PARP-1对淋巴瘤Raji细胞增殖与凋亡的影响及机制探讨

发布时间：2018-03-20 19:22

  本文选题：淋巴瘤　切入点：Raji细胞　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的运用转染慢病毒介导的si RNA干扰的方法,抑制伯基特淋巴瘤Raji细胞中PARP-1基因的表达,研究其对肿瘤细胞增殖与凋亡的影响,探讨PARP-1基因在淋巴瘤中的作用机制,为恶性淋巴瘤的靶向治疗提供理论和实验依据。方法(1)细胞培养:将人伯基特淋巴瘤Raji细胞株培养于含1%青霉素和1%链霉素、15%的胎牛血清的RPMI1640培养液中,并置于37℃、5%CO2的培养箱中培养,以及后续的细胞传代、换液、冻存、复苏。采用细胞活力数90%、对数生长期的细胞用于本实验。(2)分组:将细胞随机分为三组:空白对照组;阴性对照组(NC-si RNA):转染PARP-1小干扰RNA阴性对照序列的细胞;实验组:转染PARP-1小干扰RNA的细胞。(3)转染慢病毒介导的si RNA抑制PARP-1基因在淋巴瘤细胞中的表达。(4)运用实时荧光定量PCR的方法分别对空白对照组、阴性对照组和实验组细胞中PARP-1 m RNA的表达情况进行检测,并计算出其抑制效率。(5)运用CCK-8的方法分别于12h、24h、36h、48h检测三组细胞的增殖活性。(6)运用流式细胞术分别于细胞培养12h、24h、36h、48h后检测三组细胞的凋亡率。结果(1)荧光显微镜下见到绿色荧光视为转染成功,转染后我们于24h、48h、72h分别进行荧光计数,比较绿色荧光细胞的数量,随着时间的延长,绿色荧光的表达量逐渐增高,于72h绿色荧光的表达量最高,NC-si RNA组、PARP-1—si RNA组Raji细胞的转染效率达80%以上,用来进行后续实验的研究。(2)运用实时荧光定量PCR的方法检测三组细胞中si RNA对PARP-1 m RNA的表达水平。以对照组细胞中PARP-1基因的m RNA表达水平为100%,NC-si RNA组和实验组细胞中PARP-1基因m RNA表达水平分别为(94.8±0.5)%、(54.6±0.3)%。与未转染的空白对照组相比,转染无关序列的NC组对PARP-1 m RNA的表达无影响,差异无统计学意义(P0.05),而实验组PARP-1 m RNA的表达明显受抑制,差异具有显著统计学意义(P0.01)。(3)CCK-8法检测细胞增殖活性,结果显示,随着时间的延长,细胞增殖活性抑制率逐渐升高,在48h时,三组细胞增殖活性抑制率分别为(22±0.4)%、(24±0.2)%、(58±0.7)%,对照组与阴性对照组相比,差异无统计学意义(P0.05),而实验组Raji细胞的增殖活性明显受抑制,与对照组相比,差异有统计学意义(P0.05)。(4)运用流式细胞术检测细胞凋亡率,结果显示,随着时间的延长,凋亡率逐渐增加,于48h时细胞凋亡率增高,实验组细胞凋亡率达到(37±0.4)%,而空白对照组、阴性对照组细胞凋亡率分别为(11.4±0.3)%、(13.8±0.3)%,实验组与对照组相比,差异具有显著统计学意义(P0.01),而阴性对照组与空白对照组相比,差异无统计学意义(P0.05)。结论抑制PARP-1基因在淋巴瘤Raji细胞中的表达,可抑制细胞的增殖,促进细胞的凋亡。其作用机制较为复杂,可能为不仅通过调控转录因子对恶性肿瘤的增殖产生抑制作用,而且通过激活“死亡底物”—Caspase促进其凋亡,起到促进细胞凋亡的作用,该方法可以提高治疗肿瘤细胞的针对性和敏感性,为恶性淋巴瘤的靶向治疗提供理论和实验依据。
[Abstract]:Methods Si RNA interference by lentivirus mediated transfection, inhibit the expression of PARP-1 gene in Burkitt's lymphoma Raji cells, to study its effect on tumor cell proliferation and apoptosis, to explore the mechanism of PARP-1 gene in lymphoma, malignant lymphoma of the target and provide theoretical and experimental basis to the treatment method (1). Cell culture: the human Burkitt lymphoma cell line Raji cultured in 1% and 1% penicillin streptomycin, 15% fetal bovine serum RPMI1640 culture medium, and at 37 DEG C, the cultivation box of 5%CO2, and the subsequent cell passage, cryopreservation, resuscitation fluid change, cell viability. The number 90%, logarithmic growth the cells used in this experiment. (2) group: the cells were randomly divided into three groups: control group; negative control group (NC-si RNA): transfection of PARP-1 small interfering RNA sequence of negative control cells; experimental group: transfection of PARP-1 small interfering RNA (cell. 3) Si RNA with lentivirus mediated inhibition of PARP-1 gene expression in lymphoma cells. (4) using the method of real-time fluorescence quantitative PCR respectively on the blank control group, negative control group and the experimental group the expression of PARP-1 in cells of M RNA were detected, and calculate the inhibition rate (5). Using the method of CCK-8 in 12h, 24h, 36h, proliferation of 48h were detected in three groups of cells. (6) the use of flow cytometry in cell culture 12h, 24h, 36h, 48h after the detection of apoptotic cells in three groups. Results (1) see green fluorescence under the fluorescence microscope as a successful transfection. After transfection, we at 24h, 48h, 72h respectively for fluorescence count, number of green fluorescent cells, with the extension of time, the expression of green fluorescence increased gradually, the expression of 72h in green fluorescence is the highest, NC-si RNA group, PARP-1 Si Group RNA Raji cell transfection efficiency was more than 80%, to 杩涜�屽悗缁�瀹為獙鐨勭爺绌，

本文编号：1640466