PLK1基因沉默抑制淋巴瘤Raji细胞增殖和侵袭

发布时间：2018-05-01 19:39

本文选题：PLK1 + 淋巴瘤　；参考：《南华大学》2016年硕士论文

【摘要】：[目的]Polo样激酶1(polo-like kinase 1,PLK1)是一种丝/苏氨酸蛋白激酶,在调节细胞周期、维持基因组稳定和修复DNA损伤中起重要作用。研究表明PLK1在人类多数肿瘤中高表达,我们前期实验也发现EBV转化淋巴母细胞中PLK1表达上调。本实验通过体外细胞实验及动物实验,观察RNA干扰PLK1表达对淋巴瘤Raji细胞增殖、迁移侵袭、细胞周期和凋亡以及裸鼠移植瘤形成等生物学行为的影响,研究PLK1在EBV相关淋巴瘤发生中的作用,为淋巴瘤的分子机制研究与靶向治疗提供实验依据。[方法]构建针对PLK1基因的慢病毒干扰载体p Len R-GPHh PLK1-sh,建立PLK1稳定低表达的Raji细胞系,倒置荧光显微镜观察细胞内绿色荧光情况,经过q RT-PCR及Western Blot技术验证PLK1干扰效果。采用CCK-8、流式细胞术、迁移侵袭实验及裸鼠成瘤实验,观察PLK1表达改变前后淋巴瘤Raji细胞生物学行为的变化。[结果]1.成功建立PLK1稳定低表达的Raji细胞系。重组质粒酶切电泳结果与预期相符,质粒测序结果经BLAST比对显示,目的基因的干扰质粒p Len R-GPH-h PLK1-sh与设计的干扰序列吻合率达100%,证明载体构建成功;荧光显微镜观察显示转染组携带绿色荧光的细胞数达80%;q RT-PCR、Western Blot检测表明转染p Len R-GPH-h PLK1-sh质粒的细胞(干扰组,Raji/PLK1 sh RNA)较转染p Len R-GPH的细胞(空载体组,Raji/GPH)和普通Raji细胞(空白组)相比,PLK1基因的m RNA表达及蛋白水平均明显降低,表明成功建立PLK1稳定低表达的Raji细胞系。且干扰组PLK1-SH1中PLK1 m RNA水平下降更为明显,提示干扰效果最好,所以选取PLK1-SH1组进入后续实验。2.干扰PLK1表达可抑制淋巴瘤Raji细胞的增殖、迁移侵袭能力,诱导G2期阻滞和凋亡,并抑制裸鼠移植瘤的形成。CCK-8结果显示:干扰组较空载体组及空白组相比,生长速度慢,且差异具有统计学意义(P0.01),表明干扰PLK1表达可抑制Raji细胞的增殖。迁移侵袭实验结果显示:干扰组较空载体组及空白组相比,迁移细胞数较少,差异有统计学意义(P0.05);干扰组穿过Matrigel基质胶的细胞数明显少于空载体组及空白组,差异有统计学意义(P0.01),表明干扰PLK1的表达可抑制淋巴瘤Raji细胞的迁移和侵袭能力。流式细胞术结果显示:干扰组较空载体组及空白组相比,G2期细胞百分比显著升高,G1期细胞百分比降低(P0.01);细胞早期凋亡率明显升高(P0.01),差异有统计学意义。表明干扰PLK1表达可诱导淋巴瘤Raji细胞G2期阻滞和凋亡。裸鼠成瘤实验结果显示:空载体组及空白组各组成瘤率达80%;而干扰组成瘤率为0%。干扰组相比于对照组,裸鼠成瘤率明显下降,这提示干扰PLK1的表达,对裸鼠移植瘤的形成有显著抑制作用。[结论]干扰PLK1的表达可抑制淋巴瘤Raji细胞增殖、迁移侵袭及移植瘤形成,诱导凋亡与周期阻滞。
[Abstract]:[objective] Polo like kinase (1(polo-like kinase 1) is a serine / threonine protein kinase, which plays an important role in regulating cell cycle, maintaining genomic stability and repairing DNA damage. Studies have shown that PLK1 is highly expressed in most human tumors, and we also found that the expression of PLK1 in transformed lymphoblastocytes of EBV was up-regulated in our previous experiments. The effects of RNA interfering PLK1 expression on the proliferation, migration, invasion, cell cycle and apoptosis of lymphoma Raji cells and the formation of xenografts in nude mice were observed by in vitro cell experiments and animal experiments. To study the role of PLK1 in the pathogenesis of EBV-associated lymphoma and to provide experimental evidence for the molecular mechanism and targeted therapy of lymphoma. [methods] the lentivirus interference vector p Len R-GPHh PLK1-shtargeted at PLK1 gene was constructed, and a stable and low expression Raji cell line with PLK1 was established. The green fluorescence was observed by inverted fluorescence microscope. The effect of PLK1 interference was verified by Q RT-PCR and Western Blot techniques. Using CCK-8, flow cytometry, migration and invasion assay and tumorigenesis in nude mice, the biological behavior of lymphoma Raji cells was observed before and after the change of PLK1 expression. [result] 1. Raji cell lines with stable and low expression of PLK1 were successfully established. The result of restriction endonuclease electrophoresis of the recombinant plasmid was consistent with the expected results. The result of plasmid sequencing showed that the interference plasmid p Len R-GPH-h PLK1-sh of the target gene coincided with