表皮生长因子受体对鼻咽癌细胞放射敏感性的影响
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摘要
鼻咽癌(Nasopharyngeal carcinoma，NPC)高发于我国南方及东南亚地区。虽然鼻咽癌的研究有了极大的发展，其5年生存率仍徘徊于50-60％。
放射治疗是鼻咽癌最主要的治疗方法。鼻咽癌患者治疗失败原因主要为局部复发或远处转移，严重影响鼻咽癌治疗生存率的提高。放射治疗是一个局部或区域治疗的手段，提高放射治疗的疗效不止是提高了局部或区域控制率，同时也影响生存及远地转移。如何提高放射治疗的疗效，如何提高放射敏感性一直困扰着肿瘤医师。
肿瘤的放射敏感性是一个十分复杂的问题，影响因素是多方面的。肿瘤的放射敏感性与肿瘤组织的来源、分化程度、病理类型、瘤床、贫血、局部是否合并感染、生活指数等有关。以往放射治疗效价的修饰措施有如下几个方面：
1．乏氧细胞放射增敏剂：目前研究的药物有美国的SR-2058和日本的AK-2133及其衍生物KU系列，但两者均属于硝基类化合物，仍无法回避限制剂量毒性的问题；2．肿瘤细胞放射增敏剂：其中以卤素类药物为主，但目前尚未在临床广泛使用；3．通过吸入不同浓度的氧来改变肿瘤氧含量的措施；4．放化疗的联合应用，正常组织的反应也有可能有所增加：5．放射治疗联合热疗、光动力学疗法等。
表皮生长因子受体(Eepidermal Growth Factor Receptor，EGFR)是一种受体酪氨酸激酶(receptor tyrosine kinase，RTK)，1982年由Cohen等首次发现。细胞膜上具有酪氨酸激酶活性(tyrosine kinase activity)的跨膜蛋白受体在细胞内信号转导中起着较为重要的作用。该激酶活性启动了一系列导致细胞增殖和分化的信号级联反应的发生，而表皮生长因子受体家族是涉及细胞增殖和肿瘤发生、发展中的极为重要的受体信号系统。许多癌基因能够编码生长因子受体或生长因子样受体蛋白，在人类的大部分上皮源性肿瘤的发展中起重要作用。表皮生长因子受体家族在肿瘤中的表达或过度表达往往引起肿瘤放、化疗敏感性的变化，而通过分子靶向治疗、基因等技术抑制其表达又可以抑制肿瘤细胞的生长，改变其对放射治疗、化学治疗、激素治疗的敏感性。表皮生长因子受体家族包括四个成员：EGFR、HER-2／P185erbB2、HER-4／P180erbB4和HER-3／P160erbB3。
EGFR(HER1，erbB1)基因定位于人染色体7q21上，编码一个分子量为170×10~3的具有酪氨酸激酶活性的受体糖蛋白。它通常与其相应的配体(EGF、TGF-α)相结合，使受体自身磷酸化，从而引起胞内一系列信号的级联放大及传递，最终影响核内基因的表达，导致细胞的增殖与分化。EGFR广泛分布于正常的哺乳动物上皮细胞表面，平均每个细胞受体个数为5×10~4-10×10~4。目前发现的EGFR配体共有6种，分别是表皮生长因子(EGF)、转化生长因子-α(TGF-α)、双向调节因子(amphiregulin)、Bctacellulin(BTC)、肝素结合表皮生长因子(heparin-binding EGF，HB-EGF)和表皮调节因子(epiregulin，EPR)，其中前三种仅与EGFR结合，后三种为EGFR与erbB家族其它受体的共同配体。EGFR活化后可激活许多的下游信号转导通路。继Hendler等首次检测出非小细胞肺癌细胞上EGFR有超表达以后，人们相继在多种实体瘤如乳腺癌、结直肠癌、头颈肿瘤、脑瘤、卵巢癌、膀胱癌、肾癌中均检到EGFR的超表达或扩增，EGFR高表达是肿瘤预后不良和对放射抗拒的重要指标。EGFR超表达还与肿瘤的侵袭和恶性转移有关。此外，EGFR还对细胞骨架的重构、移动、粘附以及蛋白酶活性有作用。
抑制EGFR为抗癌领域提供了一个理想的靶向抗癌疗法。除了分子靶向治疗，RNA干扰(RNAi，RNA interference)作为一种实用的分子生物学技术近年来发展非常迅速。该技术可高效特异性的抑制基因表达。在实用性上它比以前的基因抑制技术有更多的优点，siRNA介导的瞬时或稳定的基因沉默有高度的特异性并且无副作用。国内外尚未见应用RNAi抑制鼻咽癌细胞表皮生长因子受体表达后放射敏感性变化的相关报道。
我们的实验包括以下几个部分：
第一章鼻咽癌CNE1、CNE2细胞株表皮生长因子受体家族的表达
目的：检测鼻咽癌CNE1、CNE2细胞株EGFR、HER-2、HER-3、HER-4的表达情况。方法：1．免疫组化、Western blot检测鼻咽癌CNE1、CNE2细胞株EGFR、HER-2、HER-3、HER-4的蛋白表达情况；RT-PCR检测EGFRmRNA的表达情况；2．进行克隆形成实验，为进行细胞的照射作准备。结果：1．免疫组化、Westernblot检测发现CNE1、CNE2细胞株EGFR蛋白阳性表达，HER-2、HER-3、HER-4蛋白阴性表达；2．RT-PCR检测发现CNE1、CNE2细胞株EGFRmRNA阳性表达，HER-2、HER-3、HER-4阴性表达；3．X射线照射后12天，2Gy可达到约50％的细胞存活率，4Gy存活率CNE1和CNE2细胞分别为22.93％和3.67％。结论：CNE1、CNE2细胞株EGFR阳性表达，HER-2、HER-3、HER-4阴性表达。
第二章EGFRSiRNA表达载体的建立、转染和鉴定
