本文選題：鼻咽癌 + 腫瘤干細(xì)胞��； 參考：《南方醫(yī)科大學(xué)》2011年碩士論文

【摘要】：研究背景與目的 鼻咽癌(Nasopharyngeal carcinoma, NPC)是我國南方及東南亞地區(qū)高發(fā)的一種頭頸部惡性腫瘤,它是一種低分化、高轉(zhuǎn)移的惡性腫瘤,并且解剖位置比較隱蔽,早期癥狀不明顯而容易被忽視,因此確診的患者有60%以上都是中晚期病例,常伴有轉(zhuǎn)移發(fā)生。對(duì)于進(jìn)展期的腫瘤病人,雖然通過手術(shù)治療并結(jié)合放化療能縮小腫瘤體積,但卻不能徹底清除腫瘤,病人最終死于腫瘤的轉(zhuǎn)移或復(fù)發(fā)。因此,腫瘤治療后的高轉(zhuǎn)移及高復(fù)發(fā)已成為腫瘤臨床治療中一個(gè)亟待解決的難題。近年來,“腫瘤干細(xì)胞(cancer stem cells)”學(xué)說的提出以及其在這一領(lǐng)域所取得的研究進(jìn)展,從一個(gè)全新的角度去認(rèn)識(shí)了腫瘤。從腫瘤發(fā)生學(xué)、腫瘤轉(zhuǎn)移學(xué)以及腫瘤治療學(xué)方面對(duì)腫瘤進(jìn)行了全新的詮釋,提出腫瘤轉(zhuǎn)移和復(fù)發(fā)的根源是腫瘤干細(xì)胞,使人們?cè)谔剿鲝氐字斡[瘤的道路上大大地向前邁進(jìn)了一步。 巢蛋白(nestin)屬于第Ⅵ類中間絲蛋白,最初認(rèn)為其主要表達(dá)于胚胎發(fā)育時(shí)期的神經(jīng)組織,在成體中,nestin僅在一小部分組織和細(xì)胞中表達(dá)。長(zhǎng)期以來,nestin都作為神經(jīng)干細(xì)胞和前體細(xì)胞的標(biāo)記,nestin名字的來源即：neural stem cell protein而近年來的研究表明,在多種類型的實(shí)體瘤及相應(yīng)的細(xì)胞系中也發(fā)現(xiàn)了nestin的表達(dá),并且發(fā)現(xiàn)nestin的表達(dá)與腫瘤的惡性分型有很大的關(guān)系。在中樞神經(jīng)系統(tǒng)中,nestin的表達(dá)與細(xì)胞增殖之間的密切關(guān)系也已經(jīng)多次被證實(shí)。有文獻(xiàn)報(bào)道,在腦腫瘤中,CD133/nestin陽性細(xì)胞被認(rèn)為是潛在的腫瘤干細(xì)胞群,但是具體的機(jī)制還不是很清楚�？偟膩碚f,nestin陽性的成體細(xì)胞代表了未分化的狀態(tài),干性或者前體細(xì)胞類型及代表了病理狀態(tài)。隨著nestin作為中樞神經(jīng)系統(tǒng)腫瘤干細(xì)胞標(biāo)記證據(jù)的增多,及在越來越多的實(shí)體瘤中發(fā)現(xiàn)nestin的表達(dá),nestin在腫瘤中的返祖表現(xiàn)機(jī)制及在腫瘤干細(xì)胞中扮演了什么角色,開始引起了更多學(xué)者的關(guān)注。關(guān)于鼻咽部干細(xì)胞的研究已有一些報(bào)告,Zhang HB等在鼻咽癌細(xì)胞株5-8F的體外實(shí)驗(yàn)中發(fā)現(xiàn)了鼻咽粘膜基底部有散在干細(xì)胞,WangJ等在鼻咽癌細(xì)胞株CNE2中分離出類似干細(xì)胞的側(cè)群細(xì)胞。然而,nestin與鼻咽癌的關(guān)系如何及是否為鼻咽癌腫瘤干細(xì)胞的重要標(biāo)記物,迄今尚無相關(guān)的報(bào)道。 本課題旨在研究nestin在鼻咽癌細(xì)胞株和組織中的表達(dá)特性；進(jìn)一步通過改變nestin的表達(dá)水平觀察其對(duì)于鼻咽癌細(xì)胞生物學(xué)特性及鼻咽癌細(xì)胞干性的影響,特別是關(guān)注nestin對(duì)鼻咽癌細(xì)胞干性的影響,探討nestin在鼻咽癌細(xì)胞中的功能,為進(jìn)一步尋找nestin基因表達(dá)調(diào)控及相關(guān)的信號(hào)通路提供參考,并為闡明鼻咽癌的發(fā)病機(jī)制和尋找新的早期診斷與分子靶向治療靶點(diǎn)提供新線索。 研究?jī)?nèi)容與方法 1.Nestin在鼻咽癌中表達(dá)水平的檢測(cè) 采用Western blot、免疫熒光、免疫組化及real-time PCR方法從蛋白和mRNA水平檢測(cè)nestin在4株鼻咽癌細(xì)胞5-8F、6-10B、CNE1和CNE2及鼻咽癌組織標(biāo)本中的表達(dá)情況。 2.構(gòu)建nestin基因的shRNA表達(dá)載體及建立nestin穩(wěn)定干擾的鼻咽癌細(xì)胞株 構(gòu)建含綠色熒光蛋白(green fluorescent protein, GFP)的重組靶向nestin shRNA慢病毒表達(dá)質(zhì)粒,篩選陽性克隆,測(cè)序鑒定。