發(fā)布時間：2018-01-05 23:35

  本文關(guān)鍵詞：載有hTERT基因逆轉(zhuǎn)錄病毒感染日本血吸蟲童蟲的研究　出處：《中南大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 雙嗜性逆轉(zhuǎn)錄病毒 rRam-1受體 生物信息學(xué) 鈉離子依賴性磷酸鹽轉(zhuǎn)運體家族 血吸蟲童蟲細(xì)胞 pBABE-puro-hTERT質(zhì)粒 PA317細(xì)胞 雙嗜性逆轉(zhuǎn)錄病毒 嘌呤霉素 細(xì)胞增殖 泛嗜性逆轉(zhuǎn)錄病毒 Sj童蟲 Southern雜交 基因整合 蛋白

【摘要】： 迄今為止,應(yīng)用于血吸蟲病防治實踐的技術(shù)仍存在許多不足,尤在控制日本血吸蟲病流行與傳播方面尚無理想方法。30年前有學(xué)者提出,建立穩(wěn)定生長和連續(xù)傳代培養(yǎng)的血吸蟲細(xì)胞系,可為尋求新的防治技術(shù)提供基礎(chǔ)和條件,但通過數(shù)十年的細(xì)胞培養(yǎng)技術(shù)探索,一直未能實現(xiàn)建立可連續(xù)傳代生長的血吸蟲細(xì)胞系目標(biāo)。本研究受哺乳動物體細(xì)胞經(jīng)轉(zhuǎn)導(dǎo)外源永生化基因后成功建立永生化細(xì)胞系的啟示,開展了采用逆轉(zhuǎn)錄病毒載體對日本血吸蟲童蟲細(xì)胞進(jìn)行外源基因轉(zhuǎn)導(dǎo)的生物學(xué)理論探索,制備了載有永生化基因(hTERT)的雙嗜性逆轉(zhuǎn)錄病毒和泛嗜性逆轉(zhuǎn)錄病毒,并觀察了這2種逆轉(zhuǎn)錄病毒載體轉(zhuǎn)導(dǎo)外源基因到日本血吸蟲童蟲細(xì)胞或蟲體后所發(fā)生的整合、轉(zhuǎn)錄和表達(dá)以及對Sj細(xì)胞增殖的影響。本研究目的在于為血吸蟲細(xì)胞永生化研究提供理想的轉(zhuǎn)導(dǎo)外源基因的載體,驗證外源hTERT基因能否在血吸蟲體內(nèi)整合、表達(dá)及其表達(dá)部位,同時探索hTERT基因表達(dá)誘導(dǎo)細(xì)胞增殖的可行性。 [目的]探討用雙嗜性逆轉(zhuǎn)錄病毒載體將外源基因?qū)肴毡狙x(Sj)細(xì)胞的生物學(xué)理論與實驗依據(jù)。[方法]從GenBank中收集褐家鼠雙嗜性逆轉(zhuǎn)錄病毒受體(rRam-1)的氨基酸序列,應(yīng)用Blastp工具對其進(jìn)行序列相似性搜索,并用Cluster W2工具對相似性較高的氨基酸序列進(jìn)行同源性分析；采用RPS-blast與InterproScan在線工具對rRam-1受體及其同源的氨基酸序列進(jìn)行保守區(qū)域分析；進(jìn)一步應(yīng)用多個在線分析工具對與rRam-1同源的Sj蛋白進(jìn)行蛋白二級結(jié)構(gòu)、疏水性、跨膜性、信號肽、亞細(xì)胞定位以及翻譯后修飾點等進(jìn)行分析與預(yù)測；在生物信息學(xué)預(yù)測的基礎(chǔ)上采用含短片段外源基因的雙嗜性逆轉(zhuǎn)錄病毒載體感染Sj-12d童蟲細(xì)胞培養(yǎng)物,并應(yīng)用PCR與RT-PCR方法檢測外源基因在Sj細(xì)胞中的整合與表達(dá)。[結(jié)果]rRam-1氨基酸殘基序列相似性搜索及同源性分析顯示,其氨基酸序列與多種脊椎動物的鈉離子依賴性的磷酸鹽運載體家族的氨基酸序列有很高的同源性,一致性均在59%以上,其中,與中國倉鼠Ram-1受體(cRam-1)和人類Ram-1 (h Ram-1)受體的氨基酸序列一致性均為93%；此外,與多種無脊椎動物磷酸鹽運載體家族的氨基酸序列也有較高的同源性,氨基酸一致性在42%以上；Sj中存在2種與rRam-1受體有較高同源性的蛋白SJCHGC09605和SjCHGC05362,它們與rRam-1受體之間的氨基酸序列一致性分別為54%和61%,相似性分別為74%和72%。2種血吸蟲蛋白與人類、褐家鼠及中國倉鼠的Ram-1受體蛋白處于平行的進(jìn)化分枝上。