导　　师： 申洪

学科专业： J0104

授予学位： 博士

作　　者： ;

机构地区： 南方医科大学

摘　　要： 研究背景和目的 国内外肺癌的发病率和死亡率持续上升，在人口密度较高的工业城市尤为显著；在男性肿瘤死因中已居首位，在女性中仅次于乳腺癌居第二位，且有年轻化趋势，而肺癌的治疗和预后不容乐观。因此深入研究肺癌的病因、发病机制，探索有效的防治方法已是当前肿瘤研究的首要任务。 abcg2是abc转运蛋白超家族(atpbindingcassettetransportersuperfamily)成员之一，是1998年从耐药的乳腺癌细胞中克隆，并引起耐药的基因，所以又叫乳腺癌耐药蛋白(bcrpbreastcancerresistanceprotein)基因。与其他研究小组发现的mxr(mitoxantroneresistanceprotein)和abcp(placentalabcprotein)有着结构和序列的相似性，同属于abcg基因家族，因此人类基因组命名委员会将其统一命名为abcg2。 abcg2蛋白是半转运蛋白，由6个跨膜螺旋结构域和一个氨基端胞内核酸结合结构域以及相应的一个atp-bindingcassette组成，因此要通过与自身或其他半转运体组成同型或异型二聚体或多聚体后才能发挥作用。包括abcg2在内的转运蛋白超家族正是通过转运复合体的形成在宿主对异物的防御和肿瘤细胞对抗癌药物的耐药性中起重要作用。 研究还发现abcg2／berp1与造血干／祖细胞泵出hochest33342染料的特性有关。部分干细胞和癌细胞可以将hochest33342排出细胞，表现为核不着色或着色很少，流式细胞仪可以将不着色细胞分离，分离出的这部分细胞称为sp细胞。目前认为abcg2就是sp细胞的表型标志，是潜在的干细胞标志物。 因此，abcg2既是一个新的耐药基因，又和sp细胞、肿瘤干细胞密切相关，研究其在肺癌中的表达，分析其与临床病理特征的关系，揭示其对肺癌细胞基本生物学特性的影响，有助于深入研究abcg2与肺癌的关系，可能会为肺癌的发生和防治提供新策略。 方法： 1.abcg2在肺癌中表达的定量研究 (1)应用免疫组化(sp法)观察了86例肺癌(鳞癌42例、腺癌31例、大细胞癌5例，小细胞癌8例，其中伴淋巴结转移者33例)和24例癌周肺组织(距肿物5cm)abcg2蛋白的表达： (2)应用原位杂交观察了86例肺癌组织中(鳞癌42例、腺癌31例、大细胞癌5例，小细胞癌8例)abcg2mrna的表达： (3)应用显微图像定量分析，测试靶细胞abcg2蛋白及mrna表达的阳性单位(pu)值。 2.abcg2基因沉默对肺癌细胞体外生物学特性的影响 (1)在倒置显微镜下观察肺腺癌细胞glc-82和a549的形态学特点；应用he细胞爬片和组织切片比较二者形态异同；应用免疫细胞化学、细胞原位杂交、rt-荧光定量pcr、western-blot和hoechst33342染色比较glc-82和a549中abcg2基因表达程度。 (2)应用基因转染方法，将含有不同片段的4个干扰质粒、一个阳性对照质粒和一个阴性对照质粒，通过脂质体分别转入glc-82，用g418反复筛选纯化克隆。用rt-pcr法鉴定阳性对照中gapdh的干扰效率，再用rt-荧光定量pcr(rt-qpcr)法鉴定各干扰各隆的干扰效率，并通过免疫细胞化学和westernblot验证。 (3)应用mtt法测od值并绘制细胞生长曲线，流式细胞术检测细胞周期分布，克隆形成实验检测单细胞克隆形成率，比较干扰前后细胞生长增殖能力的改变；应用划痕实验观察细胞迁移速度，观察abcg2沉默对肺癌细胞转移能力的影响；应用倒置显微镜、细胞爬片、扫描电镜和透射电镜观察干扰前后细胞的形态学改变：应用mtt法检测干扰前后细胞对顺铂耐药性的改变。 3.abcg2基因沉默对肺癌细胞皮下成瘤的影响 通过动物实验裸鼠皮下成瘤观察干扰前后细胞成瘤过程和成瘤能力的变化，绘制体内生长曲线，行he染色观察其组织学形态的变化。 结论： 1.abcg2在不同类型和分化的肺癌中表达不同，可作为肺癌分型和判断肺癌分化程度的指标之一。 2.abcg2表达与肺癌患者的性别、年龄、转移和tnm分期无关，与肺癌分化程度有关，提示abcg2在肺癌耐药中起一定作用。 3.从蛋白和mrna水平及功能方面证实了abcg2在glc-82中表达高于a549。 4.建立了稳定的abcg2基因沉默细胞株，为后续实验提供了理想的细胞模型。 5.abcg2基因沉默致肺癌细胞增殖变缓且对顺铂耐药性降低，提示其对肺癌细胞生物学特性有一定影响，这有助于进一步揭示abcg2与肺癌之间的关系。 创新之处： 1.首次从定量角度揭示了abcg2蛋白和mrna在肺癌中表达的特点及规律。 2.建立了稳定的abcg2表达沉默的肺癌细胞株，为肺癌的体外研究提供了理想的细胞模型，为各种肺癌动物实验模型的建立奠定了基础。 3.首次系统性地研究abcg2基因对肺癌细胞生物特性的影响，为深入研究肺癌的发生和治疗奠定基础。 Backgroud and objective Lung cancer is the leading cancer killer in both men and women especially in industrial city while its morbidity and mortality still continue to rise.The surest way to defeat it is to prevent it from ever happening.Since the detection,treatment and prognosis will not be optimistic without clear mechanism and effective therapy,it is urgent to explore etiopathogenisis and potent means of prevention and cure. The ATP-binding cassette transporter protein ABCG2,a new member of the supeffamily of ABC transporter proteins,also known as mitoxantrone resistance protein,ABCP,and Breast cancer resistance protein/(BCRP/),was identified in a MCF-7 breast cancer cell subline that selected for resistance to doxorubicin.The Human Genome Nomenclature Committee recently changed the gene symbol to ABCG2. Structurally ABCG2 contains a single NH2-terminal cytosolic nucleotide-binding domain/(NBD/)-an ATP binding cassette involved in ATP hydrolysis,and six transmembrane domains/(TMD/) involved in drug binding and efflux follow.so it is proposed to be a halftransporter that may be function as a homodimer or a tetramer bridged by disulphide bonds or other formations.Based on the structure it plays an important role in prevention host against extraneous matter and drug resistance. Recent