將慢病毒載體質(zhì)粒與輔助質(zhì)粒共轉(zhuǎn)染293T細(xì)胞包裝病毒,收集病毒上清,測(cè)定滴度。經(jīng)real-time PCR內(nèi)源篩靶,選擇最佳靶點(diǎn)。將重組慢病毒感染5-8F細(xì)胞,挑取單克隆,建立nestin穩(wěn)定干擾的鼻咽癌細(xì)胞株。 3.檢測(cè)nestin表達(dá)沉默對(duì)鼻咽癌細(xì)胞5-8F細(xì)胞生物學(xué)特性的影響 用MTT法檢測(cè)nestin沉默后對(duì)鼻咽癌細(xì)胞體外增殖能力的影響；利用體外遷移實(shí)驗(yàn)(Transwell)檢測(cè)nestin沉默后對(duì)鼻咽癌細(xì)胞體外遷移能力的影響；利用流式細(xì)胞術(shù)檢測(cè)nestin干擾后對(duì)鼻咽癌細(xì)胞凋亡和細(xì)胞周期的影響；利用裸鼠皮下成瘤實(shí)驗(yàn)檢測(cè)nestin干擾后對(duì)鼻咽癌細(xì)胞體內(nèi)成瘤能力的影響。 4.檢測(cè)nestin表達(dá)沉默對(duì)鼻咽癌細(xì)胞5-8F干性的影響 利用平板克隆形成實(shí)驗(yàn)、腫瘤球形成實(shí)驗(yàn)及流式細(xì)胞術(shù)分選SP細(xì)胞法檢測(cè)nestin沉默后對(duì)鼻咽癌細(xì)胞干性的影響。 結(jié)果 1.Nestin在鼻咽癌中的表達(dá)情況 利用western blot方法檢測(cè)各鼻咽癌細(xì)胞株中nestin在蛋白水平的表達(dá)情況,結(jié)果表明,與永生化的鼻咽上皮細(xì)胞NP69相比,鼻咽癌細(xì)胞株中5-8F、6-10B和CNE2細(xì)胞總蛋白在大約250KD處檢測(cè)到陽性雜交信號(hào)。利用免疫熒光方法檢測(cè)鼻咽癌細(xì)胞株nestin的表達(dá),結(jié)果顯示,5-8F、6-10B細(xì)胞及CNE2細(xì)胞可見綠色熒光,大部分在胞質(zhì)表達(dá)的,少量胞核表達(dá)的。利用real-time PCR方法檢測(cè)鼻咽癌細(xì)胞株nestin的表達(dá),結(jié)果顯示鼻咽癌細(xì)胞株中5-8F和CNE2細(xì)胞中nestin有較高的表達(dá),5-8F細(xì)胞表達(dá)最高。免疫組化法檢測(cè)鼻炎癌組織、非癌鼻咽組織nestin的表達(dá),結(jié)果顯示,nestin在炎癥組幾乎沒有陽性細(xì)胞,為陰性表達(dá)。而在低分化鱗癌和未分化癌組陽性細(xì)胞占80%左右,nestin在鼻咽癌中的表達(dá)量明顯高于炎癥組,并且nestin主要在胞核表達(dá),較少在胞漿表達(dá)。 2.成功構(gòu)建nestin基因的shRNA表達(dá)載體及建立了nestin穩(wěn)定干擾的鼻咽癌細(xì)胞株 PCR和測(cè)序證實(shí),成功構(gòu)建了nestin shRNA慢病毒載體pGCL-GFP/shnestin。包裝慢病毒,并測(cè)得病毒滴度為4×106TU/ml。慢病毒感染5-8F細(xì)胞,有限稀釋法挑取GFP陽性克隆,real-time PCR和western blot鑒定干擾效率后建立穩(wěn)定干擾nestin的鼻咽癌細(xì)胞株5-8FNESi及陰性對(duì)照細(xì)胞株5-8FNC,為進(jìn)一步對(duì)nestin功能進(jìn)行研究提供細(xì)胞模型。 3.Nestin表達(dá)沉默后對(duì)鼻咽癌細(xì)胞5-8F細(xì)胞生物學(xué)特性的影響 采用MTT法檢測(cè)nestin基因沉默后細(xì)胞的體外增殖情況,對(duì)5-8F、5-8FNESiⅢ和5-8FNC三組細(xì)胞的增殖情況采用析因設(shè)計(jì)的方差分析,三組細(xì)胞的增殖能力具有顯著差異(F=139.447, P0.001),5-8FNESi細(xì)胞的增殖速度顯著減慢。采用流式細(xì)胞術(shù)分析干擾nestin后5-8F、5-8FNESi和5-8FNC三組細(xì)胞的周期分布,結(jié)果發(fā)現(xiàn),三組細(xì)胞的S期比例有顯著差異(F=216.467,P0.001)。5-8FNESi細(xì)胞S期細(xì)胞比例比5-8F細(xì)胞顯著增加(P0.001),也就是說,在nestin干擾后細(xì)胞出現(xiàn)了S期向G2/M期轉(zhuǎn)化障礙,細(xì)胞被阻滯在S期。采用Traswell小室檢測(cè)nestin干擾后細(xì)胞體外遷移運(yùn)動(dòng)能力的改變,結(jié)果發(fā)現(xiàn)三組細(xì)胞運(yùn)動(dòng)能力的差異具有顯著性(F=298.278,P0.001)。與5-8F細(xì)胞相比,5-8FNESi細(xì)胞穿過膜的細(xì)胞數(shù)顯著減少(P0.001),其運(yùn)動(dòng)能力明顯降低。