本文關(guān)鍵詞： 銅綠假單胞菌 鼻咽癌 增殖 細(xì)胞周期 凋亡　出處：《南方醫(yī)科大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：一、研究目的 鼻咽癌是我國(guó)兩廣地區(qū)最常見(jiàn)的頭頸部惡性腫瘤,其發(fā)病可能與遺傳易感性、EBV感染、環(huán)境與飲食習(xí)慣以及免疫功能缺陷相關(guān)。該病嚴(yán)重影響著我國(guó)人民的健康。如何改進(jìn)鼻咽癌的治療技術(shù)以及方法,提高疾病治愈率以及局部控制率,對(duì)提升人民的健康水平有著其實(shí)際的意義。 鼻咽癌的治療目前公認(rèn)為是以放射治療或者以放射治療為主的綜合治療。早期鼻咽癌患者單純放療即能達(dá)到臨床治愈,晚期患者需要選擇以放射治療為主的綜合治療。 在放射治療方面,鼻咽癌的放療劑量已經(jīng)基本恒定在70Gy左右。如何在保證病灶區(qū)域劑量的同時(shí),減少周邊正常組織的受量,是目前放射治療在技術(shù)層面改進(jìn)的方向。目的是在保證局部控制率不降低的情況下,減少口干以及張口困難等并發(fā)癥的發(fā)生,提高患者的遠(yuǎn)期生活質(zhì)量�，F(xiàn)階段已經(jīng)出現(xiàn)IMRT, IGRT等新治療技術(shù),精確放射治療的目標(biāo)正在逐漸被推廣。 由于大部分鼻咽癌患者在發(fā)現(xiàn)時(shí)已經(jīng)是局部晚期或者晚期,需要接受以放療為主的綜合治療。并且惡性腫瘤本身是涉及全身性的疾病,配合放療的全身治療,如化療、靶向治療以及生物治療,在鼻咽癌的綜合治療方面一樣占據(jù)著重要地位。目前以細(xì)胞毒藥物為主的化療仍舊在晚期鼻咽癌的綜合治療中占據(jù)重要地位。但細(xì)胞毒藥物具有副反應(yīng)較重,患者耐受能力有限的缺陷,所以現(xiàn)階段有人把研究熱點(diǎn)逐漸轉(zhuǎn)向靶向治療以及生物治療。由于鼻咽癌細(xì)胞表面EGFR高表達(dá),所以針對(duì)阻斷EGFR通路的靶向藥物的研究為鼻咽癌藥物治療的研究熱點(diǎn),目前已經(jīng)上市并投入使用的西妥昔單抗正是以EGFR通路的阻斷為作用機(jī)制,并推廣應(yīng)用于臨床中晚期患者的成熟藥物。 銅綠假單胞菌毛制劑(PA-MSHA)是銅綠假單胞菌的減毒制劑。它是自然界的銅綠假單胞菌(PA)在經(jīng)過(guò)基因工程重組處理后,保留了高度的甘露糖親和力以及免疫源性,而毒性明顯下降的生物制劑。在國(guó)內(nèi)臨床應(yīng)用已經(jīng)比較廣泛,并且也觀察到明顯的抗腫瘤療效；基礎(chǔ)研究方面認(rèn)為其能夠與EGFR表面的甘露糖結(jié)合而阻滯EGFR通路,同時(shí)激活免疫系統(tǒng),兼有免疫調(diào)節(jié)功能,最終抑制靶細(xì)胞生長(zhǎng)。所以該藥物同時(shí)具有抑制EGFR通路以及免疫調(diào)節(jié)增強(qiáng)的功能。由于其價(jià)格較低,性價(jià)比較高,預(yù)計(jì)在臨床應(yīng)用方面前景比較廣闊。 本實(shí)驗(yàn)用體外實(shí)驗(yàn)的方法,研究銅綠假單胞菌毛制劑(PA-MSHA)對(duì)鼻咽癌細(xì)胞株5-8F以及6-10B的生長(zhǎng)抑制作用,并且初步探討其作用機(jī)制,為鼻咽癌的綜合治療新藥選擇提供依據(jù)。 二、實(shí)驗(yàn)方法 1.體外培養(yǎng)人鼻咽癌細(xì)胞株5-8F及6-10B,分為空白對(duì)照組及不同藥物濃度處理組。不同濃度藥物處理組的PA-MSHA濃度分別選擇為：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。處理時(shí)間1-7d。 2.采用MTT法測(cè)定PA-MSHA對(duì)鼻咽癌5-8F以及6-10B的生長(zhǎng)抑制作用。 上述不同濃度的梯度的PA-MSHA分別與兩組細(xì)胞株作用48h后,測(cè)定其吸光值,計(jì)算抑制率(IR),并繪制濃度-抑制率相關(guān)曲線。根據(jù)曲線分別計(jì)算半數(shù)抑制濃度(48hIC50)。 分別根據(jù)兩組細(xì)胞的IC50選擇適宜的藥物濃度(1×108/ml和2×108/ml)再次與兩組細(xì)胞株相互作用1-7d,每天測(cè)定相應(yīng)時(shí)間點(diǎn)的吸光值,繪制生長(zhǎng)曲線,分析細(xì)胞增殖抑制情況與藥物濃度及作用時(shí)間的關(guān)系。 