保守性分析顯示,2種Sj蛋白與人類、褐家鼠及中國倉鼠的Ram-1受體存在相同的PH04 Superfamily保守結(jié)構(gòu)域,均為磷酸鹽運載體超家族成員；二級結(jié)構(gòu)顯示,SJCHGC09605和SJCHGC05362蛋白中,α螺旋分別占68.97%和39.22%,跨膜區(qū)預(yù)測顯示分別有7個和5個可能的區(qū)域,與疏水性預(yù)測結(jié)果完全一致；亞細(xì)胞定位及翻譯后修飾位點分析顯示,2種蛋白均不含信號肽序列和亞細(xì)胞定位信號,也不含糖基化、磷酸化和脂�；确g后修飾位點。利用攜帶外源E77.43基因的雙嗜性逆轉(zhuǎn)錄病毒感染Sj童蟲細(xì)胞后,經(jīng)PCR與RT-PCR檢測到目的基因存在與表達(dá),擴(kuò)增的目的片段大小為330 bp,與理論值相符。[結(jié)論]雙嗜性逆轉(zhuǎn)錄病毒rRam-1受體與日本血吸蟲細(xì)胞膜上起離子轉(zhuǎn)運通道或受體蛋白作用的SjCHGC09605和SjCHGC05362兩種跨膜蛋白成分存在較高同源性；用載有E77.43基因的雙嗜性逆轉(zhuǎn)錄病毒感染Sj童蟲細(xì)胞獲得成功,推測SjCHGC09605和SjCHGC05362兩種與rRam-1受體同源的蛋白可能是Sj感染過程中起作用的分子。該結(jié)果為下一步用雙嗜性逆轉(zhuǎn)錄病毒載體轉(zhuǎn)導(dǎo)永生化基因至Sj細(xì)胞提供了生物學(xué)理論與實驗依據(jù)。 [目的]建立含永生化基因(hTERT)的穩(wěn)定產(chǎn)逆轉(zhuǎn)錄病毒細(xì)胞株,觀察hTERT基因轉(zhuǎn)導(dǎo)Sj童蟲細(xì)胞后的整合和表達(dá)情況以及對細(xì)胞增殖作用的影響。[方法]將從美國引進(jìn)的pBABE-puro-hTERT質(zhì)粒經(jīng)核酸內(nèi)切酶酶切、PCR擴(kuò)增和測序鑒定確認(rèn)；倍比稀釋測定PA317細(xì)胞和NIH3T3細(xì)胞對嘌呤霉素的最高耐受濃度；用脂質(zhì)體將質(zhì)粒轉(zhuǎn)染至PA317細(xì)胞內(nèi),經(jīng)嘌呤霉素篩選獲得抗性克隆并擴(kuò)大培養(yǎng),并通過PCR、測序、免疫熒光、Western-blot及透射電鏡對抗性細(xì)胞株進(jìn)行鑒定,并以NIH3T3細(xì)胞測定收集的逆轉(zhuǎn)錄病毒液滴度。常規(guī)制備Sj-12d童蟲細(xì)胞,并在體外培養(yǎng)中用BrudU-ELISA法檢測細(xì)胞增殖情況,PCR法檢測兔線粒體特異性基因確定無宿主來源細(xì)胞污染,倍比稀釋法測定Sj細(xì)胞對嘌呤霉素的最高耐受濃度；用濃縮的雙嗜性逆轉(zhuǎn)錄病毒感染Sj-12d童蟲細(xì)胞并以嘌呤霉素連續(xù)篩選培養(yǎng)獲得抗性Sj細(xì)胞克隆,擴(kuò)大培養(yǎng)后用PCR、RT-PCR、Western-blot檢測外源hTERT基因和puror基因在細(xì)胞內(nèi)的整合與表達(dá)；用3H-TdR摻入法檢測嘌呤霉素抗性Sj-12d細(xì)胞的增殖能力,利用細(xì)胞計數(shù)法繪制其生長曲線,并應(yīng)用TRAP-ELISA法測定其端粒酶活性。[結(jié)果]pBABE-puro-hTERT質(zhì)粒經(jīng)酶切、PCR和測序鑒定為目的質(zhì)粒；PA317細(xì)胞和NIH3T3細(xì)胞對嘌呤霉素的最高耐受濃度為6μg/ml和3μg/ml；嘌呤霉素抗性PA317克隆擴(kuò)大培養(yǎng)物經(jīng)PCR、測序、免疫熒光以及Western-blot檢測到外源hTERT基因和puror基因的整合、轉(zhuǎn)錄及蛋白質(zhì)表達(dá)；透射電鏡檢測到抗性PA317細(xì)胞的培養(yǎng)上清及胞漿內(nèi)有逆轉(zhuǎn)錄病毒顆粒的存在,經(jīng)濃縮后測得其滴度為2×105cfu/ml。