Effect of luteolin on apoptosis and vascular endothelial growth factor in human choroidal melanoma cells

AIM:To investigate the effects of luteolin on apoptosis,the cell cycle,and the expression and secretion of vascular endothelial growth factor(VEGF)in human choroidal melanoma cells(C918 and OCM-1).METHODS:C918 and OCM-1 cells cultured in vitro were treated with various concentrations of luteolin(0,5,10,15μmol/L).Cell growth was observed with an inverted microscope,and cell cycle arrest was detected by propidium iodide(PI)staining using flow cytometry.Apoptosis was detected by Hoechst33342 staining,and apoptosis rate was determined by Annexin V-FITC/PI experiments using flow cytometry.The expression of apoptosis-related proteins Bcl-2,Bax and VEGF was analyzed using Western blots.The levels of VEGF secreted by the cells into the supernatant was analyzed using ELISA.RESULTS:After treating with 5 to 15μmol/L luteolin for 48 h,the fusion degree of C918 and OCM-1 cells decreased,and more floating apoptotic cells appeared.Luteolin treatment increased the G0-G1 phase ratio of the C918 and OCM-1 cells,blocked cell cycle progression,and increased the apoptosis rate of the C918 and OCM-1 cells.Western blot showed that luteolin decreased the expression of Bcl-2 and VEGF in the C918 and OCM-1 cells and increased the expression of Bax protein.The ELISA results showed that 10 to 15μmol/L luteolin decreased the cell secretion of VEGF.CONCLUSION:Luteolin may induce apoptosis by regulating the levels of apoptosis-related proteins in C918 and OCM-1 cells.Luteolin can induce cell cycle arrest,decrease the expression of VEGF.

Expression and significance of factors related to angiogenesis in choroidal melanoma

AIM: To investigate expression of factors related to angiogenesis: HIF-1 alpha, iNOS, COX-2 and VEGF in choroidal melanoma and its clinical significance. METHODS: Fifty samples of choroidal melanoma and 15 samples of melanocytic nevi of the eyelid identified by pathology were collected. Immunohistochemistry SP method was used to examine the expression of HIF-1 alpha, iNOS, COX-2 and VEGF in these samples. The comparison among groups was done by SPSS 13.0 software. RESULTS: The positive expression rates of HIF-1 alpha, iNOS, COX-2 and VEGF in choroidal melanoma group were significantly higher than those in eyelid nevi group (chi(2)= 6.5542, 7.7224, 8.5828, 15.1749). The positive expression rate of VEGF was associated with the tumor size (chi(2)= 10.9194), but was not associated with pathological type (chi(2)=2.0712) and the situation of scleral invasion (chi(2)= 5.4289). The positive expression rate of HIF-1 alpha was associated with the tumor size (chi(2)=7.1216) and pathological type (chi(2)=9.0889), but was not associated with the situation of scleral invasion (chi(2)=3.3586). The positive expression rate of iNOS was associated with the tumor size (chi(2)=9.5503), but was not associated with pathological type (chi(2)=1.9450) and the situation of scleral invasion (chi(2)=2.3810). The positive expression rate of COX-2 was associated with the tumor size (chi(2)=7.2970), but was not associated with pathological type (chi(2)=1.8421) and the situation of sclera! invasion (chi(2)= 0.4018). The expression of HIF-1 alpha, iNOS and COX-2 were significantly associated with the expression of VEGF (r = 0.9429, 1, 0.9857). The expression of COX-2 was significantly associated with the expression of iNOS (r=0.9857). The expression of HIF-1 alpha was significantly associated with the expression of COX-2 ( r=0.9857). The expression of HIF-1 alpha was significantly associated with the expression of iNOS (r= 0. 9429). CONCLUSION: The expression of HIF-1 alpha, iNOS and COX-2 protein in choroidal melanoma were higher and may relate to angiogenesis and stimulate tumor growth. Determination of HIF-1 alpha, iNOS and COX-2 may be helpful for the diagnosis and therapy of this tumor.