the designed interference sequence by 100%, which proved that the vector was successfully constructed. Fluorescence microscopy showed that the number of cells carrying green fluorescence in the transfected group was 80% and that the cells transfected with p Len R-GPH-h PLK1-sh plasmid (interference group Raji / PLK1 sh) were higher than those transfected with p Len R-GPH (empty vector group) and ordinary Raji cells. Compared with the control group, the expression of m RNA and the protein level of PLK1 gene were significantly decreased. The results showed that Raji cell line with stable and low expression of PLK1 was successfully established. The decrease of PLK1 m RNA level in PLK1-SH1 was more obvious in the interference group, indicating that the interference effect was the best, so the PLK1-SH1 group was selected to enter the follow-up experiment. 2. Interfering the expression of PLK1 could inhibit the proliferation, migration and invasion of Raji cells, induce G2 phase arrest and apoptosis, and inhibit the formation of xenografts in nude mice. The results showed that the growth rate of interference group was slower than that of empty vector group and blank group. The difference was statistically significant (P 0.01), indicating that interfering with PLK1 expression could inhibit the proliferation of Raji cells. The results of migration and invasion experiments showed that the number of migration cells in the interference group was less than that in the empty carrier group and the blank group, the difference was statistically significant (P 0.05), and the number of cells passing through the Matrigel matrix in the interference group was significantly less than that in the empty carrier group and blank group. The difference was statistically significant (P 0.01), suggesting that interfering with the expression of PLK1 could inhibit the migration and invasion of lymphoma Raji cells. The results of flow cytometry showed that the percentage of cells in G _ 2 phase in the interference group was significantly higher than that in the empty carrier group and the blank group, and the percentage of cells in the G _ 1 phase was lower than that in the control group, and the apoptosis rate in the early stage was significantly higher than that in the control group (P _ (0.01). It was suggested that interfering with PLK1 expression could induce G 2 arrest and apoptosis in lymphoma Raji cells. The results of nude mice tumorigenesis test showed that the tumor rate of empty carrier group and blank group was 80%, while that of interference group was 0%. Compared with the control group, the tumorigenesis rate in the interference group was significantly lower than that in the control group, which suggested that interfering with the expression of PLK1 could significantly inhibit the formation of xenografts in nude mice. [conclusion] interfering with the expression of PLK1 can inhibit the proliferation, migration and invasion of Raji cells, induce apoptosis and cell cycle arrest.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R733.1

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