3.采用流式細(xì)胞技術(shù)分析經(jīng)過(guò)PA-MSHA處理后的鼻咽癌5-8F的細(xì)胞周期分布情況。 以1×108/ml濃度的PA-MSHA作用鼻咽癌5-8F細(xì)胞株24h,PI染色,上流式細(xì)胞儀檢測(cè)細(xì)胞周期分布情況,分析PI熒光直方圖上細(xì)胞各周期的百分率與空白對(duì)照組的區(qū)別。 4. Westerm blot法檢測(cè)經(jīng)過(guò)不同濃度藥物處理24h以及48h后的鼻咽癌5-8F以及6-10B的周期、凋亡相關(guān)蛋白級(jí)聯(lián)水平變化。 5. SPSS16.0統(tǒng)計(jì)軟件,比較不同處理組細(xì)胞生長(zhǎng)曲線采用析因分析的方法,不同處理組的細(xì)胞周期Go/G1比率用兩獨(dú)立樣本t檢驗(yàn)方法,P0.05有統(tǒng)計(jì)學(xué)意義。 三、實(shí)驗(yàn)結(jié)果 1.細(xì)胞培養(yǎng)可見(jiàn)空白對(duì)照組鼻咽癌細(xì)胞呈短梭形,部分為類圓形,輪廓清晰,核仁明顯,胞漿少。細(xì)胞網(wǎng)格狀排列均勻,無(wú)胞突,飽滿明亮,生長(zhǎng)旺盛,一般在接種12小時(shí)后,能夠適應(yīng)新的生存環(huán)境,生長(zhǎng)逐漸旺盛,并貼壁良好。兩組細(xì)胞48小時(shí)IC50分別為(1.62+0.31)×108/ml和(1.95+0.35)×108/ml。在細(xì)胞培養(yǎng)的7天時(shí)間里,每天對(duì)兩組細(xì)胞株的生長(zhǎng)情況進(jìn)行比較分析,可以發(fā)現(xiàn)PA-MSHA對(duì)鼻咽癌5-8F以及6-10B細(xì)胞的生長(zhǎng)抑制作用呈劑量依賴性。相同藥物濃度組比較亦可發(fā)現(xiàn)發(fā)現(xiàn)PA-MSHA對(duì)鼻咽癌5-8F以及6-10B細(xì)胞的生長(zhǎng)抑制作用呈時(shí)間依賴性。綜合分析發(fā)現(xiàn)：銅綠假單胞菌毛制劑(PA-MSHA)明顯抑制鼻咽癌細(xì)胞株5-8F的增殖,其增殖能力隨時(shí)間的增加出現(xiàn)顯著下降(F=665.403,P0.001),并且該細(xì)胞株的增殖能力隨PA-MSHA濃度梯度的增加而顯著下降(F=151.607,P0.001)。同樣,PA-MSHA能夠明顯抑制鼻咽癌6-10B細(xì)胞株的增殖能力,其增殖能力隨時(shí)間的增加出現(xiàn)顯著下降(F=597.422,P0.001)；并且該細(xì)胞株的增殖能力隨PA-MSHA濃度梯度的增加而顯著下降(F=74.481,P0.001)。 2.經(jīng)過(guò)PA-MSHA處理后的鼻咽癌細(xì)胞株5-8F的細(xì)胞周期發(fā)生再分布。 與對(duì)照組比較,藥物處理后的細(xì)胞株細(xì)胞Go/G1期比例增高,差異具有統(tǒng)計(jì)學(xué)意義(t=5.150,P=0.007),而其S期比例顯著降低(t=-6.820,P=0.002)。 3. PA-MSHA處理后的兩組細(xì)胞的周期相關(guān)蛋白cyclinD1、CDK4、CDK6表達(dá)下調(diào),凋亡促進(jìn)相關(guān)蛋白Bax表達(dá)上調(diào),凋亡抑制蛋白Bcl-2的表達(dá)下調(diào)。并且在部分蛋白表達(dá)上體現(xiàn)出劑量-時(shí)間依賴的特點(diǎn)。 結(jié)論 1. PA-MSHA對(duì)鼻咽癌細(xì)胞生長(zhǎng)有顯著的抑制作用,并且其抑制的效應(yīng)呈時(shí)間-濃度依賴性。 2. PA-MSHA抑制鼻咽癌細(xì)胞增殖可能是通過(guò)抑制腫瘤細(xì)胞G1向S期轉(zhuǎn)化以及誘導(dǎo)凋亡的發(fā)生而實(shí)現(xiàn)的。 3. PA-MSHA可以考慮作為鼻咽癌綜合治療的輔助用藥,具有一定的臨床應(yīng)用價(jià)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.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R739.63