Inhibition on Telomerase Activity and Cytotoxic Effects by Cisplatin in Cultured Human Choroidal Melanoma Cells

Purpose: To study the changes of telomerase activity and cytotoxic effects by Cisplatin;cis-dichlorodiamine platinum (CDDP) in cultured human choroidal melanoma.Material and Methods: The primary cultured human choroidal melanoma cells werecultured in the presence and absence of CDDP with different concentration and timerespectively. The toxic effects were evaluated by MTT and the level of telormarse wasdetected by PCR-ELISA assay. And the relationship between telomerase activity andcytotoxic effects were analyzed by a correlation analysis.Results: Following the increase of the concentration and the time of CDDP, graduallyrepressed telomerase activity was detected in cultured cells. Meanwhile, the restrain rateof the cells increased. The telomerase activity at 24h and 1μg/ml was repressedsignificantly compared with the control cells. However, the appearance of cell deathlagged behind the decreasing of telomerase.Conclusions: CDDP is an effective telomerase inhibitor in cultured choroidal melanomacells of human eyes, which presents concentration and time dependency and can causethe death of cultured cells.

Secondary endoresection for previously treated choroidal melanomas with a non-responsive course and persistent exudative retinal detachment

AIM:To evaluate the results and complications of secondary endoresection via pars plana vitrectomy for choroidal melanoma and review the previously reported endoresection studies on the treatment of choroidal melanoma.METHODS:The medical records of 6 patients with choroidal melanoma who underwent secondary endoresection between March 2012 and March 2020 were retrospectively reviewed.The indications for secondary endoresection were progressive or recurrent tumor and severe exudative retinal detachment after previous treatment with plaque radiotherapy/Cyberknife radiosurgery/transpupillary thermotherapy(TTT).RESULTS:Before endoresection,2 eyes had Iodine-125 plaque radiotherapy and TTT,1 eye had Ruthenium-106 plaque radiotherapy and TTT,1 eye had Cyberknife radiosurgery and TTT,1 eye had Cyberknife radiosurgery,and 1 eye had TTT only.Preoperative visual acuity ranged from 20/63 to 20/1600(Snellen) and from 0.5 to 1.9(mean:1.1) on the log MAR scale.The mean tumor base diameters were 9.5×8.7 mm and the mean tumor thickness was 5.4 mm.After secondary endoresection,transient vitreous hemorrhage developed in 2(33.3%) eyes and retinal detachment in 1(16.7%) eye.Cytopathological examination revealed epithelioid cell melanoma in 4(66.7%) eyes and mixed cell melanoma in 1(16.7%).Melanoma cell type was not specified in 1(16.7%) eye.At a mean follow-up of 49.6 mo(range:16-90 mo),mean visual acuity did not improve and 1 eye was enucleated due to tumor recurrence.Final visual acuity ranged from 20/63 to 20/1600(Snellen) and from 0.5 to 1.9(mean:1.2) on the log MAR scale.Two patients with choroidal melanoma developed metastasis and eventually expired.CONCLUSION:Secondary endoresection seems to be an effective treatment option for globe salvage in choroidal melanoma not responsive to conventional treatment and displaying persistent exudative retinal detachment.There was no visual acuity increase among the treated eyes but globe salvage was possible in most cases in this study.

Distant metastasis in choroidal melanoma with spontaneous corneal perforation and intratumoral calcification: A case report

BACKGROUND Uveal melanoma is the most common primary intraocular malignancy in adults,but its incidence is low in Asian populations.Spontaneous corneal perforation and intratumoral calcification are rare presentations of choroidal melanoma(CM),and reports regarding these presentations have been limited.Even after complete surgical treatment,the prognosis of CM patients is usually poor if distant metastasis is present.We here present a case of CM with unique presentations and early distant metastasis to the liver.CASE SUMMARY A 63-year-old Asian woman presented to our hospital with complaint of pain and brownish discharge from her left eye for 3 d.Imaging studies revealed intratumoral calcification within the left eye with eyeball rupture.Enucleation of the left eye was performed and pathological examination confirmed the diagnosis of CM.Systemic surveillance revealed no metastatic diseases.However,the patient was lost to follow-up 3 mo after surgery.At 1.5 years after the operation,she presented to our emergency department with complaint of dull epigastric pain that radiated to the back for 1 d.Imaging studies revealed a large mass at the upper abdomen abutting the pancreatic neck and body as well as several nodular lesions in the liver.Fine needle biopsy was performed and findings confirmed liver and pancreatic metastases.CONCLUSION This case highlights the importance of continued follow-up of patients with CM.