目的：建立EGFRSiRNA表达载体，完成转染和鉴定；并检测转染后EGFR的表达。方法：除转染EGFR干扰质粒外，还转染空白质粒和无义质粒作为对照；采用DNA测序；实时荧光定量PCR检测转染前后及转染不同质粒后EGFRmRNA的表达；Western blot检测转染不同质粒后EGFR蛋白的表达。结果：1．成功构建了EGFR干扰质粒。2．实时荧光定量PCR检测CNE1、CNE2细胞EGFRmRNA的表达：转染EGFR干扰质粒组与转染无义质粒组及转染空载体组比较EGFRmRNA的表达降低。统计学分析：实时荧光定量RT-PCR检测CNE1细胞EGFRmRNA的表达：CNE1细胞转染EGFR干扰质粒组与转染无义质粒组比较有统计学差异(P=0.000)；转染EGFR干扰质粒组与转染空载体组比较也有统计学差异(P=0.000)；转染EGFR干扰质粒组：转染后0、12、24、48、60、72小时EGFR基因拷贝逐渐减少，转染前(即0小时)与转染后48、60、72小时比较有统计学差异(P=0.048、0.011、0.013)。实时荧光定量RT-PCR检测CNE2细胞EGFRmRNA的表达：CNE2细胞转染EGFR干扰质粒组与转染无义质粒组比较有统计学差异(P=0.000)；转染EGFR干扰质粒组与转染空载体组比较也有统计学差异(P=0.000)；转染EGFR干扰质粒组：转染后0、12、24、48、60、72小时EGFR基因拷贝逐渐减少，转染前(即0小时)与转染后12、24、48、60、72小时比较均有统计学差异(P=0.004、0.003、0.000、0.000、0.000)。3．Western blot检测蛋白表达情况：CNE1、CNE2细胞转染EGFR干扰质粒后与转染β-actin、空载体、无义质粒组比较，EGFR蛋白的表达减少。结论：1．成功建立EGFRSiRNA表达质粒并进行转染；2．CNE1、CNE2细胞转染EGFR干扰质粒后EGFRmRNA的表达降低；3．CNE1、CNE2细胞转染EGFR干扰质粒后EGFR蛋白的表达减少。
第三章EGFR干扰后CNE1、CNE2细胞放射敏感性的变化
目的：研究RNA干扰后放射敏感性的变化。方法：1．应用X射线照射转染及未转染EGFRSiRNA的细胞，TUNEL、DNA LADDER、电镜、DAPI等观察细胞凋亡。2．Annexin V／PI双标记法检测凋亡及坏死，并进行转染干扰质粒前后照射后凋亡及坏死率的比较。3．MTT实验比较转染干扰质粒前后照射后细胞增殖变化。结果：1．DAPI染色，TUNEL法，电子显微镜，凝胶电泳DNA LADDER均观察到ONE1和ONE2细胞照射后或转染后照射后的凋亡。
2．Annexin V／PI双标记法检测：ONE1、ONE2干扰后照射较干扰前照射凋亡及坏死均增加；凋亡所占比例高过坏死。统计学分析：CNE1干扰前照射组与干扰后照射组两组间的凋亡率比较有显著性差异(P=0.008)，照射前及照射后各时间点的凋亡率之间均有显著性差异(P＜0.05)，CNE1干扰前照射组与干扰后照射组两组间的坏死率比较无显著性差异(P=0.082)，照射前及照射后各时间点的坏死率之间均有显著性差异(P＜0.05)；CNE2干扰前照射组与干扰后照射组两组间的凋亡率比较有显著性差异(P=0.020)，照射前及照射后各时间点的凋亡率之间均有显著性差异(P＜0.05)，CNE2干扰前照射组与干扰后照射组两组间的坏死率比较有显著性差异(P=0.005)，除干扰前照射后24h与48h之间无显著性差异(P=0.170)外，照射前及照射后各时间点的坏死率之间均有显著性差异(P＜0.05)。3．应用MTT实验检测：CNE1、CNE2细胞干扰后照射较干扰前照射肿瘤增殖降低。统计学分析：CNE1细胞干扰前照射组与干扰后照射组之间有显著性差异(P＜0.001)。干扰前：照射前与照射后30、36、48、60、84、96小时比较有显著性差异(P=0.037、0.002、0.017、0.003、0.006、0.013)。干扰后：照射前与照射后24、36、48、60、72、84、96小时比较有显著性差异(P=0.037、0.037、0.002、0.001、0.001、0.001、0.003)。CNE2细胞干扰前照射组与干扰后照射组之间有显著性差异(P＜0.001)。干扰前：照射前与照射后30、48、60、84、96小时比较有显著性差异(P=0.002、0.031、0.005、0.010、0.033)。干扰后：照射前与照射后36、48、60、72、84、96小时比较有显著性差异(P=0.047、0.002、0.001、0.001、0.001、0.003)。结论：1．应用DAPI染色，TUNEL法，电子显微镜，DNA ladder均观察到CNE1、CNE2细胞X射线照射后细胞凋亡。2．Annexin V／PI双标记法检测：CNE1、CNE2细胞干扰后行X射线照射较干扰前行X射线照射凋亡及坏死均增加；凋亡所占比例高过坏死。应用RNA干扰技术抑制EGFR表达后进行照射，促进了肿瘤细胞的凋亡及坏死，凋亡占主导作用。3．MTT实验检测：CNE1、CNE2细胞干扰后行X射线照射较干扰前行X射线照射细胞增殖降低，应用RNA干扰技术抑制EGFR表达后进行照射，抑制肿瘤细胞增殖。应用RNA干扰技术抑制EGFR表达后提高了鼻咽癌CNE1、CNE2细胞放射敏感性。
Nasopharyngeal carcinoma (NPC) is an endemic cancer with high incidence inSoutheast Asia and Southern china. Although great progress in the investigation ofNPC, such as radiotherapy and imaging diagnosis and so on, have been made in thelate 90s,the cure rate of NPC has not been improved yet and 5 year survival rate stillremained around 50-60％.