Sj-12d童蟲細(xì)胞培養(yǎng)3d后即可見部分細(xì)胞分裂相,10-14d后可見較多分裂相,BrdU-ELISA也顯示培養(yǎng)14d后有明顯的DNA合成與增殖,其對嘌呤霉素最高耐受濃度為0.5μg/ml;雙嗜性逆轉(zhuǎn)錄病毒感染Sj-12d細(xì)胞后經(jīng)嘌呤霉素連續(xù)篩選21d可見抗性克隆形成,對擴(kuò)大培養(yǎng)后的抗性細(xì)胞做PCR、RT-PCR和Western blot,檢測到外源hTERT基因和puror基因在抗性Sj-12d細(xì)胞內(nèi)的整合、轉(zhuǎn)錄及蛋白表達(dá),但整合的拷貝數(shù)少,轉(zhuǎn)錄水平低下；3H-TdR摻入法檢測顯示抗性Sj-12d細(xì)胞與常規(guī)培養(yǎng)的Sj-12d細(xì)胞均有一定增殖能力,但二者間差異無顯著性(P0.05)；TRAP-ELISA實驗未能從抗性Sj-12d細(xì)胞內(nèi)檢測到端粒酶活性；在培養(yǎng)4周內(nèi)的抗性Sj-12d細(xì)胞生長相對較快,此后生長逐漸減慢,死亡細(xì)胞和退變細(xì)胞的數(shù)目逐漸增多,最后全部死亡。[結(jié)論]用pBABE-puro-hTERT逆轉(zhuǎn)錄病毒質(zhì)粒轉(zhuǎn)染PA317細(xì)胞后,成功建立含hTERT基因穩(wěn)定產(chǎn)雙嗜性逆轉(zhuǎn)錄病毒顆粒的細(xì)胞株；該病毒感染Sj-12d童蟲細(xì)胞后可檢測到外源hTERT基因和puror基因的整合、轉(zhuǎn)錄及蛋白表達(dá),但整合拷貝數(shù)少,轉(zhuǎn)錄水平低下,未能激活Sj-12d細(xì)胞的端粒酶活性和改善細(xì)胞的增殖能力。 [目的]為提高逆轉(zhuǎn)錄病毒對血吸蟲的感染能力,探討應(yīng)用pVSV-G質(zhì)粒和pBABE-puro-hTERT質(zhì)粒共轉(zhuǎn)染包裝細(xì)胞制備泛嗜性逆轉(zhuǎn)錄病毒的可行性,并觀察該病毒感染Sj童蟲后外源基因在蟲體內(nèi)的整合、轉(zhuǎn)錄、表達(dá)及具體的表達(dá)部位情況。[方法]將pVSV-G質(zhì)粒和pBABE-puro-hTERT質(zhì)粒共轉(zhuǎn)染GP2-293包裝細(xì)胞,轉(zhuǎn)染后48h收集細(xì)胞培養(yǎng)上清,用濃縮的上清液與Polybrene混合液感染NIH3T3細(xì)胞系,經(jīng)嘌呤霉素連續(xù)篩選12d后獲得抗性克隆,計數(shù)抗性克隆數(shù)目并計算病毒滴度；挑取抗性NIH3T3克隆擴(kuò)大培養(yǎng),以PCR檢測外源hTERT基因和puror基因在細(xì)胞內(nèi)的整合,采用免疫細(xì)胞化學(xué)染色法檢測hTERT基因在細(xì)胞內(nèi)的表達(dá)情況；將泛嗜性逆轉(zhuǎn)錄病毒加入到體外培養(yǎng)的Sj-12d童蟲,感染24h后更換培養(yǎng)基,將蟲體連續(xù)培養(yǎng)6d；采用PCR和Southern雜交檢測外源hTERT基因在蟲體內(nèi)的整合,同時應(yīng)用RT-PCR、Western blot及免疫組織化學(xué)染色檢測外源hTERT基因在蟲體內(nèi)的轉(zhuǎn)錄、表達(dá)及定位情況。[結(jié)果]經(jīng)計數(shù)后,泛嗜性逆轉(zhuǎn)錄病毒顆粒滴度為3.2×108,抗性NIH3T3細(xì)胞經(jīng)PCR擴(kuò)增出外源hTERT基因和puror基因特異性145bp和204bp目的條帶,免疫細(xì)胞化學(xué)染色法檢測到hTERT基因在細(xì)胞內(nèi)發(fā)生了蛋白質(zhì)表達(dá),表達(dá)部位以細(xì)胞核內(nèi)為主；泛嗜性逆轉(zhuǎn)錄病毒感染后的蟲體經(jīng)PCR和RT-PCR擴(kuò)增出了外源hTERT基因和puror基因特異性145bp和204bp目的產(chǎn)物,其中,hTERT基因的轉(zhuǎn)錄水平較高,而puror基因的轉(zhuǎn)錄水平則較低,Southern雜交也顯示出蟲體內(nèi)有外源hTERT基因的多個拷貝整合；Western blot實驗顯示病毒感染后的蟲體內(nèi)有外源hTERT基因的表達(dá),其表達(dá)部位經(jīng)免疫組織化學(xué)染色確定為在蟲體的口吸盤、腹吸盤和后部體壁皮層下的表達(dá)量最多。