靶向MYCN干扰稳转株的构建及联合TAE684对神经母细胞瘤生物学行为的影响

发布时间：2018-06-04 03:42

  本文选题：神经母细胞瘤 + MYCN　； 参考：《郑州大学》2014年博士论文

【摘要】：目的神经母细胞瘤是小儿时期最为常见的颅外实体肿瘤,高危病例临床进展迅速,且易早期发生远处转移,其治疗手段虽经不断改进和完善,但仍预后不良。目前多项研究表明相当比例的高危神经母细胞瘤同时存在MYCN和ALK基因异常改变,且二者显示出强大的协同作用,与肿瘤的迅速增殖、恶性转移和不良预后密切相关。RNAi具有强大的特定靶基因沉默特性,可通过下调和关闭基因表达调控细胞的各项活动及进行基因表达缺失下的功能研究。TAE684是最早发现的小分子ALK激酶抑制剂,目前部分ALK激酶抑制剂已用于肿瘤的分子靶向治疗并取得良好的抗肿瘤作用。但针对人神经母细胞瘤MYCN和ALK两种异常改变基因应用RNAi与ALK抑制剂联合治疗的研究尚未见报道。本研究拟在人神经母细胞瘤细胞系中选择出高表达MYCN和ALK的细胞系作为研究对象,以MYCN和ALK作为靶基因,利用RNAi和稳转株构建技术,以慢病毒作为基因载体建立靶向MYCN的干扰稳转人神经母细胞瘤细胞株；研究MYCN敲减对体外培养的人神经母细胞瘤细胞增殖和侵袭能力的影响；以及MYCN敲减与ALK抑制剂TAE684联合对人神经母细胞瘤细胞凋亡水平的影响,MYCN, ALK蛋白及部分凋亡相关蛋白Bcl-2,Bax和Cleaved Caspase 3表达水平的改变,并分析其可能机制；同时应用裸鼠成瘤技术建立人源性神经母细胞瘤成瘤动物模型,检测移植瘤生长情况,在体内进一步检测MYCN敲减与TAE684的联合作用,旨在探讨MYCN和ALK在人神经母细胞瘤细胞发生发展中的可能作用,为MYCN和ALK基因作为联合治疗靶点提供实验依据,为多基因改变的神经母细胞瘤的治疗提供新的思路,使肿瘤的分子靶向治疗更加高效和完善。方法1.应用荧光定量PCR和Western blot检测三株人神经母细胞瘤SH-SY5Y, SK-N-SH,SK-N-BE (2)细胞系MYCN mRNA和蛋白,ALK蛋白表达水平,从而筛选出高表达MYCN和ALK的人神经母细胞瘤细胞系。2.根据人MYCN基因信息设计并构建四个靶向MYCN的干扰质粒,筛选鉴定后与pLenti6.3/V5 Dest载体重组,并行慢病毒包装构建成为Lenti-EGFP-MYCN-miR'幔病毒表达载体,感染高表达MYCN和ALK的人神经母细胞瘤细胞,应用荧光定量PCR和Western blot检测四个Lenti-EGFP-MYCN-miR慢病毒表达载体对MYCN mRNA和蛋白表达的沉默效应,筛选出最佳干扰靶序列慢病毒表达载体。3.通过Blasticidin药物抗性筛选获得MYCN干扰稳转人神经母细胞瘤细胞株,并应用荧光定量PCR, Western blot检测MYCN干扰稳转株MYCN mRNA和蛋白表达水平,鉴定稳转株成功建立。4.体外实验中,应用CCK-8检测MYCN干扰稳转株MYCN基因敲减后人神经母细胞瘤细胞增殖水平,Transwell小室结合结晶紫染色检测细胞侵袭能力。5.体外实验中,MYCN敲减与TAE684联合作用下,应用流式细胞术检测人神经母细胞瘤细胞凋亡水平,应用Western blot检测MYCN,ALK蛋白和Bcl-2, Bax, Cleaved Caspase -3蛋白表达情况,并分析其可能机制。6.应用高表达MYCN和ALK的人神经母细胞瘤细胞系建立人源性神经母细胞瘤荷瘤裸鼠模型,检测MYCN敲减与TAE684联合作用下肿瘤生长情况,并应用Western blot检测肿瘤组织MYCN,ALK蛋白表达水平。结果1.三株SH-SY5Y,SK-N-SH,SK-N-BE (2)细胞系细胞的MYCN mRNA及蛋白表达水平分别为0.090±0.010,0.340±0.026,87.867±2.316和0.0064±0.0003,0.3887±0.0027,1.2447±0.0301；ALK蛋白表达分别为0.0175±0.0005,0.0173±0.0006,0.2100±0.0200；SK-N-BE(2)细胞株为高表达MYCN和ALK的人神经母细胞瘤细胞系,可用于后续实验。2.酶切和测序证实四个靶向MYCN的Lenti-EGFP-MYCN-miR慢病毒载体构建成功,四个慢病毒表达载体感染SK-N-BE(2)细胞,应用荧光定量PCR和Western blot检测后,MYCN mRNA和蛋白的表达水平分别为0.577±0.015,0.333±0.016,0.090±0.010,0.197±0.015和0.833±0.049,0.563±0.040,0.300±0.020,0.387±0.015,其中Lenti-EGFP-MYCN-3-miR对MYCN抑制作用最为显著,为最佳干扰靶序列慢病毒表达载体。3.通过Blasticidin药物抗性进行靶向MYCN干扰SK-N-BE(2)稳转株筛选,并且用荧光定量PCR和Western blot检测稳转株MYCN mRNA及蛋白表达,结果显示分别为0.363±0.012,0.467±0.035,均较正常对照组和阴性对照慢病毒稳转组明显下降(P0.05),成功建立MYCN干扰SK-N-BE(2)稳转株。4.体外实验中,CCK-8检测细胞增殖水平,结果显示MYCN干扰SK-N-BE(2)稳转株OD值自感染后第1d至第6d分别为0.258±0.012,0.335±0.016,0.383±0.076,0.597±0.023,0.767±0.021,0.989±0.060,其中第2d至第6d OD值较正常对照组和阴性对照慢病毒稳转组明显下降(P0.05),阴性对照慢病毒稳转组较正常对照组轻微下降,但差异不具有统计学意义(P0.05)。细胞侵袭实验结果显示,MYCN干扰SK-N-BE(2)稳转组OD值为0.042±0.009,较正常对照组和阴性对照慢病毒稳转组明显下降(P0.05)。5.体外实验中,在分别应用MYCN敲减,TAE684以及MYCN敲减联合TAE684作用下,流式细胞术检测细胞凋亡水平分别为28.867±0.764,22.593±0.075,51.623±0.137,Western blot检测MYCN蛋白为0.247±0.006,0.403±0.012,0.130±0.002,ALK蛋白为0.068±0.007,0.036±0.003,0.021±0.001,Bcl-2蛋白为0.192±0.005,0.190±0.015,0.056±0.010,Bax蛋白为0.314±0.008,0.161±0.003,0.435±0.024,Cleaved Caspase-3蛋白为0.807±0.041,0.949±0.003,1.452±0.020,与正常对照组、DMSO组、阴性慢病毒稳转组+DMSO组相比差异有统计学意义(P0.05),MYCN敲减联合TAE684与分别应用MYCN敲减、TAE684组相比变化更为显著,差异有统计学意义(P0.05)。6.应用SK-N-BE(2)成功建立裸鼠荷瘤模型,检测成瘤体积结果显示MYCN敲减组、TAE684组以及MYCN敲减联合TAE684组移植瘤体积分别为0.1097±0.008cm3,0.0930±0.0060cm3和0.0024±0.0003cm3,Western blot检测MYCN蛋白分别为0.345±0.020,0.650±0.033和0.217±0.023；ALK蛋白分别为0.054±0.002,0.042±0.005和0.010±0.001,与正常对照组、DMSO组、阴性慢病毒稳转+DMSO组相比明显下降(P0.05),MYCN敲减联合TAE684与分别应用MYCN敲减、TAE684组相比变化更为显著,差异有统计学意义(P0.05)。