Radiotherapy is the most important treatment method for NPC. Treatmentfailure is mainly because of local relapse and metastasis, which decreases thesurvival rate obviously. Radiotherapy is a regional treatment.A better local curativeeffect of radiotherapy not only increases local contral rate, but also affects survivalrate and metastasis. Oncologists have been investigating the approach to increase thecurative effect of radiotherapy and radiation sensitivity.
The radiosensitivity of tumor is a very complicated problem and the impactfactor involves in many aspects. It is decided by organize source, differentiationdegree, pathological type and the bed of tumor, anemia and partial infection, lifeindex…etc. The messures to modify the radiotherapy effects before are as follows: 1.anoxic cell radiosensitizer. 2. radiosensitizer of tumor cell, halogen as principleamong them, but are't used widely. 3. to change oxygen content of tumor by inhaling different density of oxygen 4.radiotherapy combined with chemotherapy,but thismight increase the side effects of normal tissue 5.radiotherapy combined with othermeasures such as thermotherapy,photodynamic therapy and so on.
Epidermal growth factor receptor (EGFR) is a sort of receptor tyrosine kinase(RTK). It was first discovered by Cohen and his colleagues in 1982. Thetransmembrane protein receptor with tyrosine kinase activity in cellular membraneplays an important role in cell signal transduction.It breakdowns a series of signalcascade reactions that induce cell proliferation and differentiation. Epidermal growthfactor receptor family is a very important receptor signal systerm which involves inthe cell proliferation and tumorigenesis. Many oncogenes can encode EGFR or thesimilar protein that lead an important function in the growth of most of tumororiginated from epithelium tissue of mankind. The overexpression of EGFR familyalways causes the sensitivity changes of radiotherapy and chemotherapy of tumor.We can restrain the growth of tumor and change the sensitivity of radiotherapy,chemotherapy and hormone therapy through inhibiting the expression of EGFR bymolecular targeted therapy or gene technology. Epidermal growth factor receptorfamily includes four members: EGFR、HER-2/P185erbB2、HER-4/P180erbB4 andHER-3/P160erbB3.
EGFR (HER1,erbB1) gene,localizing in chromosome 7q21, encodes aglycoprotein receptor with tyrosine kinase activity. The phosphorylation of itself afterintegrating with corresponding ligand results in a trial of signal cascade amplificationand transmitting. At last, it affects the expression of intranuclear genes and inducescell proliferation and differentiation. EGFR are widely distributed on the surface ofendothelial cell of normal mammal, 5×10~4～10×10~4 receptors in every cell average.There are six kind of ligands of EGFR at present:EGF, TGF-a, amphiregulin,BTC,HB-EGF and EPR. BTC,HB-EGF and EPR are the co-ligand oferbB receptor family,but EGF, TGF-a and amphiregulin just bind on EGFR.ActivitedEGFR can activate several downstream signal transduction pathway. After Hendlerhad found the overexpression of EGFR of non small cell lung cancer at the firsttime,the overexpression or amplification was detected in several solid tumor asbreast,colorectal, ovarian, head and neck cancer, cerebroma,carcinoma of bladderand renal carcinoma. The overexpression of EGFR of tumor is an important index ofunfavourable prognosis and radiation resistance. EGFR is associated with invasionand metastasis of tumor also. In addition, EGFR can affect the reconstitution, shift,sticking of cystoskeleton and protease activity.
Can radiosensitivity of tumor be changed by blocking up or restraining theexpression of EGFR? It is an ideal targeted anticancer therapy method to restrainEGFR. In recent years, RNA interference(RNAi) develops very quickly as apragmatic molecular biology technology besides molecular targeted therapy. RNAican inhibit gene expression specificly in high performance. In practically, it is morevaluable than other technologis of intergenic suppression. The transient or stabilegene silencing mediated by siRNA has a high specificity and no side effect. There isno report about the changes of radiosensitivity of nasopharyngeal carcinoma cell afterEGFR was restrained with RNAi up to now in literatures.
Our studies included:
Part One: The expression of epidermal growth factor receptorfamily of nasopharyngeal carcinoma CNE1 and CNE2 cell lines
Objiective: To detect the expression of EGFR, HER-2, HER-3 and HER-4 ofnasopharyngeal carcinoma CNE1 and CNE2 cell lines. Methods: 1.The expression ofEGFR, HER-2, HER-3 and HER-4 of nasopharyngeal carcinoma CNE1 and CNE2 lines were tested by immunohistochemistry(IHC)、Western blot and RT-PCR. 2. Therelation of radiation dose and survival rate of cell was study by colony formationexperiment to prepare the conditions of the irradiation. Result: 1. The expressionof EGFR of CNE1 and CNE2 protien was positive, HER-2, HER-3 and HER-4negative detected by IHC and Western blot. 2. The expression of EGFRmRNA ofCNE1 and CNE2 was positive, HER-2, HER-3 and HER-4 negative detected byRT-PCR. 3. After CNE1 and CNE2 were irradiated 2Gy with X-ray, the survivalrate is about 50% on the 12th day; after were irradiated 4Gy, 22.93% and 3.67%respectively. Conclusion: The expression of EGFR of CNE1 and CNE2 was positive,HER-2, HER-3 and HER-4 negative.