[結(jié)論]用pVSV-G質(zhì)粒和pBABE-puro-hTERT質(zhì)粒共轉(zhuǎn)染包裝細(xì)胞后成功制備攜帶外源hTERT基因泛嗜性逆轉(zhuǎn)錄病毒,并證明該病毒感染Sj童蟲活蟲體后可在吸盤和后部體壁皮層下產(chǎn)生外源hTERT基因的多拷貝整合、轉(zhuǎn)錄與蛋白質(zhì)表達(dá)。
[Abstract]:So far, there are still many problems in the technologies used for schistosomiasis control, especially in controlling the spread of schistosomiasis japonica is the ideal method of.30 years ago, some scholars put forward the establishment of schistosome cell lines with stable growth and continuous passage, can provide the basis and conditions for prevention of new technology, but through decades of cells culture exploration, has failed to achieve a continuous passage of growth of schistosome cells target. This study by mammalian somatic cell transduction by exogenous immortalized gene successfully established immortalized cell lines of enlightenment, to carry out exploration of the biological theory of exogenous gene transduction on schistosomulum cells by retroviral vector, preparation an immortalized gene (hTERT) of the amphotropic retrovirus and retroviral, and observe the 2 kinds of reverse transcriptase disease Virus vector exogenous gene to integrate the schistosomulum cells or body of transcription and expression and effects on proliferation of Sj cells. The purpose of this study is to provide an ideal carrier exogenous gene for the study of schistosome cell immortalization, verify whether exogenous hTERT gene in schistosome integration, expression and expression at the same time, the expression of hTERT gene to explore the feasibility of inducing cell proliferation.
