本文選題：神經(jīng)母細(xì)胞瘤 + MYCN��； 參考：《鄭州大學(xué)》2014年博士論文

【摘要】：目的神經(jīng)母細(xì)胞瘤是小兒時期最為常見的顱外實體腫瘤,高危病例臨床進(jìn)展迅速,且易早期發(fā)生遠(yuǎn)處轉(zhuǎn)移,其治療手段雖經(jīng)不斷改進(jìn)和完善,但仍預(yù)后不良。目前多項研究表明相當(dāng)比例的高危神經(jīng)母細(xì)胞瘤同時存在MYCN和ALK基因異常改變,且二者顯示出強大的協(xié)同作用,與腫瘤的迅速增殖、惡性轉(zhuǎn)移和不良預(yù)后密切相關(guān)。RNAi具有強大的特定靶基因沉默特性,可通過下調(diào)和關(guān)閉基因表達(dá)調(diào)控細(xì)胞的各項活動及進(jìn)行基因表達(dá)缺失下的功能研究。TAE684是最早發(fā)現(xiàn)的小分子ALK激酶抑制劑,目前部分ALK激酶抑制劑已用于腫瘤的分子靶向治療并取得良好的抗腫瘤作用。但針對人神經(jīng)母細(xì)胞瘤MYCN和ALK兩種異常改變基因應(yīng)用RNAi與ALK抑制劑聯(lián)合治療的研究尚未見報道。本研究擬在人神經(jīng)母細(xì)胞瘤細(xì)胞系中選擇出高表達(dá)MYCN和ALK的細(xì)胞系作為研究對象,以MYCN和ALK作為靶基因,利用RNAi和穩(wěn)轉(zhuǎn)株構(gòu)建技術(shù),以慢病毒作為基因載體建立靶向MYCN的干擾穩(wěn)轉(zhuǎn)人神經(jīng)母細(xì)胞瘤細(xì)胞株；研究MYCN敲減對體外培養(yǎng)的人神經(jīng)母細(xì)胞瘤細(xì)胞增殖和侵襲能力的影響；以及MYCN敲減與ALK抑制劑TAE684聯(lián)合對人神經(jīng)母細(xì)胞瘤細(xì)胞凋亡水平的影響,MYCN, ALK蛋白及部分凋亡相關(guān)蛋白Bcl-2,Bax和Cleaved Caspase 3表達(dá)水平的改變,并分析其可能機制；同時應(yīng)用裸鼠成瘤技術(shù)建立人源性神經(jīng)母細(xì)胞瘤成瘤動物模型,檢測移植瘤生長情況,在體內(nèi)進(jìn)一步檢測MYCN敲減與TAE684的聯(lián)合作用,旨在探討MYCN和ALK在人神經(jīng)母細(xì)胞瘤細(xì)胞發(fā)生發(fā)展中的可能作用,為MYCN和ALK基因作為聯(lián)合治療靶點提供實驗依據(jù),為多基因改變的神經(jīng)母細(xì)胞瘤的治療提供新的思路,使腫瘤的分子靶向治療更加高效和完善。方法1.應(yīng)用熒光定量PCR和Western blot檢測三株人神經(jīng)母細(xì)胞瘤SH-SY5Y, SK-N-SH,SK-N-BE (2)細(xì)胞系MYCN mRNA和蛋白,ALK蛋白表達(dá)水平,從而篩選出高表達(dá)MYCN和ALK的人神經(jīng)母細(xì)胞瘤細(xì)胞系。2.根據(jù)人MYCN基因信息設(shè)計并構(gòu)建四個靶向MYCN的干擾質(zhì)粒,篩選鑒定后與pLenti6.3/V5 Dest載體重組,并行慢病毒包裝構(gòu)建成為Lenti-EGFP-MYCN-miR'幔病毒表達(dá)載體,感染高表達(dá)MYCN和ALK的人神經(jīng)母細(xì)胞瘤細(xì)胞,應(yīng)用熒光定量PCR和Western blot檢測四個Lenti-EGFP-MYCN-miR慢病毒表達(dá)載體對MYCN mRNA和蛋白表達(dá)的沉默效應(yīng),篩選出最佳干擾靶序列慢病毒表達(dá)載體。3.通過Blasticidin藥物抗性篩選獲得MYCN干擾穩(wěn)轉(zhuǎn)人神經(jīng)母細(xì)胞瘤細(xì)胞株,并應(yīng)用熒光定量PCR, Western blot檢測MYCN干擾穩(wěn)轉(zhuǎn)株MYCN mRNA和蛋白表達(dá)水平,鑒定穩(wěn)轉(zhuǎn)株成功建立。