Part Two: To construct, transfect and identity the EGFRSiRNAexpression vector
Objective: To construct the EGFRSiRNA expression vector and complete thetransfection and identity, and detect the expression of EGFR after tranfection.Methods: The nonsense and vacant vector were transfected as control besides EGFRinterference plasmid. DNA sequencing was deployed. Expression of EGFRmRNAwas detected before and after transfection by real-time fluorescence quantitive PCR.Expression of EGFR protein after having tansfected different vector was detected byWestern blotting. Result: 1. EGFR interference vector was constructed successfully.2. Expression of EGFRmRNA of CNE1 and CNE2 was detected by real-timefluorescence quantitive PCR: expression of EGFRmRNA of CNE1 and CNE2 cellswas decreased in transfecting EGFR interference vector group in contrast to nonsenseand vacant vector group. In statistics analysis, the difference of EGFRmRNAexpression of CNE1 cells between transfecting EGFR interference vector group andnonsense group was significant(P=0.000), so was between EGFR interference vector group and vacant vector group(P=0.000). In transfecting EGFR interference vectorgroup, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72 hour aftertransefection, the difference was significant between before transfecting and 48,60,72hour after transfecting(P=0.048,0.011,0.013). In statistics analysis, the difference ofEGFRmRNA expression of CNE2 cells between transfecting EGFR interferencevector group and nonsense group was significant(P=0.000), so was between EGFRinterference vector group and vacant vector group (P=0.000). In transfecting EGFRinterference vector group, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72hour after transefection, the difference was significant between before transfectingand12,24, 48,60,72 hour after transfecting (P=0.004、0.003、0.000、0.000、0.000).3. Expression of EGFR protein was detected by Western blotting: expression ofEGFR protein of CNE1 and CNE2 cells was decreased after transfecting EGFRinterference vector compared with transfectingβ-actin, vacant and nonsense vector.Conclusion: 1. SiRNA expression plasmid was constructed and transfectedsucceesfully. 2. Expression of EGFRmRNA of CNE1 and CNE2 cells was decreasedafter transfecting EGFR interference vector. 3. Expression of EGFR protein of CNE1and CNE2 cells was decreased after transfecting EGFR interference vector.
Part Three: The change of radiosensitivity of CNE1 and CNE2 cellsafter EGFR interference
Objective: To study the change of radiosensitivity of CNE1 and CNE2 cellsafter interference. Methods: 1. The apoptosis of CNE1 and CNE2 cell before andafter interfering was observed by TUNEL, DNA LADDER, electron microscope andDAPI after irradiated by X-ray. 2. The ratio of apoptosis and necrosis of CNE1 andCNE2 cell was detected by Annexin V/PI, and was compared before and afterinterfering vector after irradiation. 3. The proliferation of cells irradiated was detected by MTT experiment, and was compared before and after interfering. Result: 1.Apoptosis can be found after irradiation or interference by TUNEL, DNA LADDER,electron microscope and DAPI. 2. Detected by Annexin V/PI, apoptosis and necrosisof CNE1 and CNE2 cells irradiated increased befoe and after interference; theproportion of apoptosis was more than necrosis. In statistics analysis, the difference ofapoptosis ratio of CNE1 cells treated by irradiation between before and afterinterference was significant(P=0.008), and it changed following the timesignificantly(P＜0.05); the necrosis ratio of CNE1 cells treated by irradiation betweenbefore and after interference was not significantly different(P=0.082), but it changedfollowing the time significantly(P＜0.05). The difference of apoptosis ratio of CNE2cells treated by irradiation between before and after interference was significant(P=0.020), and it changed following the time significantly (P＜0.05); the necrosis ratioof CNE2 cells irradiated between before and after interference was significantlydifferent(P＜0.05), it changed following the time significantly(P＜0.05) except between24h and 48h after irradiation(P=0.170). 3. Detected by MTT experiment: proliferationof CNE1 and CNE2 cells irradiated was decreased after interference. In statisticsanalysis, the proliferation difference of CNE1 cells irradiated was significant betweenbefore and after interference(P＜0.001); before interference: the difference betweenbefore irradiation and at 30,36,48,60,84,96 hour after irradiation was significant(P=0.037、0.002、0.017、0.003、0.006、0.013); after interference, the differencebetween before irradiation and at 24,36,48,60,72,84,96 hour after irradiation wassignificant (P=0.037、0.037、0.002、0.001、0.001、0.001、0.003). The proliferationdifference of CNE2 cells irradiated was significant between before and afterinterference(P＜0.001); before interference: the difference between before irradiationand at 36,48,60,84,96 hour after irradiationis was significant (P=0.002、0.031、0.005、0.010、0.033); after interference, the difference between before irradiation and at 36,48,60,72,84,96 hour after irradiation was significant(P=0.047、0.002、0.001、0.001、0.001、0.003). Conclusion: 1. Apoptosis of CNE1 and CNE2 cells could befound after irradiation by TUNEL, DNA LADDER, electron microscope and DAPI. 2.The ratio of apoptosis and necrosis of CNE1 and CNE2 cells irradiated was increasedafter interference, the proportion of apoptosis was higher than necrosis. 3. Theproliferation of CNE1 and CNE2 cells treated by irradiation was decreased afterinterference. The proliferation of tumor cells irradiated was restrained after theexpression of EGFR had been decreased by RNA interference. The radiosensitivity ofNPC CNE1 and CNE2 cells was increased after the expression of EGFR had beendecreased by RNA interference.