[of amphotropic retroviral vector exogenous gene of Schistosoma japonicum (Sj)] to the biological basis of theoretical and experimental methods. Cell collection of Rattus norvegicus amphotropic retrovirus receptor from GenBank (rRam-1) amino acid sequence, the application of Blastp tools for sequence similarity search, and the amino acid sequence similarity high homology analysis with Cluster W2 tools; conservative region analysis of the amino acid sequence of rRam-1 receptor and its homologous with RPS-blast online tools and InterproScan; the further application of multiple online analysis tools for protein secondary structure of two homologous with rRam-1 Sj protein, hydrophobicity, transmembrane, signal peptide, subcellular localization and post translation modification sites for analysis and prediction; in bioinformatics prediction based on the exogenous gene fragment containing short amphotropic retrovirus disease Sj-12d schistosomula cell culture virus vector infection, and]rRam-1. The amino acid sequence similarity search and homology analysis showed that the expression of the integration and application of PCR and RT-PCR for the detection of exogenous gene in Sj cells, the sodium ion of its amino acid sequence with a variety of vertebrate dependent phosphate transport carrier with amino acid sequence family high homology, consistency in more than 59%, and Chinese hamster Ram-1 receptor (cRam-1) and human Ram-1 (H Ram-1) amino acid sequence identity of the receptor was 93%; in addition, the homology of amino acid sequence with a variety of invertebrate transport phosphate carrier family also has a higher consistency in 42% amino acids. There are 2 or more; Sj and rRam-1 receptor homology protein SJCHGC09605 and SjCHGC05362 amino acid sequence consistency between them and rRam-1 receptor were 54% and 61%. It is 74% and 72%.2 respectively for schistosome proteins and human Chinese, Rattus norvegicus and hamster Ram-1 receptor protein in parallel evolutionary branches. Conservative analysis showed that 2 Sj protein and human, Rattus norvegicus and Ram-1 receptor PH04 China hamster Superfamily conserved domain of the same carrier are phosphate superfamily; two level structure, SJCHGC09605 and SJCHGC05362 protein, alpha helix accounted for 68.97% and 39.22%, transmembrane prediction showed there were 7 and 5 possible areas, was exactly consistent with hydrophobicity prediction; subcellular localization and posttranslational modification site analysis showed that all the 2 proteins without signal peptide and subcellular localization sequence signal, without glycosylation, phosphorylation and lipid acylation post-translational modification sites. By carrying exogenous E77.43 gene amphotropic retrovirus infected Sj cells of schistosomula, and by PCR The presence of RT-PCR detected and expression of target gene, the amplified fragments were 330 BP, consistent with the ion channels or the role of the SjCHGC09605 receptor protein and SjCHGC05362 two transmembrane protein has high homology up. Conclusion] amphotropic retrovirus receptor rRam-1 of Schistosoma japonicum and on the cell membrane and the theoretical value; for carrying E77.43 gene amphotropic retrovirus infection Sj schistosomula cells successfully, suggesting that SjCHGC09605 and SjCHGC05362 two and rRam-1 receptor homologous protein may play a role in the molecular process of Sj infection. The results provide a theoretical and experimental basis for the biological amphotropic retrovirus vector transduction immortalized gene into Sj cells under a step.
[Objective] to establish immortalized gene (hTERT) of the stable production of retroviral plasmid pBABE-puro-hTERT cell lines, to observe hTERT gene transduction of Sj insect cells after children integration and expression and effect on cell proliferation. Methods will be introduced from America by enzyme digestion, PCR amplification and sequencing confirmed times; dilution determination of PA317 cells and NIH3T3 cells of the highest puromycin resistant concentration; plasmid by liposome was transfected into PA317 cells by puromycin resistant clones screened and expanded in culture, and by PCR, sequencing, immunofluorescence and transmission electron microscopy, Western-blot resistant cells were identified by NIH3T3 the cell titer of retrovirus was determined. The routine preparation of Sj-12d cells of schistosomula, and in vitro cell proliferation was assessed by BrudU-ELISA method, PCR method for detection of rabbit mitochondrial specific base Because of no pollution from host cells, dilution of Sj cells was determined by the highest puromycin resistant concentration; with concentrated amphotropic retrovirus infected Sj-12d cells of schistosomula and puromycin resistance continuous screening culture Sj cell clones after culture expansion by PCR, RT-PCR, integration and expression of exogenous detection hTERT gene and puror gene in Western-blot cells; detection of puromycin resistant Sj-12d cells by 3H-TdR incorporation assay proliferation and draw the growth