本文關(guān)鍵詞： 惡性血液病 B-H 基因沉默 裸鼠　出處：《北京大學(xué)學(xué)報(bào)(醫(yī)學(xué)版)》2017年02期 　論文類型：期刊論文

【摘要】：目的:探討靶向B7-H3基因沉默對人惡性血液病細(xì)胞株在裸鼠體內(nèi)成瘤和轉(zhuǎn)移的影響及機(jī)制。方法:應(yīng)用實(shí)時熒光定量PCR(real-time quantitative polymerase chain reaction,q PCR)和流式細(xì)胞術(shù)(flow cytometry,FCM)檢測13株惡性血液病細(xì)胞中B7-H3分子的表達(dá)水平,篩選高表達(dá)的U937、Maver和Z138細(xì)胞。分別利用慢病毒轉(zhuǎn)染技術(shù)靶向敲減B7-H3,建立穩(wěn)定低表達(dá)細(xì)胞株,應(yīng)用q PCR和FCM驗(yàn)證基因沉默效果。將U937、Maver和Z138各自分為空白對照組(non-infected control group,CON)、靶向B7-H3敲減組(B7-H3 knockdown group,KD)和陰性無關(guān)序列對照組(negative nontargeted control infected group,NC),將以上9組分別構(gòu)建裸鼠皮下移植瘤模型,比較腫瘤生長和轉(zhuǎn)移情況,免疫組織化學(xué)(immunohistochemistry,IHC)染色檢測Ki-67表達(dá),蛋白質(zhì)免疫印跡法(Western blot)檢測基質(zhì)金屬蛋白酶2(matrix metalloproteinase 2,MMP-2)水平。結(jié)果:篩選B7-H3分子高表達(dá)的U937、Maver和Z138細(xì)胞進(jìn)行靶向敲減B7-H3,成功建立穩(wěn)定低表達(dá)細(xì)胞株。U937、Maver和Z138細(xì)胞各自的KD組與NC組比較,觀察終點(diǎn)的平均腫瘤生長抑制率分別為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組與CON組相比,腫瘤體積增長趨勢差異無統(tǒng)計(jì)學(xué)意義(P0.05)。在所有移植瘤裸鼠模型中肝轉(zhuǎn)移最常見,KD組裸鼠的遠(yuǎn)處轉(zhuǎn)移較相應(yīng)NC組明顯降低。3種細(xì)胞各自KD組的Ki-67指數(shù)均較相應(yīng)NC組顯著下降(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組與CON組Ki-67表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。3種細(xì)胞各自KD組腫瘤組織的MMP-2蛋白表達(dá)顯著降低(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組與CON組MMP-2表達(dá)差異無統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:靶向B7-H3基因沉默能夠抑制人惡性血液病細(xì)胞在裸鼠體內(nèi)的成瘤和轉(zhuǎn)移能力,其機(jī)制可能與Ki-67和MMP-2的表達(dá)下調(diào)有關(guān)。
[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% 鹵5.2vs.79.1% 鹵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% 鹵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.
【作者單位】： 北京大學(xué)第三醫(yī)院血液科;
【基金】：國家自然科學(xué)基金(81172245)資助~~
【分類號】：R733

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