铜绿假单胞菌制剂抑制鼻咽癌细胞体外增殖

发布时间：2018-02-20 08:03

本文关键词： 铜绿假单胞菌鼻咽癌增殖细胞周期凋亡　出处：《南方医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：一、研究目的鼻咽癌是我国两广地区最常见的头颈部恶性肿瘤,其发病可能与遗传易感性、EBV感染、环境与饮食习惯以及免疫功能缺陷相关。该病严重影响着我国人民的健康。如何改进鼻咽癌的治疗技术以及方法,提高疾病治愈率以及局部控制率,对提升人民的健康水平有着其实际的意义。鼻咽癌的治疗目前公认为是以放射治疗或者以放射治疗为主的综合治疗。早期鼻咽癌患者单纯放疗即能达到临床治愈,晚期患者需要选择以放射治疗为主的综合治疗。在放射治疗方面,鼻咽癌的放疗剂量已经基本恒定在70Gy左右。如何在保证病灶区域剂量的同时,减少周边正常组织的受量,是目前放射治疗在技术层面改进的方向。目的是在保证局部控制率不降低的情况下,减少口干以及张口困难等并发症的发生,提高患者的远期生活质量。现阶段已经出现IMRT, IGRT等新治疗技术,精确放射治疗的目标正在逐渐被推广。由于大部分鼻咽癌患者在发现时已经是局部晚期或者晚期,需要接受以放疗为主的综合治疗。并且恶性肿瘤本身是涉及全身性的疾病,配合放疗的全身治疗,如化疗、靶向治疗以及生物治疗,在鼻咽癌的综合治疗方面一样占据着重要地位。目前以细胞毒药物为主的化疗仍旧在晚期鼻咽癌的综合治疗中占据重要地位。但细胞毒药物具有副反应较重,患者耐受能力有限的缺陷,所以现阶段有人把研究热点逐渐转向靶向治疗以及生物治疗。由于鼻咽癌细胞表面EGFR高表达,所以针对阻断EGFR通路的靶向药物的研究为鼻咽癌药物治疗的研究热点,目前已经上市并投入使用的西妥昔单抗正是以EGFR通路的阻断为作用机制,并推广应用于临床中晚期患者的成熟药物。铜绿假单胞菌毛制剂(PA-MSHA)是铜绿假单胞菌的减毒制剂。它是自然界的铜绿假单胞菌(PA)在经过基因工程重组处理后,保留了高度的甘露糖亲和力以及免疫源性,而毒性明显下降的生物制剂。在国内临床应用已经比较广泛,并且也观察到明显的抗肿瘤疗效；基础研究方面认为其能够与EGFR表面的甘露糖结合而阻滞EGFR通路,同时激活免疫系统,兼有免疫调节功能,最终抑制靶细胞生长。所以该药物同时具有抑制EGFR通路以及免疫调节增强的功能。由于其价格较低,性价比较高,预计在临床应用方面前景比较广阔。本实验用体外实验的方法,研究铜绿假单胞菌毛制剂(PA-MSHA)对鼻咽癌细胞株5-8F以及6-10B的生长抑制作用,并且初步探讨其作用机制,为鼻咽癌的综合治疗新药选择提供依据。二、实验方法 1.体外培养人鼻咽癌细胞株5-8F及6-10B,分为空白对照组及不同药物浓度处理组。不同浓度药物处理组的PA-MSHA浓度分别选择为：0.02×108/mh0.05×108/ml、0.1×108/ml、0.2×108/ml、0.5×108/ml、1×108/ml、2×108/ml、5×108/ml。处理时间1-7d。 2.采用MTT法测定PA-MSHA对鼻咽癌5-8F以及6-10B的生长抑制作用。上述不同浓度的梯度的PA-MSHA分别与两组细胞株作用48h后,测定其吸光值,计算抑制率(IR),并绘制浓度-抑制率相关曲线。根据曲线分别计算半数抑制浓度(48hIC50)。分别根据两组细胞的IC50选择适宜的药物浓度(1×108/ml和2×108/ml)再次与两组细胞株相互作用1-7d,每天测定相应时间点的吸光值,绘制生长曲线,分析细胞增殖抑制情况与药物浓度及作用时间的关系。 3.采用流式细胞技术分析经过PA-MSHA处理后的鼻咽癌5-8F的细胞周期分布情况。以1×108/ml浓度的PA-MSHA作用鼻咽癌5-8F细胞株24h,PI染色,上流式细胞仪检测细胞周期分布情况,分析PI荧光直方图上细胞各周期的百分率与空白对照组的区别。 4. Westerm blot法检测经过不同浓度药物处理24h以及48h后的鼻咽癌5-8F以及6-10B的周期、凋亡相关蛋白级联水平变化。 5. SPSS16.0统计软件,比较不同处理组细胞生长曲线采用析因分析的方法,不同处理组的细胞周期Go/G1比率用两独立样本t检验方法,P0.05有统计学意义。三、实验结果 1.细胞培养可见空白对照组鼻咽癌细胞呈短梭形,部分为类圆形,轮廓清晰,核仁明显,胞浆少。细胞网格状排列均匀,无胞突,饱满明亮,生长旺盛,一般在接种12小时后,能够适应新的生存环境,生长逐渐旺盛,并贴壁良好。两组细胞48小时IC50分别为(1.62+0.31)×108/ml和(1.95+0.35)×108/ml。在细胞培养的7天时间里,每天对两组细胞株的生长情况进行比较分析,可以发现PA-MSHA对鼻咽癌5-8F以及6-10B细胞的生长抑制作用呈剂量依赖性。相同药物浓度组比较亦可发现发现PA-MSHA对鼻咽癌5-8F以及6-10B细胞的生长抑制作用呈时间依赖性。综合分析发现：铜绿假单胞菌毛制剂(PA-MSHA)明显抑制鼻咽癌细胞株5-8F的增殖,其增殖能力随时间的增加出现显著下降(F=665.403,P0.001),并且该细胞株的增殖能力随PA-MSHA浓度梯度的增加而显著下降(F=151.607,P0.001)。同样,PA-MSHA能够明显抑制鼻咽癌6-10B细胞株的增殖能力,其增殖能力随时间的增加出现显著下降(F=597.422,P0.001)；并且该细胞株的增殖能力随PA-MSHA浓度梯度的增加而显著下降(F=74.481,P0.001)。 2.经过PA-MSHA处理后的鼻咽癌细胞株5-8F的细胞周期发生再分布。与对照组比较,药物处理后的细胞株细胞Go/G1期比例增高,差异具有统计学意义(t=5.150,P=0.007),而其S期比例显著降低(t=-6.820,P=0.002)。 3. PA-MSHA处理后的两组细胞的周期相关蛋白cyclinD1、CDK4、CDK6表达下调,凋亡促进相关蛋白Bax表达上调,凋亡抑制蛋白Bcl-2的表达下调。并且在部分蛋白表达上体现出剂量-时间依赖的特点。结论 1. PA-MSHA对鼻咽癌细胞生长有显著的抑制作用,并且其抑制的效应呈时间-浓度依赖性。 2. PA-MSHA抑制鼻咽癌细胞增殖可能是通过抑制肿瘤细胞G1向S期转化以及诱导凋亡的发生而实现的。 3. PA-MSHA可以考虑作为鼻咽癌综合治疗的辅助用药,具有一定的临床应用价值。
[Abstract]:First, the purpose of the study
Nasopharyngeal carcinoma is the most common malignant tumor of the head and neck of the Guangdong and Guangxi region of our country, the incidence of EBV infection and genetic susceptibility, environmental and dietary habits and immune dysfunction. The disease seriously affects people's health in China. How to improve the technique for nasopharyngeal carcinoma and the method of improving the curative rate and local control rate. Has its practical significance to improve the level of people's health.
