment, and visual acuity prognosis were observed and analyzed. In 44 eyes treated by eye-retaining therapy, transpupillary thermotherapy (TTT) was performed primaryly on 7 (15.9%), 106 Ru brachytherapy on 25 (56.8%), and local resection of tumor combined with 106 Ru brachytherapy on 12 (27.3%).The average follow-up period was 13.3 months. Results Forty-four patients had no melanoma metastasis during the follow-up period. In 39 patients (88.6%) who had their eyes retained successfully, the retention rate of eyeball was 100%, 92.9%, and 83.3% in 6, 14, and 24 eyes with small, middle, and large tumor, respectively. In the patients treated by eye-retaining therapy, the visual acuity was ge;0.3 in 11 (28.2%), ge;0.05-＜0.3 in 18 (46.2%), and ＜0.05 (25.6%) in 10 eyes. Conclusions 106 Ru brachytherapy and transpupillary thermotherapy are effective treatments for small and medium-sized choroidal melanomas; some selected cases with large choroidal melanomas was treated with local resection of tumor combined with106 Rubrachytherapy. However, longer followup will be necessary to assess if this treatment has a better comprehensive outcome, compared with enucleation surgery. (Chin J Ocul Fundus Dis, 2006, 22: 150-153)