结论1.人SK-N-BE (2)细胞为高表达MYCN和ALK神经母细胞瘤系。2.成功构建的靶向MYCN基因的Lenti-EGFP-MYCN-3-miR幔病毒表达载体可高效感染SK-N-BE (2)细胞及抑制目的基因MYCN表达,经过Blasticidin药物筛选可建立MYCN干扰稳转SK-N-BE (2)细胞株。3. MYCN干扰稳转SK-N-BE (2)细胞株MYCN敲减后,细胞增殖能力和侵袭能力明显抑制。4. MYCN敲减联合TAE684可明显下调体外培养的SK-N-BE(2)细胞MYCN, ALK, Bcl-2蛋白表达,上调BAX, Cleaved Caspase-3蛋白表达,细胞凋亡水平明显增加,MYCN敲减联合TAE684可能通过对凋亡分子的调控来实现对细胞凋亡的诱导促进作用。5.应用SK-N-BE (2)细胞系可成功建立裸鼠荷瘤模型,MYCN干扰稳转SK-N-BE (2)细胞株MYCN敲减与TAE684联合作用可增强对靶基因MYCN,ALK蛋白表达的抑制作用,抑制裸鼠移植瘤的生长,具有体内抗肿瘤作用。
[Abstract]:Objective neuroblastoma is the most common extracranial solid tumor in childhood. The high risk cases are progressing rapidly, and the distant metastasis is easy to occur early. Although the treatment is improved and perfected, the prognosis is poor. At present, a number of studies have shown that a considerable proportion of Gao Weishen and ALK genes have abnormal changes of MYCN and ALK genes. Change, and the two show strong synergy, which is closely related to the rapid proliferation of tumor, malignant metastasis and poor prognosis..RNAi has strong specific target gene silencing characteristics, and.TAE684 is the earliest small molecule A to be found by regulating the activities of cells and the function of gene expression deletion by down and off gene expression. LK kinase inhibitors, some of the ALK kinase inhibitors have been used for molecular targeting and anti-tumor effects of tumor, but the study of the combination of RNAi and ALK inhibitors for the two abnormal changes of human neuroblastoma MYCN and ALK has not yet been reported. This study is intended to be selected in the human neuroblastoma cell line A cell line with high expression of MYCN and ALK was used as a target gene, using MYCN and ALK as the target gene, using RNAi and stable transgenic plants to construct a targeted MYCN mediated human neuroblastoma cell line with the lentivirus as a gene carrier, and to study the effect of MYCN knockout on the proliferation and invasion ability of human neuroblastoma cells cultured in vitro. The effect of MYCN knockout and ALK inhibitor TAE684 on the apoptosis of human neuroblastoma cells, the changes of MYCN, ALK protein and partial apoptosis related protein Bcl-2, Bax and Cleaved Caspase 3 expression level, and the analysis of its possible mechanism, and the establishment of a human neuroblastoma tumor model in nude mice. In order to explore the possible role of MYCN and ALK in the development of human neuroblastoma cells in vivo, the possible role of MYCN and ALK in the development of human neuroblastoma cells was examined in vivo, and the experimental basis for the combination of MYCN and ALK genes was provided to provide a new thought for the treatment of neuroblastoma with multiple changes of the neuroblastoma. Methods to make the molecular targeting therapy of tumor more efficient and perfect. Method 1. using fluorescence quantitative PCR and Western blot to detect the MYCN mRNA and protein of three human neuroblastoma, MYCN mRNA and protein in SK-N-SH, SK-N-BE (2) cell lines, and the expression level of ALK protein, thus screening