The treatment of nasopharyngeal carcinoma is generally considered to be a comprehensive therapy mainly based on radiotherapy or radiotherapy. Early radiotherapy for nasopharyngeal carcinoma can achieve clinical cure. Advanced patients need radiotherapy combined with radiotherapy.
In the treatment of nasopharyngeal carcinoma radiotherapy, radiotherapy dose has been basically constant at about 70Gy. How to ensure the lesion area dose at the same time, reduce the surrounding normal tissue dose, radiation therapy is currently improving at the technical level of the direction. The purpose is to ensure that the local control rate is lower, reduce the complications such as dry mouth and trismus the occurrence, improve the long-term quality of life of patients. At present there have been IMRT, IGRT and other new treatment technology, precise radiotherapy target is being extended.
Because most patients with nasopharyngeal carcinoma has been found in locally advanced or late, need to receive comprehensive treatment including radiotherapy. And the malignant tumor is related to systemic diseases, such as systemic treatment with radiotherapy, chemotherapy, targeted therapy and biological treatment, in the comprehensive treatment of nasopharyngeal carcinoma as occupies an important position to occupy an important. The current status of cytotoxic drug based chemotherapy is still in the comprehensive treatment of advanced nasopharyngeal carcinoma. But the cytotoxic drugs have severe side effects, defect tolerance of patients with limited stage, so the research focuses are increasingly turning to targeted therapy and biological therapy. Because of the high expression of nasopharyngeal carcinoma cell surface EGFR, so for blocking EGFR pathway targeted drug research as the research hotspot of nasopharyngeal carcinoma drug therapy, is already on the market and put into use of cetuximab is based on the EG The blocking of the FR pathway is the mechanism of action and is applied to the mature drugs used in the patients in the middle and late clinical stages.
Piliated Pseudomonas aeruginosa (PA-MSHA) is attenuated preparations of Pseudomonas aeruginosa. It is Pseudomonas aeruginosa in nature (PA) after treatment of recombinant gene engineering, retain a high degree of mannose affinity and immunogenicity, and toxicity of biological agents decreased significantly. In domestic clinical applications have been more widely and also observed obvious anti-tumor effect; basic research it can with the mannose binding surface of EGFR and block EGFR pathway and activate the immune system with immune function, inhibit cell growth. So the final target of the drug and inhibition of the EGFR pathway and the immune enhancement function. Because of its low price. The higher price, is expected in clinical application prospect is broad.