Expression of connexin 43 and E-cadherin in choroidal melanoma

AIM: To investigate the expression of connexin 43 and epithelial cadherin (E-cadherin) in choroidal melanoma, to explore the clinical and pathological implications of expression of these proteins, and to determine their relations with malignant features. METHODS: The Expression of connexin 43 and E-cadherin in choroidal melanoma were detected by immunohistochemistry and correlated with clinicopathological features. RESULTS: Positive rates of connexin 43 in choroidal melanomas and benign pigmented nevus tissues were 75% and 40% respectively with significant differences between the two groups (X(2)=5.607, P =0.009). Positive rates of E-cadherin in choroidal melanomas and benign pigmented nevus tissues were 40% and 75% respectively with significant differences between the two groups (X(2)=5.214, P = 0.010). Significant overexpression of connexin 43 and reduction of E-cadherin expression was associated with the invasion to the sclera, and there were respectively significant differences between without and with scleral invasion groups (X(2)=2.880, P=0.040; X(2)=2.778, P=0.046). Overexpression of connexin 43 were correlated with tumor cell types and the expression of connexin 43 and E-cadherin may be correlated with each other. CONCLUSION: The increased expression of connexin 43 and the decreased expression of E-cadherin may be involved in the process of invasion of choroidal melanoma. The overepression of connexin 43 and reduction of E-cadherin may contribute to the development of choroidal melanoma.

In Vitro Study on Lethal Effect of Human Choroidal Melanoma OCM-1 Cell Line by Repeating -70℃ Freeze Thawing

Objective: To investigate the effects of repeating-70℃freeze thawing on human choroidal melanoma cell line OCM-1. Methods: OCM-1 cells were frozen by repeating -70℃ freeze thawing with various durations and frequencies. Then the inhibit rate of cells was examined by MTT essay. The cell viability was measured by monoclonal formation assay. We also used the HE staining, immunohistochemistry staining and the laser-scanning confocal microscopy (LSCM) to investigate the morphological changes of the cells. Results: The growth of OCM-1 cells was inhibited by repeating -70℃ freeze thawing in time-dependent and frequency-dependent manners (P < 0.01). Different morphous including necrosis and apoptosis of the cells could be observed after -70℃ freeze thawing by the LSCM. Conclusion: Repeating -70℃ freeze thawing can not only kill cells directly and induce considerable cells to apoptosis, but also inhibit the growth of the survivals. The kill and wound ratio of the cells disposal with different times and frequencies present variance. And the distinction when treated with different frequencies during the same time is much more significant than different times with the same frequency, which guide clinical workers to choose repeating cryotherapy with short term method instead of single cryotherapy with long term in choroidal melanoma treatment.

Complications from Plaque versus Proton Beam Therapy for Choroidal Melanoma: A Qualitative Systematic Review

Plaque brachytherapy has been a mainstay of treatment for choroidal melanoma to achieve intraocular tumor control. The most common radioisotopes used for treating smaller sized tumors are Iodine-125 in North America and Ruthenium-106 in Europe. Proton beam radiotherapy is available at a few centers and may also be used to achieve local tumor control. Both plaque and proton beam therapy are known to be associated with a range of complications that may affect visual outcome and quality of life. These include radiation retinopathy, optic neuropathy, neovascular glaucoma and local treatment failure, requiring enucleation. While differences in the rates of these complications have not been well established in the literature for patients treated with plaque versus proton beam therapy for choroidal melanoma, certain geographic regions prefer one treatment modality over the other. The purpose of this qualitative systematic review was to compare and contrast reported complications that developed with plaque and proton beam therapy for the treatment of choroidal melanoma in studies published over a ten-year period. Reported rates suggest that patients with proton beam therapy had potentially higher rates of complications, including vision loss, enucleation, and neovascular glaucoma compared to those with plaque therapy. The rates of optic neuropathy, radiation retinopathy, and cataract formation were widely variable for the two treatment modalities and rates of metastasis and metastasis-free survival appeared similar with both treatments. The most common reported predictors of ocular complications following both types of therapy were tumor distance from the optic nerve, tumor thickness, and radiation dose, suggesting that inherent tumor characteristics play a role in visual prognosis.