evidence suggests that ABCG2 as the transporter is responsible for the Hoechst 33342 dye efflux pattern in cells.Side population/(SP/) cells are enriched for stem cell activity and are defined by their ability to efficiently efflux Hoechst 33342.SP cell can be isolated by which can rapidly efflux the Hoechst dye to produce a characteristic SP profile based on fluorescence-activated flow cytometric analysis. ABCG2 was subsequently characterized as a novel stem cell transporter and a promising/(tumor/) stem cell maker. The importance of ABCG2 in chemotherapy and pharmacokinetics and the recent discoveries of ABCG2 role in stem cell regulation encourage further investigation of the relationship between this transporter and lung cancer,which might provide a new discovery to the develeopment and a new strategy for the prevention and cure. Methods 1.The quantitative study of ABCG2 expression in lung cancer /(1/) By immunohistochemical method/(S-P/) the expression of ABCG2 protein was detected in specimens of 86 lung cancer including 42 squamous carcinoma/(Sq Ca/), 31adenocarcinoma/(AC/),5 large cell carcinoma/(LC/) and 8 small cell carcinoma/(SC/), 33 with regional lymphatic metastasis,and 24 normal lung tissue far from the focus. /(2/)By hybridization in situ the expression of ABCG2 mRNA was deteceted in specimens of 86 lung cancer including 42 Sq Ca,31AC,5 large cell carcinoma LC and 8 small cell carcinoma SC. /(3/)By image quantitive analysis,the gray scale of the target cell was measured and positive unit/(PU/)of ABCG2 protein or mRNA was calculated. 2.The effect of ABCG2 silence on the biological behaviors of lung cancer cell line in vitro /(1/)Morphological feature of GLC-82 and A549 was observed by inverted microscope and HE staining of cell slide and tissue section.The expression of ABCG2 protein and mRNA GLC-82 and A549 was detected and compared by immunocytochemstry, hybridization in situ,Quantitative reverse transcriptase polymerase chain reaction /(RT-QPCR/),western blot and Hoechst33342. /(2/)4 RNAi vector,1 positive control vector and 1 negative control vector were separately tranfected into GLC-82 with lipofectamin2000 to establish the stable ABCG2 silencing lung caner cell line with G418.The ABCG2 expression in transfectant was determined by RT-PCR or RT-QPCR,immunocytochemistry and Western blot. /(3/) MTT assay,colony formation assay,cell cycle distribution was used to assess growth ability in vitro.Scratching experiment was applied to compare the migration speed.The resisitance to cisplatin was assessed by MTT.Morphological feature of ABCG2 slienced GLC-82 was observed by light microscope and electron microscope. 3.The effect of ABCG2 silence on tumorgenesis of lung cancer cell line in vivo The process of tumorgenesis was observed by nude eye and Whole-body optical images and the proliferation change of GLC-82 after ABCG2 silencing was compared. The histological morphology was observed by HE staining tissue section. Results 1.ABCG2 monoclonal antibody shows specific plasma membrane staining,prominent staining was observed in AC and SqC of primary focus and lymphatic metastasis,the apical membrane of the bronchial layer and the epithelium of mucus glands..It was not present in LC and SC. /(1/) in primary focus PU difference of ABCG2 protein was found among the four type of lung cancer/(F=15.857,P＜0.001/).The differnce was also found