采用流式細(xì)胞術(shù)檢測(cè)nestin干擾后對(duì)鼻咽癌細(xì)胞凋亡的影響,結(jié)果發(fā)現(xiàn)三組細(xì)胞凋亡細(xì)胞比例的差異具有顯著性(F=603.612,P0.001)。與5-8F和5-8FNC細(xì)胞相比,5-8FNESi細(xì)胞凋亡細(xì)胞比例明顯升高(P=0.001)。裸鼠皮下成瘤接種后連續(xù)觀察,于第21天處理5-8F組(接種5-8F細(xì)胞)、5-8FNC組(接種5-8FNC細(xì)胞)和5-8FNESi組(接種5-8FNESi細(xì)胞)各6只裸鼠。析因設(shè)計(jì)的方差分析結(jié)果表明,三組裸鼠移植瘤的體積增長(zhǎng)有顯著差異(F=14.670,P0.001),5-8F細(xì)胞慢病毒干擾nestin后,移植瘤的體積增長(zhǎng)明顯減慢。單因素方差分析(One-way ANOVA)結(jié)果表明,三組移植瘤的平均瘤重量也有顯著差異(F=19.089,P0.001),與5-8F細(xì)胞相比,5-8FNESi細(xì)胞形成移植瘤的重量顯著降低(P0.001)。 4.Nestin表達(dá)沉默后影響了鼻咽癌細(xì)胞5-8F的干性 利用平板克隆形成實(shí)驗(yàn)、腫瘤球形成實(shí)驗(yàn)及流式細(xì)胞術(shù)分選SP細(xì)胞法檢測(cè)nestin沉默后對(duì)鼻咽癌細(xì)胞干性的影響,統(tǒng)計(jì)結(jié)果采用SPSS13.0統(tǒng)計(jì)軟件分析,結(jié)果發(fā)現(xiàn),三組細(xì)胞的集落形成能力具有顯著性差異(F=150.845,P0.001),SP比例有顯著差異(F=205.400,P0.001)及腫瘤球的形成數(shù)量也有顯著差異(F=830.622,P0.001)。5-8F細(xì)胞在nestin干擾后,平板克隆集落形成率明顯低于干擾前(P0.001),SP細(xì)胞的比例明顯少于干擾前(P0.001),而5-8FNESi細(xì)胞腫瘤球的形成數(shù)量也比5-8F細(xì)胞顯著減少(P0.001)。 結(jié)論 1.相對(duì)于永生化的鼻咽上皮細(xì)胞NP69和鼻咽慢性炎組織,nestin在鼻咽癌細(xì)胞株和鼻咽癌組織中的表達(dá)上調(diào)。 2.成功構(gòu)建特異性干擾nestin的慢病毒載體pGCL-GFP/shnestin,建立穩(wěn)定干擾nestin的單克隆細(xì)胞5-8FNESi及空載對(duì)照5-8FNC細(xì)胞。 3.Nestin基因沉默后顯著抑制了鼻咽癌細(xì)胞的體、內(nèi)外增殖和遷移能力,顯著增加了鼻咽癌細(xì)胞中凋亡細(xì)胞的比例,這些結(jié)果提示nestin可能是一種與鼻咽癌相關(guān)基因。 4.Nestin基因沉默后顯著減弱了鼻咽癌細(xì)胞5-8F的干性,表明nestin可能在維持鼻咽癌細(xì)胞干性中發(fā)揮重要作用。
[Abstract]:Research background and purpose
Nasopharyngeal carcinoma (NPC) is a high incidence of head and neck malignant tumor in South and Southeast Asia. It is a low differentiation, high metastasis malignant tumor, and the anatomical location is relatively concealed, the early symptoms are not obvious and easy to be ignored. Therefore, more than 60% of the patients are in the middle and late cases, often accompanied by metastasis. The tumor patients in the progressing stage, although surgical treatment and combined chemotherapy and chemotherapy can reduce the tumor volume, but can not completely remove the tumor, the patient eventually died of tumor metastasis or recurrence. Therefore, high metastasis and high recurrence after the treatment of cancer have become an urgent problem in the treatment of tumor. The advances in the theory of cancer stem cells and the progress made