液基细胞学涂片和细胞蜡块技术在脉络膜黑色素瘤诊断中的应用

目的:脉络膜黑色素瘤(melanoma of choroid,CM)是成人常见的原发性眼内恶性肿瘤,特点为恶性程度高、易侵袭转移、预后极差,严重影响患者视力,甚至威胁患者生命。目前诊断CM主要依赖影像学检查,在眼球摘除手术后加做病理诊断,缺乏明确的术前病理诊断。本研究旨在探索液基细胞学涂片和细胞蜡块技术在CM术前诊断中的应用。方法:收集南京医科大学第一附属医院2023年3至6月收治的2例临床诊断为CM患者的脉络膜下液。采用液基细胞学涂片方法进行初步诊断,改良的细胞蜡块制作技术、苏木精-伊红(hematoxylin and eosin,HE)染色、脱黑色素和免疫组织化学(immunohistochemistry,IHC)染色进行进一步的病理诊断。结果:2例患者经过影像学检查均诊断为CM,通过液基细胞学涂片做出初步的细胞学病理诊断,1例诊断为“黑色素瘤”,另1例诊断为“见异型细胞,黑色素瘤不除外”。通过改良的细胞蜡块制作技术,HE、脱黑色素和IHC染色,病理诊断结果显示临床诊断相同的2例患者,1例明确诊断为CM,而另1例诊断为低分化神经内分泌癌,修正了原先的诊断。结论:本改良技术能够在手术前精准诊断CM,为临床眼科医生制定治疗方案提供重要依据,操作简便,值得推广。其有3个优点:1)能制作出10~20张连续切片,满足IHC染色的要求;2)在用本技术处理的切片中,可见各种异型细胞,且细胞核结构清晰;3)简单易行,成本不高,适用于各级医院,特别是中小医院。本文有2个原创点:1)将液基细胞学涂片、细胞蜡块切片及IHC染色用于诊断CM;2)对传统的细胞蜡块制作技术进行改良,使其适用于诊断CM。

Role of wide-field autofluorescence imaging and scanning laser ophthalmoscopy in differentiation of choroidal pigmented lesions

·AIM:Toevaluatethe diagnostic properties of wide-field fundus autofluorescence(FAF) scanning laser ophthalmoscope(SLO) imaging for differentiating choroidal pigmented lesions.·METHODS: A consecutive series of 139 patients were included, 101 had established choroidal melanoma with13 untreated lesions and 98 treated with radiotherapy.Thirty-eight had choroidal nevi. All patients underwent a full ophthalmological examination, undilated wide-field imaging, FAF and standardized US examination. FAF images and imaging characteristics from SLO were correlated with the structural findings in the two patient groups.·RESULTS: Mean FAF intensity of melanomas was significantly lower than the FAF of choroidal nevi. Only 1out of 38 included eyes with nevi touched the optic disc compared to 31 out of 101 eyes with melanomas. In 18 out of 101 melanomas subretinal fluid was seen at the pigmented lesion compared to none seen in eyes with confirmed choroidal nevi. In 'green laser separation', a trend towards more mixed FAF appearance of melanomas compared to nevi was observed. The mean maximal and minimal transverse and longitudinal diameters of melanomas were significantly higher than those of nevi.·CONCLUSION: Wide-field SLO and FAF imaging may be an appropriate non-invasive diagnostic screening tool to differentiate benign from malign pigmented choroidal lesions.

Combination of oridonin and TRAIL induces apoptosis in uveal melanoma cells by upregulating DR5

AIM:To investigate the inhibitory effect of the combined use of tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)and oridonin on choroidal melanoma cell lines,and to explore its underlying mechanism.METHODS:MUM-2B and C918 cells were treated with different concentrations of TRAIL and oridonin,and MTT assay used to evaluate the inhibition rate of the two compounds on cells.Then,the cell cycle distribution and apoptosis were detected by flow cytometry,and changes in apoptosis-related proteins such as death receptor 5(DR5),a-caspase-3,and x-linked inhibitor of apoptosis protein(XIAP)were detected by Western blot.MUM-2B cells were transfected with si-DR5,which interfered with the expression of the DR5 gene.MTT and Western blot assay were used to detect cell activity and apoptosis-related proteins.RESULTS:When TRAIL and oridonin were simultaneously administered to the MUM-2B cells,the apoptosis rate was significantly higher than that by the two drugs individually.However,the effect of combined use of TRAIL and oridonin on C918 cells was not significantly different from that used alone.Cell cycle analysis showed that TRAIL and oridonin could induce G2/M arrest in MUM-2B cells.The Western blot results showed that the protein expression levels of the DR5,a-caspase-3,and BAX increased,while the expression levels of the anti-apoptosis-related proteins XIAP and BCL-2 were suppressed when TRAIL and oridonin simultaneously administered to MUM-2B cells.Interfering the expression of DR5 gene in MUM-2B cells could reverse the inhibitory effect of oridonin and TRAIL on the proliferation and apoptosis induction of MUM-2B cells.CONCLUSION:The inhibitory effects of oridonin and TRAIL on MUM-2B cells are significantly enhanced when they were administered as a combined treatment,which may ascribe to up-regulation of DR5.