放射治疗是鼻咽癌最主要的治疗方法。鼻咽癌患者治疗失败原因主要为局部复发或远处转移，严重影响鼻咽癌治疗生存率的提高。放射治疗是一个局部或区域治疗的手段，提高放射治疗的疗效不止是提高了局部或区域控制率，同时也影响生存及远地转移。如何提高放射治疗的疗效，如何提高放射敏感性一直困扰着肿瘤医师。
肿瘤的放射敏感性是一个十分复杂的问题，影响因素是多方面的。肿瘤的放射敏感性与肿瘤组织的来源、分化程度、病理类型、瘤床、贫血、局部是否合并感染、生活指数等有关。以往放射治疗效价的修饰措施有如下几个方面：
1．乏氧细胞放射增敏剂：目前研究的药物有美国的SR-2058和日本的AK-2133及其衍生物KU系列，但两者均属于硝基类化合物，仍无法回避限制剂量毒性的问题；2．肿瘤细胞放射增敏剂：其中以卤素类药物为主，但目前尚未在临床广泛使用；3．通过吸入不同浓度的氧来改变肿瘤氧含量的措施；4．放化疗的联合应用，正常组织的反应也有可能有所增加：5．放射治疗联合热疗、光动力学疗法等。
表皮生长因子受体(Eepidermal Growth Factor Receptor，EGFR)是一种受体酪氨酸激酶(receptor tyrosine kinase，RTK)，1982年由Cohen等首次发现。细胞膜上具有酪氨酸激酶活性(tyrosine kinase activity)的跨膜蛋白受体在细胞内信号转导中起着较为重要的作用。该激酶活性启动了一系列导致细胞增殖和分化的信号级联反应的发生，而表皮生长因子受体家族是涉及细胞增殖和肿瘤发生、发展中的极为重要的受体信号系统。许多癌基因能够编码生长因子受体或生长因子样受体蛋白，在人类的大部分上皮源性肿瘤的发展中起重要作用。表皮生长因子受体家族在肿瘤中的表达或过度表达往往引起肿瘤放、化疗敏感性的变化，而通过分子靶向治疗、基因等技术抑制其表达又可以抑制肿瘤细胞的生长，改变其对放射治疗、化学治疗、激素治疗的敏感性。表皮生长因子受体家族包括四个成员：EGFR、HER-2／P185erbB2、HER-4／P180erbB4和HER-3／P160erbB3。
EGFR(HER1，erbB1)基因定位于人染色体7q21上，编码一个分子量为170×10~3的具有酪氨酸激酶活性的受体糖蛋白。它通常与其相应的配体(EGF、TGF-α)相结合，使受体自身磷酸化，从而引起胞内一系列信号的级联放大及传递，最终影响核内基因的表达，导致细胞的增殖与分化。EGFR广泛分布于正常的哺乳动物上皮细胞表面，平均每个细胞受体个数为5×10~4-10×10~4。目前发现的EGFR配体共有6种，分别是表皮生长因子(EGF)、转化生长因子-α(TGF-α)、双向调节因子(amphiregulin)、Bctacellulin(BTC)、肝素结合表皮生长因子(heparin-binding EGF，HB-EGF)和表皮调节因子(epiregulin，EPR)，其中前三种仅与EGFR结合，后三种为EGFR与erbB家族其它受体的共同配体。EGFR活化后可激活许多的下游信号转导通路。继Hendler等首次检测出非小细胞肺癌细胞上EGFR有超表达以后，人们相继在多种实体瘤如乳腺癌、结直肠癌、头颈肿瘤、脑瘤、卵巢癌、膀胱癌、肾癌中均检到EGFR的超表达或扩增，EGFR高表达是肿瘤预后不良和对放射抗拒的重要指标。EGFR超表达还与肿瘤的侵袭和恶性转移有关。此外，EGFR还对细胞骨架的重构、移动、粘附以及蛋白酶活性有作用。
抑制EGFR为抗癌领域提供了一个理想的靶向抗癌疗法。除了分子靶向治疗，RNA干扰(RNAi，RNA interference)作为一种实用的分子生物学技术近年来发展非常迅速。该技术可高效特异性的抑制基因表达。在实用性上它比以前的基因抑制技术有更多的优点，siRNA介导的瞬时或稳定的基因沉默有高度的特异性并且无副作用。国内外尚未见应用RNAi抑制鼻咽癌细胞表皮生长因子受体表达后放射敏感性变化的相关报道。
我们的实验包括以下几个部分：
第一章鼻咽癌CNE1、CNE2细胞株表皮生长因子受体家族的表达
目的：检测鼻咽癌CNE1、CNE2细胞株EGFR、HER-2、HER-3、HER-4的表达情况。方法：1．免疫组化、Western blot检测鼻咽癌CNE1、CNE2细胞株EGFR、HER-2、HER-3、HER-4的蛋白表达情况；RT-PCR检测EGFRmRNA的表达情况；2．进行克隆形成实验，为进行细胞的照射作准备。结果：1．免疫组化、Westernblot检测发现CNE1、CNE2细胞株EGFR蛋白阳性表达，HER-2、HER-3、HER-4蛋白阴性表达；2．RT-PCR检测发现CNE1、CNE2细胞株EGFRmRNA阳性表达，HER-2、HER-3、HER-4阴性表达；3．X射线照射后12天，2Gy可达到约50％的细胞存活率，4Gy存活率CNE1和CNE2细胞分别为22.93％和3.67％。结论：CNE1、CNE2细胞株EGFR阳性表达，HER-2、HER-3、HER-4阴性表达。
第二章EGFRSiRNA表达载体的建立、转染和鉴定