the human neuroblastoma cell lines with high expression of MYCN and ALK. According to the information design and construction of four interference plasmids targeting MYCN, after screening and identification with the recombinant pLenti6.3/V5 Dest vector, parallel lentivirus packaging construction became the expression vector of Lenti-EGFP-MYCN-miR'mantle virus, infected human neuroblastoma cells with high expression of MYCN and ALK, and detected four Lenti-EGFP-MYCN- using fluorescence quantitative PCR and Western blot. The silence effect of miR Lentivirus Expression Vector on MYCN mRNA and protein expression, the best target sequence of Lentivirus Expression Vector.3. was screened by Blasticidin drug resistance screening to obtain MYCN interference stable human neuroblastoma cell lines, and fluorescence quantitative PCR was used, Western blot detection MYCN interfered with MYCN mRNA and protein expression water. .4. was successfully established in vitro, and CCK-8 was used to detect the proliferation of human neuroblastoma cells after MYCN interfering with MYCN gene knockout, and Transwell chamber combined with crystal violet staining to detect cell invasiveness in.5. in vitro. MYCN knockout and TAE684 co operation were used to detect the human neuroma by flow cytometry. The expression of MYCN, ALK protein and Bcl-2, Bax, Cleaved Caspase -3 protein was detected by Western blot, and its possible mechanism was analyzed to establish a human neuroblastoma nude mouse model with the human neuroblastoma cell line with high expression MYCN and ALK. The growth of tumor and the expression of MYCN and ALK protein in tumor tissues were detected by Western blot. Results the mRNA and protein expression levels of MYCN in 1. three strains of SH-SY5Y, SK-N-SH, SK-N-BE (2) cell lines were 0.090 + 0.010,0.340 + 0.026,87.867 + 2.316 and 0.0064 + + + + 0.0301, respectively. The expression of protein was 0.0175, respectively. 0.0005,0.0173 + 0.0006,0.2100 + 0.0200; SK-N-BE (2) cell line is a human neuroblastoma cell line with high expression of MYCN and ALK. It can be used for subsequent experimental.2. enzyme digestion and sequencing to confirm that four Lenti-EGFP-MYCN-miR lentivirus vectors targeting MYCN are successfully constructed. Four lentivirus surface vectors are infected with SK-N-BE (2) cells and use fluorescent quantitative PCR. The expression level of MYCN mRNA and protein was 0.577 + 0.015,0.333 + 0.016,0.090 + 0.015 and 0.833 + 0.049,0.563 + 0.040,0.300 + 0.020,0.387 + 0.015 respectively after detection of MYCN mRNA and protein, and Lenti-EGFP-MYCN-3-miR on MYCN was the most significant. Drug resistance was screened by targeting MYCN interference SK-N-BE (2), and the expression of MYCN mRNA and protein was detected by fluorescence quantitative PCR and Western blot. The results showed that the results were 0.363 + 0.012,0.467 + 0.035, respectively, compared with the normal control group and the negative control lentivirus stable group (P0.05), and the MYCN interference SK-N-BE (2) was successfully established. In the experiment of.4. in vitro, CCK-8 detected the cell proliferation level. The results showed that the MYCN interference