Follow the nevus: the cost-utility of monitoring for growth of choroidal nevi

AIM: To develop a model to evaluate the cost-utility of choroidal nevi monitoring recommendations with varying clinical risk factors.METHODS: A Markov model was created to evaluate the cost-utility in cost per quality-adjusted life-year($/QALY) for monitoring patients with choroidal nevus. This probabilistic model was applied both to a hypothetically monitored and unmonitored group of patients beginning at different ages and with varying clinical risk factors of the nevus. Duration of screening was modeled for the remainder of the patients’ life expectancy. Best available clinical data on the prevalence and incidence of choroidal nevi/melanoma, and relative risk of nevus transformation were combined with the initial and downstream costs of screening, downstream costs of melanoma-related mortality, and QALY saved by monitoring, to estimate the best monitoring regimen. Main outcome measures were average $/QALY saved by consensus recommended monitoring scenarios for the duration of a patient’s remaining life expectancy in comparison with no follow-up, and the cost-utility of modified regimens. RESULTS: The $/QALY of the recommended monitoring scenarios varied substantially based on nevus clinical risk factors, patient age, frequency of follow-up, and objective testing utilized. The $/QALY for the recommended monitoring scenario of a flat nevus without risk factors in a 60-yearold patient was $77 180. The $/QALY for monitoring a nevus with 3 clinical risk factors in a 60-year-old patient was $85 393. The $/QALY values for differently-aged patients were larger, and intermediate degrees of risk factors for nevus growth varied, depending largely upon the specifics of the modeled monitoring scenarios.CONCLUSION: The average $/QALY of currently recommended monitoring scenarios fall within economically acceptable standards and could provide insight for formulating appropriate clinical strategies. Cost-utility could be enhanced by targeting higher risk groups and considering less frequent monitoring for the lower risk groups.

抑制PTBP1对脉络膜黑色素瘤细胞增殖和侵袭力的影响

目的探讨多聚嘧啶区结合蛋白1(PTBP1)对脉络膜黑色素瘤(CM)细胞增殖活性和迁移、侵袭能力的影响。方法选取人CM细胞系OCM-1,根据转染序列的不同分为si-PTBP1组(转染PTBP1特异性干扰序列)、si-Control组(转染阴性对照序列)和空白组(不作任何处理)。分别检测各组细胞PTBP1 mRNA的表达,以及PTBP1、上皮型钙黏蛋白(E-Cad)和波形蛋白的表达。通过细胞实验分析各组细胞的增殖活性和迁移、侵袭能力。结果si-PTBP1组PTBP1 mRNA相对表达量显著低于si-Control组和空白组(P<0.05),si-Control组和空白组之间差异无统计学意义(P>0.05)。培养24、48、72和96h,si-PTBP1组细胞增殖活性均低于si-Control组和空白组(P<0.05)。si-PTBP1组迁移细胞数和侵袭细胞数均少于si-Control组和空白组(P<0.05)。si-PTBP1组PTBP1和波形蛋白相对表达量低于si-Control组和空白组(P<0.05),E-Cad相对表达量高于si-Control组和空白组(P<0.05)。结论抑制OCM-1细胞PTBP1表达可抑制细胞增殖活性,削弱细胞的迁移和侵袭能力,其机制可能与抑制上皮-间质转化有关。