curve by using cell counting method, determination of the activity of]pBABE-puro-hTERT. The plasmid was verified by enzyme digestion of telomerase and the application of TRAP-ELISA method, PCR and DNA sequencing plasmids and PA317 cells; NIH3T3 cells of the highest puromycin resistant concentration was 6 g/ml and 3 g/ml; puromycin resistant clones were cultured by PA317 PCR, immunofluorescence and Western-blot sequencing. Detection of exogenous hTERT gene and puror gene expression, transcription and protein; the culture supernatant and cytoplasm by electron microscopy to resistant PA317 cells with retroviral particles exist, after concentration measured the titer was 2 * 105cfu/ml.Sj-12d schistosomula cultured 3D cells after visible cell division phase, can be seen after 10-14d more splitting phase, BrdU-ELISA showed that after 14d culture, DNA synthesis and proliferation, the highest puromycin tolerance concentration was 0.5 g/ml; amphotropic retrovirus infected Sj-12d cells by puromycin screening for 21d visible clone formation, expansion of resistant cells cultured in PCR and RT-PCR. Western blot, detection of exogenous hTERT gene and puror gene in resistant Sj-12d cells, the expression of mRNA and protein, but the integration of the copy number of small, low level of transcription; 3H-TdR incorporation assay. In resistant Sj-12d cells and normal cultured Sj-12d cells have certain ability of proliferation, but the difference between the two groups had no significant difference (P0.05); TRAP-ELISA experiment failed to detect from the resistant Sj-12d cells to telomerase activity in the culture; within 4 weeks of the resistance of Sj-12d cell growth is relatively fast, then the growth gradually slowed down, the number of cell death and cell degeneration increased gradually, finally died. Conclusion pBABE-puro-hTERT with retroviral vector transfected PA317 cells was successfully established with hTERT gene stably producing amphotropic retrovirus particles in cell lines; the virus infection Sj-12d schistosomula cells can be detected after exogenous hTERT gene and puror gene expression, transcription and protein, but copy number of integrated, low level of transcription, failed to activate Sj-12d cells telomerase activity and improve the proliferation of the cells.
[Objective] to improve the ability of retroviral infection of Schistosoma japonicum and explore the application of pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into the packaging cell preparation feasibility of retroviral infection, and observe the transcription of Sj gene in schistosomula after integration, parasite, expression and specific expression site. Methods] the pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into GP2-293 packaging cells, transfected 48h supernatants were collected to infect NIH3T3 cell lines with the supernatant and Polybrene mixed solution concentration, by puromycin resistant clones obtained after continuous screening of 12D, the number of counting resistant clones and calculate the virus titer were NIH3T3; resistant clones were cultured to detect exogenous PCR hTERT gene and puror gene in the cells of the integration, expression of staining detection of hTERT gene in cells by immunocytochemistry; will Retroviral entry into cultured Sj-12d schistosomula, 24h infection after replacement of the medium will worm continuous culture of 6D; PCR and Southern hybridization detection of exogenous hTERT gene in the integration of the parasite by simultaneous application of RT-PCR, Western blot and immunohistochemical detection of exogenous hTERT gene transcription in the parasite., the expression and localization. The results by counting after retroviral particles titer was 3.2 * 108, the resistance of NIH3T3 cells was amplified by PCR hTERT gene and puror gene specific 145bp and 204bp band, immunocytochemical staining method to detect the hTERT gene in the cell occurs in protein expression, the expression in the nucleus of the main parts of the body; retroviral infection after PCR and RT-PCR were amplified with exogenous hTERT gene and puror gene specific 145bp and 204bp products, including hTERT based Because of the high transcription level and puror gene transcription level is low, Southern hybridization also showed the parasite multiple copies of exogenous hTERT gene; Western blot experiment showed the expression of exogenous hTERT gene in infected larvae, the expression by immunohistochemical staining to determine the oral sucker in insects the expression of ventral sucker and posterior wall. Most subcortical conclusion with pVSV-G plasmid and pBABE-puro-hTERT plasmid were transfected into the packaging cell prepared successfully carrying exogenous hTERT gene retroviral, and prove that the virus infection Sj schistosomula live parasites can produce hTERT gene multi copy integration in sucker and the rear wall cortex, and protein expression of transcription.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R346

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