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳衛(wèi)東;唐中華;徐峰;;TAC方案聯(lián)合使用綠膿桿菌制劑在乳腺癌新輔助化療中的應(yīng)用研究[J];南方醫(yī)科大學(xué)學(xué)報(bào);2009年06期

2 李志平,郝德治,李麗云,周喜榮,任莉,楊玉瓊,李平,秦紅,羅德云,張洪,周清華,徐泳;PA-MSHA菌苗輔助治療肺癌臨床研究[J];肺癌雜志;1999年01期

3 謝玉權(quán);鄭朝旭;馮強(qiáng);袁興華;趙玫;黃常志;;銅綠假單胞菌制劑胃癌細(xì)胞抑制作用研究[J];中外醫(yī)療;2009年03期

4 孫杰;馬鋼;李俊彥;劉俊;高鴻;宋曉斌;;銅綠假單胞菌制劑對(duì)大鼠C6膠質(zhì)瘤細(xì)胞作用的實(shí)驗(yàn)研究[J];昆明醫(yī)學(xué)院學(xué)報(bào);2010年10期

5 張艷;梁耕田;孫廣濱;潘兆虎;吳國(guó)民;劉爭(zhēng);;鼻咽癌中表皮生長(zhǎng)因子受體、C-erbB-2表達(dá)及其與細(xì)胞增殖的關(guān)系[J];臨床耳鼻咽喉頭頸外科雜志;2011年02期

6 凌偉;劉驊;曹暉;郁豐榮;徐佳;;銅綠假單胞菌注射液對(duì)胃癌細(xì)胞體外殺傷作用的觀察[J];中華腫瘤防治雜志;2008年18期

7 郟琴;孫依萍;蔡蓉;王春燕;俞靜;;綠膿桿菌制劑輔助治療肺癌臨床研究[J];實(shí)用醫(yī)學(xué)雜志;2007年16期

8 黃崇標(biāo);崔焱;辛亮;楊彥卓;齊大亮;;銅綠假單胞菌聯(lián)合滑石粉治療惡性胸腔積液臨床觀察[J];天津醫(yī)藥;2011年09期

9 孫文平,牟希亞,高小平,陳艷君,譚梅;銅綠假單胞菌菌苗對(duì)尿路感染的免疫功能調(diào)節(jié)[J];微生物學(xué)雜志;2002年04期

10 劉懷民;楊峰;劉曉莉;唐靜雯;;PA-MSHA輔助治療晚期胃癌對(duì)外周血T細(xì)胞及NK細(xì)胞影響的研究[J];實(shí)用臨床醫(yī)藥雜志;2011年11期

，

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