基于倾向性评分逆概率加权法的脉络膜黑色素瘤患者疗效评价研究

目的 在平衡组间基线变量后,评价放射治疗与手术联合放射治疗两种治疗方式对脉络膜黑色素瘤患者的疗效,分析脉络膜黑色素瘤患者的预后影响因素,为脉络膜黑色素瘤的有效诊疗提供参考依据。方法 对2000年1月至2019年12月在SEER数据库中登记的脉络膜黑色素瘤患者进行分析,采用倾向性评分逆概率加权法(IPTW)对放射治疗和手术联合放射治疗两组患者的基线特征进行平衡。结果 本研究共筛选脉络膜黑色素瘤患者5547例,其中接受放射治疗者4422例、接受手术联合放射治疗治疗者1125例。在接受放射治疗的4422例患者中,光束辐射(BR)组1155例,放射性植入物(RI)组2608例,放射性同位素(RIT)组659例。Log-rank检验结果显示,逆概率加权前后,放射治疗组患者的预后均高于手术联合放射治疗组(P<0.000 1)。多因素Cox回归分析结果显示,在控制了种族、性别、多灶性、是否为首要恶性肿瘤、婚姻状态、从诊断到治疗的时间、年龄、眼别、诊断确认、放射治疗方法等混杂因素后,与放射治疗组患者相比,手术放射治疗组患者的特异性死亡风险为HR=4.385(95%CI=3.542~5.428)。与年龄<50岁组患者相比,50~<60岁组、60~<70岁组、70~<80岁组、≥80岁组患者的特异性死亡风险分别为HR=3.083(95%CI=0.825~4.238)、HR=4.045(95%CI=2.955~5.532)、HR=4.512(95%CI=3.115~6.529)、HR=6.877(95%CI=4.151~11.398);与恶性肿瘤数为1个组患者相比,肿瘤数为2个组和>2个组患者的特异性死亡风险分别为HR=0.698(95%CI=0.449~2.944)、HR=0.538(95%CI=0.317~0.913);从诊断到治疗的时间超过1个月的患者的风险比为2.732~4.154。Log-rank检验结果表明,IPTW前后BR、RI和RIT三组脉络膜黑色素瘤患者生存曲线间差异无统计学意义(P>0.05)。结论 放射治疗的长期疗效优于手术与放射治疗联合治疗;年龄、单病灶、诊断到治疗的时间和治疗方式可增加脉络膜黑色素瘤患者死亡的风险,是影响脉络膜黑色素瘤患者预后的独立影响因素。

基于SEER数据库数据的脉络膜黑色素瘤预后预测模型构建

目的构建脉络膜黑色素瘤预后预测模型。方法收集美国国家癌症研究所SEER数据库中2010—2015年2235例诊断为脉络膜黑色素瘤患者的临床资料数据(年龄、性别、婚姻状况、种族、TNM分期、病理学类型、骨转移、肺转移、肝转移、手术信息、放化疗情况、生存时间、生存状态等信息),将所有患者以7∶3的比例,随机分为建模组1584例及验证组651例。首先将建模组中的临床资料数据利用R4.1.2软件中“survival”包的Cox风险比例回归模型进行单因素及多因素分析,得出影响脉络膜黑色素瘤患者预后的独立影响因素。基于筛选出的影响因素使用R4.1.2软件中“rms”包构建脉络膜黑色素瘤预后预测模型。在建模组和验证组中通过C指数、ROC以及校准曲线评价该预测模型的区分度和一致性。结果年龄、病理学类型、T分期、M分期、局部放疗为影响脉络膜黑色素瘤预后的独立影响因素,基于以上因素,构建脉络膜黑色素瘤预后预测模型。在建模组和验证组中预后预测模型C指数分别为0.732(95%CI:0.710,0.761)和0.713(95%CI:0.670,0.756)。建模组中预后预测模型预测1年、2年、3年预后的ROC下面积分别为0.780、0.766、0.777;在验证组中预后预测模型预测1、2、3年预后的ROC下面积分别为0.890、0.755、0.734,两组数据均高于0.7,说明具有良好的区分度。建模组和验证组的校准曲线均与平面直角坐标系中45°的直线重合度较高,说明具有良好的一致性。结论脉络膜黑色素瘤预后预测模型建立成功,其对患者预后具有良好的预测效能。