between AC or and the others/(P＜0.05/).the PU in AC exceed the one in SqC/(P＜0.01/). /(2/) the PU of ABCG2 protein in normal lung tissue was higher than in lung cancer /(t=5.158,P＜0.001/).Significant difference of PU was found among normal lung tissue/(F=22.470,P＜0.001/) and each type of lung cancer.The difference between normal lung tissue and each type/(P＜0.05/) besides AC/(P＞0.05/) was significant. /(3/) in lymphatic metastasis PU difference of ABCG2 protein was found among the four type of lung cancer/(F=7.804,P=0.001/).The differnce was also found between AC or and the others/(P＜0.05/).the PU in AC exceed that in SqC/(P＜0.01/). Significant difference of PU was not found between primary focus and lymphatic metastasis/(t=1.388,P=0.169/).and the separate differnce was either not found between primary focus and lymphatic metastasis of each type/(P＞0.05/). /(4/) the PU of ABCG2 protein was not associated with sex,old,metastasis and TNM stage/(P＞0.05/),but associated with differentiation/(F=65.385 P＜0.001/).the PU was higher when the cell is fully differentiated. 2.ABCG2 mRNA was expressed in the cell plasma of AC and SqC,and it was not found in LC and SC. /(1/) PU difference of ABCG2 mRNA was found among the four type of lung cancer /(F=12.155,P＜0.001/).The differnce was also found between AC or SqC and the others/(P＜0.05/).PU in AC exceed one in SqC/(P＜0.01/). /(2/) the PU of ABCG2 mRNA was not associated with sex,old,metastasis and TNM stage/(P＞0.05/),but associated with differentiation/(F=57.895 P＜0.001/).The PU was higher when the cell is fully differentiated. 3.Comparision between GLC-82 and A549 /(1/)cells of GLC-82 and A549 have visible difference in size and appearance.The majority of GLC-82 is epitheliod cell lined like a gland,and scattered spindle cell is the main point of A549. /(2/)Positive stainning of ABCG2 protein was detected in the plasma membrane of GLC-82 and A549.Quantitative analysis show PU of GLC-82 was higher than that of A549/(t=7.860,P＜0.001/). /(3/)Positive signal of ABCG2 mRNA existed at the plasma of GLC-82 and A549. Quantitative analysis show PU of GLC-82 was higher than that of A549/(t=8.958,P＜0.001/). /(4/) ABCG2 mRNA expression in A549 was 32/%of that in GLC-82 byRT-QPCR. /(5/)Much more ABCG2 protein was detected in GLC-82 than in A549 by western blot t=20.839,P＜0.001/) /(6/)Nucleus staining of hoechst33342 in GLC-82 was dimmer than in A549. Quantitative analysis show PU of GLC-82 was lower than that of A549/(t=17.223, P＜0.001/) 4.10 clone of RNAi,lclone of positive clone/(GLC-82//P/) and lclone of negative clone/(GLC-82//N/) were purified after transfection. /(1/)in positive control the expression of GAPDH mRNA was 19/%of that of GLC-82 /(t=11.490,P＜0.001/) by RT-QPCR. /(2/)in clone 1 and clone 2 the expression of GAPDH mRNA was 20.11/%and 27.59/% of that of GLC-82//N.the interfering ratio was 79.99/%and 72.49/%. /(3/) The difference of PU of ABCG2 protein expression among GLC-82,clone 1 /(GLC-82//R/) and GLC-82//N was detected by immunocytochemistry and quantitative analysis/(F=242.536,P＜0.001/).There was no difference between GLC-82 and GLC-82//N/(P＞0.05/).Compared with GLC-82 and GLC-82//N,PU of ABCG2 protein expression decreased/(P＜0.01/). /(4/) The difference of ABCG2 protein expression among GLC-82,clone 1 /(GLC-82//R/) and GLC-82//N was detected by western blot/(F=77.190,P＜0.001/). There was no difference between