in this field have been made to understand the tumor from a new perspective. From the oncology, tumor metastasis and tumor therapy, a new interpretation has been made to the tumor. It is suggested that the origin of tumor metastasis and recurrence is the cancer stem cells. A great step forward in exploring the way to cure cancer thoroughly.
Nestin (nestin) belongs to class VI intermediate silk protein, which was originally thought to be mainly expressed in the embryonic neural tissue during embryonic development. In adult, nestin is expressed only in a small portion of tissue and cells. For a long time, nestin has been used as a marker for neural stem cells and precursor cells, and the source of the name of nestin, that is, neural stem cell protein. Recent studies have shown that the expression of nestin is also found in various types of solid tumors and corresponding cell lines, and that the expression of nestin is closely related to the malignant typing of the tumor. In the central nervous system, the close relationship between the expression of nestin and the proliferation of cells has also been confirmed. In the tumor, CD133/nestin positive cells are considered as potential tumor stem cell groups, but the specific mechanisms are not clear. Generally, nestin positive adult cells represent undifferentiated state, dry or precursor cell types and pathological status. With nestin as the evidence for CNS tumor stem cells The increase in the expression of nestin in more and more solid tumors, the mechanism of the reversion of nestin in the tumor and what role it plays in the tumor stem cells began to attract more attention. Some reports on the stem cells of the nasopharynx have been reported, and Zhang HB has been found in the in vitro experiment of the nasopharyngeal carcinoma cell line 5-8F. In the nasopharyngeal mucosa, the basal part of the nasopharyngeal mucosa is scattered in the stem cells, WangJ and other side groups in the nasopharyngeal carcinoma cell line CNE2. However, the relationship between nestin and nasopharyngeal carcinoma and whether it is an important marker for nasopharyngeal cancer stem cells has not yet been reported.
