甲基莲心碱对视网膜母细胞瘤WERI-Rb1细胞增殖和侵袭的影响

Effects of Neferine on proliferation and invasion of WERi-Rb1 retinoblastoma cells

目的本研究旨在探讨甲基莲心碱(Nef)对视网膜母细胞瘤(RB)细胞WERI-Rb1增殖和侵袭能力的影响。方法以RB细胞系WERI-Rb1为体外模型,以4周雌性无胸腺裸鼠为异种移植模型。细胞计数试剂-8(CCK-8)测定不同浓度Nef对细胞活力的影响。5-溴脱氧尿嘧啶核苷(BrdU)染色测定细胞增殖能力,Transwell测定细胞侵袭能力,免疫组织化学法检测裸鼠移植瘤肿瘤组织中细胞增殖标志物Ki-67抗原(Ki67)和基质金属蛋白酶-9(MMP-9)表达量,Western Blot法检测WERI-Rb1细胞和肿瘤组织中Ki67、增殖细胞核抗原(PCNA)、MMP-9、基质金属蛋白酶-14(MMP-14)蛋白表达水平。结果(1)细胞活力:与Nef 0.5μM组相比,5.0、10.0、20.0、40.0、80.0、160.0μM组的细胞活力均降低,差异具有统计学意义(t_(5.0)=8.298,P=0.001;t_(10.0)=11.354,t_(20.0)=12.713,t_(40.0)=16.228,t_(80.0)=23.380,t_(160.0)=23.765,均P=0.000),选择0、2.5、5.0、10.0μM进行后续试验。(2)细胞增殖能力:与Nef 0μM组相比,5.0、10.0μM组的阳性细胞数和Ki67、PCNA蛋白表达水平均降低,差异具有统计学意义(阳性细胞数:t_(5.0)=5.857,P=0.004;t_(10.0)=8.770,P=0.001。Ki67:t_(5.0)=3.286,P=0.030;t_(10.0)=5.060,P=0.007;PCNA:t_(5.0)=6.059,P=0.004;t_(10.0)=8.109,P=0.001)。(3)细胞侵袭能力:与Nef 0μM组相比,5.0、10.0μM组的细胞侵袭数目和MMP-9蛋白表达水平均降低,差异具有统计学意义(细胞侵袭数目:t_(5.0)=7.203,P=0.002;t_(10.0)=10.847,P=0.000。MMP-9:t_(5.0)=4.323,P=0.012;t_(10.0)=8.216,P=0.001),10.0μM组的MMP-14蛋白表达水平降低(t=5.051,P=0.007),5.0μM组的MMP-14蛋白表达水平差异无统计学意义(P>0.05)。(4)裸鼠移植瘤肿瘤组织:与Nef 0 mg/kg组相比,1.0、2.0 mg/kg组的肿瘤重量、肿瘤体积和Ki67、MMP-9阳性细胞数均降低,差异具有统计学意义(肿瘤重量:t_(1.0)=6.261,t_(2.0)=12.969,均P=0.000。肿瘤体积:t_(1.0)=10.023,t_(2.0)=18.952,均P=0.000。Ki67:t_(1.0)=5.599,P=0.001;t_(2.0)=8.188,P=0.000。MMP-9:t_(1.0)=13.461,t_(2.0)=19.931,均P=0.000)。结论Nef可抑制RB细胞WERI-Rb1的增殖和侵袭能力。

OBJECTIVE To investigate the effects of neferine(Nef)on proliferation and invasion of WERI-Rb1 retinoblastoma(RB)cells.METHODS RB cell line WERI-Rb1 was used as the xenotransplantation model in vitro,and four-week-old female nude mice without thymus were used as the xenotransplantation model.Cell counting kit-8(CCk-8)was used to determine the effect of different Nef concentrations on cell viability.5-Bromodeoxyuridinc(BrdU)staining was used to measure cell proliferation,Transwell was used to measure cell invasion,and immunohistochemistry was used to detect the expression levels of cell proliferation marker Ki-67 antigen(Ki67)and matrix metallopeptidase-9(MMP-9)in the tumor tissues of nude mice transplanted tumor.Western Blot was used to detect the protein expression levels of Ki67,proliferating cell nuclear antigen(PCNA),MMP-9 and matrix metallopeptidase-14(MMP-14)in WERI-Rb1 cells and tumor tissues.RESULTS(1)Cell viability:Compared with Nef 0.5μM group,the cell viability of 5.0,10.0,20.0,40.0,80.0 and 160.0μM groups were decreased,and the differences were all statistically significant(t_(5.0)=8.298,P=0.001;t_(10.0)=11.354,t_(20.0)=12.713,t_(40.0)=16.228,t_(80.0)=23.380,t_(160.0)=23.765,all P=0.000).And 0,2.5,5.0,10.0μM were selected for the follow-up test.(2)Cell proliferation:Compared with Nef 0μM group,the positive cell number and Ki67 and PCNA protein expression levels in 5.0 and 10.0μM groups were decreased,and the differences were statistically significant(positive cell number:t_(5.0)=5.857,P=0.004;t_(10.0)=8.770,P=0.001.Ki67:t_(5.0)=3.286,P=0.030;t_(10.0)=5.060,P=0.007.PCNA:t_(5.0)=6.059,P=0.004;t_(10.0)=8.109,P=0.001).(3)Cell invasion ability:Compared with Nef 0μM group,the number of cell invasion and the expression level of MMP-9 protein in 5.0 and 10.0μM groups were decreased,the differences were statistically significant(number of cell invasion:t_(5.0)=7.203,P=0.002;t_(10.0)=10.847,P=0.000.MMP-9:t_(5.0)=4.323,P=0.012;t_(10.0)=8.216,P=0.001);The expression level of MMP-14 protein in the 10.0μM group was decreased(t=5.051,P=0.007);There was no significant difference in the expression level of MMP-14 protein in the 5.0μM group(P>0.05).(4)Tumor tissue of nude mice transplanted tumor:Compared with Nef 0 mg/kg group,tumor weight,tumor volume and number of Ki67 and MMP-9 positive cells were decreased in 1.0 and 2.0 mg/kg groups,the differences were statistically significant(t_(1.0)=6.261,t_(2.0)=12.969,all P=0.000.Tumor volume:t_(1.0)=10.023,t_(2.0)=18.952,all P=0.000.Ki67:t_(1.0)=5.599,P=0.001;t_(2.0)=8.188,P=0.000.MMP-9:t_(1.0)=13.461,t_(2.0)=19.931,all P=0.000).CONCLUSIONS Nef can inhibit the proliferation and invasion of WERI-Rb1 RB cell.