miR-375表达对脉络膜黑色素瘤细胞增殖和侵袭的影响

目的:探讨miR-375表达对脉络膜黑色素瘤MUM-2B细胞增殖和侵袭的影响。方法:培养MUM-2B细胞,分别转染miR-375模拟物序列(模拟物组)、miR-375抑制物序列(抑制物组)、阴性对照序列(阴性对照组)和不做任何处理(空白组),分别采用qRT-PCR实验、CCK-8实验、细胞凋亡实验、Transwell实验检测细胞中miR-375、细胞增殖活性、细胞凋亡情况、细胞迁移和侵袭情况。结果:相比于阴性对照组(1.01±0.10)和空白组(1.03±0.07),模拟物组细胞中miR-375表达量(2.65±0.15)升高,而抑制物组细胞中miR-375表达量(0.28±0.06)降低(P<0.05);与空白组和阴性对照组比较,模拟物组细胞24、48、72和96h时OD值均降低(P<0.05),而抑制物组细胞24、48、72和96h时OD值均升高(P<0.05);与空白组细胞凋亡率(20.54±4.01)%和阴性对照组细胞凋亡率(22.80±4.28)%比较,模拟物组细胞凋亡率(39.11±3.37)%升高(P<0.05),而抑制物组细胞凋亡率(10.13±2.17)%降低(P<0.05);与空白组和阴性对照组比较,模拟物组细胞迁移数和细胞侵袭数均降低(P<0.05),而抑制物组细胞迁移数和细胞侵袭数均升高(P<0.05)。结论:上调MUM-2B细胞中miR-375表达可降低细胞增殖活性,加速细胞凋亡,抑制细胞迁移和侵袭,下调miR-375表达则发挥相反的作用,表明miR-375可能在脉胳膜黑色素瘤病程中发挥抑癌功能。

贝伐单抗对人眼脉络膜黑色素瘤MUM-2B细胞血管生成拟态的影响

目的:通过体内外实验探讨贝伐单抗对人眼脉络膜黑色素瘤细胞(MUM-2B)血管生成拟态的影响及可能相关的分子通路。方法:通过不同浓度的贝伐单抗(0,0.1,1,2,3 mg/mL)处理MUM-2B细胞,观察其对MUM-2B细胞成管能力的影响。通过CCK-8法及Transwell法检测其对MUM-2B细胞增殖及侵袭的影响。然后,建立裸鼠皮下移植瘤模型,通过不同剂量的贝伐单抗(5 mg/kg,7.5 mg/kg,10 mg/kg)干预,探究贝伐单抗对裸鼠皮下移植瘤VM形成的影响,并通过CD31/PAS免疫组化双重染色法检测VM的表达情况,免疫组化法检测HIF-1a、VE-cadherin、EphA2、PI3K蛋白表达水平变化。结果:体外实验中,不同浓度的贝伐单抗(0,0.1,1,2,3 mg/mL)处理MUM-2B细胞24小时后,对MUM-2B细胞的成管能力、增殖、侵袭能力并没有表现出明显的抑制或促进作用。实验组与对照组相比差异无统计学意义(P>0.05)。在体内实验中,贝伐单抗实验组(5 mg/kg,7.5 mg/kg,10 mg/kg)与对照组相比较能显著的促进MUM-2B细胞形成VM的能力(P<0.05),且形成管道的数量与贝伐单抗的浓度呈正相关(r=0.942,P<0.05)。实验组HIF-1a、VE-cadherin、EphA2和PI3K-Akt蛋白的表达水平均明显高于对照组(P<0.05),且各分子的表达量随着贝伐单抗浓度的升高呈浓度依赖性的升高。结论:体外实验中,贝伐单抗对MUM-2B细胞的成管能力,增殖,侵袭能力并没有表现出明显的抑制或促进作用。体内实验中,贝伐单抗的使用能促进HIF-1a的表达、上调VE-cadherin/EphA2/PI3K-Akt后续的级连信号通路,从而加速VM的生成。