目的：建立EGFRSiRNA表达载体，完成转染和鉴定；并检测转染后EGFR的表达。方法：除转染EGFR干扰质粒外，还转染空白质粒和无义质粒作为对照；采用DNA测序；实时荧光定量PCR检测转染前后及转染不同质粒后EGFRmRNA的表达；Western blot检测转染不同质粒后EGFR蛋白的表达。结果：1．成功构建了EGFR干扰质粒。2．实时荧光定量PCR检测CNE1、CNE2细胞EGFRmRNA的表达：转染EGFR干扰质粒组与转染无义质粒组及转染空载体组比较EGFRmRNA的表达降低。统计学分析：实时荧光定量RT-PCR检测CNE1细胞EGFRmRNA的表达：CNE1细胞转染EGFR干扰质粒组与转染无义质粒组比较有统计学差异(P=0.000)；转染EGFR干扰质粒组与转染空载体组比较也有统计学差异(P=0.000)；转染EGFR干扰质粒组：转染后0、12、24、48、60、72小时EGFR基因拷贝逐渐减少，转染前(即0小时)与转染后48、60、72小时比较有统计学差异(P=0.048、0.011、0.013)。实时荧光定量RT-PCR检测CNE2细胞EGFRmRNA的表达：CNE2细胞转染EGFR干扰质粒组与转染无义质粒组比较有统计学差异(P=0.000)；转染EGFR干扰质粒组与转染空载体组比较也有统计学差异(P=0.000)；转染EGFR干扰质粒组：转染后0、12、24、48、60、72小时EGFR基因拷贝逐渐减少，转染前(即0小时)与转染后12、24、48、60、72小时比较均有统计学差异(P=0.004、0.003、0.000、0.000、0.000)。3．Western blot检测蛋白表达情况：CNE1、CNE2细胞转染EGFR干扰质粒后与转染β-actin、空载体、无义质粒组比较，EGFR蛋白的表达减少。结论：1．成功建立EGFRSiRNA表达质粒并进行转染；2．CNE1、CNE2细胞转染EGFR干扰质粒后EGFRmRNA的表达降低；3．CNE1、CNE2细胞转染EGFR干扰质粒后EGFR蛋白的表达减少。
第三章EGFR干扰后CNE1、CNE2细胞放射敏感性的变化
目的：研究RNA干扰后放射敏感性的变化。方法：1．应用X射线照射转染及未转染EGFRSiRNA的细胞，TUNEL、DNA LADDER、电镜、DAPI等观察细胞凋亡。2．Annexin V／PI双标记法检测凋亡及坏死，并进行转染干扰质粒前后照射后凋亡及坏死率的比较。3．MTT实验比较转染干扰质粒前后照射后细胞增殖变化。结果：1．DAPI染色，TUNEL法，电子显微镜，凝胶电泳DNA LADDER均观察到ONE1和ONE2细胞照射后或转染后照射后的凋亡。
2．Annexin V／PI双标记法检测：ONE1、ONE2干扰后照射较干扰前照射凋亡及坏死均增加；凋亡所占比例高过坏死。统计学分析：CNE1干扰前照射组与干扰后照射组两组间的凋亡率比较有显著性差异(P=0.008)，照射前及照射后各时间点的凋亡率之间均有显著性差异(P＜0.05)，CNE1干扰前照射组与干扰后照射组两组间的坏死率比较无显著性差异(P=0.082)，照射前及照射后各时间点的坏死率之间均有显著性差异(P＜0.05)；CNE2干扰前照射组与干扰后照射组两组间的凋亡率比较有显著性差异(P=0.020)，照射前及照射后各时间点的凋亡率之间均有显著性差异(P＜0.05)，CNE2干扰前照射组与干扰后照射组两组间的坏死率比较有显著性差异(P=0.005)，除干扰前照射后24h与48h之间无显著性差异(P=0.170)外，照射前及照射后各时间点的坏死率之间均有显著性差异(P＜0.05)。3．应用MTT实验检测：CNE1、CNE2细胞干扰后照射较干扰前照射肿瘤增殖降低。统计学分析：CNE1细胞干扰前照射组与干扰后照射组之间有显著性差异(P＜0.001)。干扰前：照射前与照射后30、36、48、60、84、96小时比较有显著性差异(P=0.037、0.002、0.017、0.003、0.006、0.013)。干扰后：照射前与照射后24、36、48、60、72、84、96小时比较有显著性差异(P=0.037、0.037、0.002、0.001、0.001、0.001、0.003)。CNE2细胞干扰前照射组与干扰后照射组之间有显著性差异(P＜0.001)。干扰前：照射前与照射后30、48、60、84、96小时比较有显著性差异(P=0.002、0.031、0.005、0.010、0.033)。干扰后：照射前与照射后36、48、60、72、84、96小时比较有显著性差异(P=0.047、0.002、0.001、0.001、0.001、0.003)。结论：1．应用DAPI染色，TUNEL法，电子显微镜，DNA ladder均观察到CNE1、CNE2细胞X射线照射后细胞凋亡。2．Annexin V／PI双标记法检测：CNE1、CNE2细胞干扰后行X射线照射较干扰前行X射线照射凋亡及坏死均增加；凋亡所占比例高过坏死。应用RNA干扰技术抑制EGFR表达后进行照射，促进了肿瘤细胞的凋亡及坏死，凋亡占主导作用。3．MTT实验检测：CNE1、CNE2细胞干扰后行X射线照射较干扰前行X射线照射细胞增殖降低，应用RNA干扰技术抑制EGFR表达后进行照射，抑制肿瘤细胞增殖。应用RNA干扰技术抑制EGFR表达后提高了鼻咽癌CNE1、CNE2细胞放射敏感性。
Nasopharyngeal carcinoma (NPC) is an endemic cancer with high incidence inSoutheast Asia and Southern china. Although great progress in the investigation ofNPC, such as radiotherapy and imaging diagnosis and so on, have been made in thelate 90s,the cure rate of NPC has not been improved yet and 5 year survival rate stillremained around 50-60％.