In this study, we studied the inhibitory effect of Pseudomonas aeruginosa preparation (PA-MSHA) on nasopharyngeal carcinoma cell line 5-8F and 6-10B by in vitro experiment, and preliminarily explored the mechanism of its action, so as to provide evidence for the comprehensive treatment of nasopharyngeal carcinoma.
Two, experimental method
Human nasopharyngeal carcinoma cell line 5-8F and 6-10B cultured in vitro for 1., divided into blank control group and different concentrations of PA-MSHA in different concentration groups. The treatment group were selected: 0.02 * 108/mh0.05 * 108/ml, 0.1 * 108/ml, 0.2 * 108/ml, 0.5 * 108/ml, 1 * 108/ml, 2 * 108/ml, 5 * 108/ml. the processing time of 1-7d.
2. the inhibitory effect of PA-MSHA on the growth of 5-8F and 6-10B in nasopharyngeal carcinoma was measured by MTT.
The PA-MSHA of the above concentration gradient was respectively treated with 48h and two groups of cells. The absorbance value was calculated, the inhibition rate (IR) was calculated, and the concentration inhibition rate correlation curve was drawn. The half inhibitory concentration (48hIC50) was calculated according to the curve.
According to the concentration of cells in two groups of IC50 to choose the appropriate (1 * 108/ml and 2 * 108/ml) again with two group cells interaction 1-7d, measured every day at the same time the light absorption value, growth curve, analysis of the relationship between inhibition and drug concentration and action time of cell proliferation.
3. the cell cycle distribution of nasopharyngeal carcinoma (NPC) 5-8F after PA-MSHA treatment was analyzed by flow cytometry.
The cell cycle distribution of nasopharyngeal carcinoma 5-8F cell line 24h was detected by PI at 1 * 108/ml concentration of PA-MSHA, and the cell cycle distribution was detected by upflow cytometry. The difference between the percentage of cell cycle in PI histogram and blank control group was analyzed.
4. Westerm blot method was used to detect the cycle of 5-8F and 6-10B in nasopharyngeal carcinoma after different concentrations of drugs treated with 24h and 48h, and the cascade level of apoptosis related protein was changed.
5. SPSS16.0 statistical software, compared the growth curve of different treatment groups by factorial analysis. The cell cycle Go/G1 ratio of different treatment groups was two independent sample t test, P0.05 had statistical significance.
Three, experimental results
1. cell culture visible blank control group of nasopharyngeal carcinoma cells showed short fusiform shape, is round, clear outline, obvious nucleolus, cytoplasm. Cell grid arranged uniformly and no cytoplasmic processes, full of bright, vigorous growth, generally in 12 hours after inoculation, can survive in the new environment to grow gradually strong, and good for 48 hours. The adherent cells in two groups respectively (IC50 1.62+0.31) and 108/ml * (1.95+0.35) * 108/ml. in cell culture for 7 days, every day on the growth of two groups of cell lines were compared, can be found in PA-MSHA and 6-10B on nasopharyngeal carcinoma 5-8F cell growth inhibition in a dose-dependent manner. The same drug concentration group also found PA-MSHA on nasopharyngeal carcinoma 5-8F and 6-10B cell growth inhibition was time dependent. Comprehensive analysis shows that Pseudomonas aeruginosa pilus preparations (PA-MSHA) inhibition of nasopharyngeal carcinoma cell line 5-8F The proliferation, increase its proliferation ability with time decreased significantly (F=665.403, P0.001), and the cell proliferation ability with PA-MSHA concentration gradient decreased significantly (F=151.607, P0.001). Similarly, PA-MSHA can inhibit the growth of nasopharyngeal carcinoma cell line 6-10B proliferation, increase the proliferation ability with time significantly decreased (F=597.422, P0.001); and the cell proliferation ability with PA-MSHA concentration gradient decreased significantly (F=74.481, P0.001).
2. the cell cycle of the nasopharyngeal carcinoma cell line, 5-8F, was redistributed after PA-MSHA treatment.
Compared with the control group, the proportion of Go/G1 phase in cells treated with drugs increased, the difference was statistically significant (t=5.150, P=0.007), and the proportion of S phase was significantly decreased (t=-6.820, P=0.002).
After 3. PA-MSHA, the expressions of cyclinD1, CDK4 and CDK6 were down regulated in the two groups of cells. The expression of Bax was up-regulated by apoptosis, and the expression of Bcl-2 was down regulated.
conclusion
1. PA-MSHA has a significant inhibitory effect on the growth of nasopharyngeal carcinoma cells, and its inhibitory effect is time dependent.
2. PA-MSHA inhibition of nasopharyngeal carcinoma cell proliferation may be achieved by inhibiting the transformation of tumor cells G1 into S phase and inducing the occurrence of apoptosis.
3. PA-MSHA can be considered as a supplementary medication for the comprehensive treatment of nasopharyngeal carcinoma and has a certain clinical value.

【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

【参考文献】