The purpose of this study is to investigate the expression of nestin in nasopharyngeal carcinoma cell lines and tissues, and to further observe the effects of nestin on the biological characteristics of nasopharyngeal carcinoma cells and the dry nature of nasopharyngeal carcinoma cells, especially the effect of nestin on the dry nature of nasopharyngeal carcinoma cells, and to explore the function of nestin in nasopharyngeal carcinoma cells. It provides a reference for further searching for the regulation of nestin gene expression and related signaling pathways, and provides new clues for elucidating the pathogenesis of nasopharyngeal carcinoma and finding new early diagnosis and target targeting therapy targets.
Research content and method
Detection of expression level of 1.Nestin in nasopharyngeal carcinoma
Western blot, immunofluorescence, immunohistochemistry and real-time PCR were used to detect the expression of nestin in 4 nasopharyngeal carcinoma cells 5-8F, 6-10B, CNE1 and CNE2 and nasopharyngeal carcinoma tissue samples from the protein and mRNA levels.
2. construction of shRNA expression vector of nestin gene and establishment of nasopharyngeal carcinoma cell line with stable nestin interference
The recombinant target of green fluorescent protein (GFP) was constructed to the nestin shRNA Lentivirus Expression Plasmid, and the positive clones were screened and sequenced. The plasmid with the lentivirus vector and the auxiliary plasmid were co transfected to the 293T cell package virus, and the virus supernatant was collected and the drop degree was measured. The optimum target was selected by the real-time PCR internal screening target. The weight of the target was selected. A group of lentivirus infected 5-8F cells, picked up monoclonal antibodies, and established nestin stable interference nasopharyngeal carcinoma cell lines.
3. detecting the effect of nestin silencing on the biological characteristics of nasopharyngeal carcinoma cell 5-8F cells
The effect of nestin silence on the proliferation of nasopharyngeal carcinoma cells in vitro was detected by MTT, and the effect of nestin silencing on the migration ability of nasopharyngeal carcinoma cells in vitro was detected by using in vitro migration test (Transwell); the effect of nestin interference on the apoptosis and cell cycle of nasopharyngeal carcinoma cells was detected by flow cytometry; the subcutaneous tumor formation of nude mice was used. The effect of nestin interference on the tumorigenic ability of nasopharyngeal carcinoma cells in vivo was tested.
4. detect the effect of nestin silencing on the 5-8F dry of nasopharyngeal carcinoma cells.
The effects of nestin silencing on the dry matter of nasopharyngeal carcinoma cells were detected by plate colony formation assay, tumor spheroid formation test and flow cytometry sorting SP cell assay.
Result
Expression of 1.Nestin in nasopharyngeal carcinoma
The Western blot method was used to detect the expression of nestin in each nasopharyngeal carcinoma cell line. The results showed that the positive hybridization signal was detected in the nasopharyngeal carcinoma cell lines 5-8F, 6-10B and CNE2 cells in the nasopharyngeal carcinoma cell lines, compared with the immortalized nasopharyngeal epithelial cell NP69. The immunofluorescence method was used to detect the nesti cell strain of nasopharyngeal carcinoma cell line. The expression of N showed that 5-8F, 6-10B cells and CNE2 cells were visible in green fluorescence, most of them were expressed in cytoplasm and expressed in a small number of nuclei. Real-time PCR method was used to detect the expression of nestin in nasopharyngeal carcinoma cell lines. The results showed that nestin in 5-8F and CNE2 cells in nasopharyngeal carcinoma cell lines was highly expressed and the expression of 5-8F cells was the highest. The expression of nestin in nasopharyngeal tissues and noncancerous nasopharynx tissues was detected by chemical method. The results showed that there were almost no positive cells in the inflammatory group and negative expression in nestin. The positive cells in the low differentiated and undifferentiated carcinoma group were about 80%, and the expression of nestin in nasopharyngeal carcinoma was significantly higher than that in the inflammatory group, and the nestin was mainly expressed in the nucleus and less in the cell. Pulp expression.
2. shRNA expression vector of nestin gene was successfully constructed and nasopharyngeal carcinoma cell line with stable nestin interference was established.