Radiotherapy is the most important treatment method for NPC. Treatmentfailure is mainly because of local relapse and metastasis, which decreases thesurvival rate obviously. Radiotherapy is a regional treatment.A better local curativeeffect of radiotherapy not only increases local contral rate, but also affects survivalrate and metastasis. Oncologists have been investigating the approach to increase thecurative effect of radiotherapy and radiation sensitivity.
The radiosensitivity of tumor is a very complicated problem and the impactfactor involves in many aspects. It is decided by organize source, differentiationdegree, pathological type and the bed of tumor, anemia and partial infection, lifeindex…etc. The messures to modify the radiotherapy effects before are as follows: 1.anoxic cell radiosensitizer. 2. radiosensitizer of tumor cell, halogen as principleamong them, but are't used widely. 3. to change oxygen content of tumor by inhaling different density of oxygen 4.radiotherapy combined with chemotherapy,but thismight increase the side effects of normal tissue 5.radiotherapy combined with othermeasures such as thermotherapy,photodynamic therapy and so on.
Epidermal growth factor receptor (EGFR) is a sort of receptor tyrosine kinase(RTK). It was first discovered by Cohen and his colleagues in 1982. Thetransmembrane protein receptor with tyrosine kinase activity in cellular membraneplays an important role in cell signal transduction.It breakdowns a series of signalcascade reactions that induce cell proliferation and differentiation. Epidermal growthfactor receptor family is a very important receptor signal systerm which involves inthe cell proliferation and tumorigenesis. Many oncogenes can encode EGFR or thesimilar protein that lead an important function in the growth of most of tumororiginated from epithelium tissue of mankind. The overexpression of EGFR familyalways causes the sensitivity changes of radiotherapy and chemotherapy of tumor.We can restrain the growth of tumor and change the sensitivity of radiotherapy,chemotherapy and hormone therapy through inhibiting the expression of EGFR bymolecular targeted therapy or gene technology. Epidermal growth factor receptorfamily includes four members: EGFR、HER-2/P185erbB2、HER-4/P180erbB4 andHER-3/P160erbB3.
EGFR (HER1,erbB1) gene,localizing in chromosome 7q21, encodes aglycoprotein receptor with tyrosine kinase activity. The phosphorylation of itself afterintegrating with corresponding ligand results in a trial of signal cascade amplificationand transmitting. At last, it affects the expression of intranuclear genes and inducescell proliferation and differentiation. EGFR are widely distributed on the surface ofendothelial cell of normal mammal, 5×10~4～10×10~4 receptors in every cell average.There are six kind of ligands of EGFR at present:EGF, TGF-a, amphiregulin,BTC,HB-EGF and EPR. BTC,HB-EGF and EPR are the co-ligand oferbB receptor family,but EGF, TGF-a and amphiregulin just bind on EGFR.ActivitedEGFR can activate several downstream signal transduction pathway. After Hendlerhad found the overexpression of EGFR of non small cell lung cancer at the firsttime,the overexpression or amplification was detected in several solid tumor asbreast,colorectal, ovarian, head and neck cancer, cerebroma,carcinoma of bladderand renal carcinoma. The overexpression of EGFR of tumor is an important index ofunfavourable prognosis and radiation resistance. EGFR is associated with invasionand metastasis of tumor also. In addition, EGFR can affect the reconstitution, shift,sticking of cystoskeleton and protease activity.
Can radiosensitivity of tumor be changed by blocking up or restraining theexpression of EGFR? It is an ideal targeted anticancer therapy method to restrainEGFR. In recent years, RNA interference(RNAi) develops very quickly as apragmatic molecular biology technology besides molecular targeted therapy. RNAican inhibit gene expression specificly in high performance. In practically, it is morevaluable than other technologis of intergenic suppression. The transient or stabilegene silencing mediated by siRNA has a high specificity and no side effect. There isno report about the changes of radiosensitivity of nasopharyngeal carcinoma cell afterEGFR was restrained with RNAi up to now in literatures.
Our studies included:
Part One: The expression of epidermal growth factor receptorfamily of nasopharyngeal carcinoma CNE1 and CNE2 cell lines
Objiective: To detect the expression of EGFR, HER-2, HER-3 and HER-4 ofnasopharyngeal carcinoma CNE1 and CNE2 cell lines. Methods: 1.The expression ofEGFR, HER-2, HER-3 and HER-4 of nasopharyngeal carcinoma CNE1 and CNE2 lines were tested by immunohistochemistry(IHC)、Western blot and RT-PCR. 2. Therelation of radiation dose and survival rate of cell was study by colony formationexperiment to prepare the conditions of the irradiation. Result: 1. The expressionof EGFR of CNE1 and CNE2 protien was positive, HER-2, HER-3 and HER-4negative detected by IHC and Western blot. 2. The expression of EGFRmRNA ofCNE1 and CNE2 was positive, HER-2, HER-3 and HER-4 negative detected byRT-PCR. 3. After CNE1 and CNE2 were irradiated 2Gy with X-ray, the survivalrate is about 50% on the 12th day; after were irradiated 4Gy, 22.93% and 3.67%respectively. Conclusion: The expression of EGFR of CNE1 and CNE2 was positive,HER-2, HER-3 and HER-4 negative.
