沉默B7-H3基因抑制人恶性血液病裸鼠移植瘤的体内成瘤和转移

发布时间：2018-02-27 21:38

  本文关键词： 恶性血液病 B-H 基因沉默 裸鼠　出处：《北京大学学报(医学版)》2017年02期 　论文类型：期刊论文

【摘要】：目的:探讨靶向B7-H3基因沉默对人恶性血液病细胞株在裸鼠体内成瘤和转移的影响及机制。方法:应用实时荧光定量PCR(real-time quantitative polymerase chain reaction,q PCR)和流式细胞术(flow cytometry,FCM)检测13株恶性血液病细胞中B7-H3分子的表达水平,筛选高表达的U937、Maver和Z138细胞。分别利用慢病毒转染技术靶向敲减B7-H3,建立稳定低表达细胞株,应用q PCR和FCM验证基因沉默效果。将U937、Maver和Z138各自分为空白对照组(non-infected control group,CON)、靶向B7-H3敲减组(B7-H3 knockdown group,KD)和阴性无关序列对照组(negative nontargeted control infected group,NC),将以上9组分别构建裸鼠皮下移植瘤模型,比较肿瘤生长和转移情况,免疫组织化学(immunohistochemistry,IHC)染色检测Ki-67表达,蛋白质免疫印迹法(Western blot)检测基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)水平。结果:筛选B7-H3分子高表达的U937、Maver和Z138细胞进行靶向敲减B7-H3,成功建立稳定低表达细胞株。U937、Maver和Z138细胞各自的KD组与NC组比较,观察终点的平均肿瘤生长抑制率分别为61.83%(F=43.78,P0.05)、59.12%(F=36.51,P0.05)和67.37%(F=40.29,P0.05),而各自NC组与CON组相比,肿瘤体积增长趋势差异无统计学意义(P0.05)。在所有移植瘤裸鼠模型中肝转移最常见,KD组裸鼠的远处转移较相应NC组明显降低。3种细胞各自KD组的Ki-67指数均较相应NC组显著下降(U937移植瘤:40.3%±5.2%vs.79.1%±6.3%,q=30.31,P0.05;Maver移植瘤:35.2%±6.4%vs.69.6%±5.1%,q=24.82,P0.05;Z138移植瘤:38.4%±7.1%vs.75.7%±4.8%,q=28.07,P0.05),而NC组与CON组Ki-67表达差异无统计学意义(P0.05)。3种细胞各自KD组肿瘤组织的MMP-2蛋白表达显著降低(U937移植瘤:q=14.59,P0.05;Maver移植瘤:q=9.25,P0.05;Z138移植瘤:q=11.04,P0.05),NC组与CON组MMP-2表达差异无统计学意义(P0.05)。结论:靶向B7-H3基因沉默能够抑制人恶性血液病细胞在裸鼠体内的成瘤和转移能力,其机制可能与Ki-67和MMP-2的表达下调有关。
[Abstract]:Objective: to investigate the effect and mechanism of targeting B7-H3 gene silencing on tumorigenesis and metastasis of human malignant hematological disease cell lines in nude mice. Methods: thirteen malignant hematological cell lines were detected by real-time fluorescence quantitative PCR(real-time quantitative polymerase chain reactionQ PCRs and flow cytometry (FCM). The expression level of B7-H3 in hematological cells, The high expression U937 Maver and Z138 cells were screened, and the stable low expression cell lines were established by using lentivirus transfection technique to target and reduce B7-H3, respectively. Q PCR and FCM were used to verify the effect of gene silencing. U937 control Maver and Z138 were divided into control group, non-infected control group, B7-H3 knockdown group (KD) and negative nontargeted control infected group (negative nontargeted control infected group) respectively. To compare the growth and metastasis of tumor, immunohistochemical immunohistochemical staining was used to detect the expression of Ki-67. Results: U937 Maver and Z138 cells with high expression of B7-H3 were screened for targeting knock down B7-H3, and stable low expression cell lines. U937 Maver and Z138 cells were successfully established and compared with NC group. The average tumor growth inhibition rates at the end point of observation were 61.83% and 43.78%, respectively, and 59.12% (59.12%) and 67.37% (40.29% P0.05), respectively, between the NC group and the CON group. There was no significant difference in tumor volume growth trend (P 0.05). The distant metastasis of KD group was significantly lower than that of NC group. The Ki-67 index of KD group was significantly lower than that of NC group. 40.3% 卤5.2vs.79.1% 卤6.3qng / s. There was no significant difference in the expression of MMP-2 protein between the NC group and the CON group. There was no significant difference in the expression of MMP-2 protein between the NC group and the CON group. There was a significant decrease in the expression of MMP-2 protein in the tumor tissues of the respective KD group. The expression of MMP-2 protein was significantly decreased in the U937 transplanted tumor group. The expression of MMP-2 protein was significantly reduced in the U937 transplanted tumor group QQN 14.59. P50.09P50.09. the tumor grafted tumor group was 14.599.25P50.0Z138Q44.01vs.75.75.7% 卤4.85.07P0.05ng. there was no significant difference between the NC group and the CON group. There was no significant difference in the expression of MMP-2 protein between the NC group and the CON group. The expression of MMP-2 protein in the tumor tissues of the respective KD group was significantly lower than that of the control group (P 0.053.The expression of MMP-2 protein in the tumor tissue of the respective KD group was 14.5909%) and that of the control group was 14.590% (P 50.09). Conclusion: targeting B7-H3 gene silencing can inhibit the tumorigenesis and metastasis of human malignant hematological cells in nude mice. The mechanism may be related to the down-regulation of Ki-67 and MMP-2 expression.
【作者单位】： 北京大学第三医院血液科;
【基金】：国家自然科学基金(81172245)资助~~
【分类号】：R733

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