4.體外實驗中,應(yīng)用CCK-8檢測MYCN干擾穩(wěn)轉(zhuǎn)株MYCN基因敲減后人神經(jīng)母細(xì)胞瘤細(xì)胞增殖水平,Transwell小室結(jié)合結(jié)晶紫染色檢測細(xì)胞侵襲能力。5.體外實驗中,MYCN敲減與TAE684聯(lián)合作用下,應(yīng)用流式細(xì)胞術(shù)檢測人神經(jīng)母細(xì)胞瘤細(xì)胞凋亡水平,應(yīng)用Western blot檢測MYCN,ALK蛋白和Bcl-2, Bax, Cleaved Caspase -3蛋白表達(dá)情況,并分析其可能機制。6.應(yīng)用高表達(dá)MYCN和ALK的人神經(jīng)母細(xì)胞瘤細(xì)胞系建立人源性神經(jīng)母細(xì)胞瘤荷瘤裸鼠模型,檢測MYCN敲減與TAE684聯(lián)合作用下腫瘤生長情況,并應(yīng)用Western blot檢測腫瘤組織MYCN,ALK蛋白表達(dá)水平。結(jié)果1.三株SH-SY5Y,SK-N-SH,SK-N-BE (2)細(xì)胞系細(xì)胞的MYCN mRNA及蛋白表達(dá)水平分別為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蛋白表達(dá)分別為0.0175±0.0005,0.0173±0.0006,0.2100±0.0200；SK-N-BE(2)細(xì)胞株為高表達(dá)MYCN和ALK的人神經(jīng)母細(xì)胞瘤細(xì)胞系,可用于后續(xù)實驗。2.酶切和測序證實四個靶向MYCN的Lenti-EGFP-MYCN-miR慢病毒載體構(gòu)建成功,四個慢病毒表達(dá)載體感染SK-N-BE(2)細(xì)胞,應(yīng)用熒光定量PCR和Western blot檢測后,MYCN mRNA和蛋白的表達(dá)水平分別為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對MYCN抑制作用最為顯著,為最佳干擾靶序列慢病毒表達(dá)載體。3.通過Blasticidin藥物抗性進(jìn)行靶向MYCN干擾SK-N-BE(2)穩(wěn)轉(zhuǎn)株篩選,并且用熒光定量PCR和Western blot檢測穩(wěn)轉(zhuǎn)株MYCN mRNA及蛋白表達(dá),結(jié)果顯示分別為0.363±0.012,0.467±0.035,均較正常對照組和陰性對照慢病毒穩(wěn)轉(zhuǎn)組明顯下降(P0.05),成功建立MYCN干擾SK-N-BE(2)穩(wěn)轉(zhuǎn)株。4.體外實驗中,CCK-8檢測細(xì)胞增殖水平,結(jié)果顯示MYCN干擾SK-N-BE(2)穩(wěn)轉(zhuǎn)株OD值自感染后第1d至第6d分別為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值較正常對照組和陰性對照慢病毒穩(wěn)轉(zhuǎn)組明顯下降(P0.05),陰性對照慢病毒穩(wěn)轉(zhuǎn)組較正常對照組輕微下降,但差異不具有統(tǒng)計學(xué)意義(P0.05)。細(xì)胞侵襲實驗結(jié)果顯示,MYCN干擾SK-N-BE(2)穩(wěn)轉(zhuǎn)組OD值為0.042±0.009,較正常對照組和陰性對照慢病毒穩(wěn)轉(zhuǎn)組明顯下降(P0.05)。5.