HDAC抑制剂SAHA对脉络膜黑色素瘤细胞OCM1的作用

目的探讨组蛋白去乙酰化酶(histone deacetylase,HDAC)抑制剂辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid,SAHA)对脉络膜黑色素瘤(choroidal melanoma,CM)细胞系OCM1增殖侵袭的机制。方法使用显微镜观察不同浓度SAHA(0.625,1.25,2.5μmol/L)对OCM1细胞形态结构的影响,利用CCK-8实验观察不同浓度SAHA(0.625,1.25,2.5μmol/L)对OCM1细胞活力的影响,通过EdU染色法和克隆形成实验观察control组(0μmol/L SAHA)和1.25μmol/L SAHA组OCM1细胞增殖的变化,通过划痕实验和迁移侵袭实验观察control组和1.25μmol/L SAHA组OCM1细胞迁移侵袭能力的变化。采用Western blot实验检测不同浓度SAHA(0.625,1.25,2.5μmol/L)组中HDAC4、CyclinD1、c-Myc、E-cadherin和Snail蛋白表达水平。结果与control组相比较,光镜下明显可见0.625μmol/L SAHA组、1.25μmol/L SAHA组、2.5μmol/L SAHA组OCM1细胞增殖能力减弱,促进细胞皱缩和死亡,且细胞增殖活力降低(P<0.05)。与control组比较,1.25μmol/L SAHA组OCM1细胞增殖和迁移侵袭能力明显降低(P<0.05)。此外,与control组相比较,0.625μmol/L SAHA组、1.25μmol/L SAHA组、2.5μmol/L SAHA组E-cadherin蛋白表达逐渐升高,而HDAC4和Snail蛋白表达逐渐降低(P<0.05)。结论SAHA可通过抑制HDAC4明显降低OCM1细胞增殖活力和迁移侵袭能力。

木犀草素对脉络膜恶性黑色素瘤细胞的影响

为探讨木犀草素调节Wnt/β-catenin信号通路对人脉络膜恶性黑色素瘤细胞(MuM-2C)凋亡、迁移和侵袭的影响,采用CCK 8法测定0,5,10,15,20,25μmol/L浓度木犀草素处理24 h后的MuM-2C活力,以筛选合适的药物作用浓度.将体外培养的MuM-2C随机分为3组:对照组、木犀草素组及木犀草素+氯化锂组,木犀草素组以15μmol/L木犀草素处理,木犀草素+氯化锂组以15μmol/L木犀草素和10μmol/L的氯化锂联合处理.采用流式细胞技术和Hoechst 33258染色检测MuM-2C的凋亡情况,采用划痕实验和Transwell实验检测各组MuM-2C的迁移、侵袭情况,采用免疫荧光检测各组MuM-2C中凋亡蛋白BAX与BCL-2表达,采用免疫印记检测各组MuM-2C中EMT标志蛋白(E-cadherin,N-cadherin,Vimentin)和Wnt/β-catenin信号相关蛋白(Wnt1,β-catenin)表达,结果表明:不同剂量木犀草素均可抑制MuM-2C生长,并在一定范围内随剂量升高而作用增强;与对照组比较,木犀草素组细胞核形态固缩而大小不一,着色不均匀,部分呈现明亮的蓝色荧光,表现为明显的凋亡病理现象,细胞凋亡率、细胞BAX/BCL-2比值与E-cadherin蛋白表达显著升高(P<0.05),细胞迁移率与侵袭数、细胞蛋白(N-cadherin,Vimentin,Wnt1,β-catenin)表达显著降低(P<0.05);与木犀草素组比较,木犀草素+氯化锂组细胞核的凋亡病理现象明显减轻,细胞凋亡率、细胞BAX/BCL-2比值与E-cadherin蛋白表达显著降低(P<0.05),细胞迁移率与侵袭数、细胞蛋白(N-cadherin,Vimentin,Wnt1,β-catenin)表达显著升高(P<0.05).说明木犀草素可通过抑制Wnt/β-catenin信号途径传导而降低MuM-2C活力,促进其凋亡,并抑制其迁移和侵袭.

眼脉络膜黑色素瘤的CT表现及其病理基础探讨

本文对比分析９例眼内期脉络膜黑色素瘤的ＣＴ表现和病理学改变。ＣＴ图像上脉络膜黑色素瘤表现为突向玻璃体腔的高密度肿块或眼环局限性梭形增厚。肿块较小，未破坏Ｂｒｕｃｈ膜时，呈扁平梭形。破坏Ｂｒｕｃｈ膜后，ＣＴ上表现为半圆形、圆形病灶。肿瘤密度接近眼环，多有中度强化。文中还对继发性视网膜脱离的ＣＴ表现及诊断价值进行了探讨。