GLC-82 and GLC-82//N/(P＞0.05/).Compared with GLC-82 and GLC-82//N,ABCG2 protein expression decreased/(P＜0.01/). /(5/) Nucleus staining of hoechst33342 in GLC-82//R was stronger than in GLC-82//N. /(t=35.107,P＜0.001/) 5.To observe the change of the biobehavior after ABCG2 silence in GLC-82 in vitro /(1/)GLC-82//R showed a significantly slow proliferation compared with GLC-82 and GLC-82//N by MTT assay in vitro from day3/(F=91.333,P＜0.001/). /(2/) The percent of cell numbers in G2,S and G1//G2 show no difference among GLC-82,GLC-82//R and GLC-82//N/(P＞0.05/) by flow cytometry,compared with GLC-82 and GLC-82//N,the percent of cell numbers in G1 decreased in GLC-82//R /(F=5.208,P=0.49/). /(3/) The ratio of colony formation in GLC-82//R was lower than that of GLC-82 and GLC-82//N/(F=31.869,P＜0.001/). /(4/)No interaction was found between group and time/(F=0.511,P=0.780/).The difference of the healing rate was not found among GLC-82,GLC-82//R and GLC-82//N/(F=0.511 P＞0.05/). /(5/) No differnce had been observed by microscope and electron microscope. /(6/)IC50 difference was found among GLC-82,GLC-82//R and GLC-82//N /(F=32.828,P=0.001/).Compared with GLC-82,the resisitance index of GLC-82//R and GLC-82//N decreased to 0.314 and 0.589 6.To observe the process of tumorgenesis of GLC-82,GLC-82//R and GLC-82//N and compare the proliferative ability in vivo. /(1/)Compared with the tumor from GLC-82 and GLC-82//N,the tumor from GLC-82 grow slowly from day 17 after injection/(F=26.102,P＜0.001/). /(2/)The tumor from GLC-82 is smaller/(F=5.920,P=0.013/) and lighter/(F=4.114, P=0.038/) than that from GLC-82 and GLC-82//N. /(3/)The gross appearance of the tumors was gray and nodose.Histologically the pleomorphic cancer cells grow infiltrative widespreadly with hemorrhage and necrosis,and surrounded by slim fibrous connective tissue,no obvious glandular differentiation is seen on light macroscopy.Tumor cells with monocaryon and dikaryon,polykaryocyte and multinucleated giant cells were visible.There was nuclear pleomorphism with numerous bizarre mitoses.Histology and morphology of tumor cell were similar in the three group. Conclusion 1.ABCG2 might be taken as a marker to diagnose the type and differenciated extent of lung caner. 2.The expression of ABCG2 was not involved in sex,old,mestastasis and TNM stage of lung cancer,but associated with the extent of differciate,which indicated that ABCG2 might be play a role in drug resistant of lung cancer. 3.The expression of ABCG2 was higher in GLC-82 than in A549 in terms of mRNA and protein expression of ABCG2 and its function. 4.we established the stable lung cancer cell line of ABCG2 silence,which will provide an ideal cell model for the ulterior experiment. 5.Since ABCG2 was associated with proliferattion and drug resistance of lung caner, deep research into the correlation between ABCG2 and lung cancer will provide a new clue for carcinogenesis,development and treatment. New point 1.To study the expression of ABCG2 protein and mRNA from the quantitative point for the first time 2.To establish the lung cancer cell line of ABCG2 silence,to provide an ideal cell model for the reserch of lung cancer in vitro and establish the foundation of animal model. 3.For the first time systematically to study the effect on the biological behavior of ABCG2 silence in lung cancer cell line.

关 键 词： 肺癌 耐药基因 生物学行为

分 类 号： [Q279]

领　　域： [生物学]