體外實驗中,在分別應(yīng)用MYCN敲減,TAE684以及MYCN敲減聯(lián)合TAE684作用下,流式細(xì)胞術(shù)檢測細(xì)胞凋亡水平分別為28.867±0.764,22.593±0.075,51.623±0.137,Western blot檢測MYCN蛋白為0.247±0.006,0.403±0.012,0.130±0.002,ALK蛋白為0.068±0.007,0.036±0.003,0.021±0.001,Bcl-2蛋白為0.192±0.005,0.190±0.015,0.056±0.010,Bax蛋白為0.314±0.008,0.161±0.003,0.435±0.024,Cleaved Caspase-3蛋白為0.807±0.041,0.949±0.003,1.452±0.020,與正常對照組、DMSO組、陰性慢病毒穩(wěn)轉(zhuǎn)組+DMSO組相比差異有統(tǒng)計學(xué)意義(P0.05),MYCN敲減聯(lián)合TAE684與分別應(yīng)用MYCN敲減、TAE684組相比變化更為顯著,差異有統(tǒng)計學(xué)意義(P0.05)。6.應(yīng)用SK-N-BE(2)成功建立裸鼠荷瘤模型,檢測成瘤體積結(jié)果顯示MYCN敲減組、TAE684組以及MYCN敲減聯(lián)合TAE684組移植瘤體積分別為0.1097±0.008cm3,0.0930±0.0060cm3和0.0024±0.0003cm3,Western blot檢測MYCN蛋白分別為0.345±0.020,0.650±0.033和0.217±0.023；ALK蛋白分別為0.054±0.002,0.042±0.005和0.010±0.001,與正常對照組、DMSO組、陰性慢病毒穩(wěn)轉(zhuǎn)+DMSO組相比明顯下降(P0.05),MYCN敲減聯(lián)合TAE684與分別應(yīng)用MYCN敲減、TAE684組相比變化更為顯著,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論1.人SK-N-BE (2)細(xì)胞為高表達(dá)MYCN和ALK神經(jīng)母細(xì)胞瘤系。2.成功構(gòu)建的靶向MYCN基因的Lenti-EGFP-MYCN-3-miR幔病毒表達(dá)載體可高效感染SK-N-BE (2)細(xì)胞及抑制目的基因MYCN表達(dá),經(jīng)過Blasticidin藥物篩選可建立MYCN干擾穩(wěn)轉(zhuǎn)SK-N-BE (2)細(xì)胞株。3. MYCN干擾穩(wěn)轉(zhuǎn)SK-N-BE (2)細(xì)胞株MYCN敲減后,細(xì)胞增殖能力和侵襲能力明顯抑制。4. MYCN敲減聯(lián)合TAE684可明顯下調(diào)體外培養(yǎng)的SK-N-BE(2)細(xì)胞MYCN, ALK, Bcl-2蛋白表達(dá),上調(diào)BAX, Cleaved Caspase-3蛋白表達(dá),細(xì)胞凋亡水平明顯增加,MYCN敲減聯(lián)合TAE684可能通過對凋亡分子的調(diào)控來實現(xiàn)對細(xì)胞凋亡的誘導(dǎo)促進(jìn)作用。5.應(yīng)用SK-N-BE (2)細(xì)胞系可成功建立裸鼠荷瘤模型,MYCN干擾穩(wěn)轉(zhuǎn)SK-N-BE (2)細(xì)胞株MYCN敲減與TAE684聯(lián)合作用可增強對靶基因MYCN,ALK蛋白表達(dá)的抑制作用,抑制裸鼠移植瘤的生長,具有體內(nèi)抗腫瘤作用。
[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.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R739.4
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本文編號：1975735