SK-N-BE (2) ood value was 0.258 + 0.012,0.335 + 0.016,0.383 + 0.076,0.597 + 0.023,0.767 + 0.060 respectively after infection, respectively, which was significantly lower than that of the normal control group and the negative control lentivirus. P0.05), the negative control lentivirus stable group was slightly lower than the normal control group, but the difference was not statistically significant (P0.05). The results of cell invasion experiment showed that MYCN interference in SK-N-BE (2) stable group was 0.042 + 0.009, compared with the normal control group and the negative control lentivirus stable group (P0.05).5. in vitro experiment, respectively, using MYCN Under the action of knockout, TAE684 and MYCN knockout combined with TAE684, the level of cell apoptosis detected by flow cytometry was 28.867 + 0.764,22.593 + 0.075,51.623 + 0.137 respectively. Western blot was used to detect MYCN protein 0.247 + 0.006,0.403 + 0.012,0.130 + 0.002, ALK protein was 0.068 + 0.001 + 0.001, and 0.192 + + 0 15,0.056 + 0.010, Bax protein was 0.314 + 0.008,0.161 + 0.003,0.435 + 0.024, Cleaved Caspase-3 protein was 0.807 + 0.041,0.949 + 0.003,1.452 + 0.020, compared with normal control group, DMSO group and negative lentivirus stable group +DMSO group, the difference was statistically significant (P0.05). More significant, the difference was statistically significant (P0.05).6. application SK-N-BE (2) successfully established nude mice model of tumor bearing tumor, detection of tumor volume results showed MYCN knockout group, TAE684 group and MYCN knockout combined TAE684 group transplanted tumor volume was 0.1097 + 0.008cm3,0.0930 + 0.0060cm3 and 0.0024 + 0.0003cm3, Western blot detection of 0.345 0.020,0.650 + 0.033 and 0.217 + 0.023, ALK protein 0.054 + 0.002,0.042 + 0.005 and 0.010 + 0.001 respectively, compared with normal control group, DMSO group and negative lentivirus in +DMSO group (P0.05), MYCN knockout combined TAE684 and MYCN knockout respectively, the phase ratio of TAE684 group was more significant, the difference was statistically significant (P0.05). 1. SK-N-BE (2) cells are high expression of MYCN and ALK neuroblastoma.2. target MYCN gene successfully constructed by Lenti-EGFP-MYCN-3-miR mantle virus expression vector, which can efficiently infect SK-N-BE (2) cells and inhibit MYCN expression of target genes. SK-N-BE (2) cell line MYCN knockdown, cell proliferation and invasion ability obviously inhibit.4. MYCN knockout combined with TAE684 can significantly reduce the expression of SK-N-BE (2) cells in vitro, MYCN, ALK, Bcl-2 protein expression, up regulation BAX, Cleaved Caspase-3 protein expression, cell withering level obviously increased. Molecular regulation to promote the induction of apoptosis,.5. application SK-N-BE (2) can successfully establish nude mice bearing tumor model. MYCN interfering with SK-N-BE (2) cell line MYCN knockout and TAE684 can enhance the inhibition of the expression of target gene MYCN, ALK protein, inhibit the growth of xenografts in nude mice and have anti tumor in vivo. Effect.
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.4
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本文编号：1975735