PCR and sequencing confirmed that the nestin shRNA lentivirus carrier pGCL-GFP/shnestin. package lentivirus was successfully constructed and the virus titer was 4 * 106TU/ml. lentivirus infected 5-8F cells. The GFP positive clones were selected by the finite dilution method, and real-time PCR and Western blot were used to establish the nasopharyngeal carcinoma cell lines that stabilized the interference nestin. The negative control cell line 5-8FNC provides a cell model for further study of nestin function.
Effect of 3.Nestin silencing on biological characteristics of nasopharyngeal carcinoma cell line 5-8F
MTT method was used to detect the proliferation of cells in vitro after nestin gene silencing. The proliferation of 5-8F, 5-8FNESi III and 5-8FNC three groups was analyzed by variance analysis of factorial design. The proliferation ability of the three groups was significantly different (F=139.447, P0.001), and the proliferation rate of 5-8FNESi cells was significantly slowed down. Flow cytometry was used to analyze the interference of NES. After tin 5-8F, 5-8FNESi and 5-8FNC three groups of cell cycle distribution, the results showed that the proportion of S phase in the three groups was significantly different (F=216.467, P0.001).5-8FNESi cells increased significantly in S phase ratio than 5-8F cells (P0.001). Ell cells detected the changes in the ability of cell migration and movement in vitro after nestin interference. The results showed that the difference of cell motility in three groups was significant (F=298.278, P0.001). Compared with 5-8F cells, the number of cells passing through the membrane of 5-8FNESi cells decreased significantly (P0.001), and its motor energy decreased obviously. The flow cytometry was used to detect nestin interference. The effect of apoptosis in nasopharyngeal carcinoma cells showed significant difference in the proportion of apoptotic cells in three groups (F=603.612, P0.001). Compared with 5-8F and 5-8FNC cells, the proportion of apoptotic cells in 5-8FNESi cells increased significantly (P=0.001). After subcutaneous tumor inoculation in nude mice, the group of 5-8F groups (inoculated 5-8F cells) and 5-8FNC group (inoculated 5) were treated on the twenty-first day. -8FNC cells and group 5-8FNESi (inoculated 5-8FNESi cells) were 6 nude mice. The analysis of variance analysis of the factorial design showed that the volume growth of the three groups of nude mice was significantly different (F=14.670, P0.001). After the 5-8F cell lentivirus interfered with nestin, the volume growth of the transplanted tumor was significantly slowed. The result of single factor analysis of variance analysis (One-way ANOVA) showed that three groups The average tumor weight of xenografts was also significantly different (F=19.089, P0.001). Compared with 5-8F cells, the weight of transplanted 5-8FNESi cells decreased significantly (P0.001).
The silencing of 4.Nestin expression affects the 5-8F dry of nasopharyngeal carcinoma cells.
The effect of nestin silence on the dry character of nasopharyngeal carcinoma cells was detected by SP cell assay and SP cell method. The results showed that the colony forming ability of the three groups was significantly different (F=150.845, P0.001), and the proportion of SP was significantly different (F= The number of 205.400, P0.001) and the formation of tumor balls was also significantly different (F=830.622, P0.001).5-8F cells after nestin interference, the formation of colony colony colony formation rate was significantly lower than before (P0.001), the proportion of SP cells was significantly less than before (P0.001), and the number of 5-8FNESi cell tumor balls was significantly lower than 5-8F cells (P0.001).
conclusion
1. compared with immortalized nasopharyngeal epithelial cells NP69 and nasopharyngeal chronic inflammation, nestin expression was up-regulated in nasopharyngeal carcinoma cell lines and nasopharyngeal carcinoma tissues.
2. we successfully constructed the lentiviral vector pGCL-GFP/shnestin which specifically interfered with nestin, and established the monoclonal nestin 5-8FNESi and the no-load control 5-8FNC cells which were stable.
3.Nestin gene silencing significantly inhibits the body, proliferation and migration of nasopharyngeal carcinoma cells, which significantly increases the proportion of apoptotic cells in nasopharyngeal carcinoma cells, which suggests that nestin may be a gene associated with nasopharyngeal carcinoma.
The silencing of 4.Nestin gene significantly attenuated the dryness of 5-8F in nasopharyngeal carcinoma cells, suggesting that nestin may play an important role in maintaining the nasopharyngeal carcinoma cell dry.
【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】：2011
【分類號(hào)】：R739.63

【共引文獻(xiàn)】

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本文編號(hào)：1972023