Part Two: To construct, transfect and identity the EGFRSiRNAexpression vector
Objective: To construct the EGFRSiRNA expression vector and complete thetransfection and identity, and detect the expression of EGFR after tranfection.Methods: The nonsense and vacant vector were transfected as control besides EGFRinterference plasmid. DNA sequencing was deployed. Expression of EGFRmRNAwas detected before and after transfection by real-time fluorescence quantitive PCR.Expression of EGFR protein after having tansfected different vector was detected byWestern blotting. Result: 1. EGFR interference vector was constructed successfully.2. Expression of EGFRmRNA of CNE1 and CNE2 was detected by real-timefluorescence quantitive PCR: expression of EGFRmRNA of CNE1 and CNE2 cellswas decreased in transfecting EGFR interference vector group in contrast to nonsenseand vacant vector group. In statistics analysis, the difference of EGFRmRNAexpression of CNE1 cells between transfecting EGFR interference vector group andnonsense group was significant(P=0.000), so was between EGFR interference vector group and vacant vector group(P=0.000). In transfecting EGFR interference vectorgroup, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72 hour aftertransefection, the difference was significant between before transfecting and 48,60,72hour after transfecting(P=0.048,0.011,0.013). In statistics analysis, the difference ofEGFRmRNA expression of CNE2 cells between transfecting EGFR interferencevector group and nonsense group was significant(P=0.000), so was between EGFRinterference vector group and vacant vector group (P=0.000). In transfecting EGFRinterference vector group, EGFRmRNA was decreaced by degree at 0,12,24,48,60,72hour after transefection, the difference was significant between before transfectingand12,24, 48,60,72 hour after transfecting (P=0.004、0.003、0.000、0.000、0.000).3. Expression of EGFR protein was detected by Western blotting: expression ofEGFR protein of CNE1 and CNE2 cells was decreased after transfecting EGFRinterference vector compared with transfectingβ-actin, vacant and nonsense vector.Conclusion: 1. SiRNA expression plasmid was constructed and transfectedsucceesfully. 2. Expression of EGFRmRNA of CNE1 and CNE2 cells was decreasedafter transfecting EGFR interference vector. 3. Expression of EGFR protein of CNE1and CNE2 cells was decreased after transfecting EGFR interference vector.
Part Three: The change of radiosensitivity of CNE1 and CNE2 cellsafter EGFR interference
Objective: To study the change of radiosensitivity of CNE1 and CNE2 cellsafter interference. Methods: 1. The apoptosis of CNE1 and CNE2 cell before andafter interfering was observed by TUNEL, DNA LADDER, electron microscope andDAPI after irradiated by X-ray. 2. The ratio of apoptosis and necrosis of CNE1 andCNE2 cell was detected by Annexin V/PI, and was compared before and afterinterfering vector after irradiation. 3. The proliferation of cells irradiated was detected by MTT experiment, and was compared before and after interfering. Result: 1.Apoptosis can be found after irradiation or interference by TUNEL, DNA LADDER,electron microscope and DAPI. 2. Detected by Annexin V/PI, apoptosis and necrosisof CNE1 and CNE2 cells irradiated increased befoe and after interference; theproportion of apoptosis was more than necrosis. In statistics analysis, the difference ofapoptosis ratio of CNE1 cells treated by irradiation between before and afterinterference was significant(P=0.008), and it changed following the timesignificantly(P＜0.05); the necrosis ratio of CNE1 cells treated by irradiation betweenbefore and after interference was not significantly different(P=0.082), but it changedfollowing the time significantly(P＜0.05). The difference of apoptosis ratio of CNE2cells treated by irradiation between before and after interference was significant(P=0.020), and it changed following the time significantly (P＜0.05); the necrosis ratioof CNE2 cells irradiated between before and after interference was significantlydifferent(P＜0.05), it changed following the time significantly(P＜0.05) except between24h and 48h after irradiation(P=0.170). 3. Detected by MTT experiment: proliferationof CNE1 and CNE2 cells irradiated was decreased after interference. In statisticsanalysis, the proliferation difference of CNE1 cells irradiated was significant betweenbefore and after interference(P＜0.001); before interference: the difference betweenbefore irradiation and at 30,36,48,60,84,96 hour after irradiation was significant(P=0.037、0.002、0.017、0.003、0.006、0.013); after interference, the differencebetween before irradiation and at 24,36,48,60,72,84,96 hour after irradiation wassignificant (P=0.037、0.037、0.002、0.001、0.001、0.001、0.003). The proliferationdifference of CNE2 cells irradiated was significant between before and afterinterference(P＜0.001); before interference: the difference between before irradiationand at 36,48,60,84,96 hour after irradiationis was significant (P=0.002、0.031、0.005、0.010、0.033); after interference, the difference between before irradiation and at 36,48,60,72,84,96 hour after irradiation was significant(P=0.047、0.002、0.001、0.001、0.001、0.003). Conclusion: 1. Apoptosis of CNE1 and CNE2 cells could befound after irradiation by TUNEL, DNA LADDER, electron microscope and DAPI. 2.The ratio of apoptosis and necrosis of CNE1 and CNE2 cells irradiated was increasedafter interference, the proportion of apoptosis was higher than necrosis. 3. Theproliferation of CNE1 and CNE2 cells treated by irradiation was decreased afterinterference. The proliferation of tumor cells irradiated was restrained after theexpression of EGFR had been decreased by RNA interference. The radiosensitivity ofNPC CNE1 and CNE2 cells was increased after the expression of EGFR had beendecreased by RNA interference.
引文
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