nestin在维持鼻咽癌细胞干性中的作用研究

发布时间：2018-06-03 07:43

本文选题：鼻咽癌 + 肿瘤干细胞　；参考：《南方医科大学》2011年硕士论文

【摘要】：研究背景与目的鼻咽癌(Nasopharyngeal carcinoma, NPC)是我国南方及东南亚地区高发的一种头颈部恶性肿瘤,它是一种低分化、高转移的恶性肿瘤,并且解剖位置比较隐蔽,早期症状不明显而容易被忽视,因此确诊的患者有60%以上都是中晚期病例,常伴有转移发生。对于进展期的肿瘤病人,虽然通过手术治疗并结合放化疗能缩小肿瘤体积,但却不能彻底清除肿瘤,病人最终死于肿瘤的转移或复发。因此,肿瘤治疗后的高转移及高复发已成为肿瘤临床治疗中一个亟待解决的难题。近年来,“肿瘤干细胞(cancer stem cells)”学说的提出以及其在这一领域所取得的研究进展,从一个全新的角度去认识了肿瘤。从肿瘤发生学、肿瘤转移学以及肿瘤治疗学方面对肿瘤进行了全新的诠释,提出肿瘤转移和复发的根源是肿瘤干细胞,使人们在探索彻底治愈肿瘤的道路上大大地向前迈进了一步。巢蛋白(nestin)属于第Ⅵ类中间丝蛋白,最初认为其主要表达于胚胎发育时期的神经组织,在成体中,nestin仅在一小部分组织和细胞中表达。长期以来,nestin都作为神经干细胞和前体细胞的标记,nestin名字的来源即：neural stem cell protein而近年来的研究表明,在多种类型的实体瘤及相应的细胞系中也发现了nestin的表达,并且发现nestin的表达与肿瘤的恶性分型有很大的关系。在中枢神经系统中,nestin的表达与细胞增殖之间的密切关系也已经多次被证实。有文献报道,在脑肿瘤中,CD133/nestin阳性细胞被认为是潜在的肿瘤干细胞群,但是具体的机制还不是很清楚。总的来说,nestin阳性的成体细胞代表了未分化的状态,干性或者前体细胞类型及代表了病理状态。随着nestin作为中枢神经系统肿瘤干细胞标记证据的增多,及在越来越多的实体瘤中发现nestin的表达,nestin在肿瘤中的返祖表现机制及在肿瘤干细胞中扮演了什么角色,开始引起了更多学者的关注。关于鼻咽部干细胞的研究已有一些报告,Zhang HB等在鼻咽癌细胞株5-8F的体外实验中发现了鼻咽粘膜基底部有散在干细胞,WangJ等在鼻咽癌细胞株CNE2中分离出类似干细胞的侧群细胞。然而,nestin与鼻咽癌的关系如何及是否为鼻咽癌肿瘤干细胞的重要标记物,迄今尚无相关的报道。本课题旨在研究nestin在鼻咽癌细胞株和组织中的表达特性；进一步通过改变nestin的表达水平观察其对于鼻咽癌细胞生物学特性及鼻咽癌细胞干性的影响,特别是关注nestin对鼻咽癌细胞干性的影响,探讨nestin在鼻咽癌细胞中的功能,为进一步寻找nestin基因表达调控及相关的信号通路提供参考,并为阐明鼻咽癌的发病机制和寻找新的早期诊断与分子靶向治疗靶点提供新线索。研究内容与方法 1.Nestin在鼻咽癌中表达水平的检测采用Western blot、免疫荧光、免疫组化及real-time PCR方法从蛋白和mRNA水平检测nestin在4株鼻咽癌细胞5-8F、6-10B、CNE1和CNE2及鼻咽癌组织标本中的表达情况。 2.构建nestin基因的shRNA表达载体及建立nestin稳定干扰的鼻咽癌细胞株构建含绿色荧光蛋白(green fluorescent protein, GFP)的重组靶向nestin shRNA慢病毒表达质粒,筛选阳性克隆,测序鉴定。将慢病毒载体质粒与辅助质粒共转染293T细胞包装病毒,收集病毒上清,测定滴度。经real-time PCR内源筛靶,选择最佳靶点。将重组慢病毒感染5-8F细胞,挑取单克隆,建立nestin稳定干扰的鼻咽癌细胞株。 3.检测nestin表达沉默对鼻咽癌细胞5-8F细胞生物学特性的影响用MTT法检测nestin沉默后对鼻咽癌细胞体外增殖能力的影响；利用体外迁移实验(Transwell)检测nestin沉默后对鼻咽癌细胞体外迁移能力的影响；利用流式细胞术检测nestin干扰后对鼻咽癌细胞凋亡和细胞周期的影响；利用裸鼠皮下成瘤实验检测nestin干扰后对鼻咽癌细胞体内成瘤能力的影响。 4.检测nestin表达沉默对鼻咽癌细胞5-8F干性的影响利用平板克隆形成实验、肿瘤球形成实验及流式细胞术分选SP细胞法检测nestin沉默后对鼻咽癌细胞干性的影响。结果 1.Nestin在鼻咽癌中的表达情况利用western blot方法检测各鼻咽癌细胞株中nestin在蛋白水平的表达情况,结果表明,与永生化的鼻咽上皮细胞NP69相比,鼻咽癌细胞株中5-8F、6-10B和CNE2细胞总蛋白在大约250KD处检测到阳性杂交信号。利用免疫荧光方法检测鼻咽癌细胞株nestin的表达,结果显示,5-8F、6-10B细胞及CNE2细胞可见绿色荧光,大部分在胞质表达的,少量胞核表达的。利用real-time PCR方法检测鼻咽癌细胞株nestin的表达,结果显示鼻咽癌细胞株中5-8F和CNE2细胞中nestin有较高的表达,5-8F细胞表达最高。免疫组化法检测鼻炎癌组织、非癌鼻咽组织nestin的表达,结果显示,nestin在炎症组几乎没有阳性细胞,为阴性表达。而在低分化鳞癌和未分化癌组阳性细胞占80%左右,nestin在鼻咽癌中的表达量明显高于炎症组,并且nestin主要在胞核表达,较少在胞浆表达。 2.成功构建nestin基因的shRNA表达载体及建立了nestin稳定干扰的鼻咽癌细胞株 PCR和测序证实,成功构建了nestin shRNA慢病毒载体pGCL-GFP/shnestin。包装慢病毒,并测得病毒滴度为4×106TU/ml。慢病毒感染5-8F细胞,有限稀释法挑取GFP阳性克隆,real-time PCR和western blot鉴定干扰效率后建立稳定干扰nestin的鼻咽癌细胞株5-8FNESi及阴性对照细胞株5-8FNC,为进一步对nestin功能进行研究提供细胞模型。 3.Nestin表达沉默后对鼻咽癌细胞5-8F细胞生物学特性的影响采用MTT法检测nestin基因沉默后细胞的体外增殖情况,对5-8F、5-8FNESiⅢ和5-8FNC三组细胞的增殖情况采用析因设计的方差分析,三组细胞的增殖能力具有显著差异(F=139.447, P0.001),5-8FNESi细胞的增殖速度显著减慢。采用流式细胞术分析干扰nestin后5-8F、5-8FNESi和5-8FNC三组细胞的周期分布,结果发现,三组细胞的S期比例有显著差异(F=216.467,P0.001)。5-8FNESi细胞S期细胞比例比5-8F细胞显著增加(P0.001),也就是说,在nestin干扰后细胞出现了S期向G2/M期转化障碍,细胞被阻滞在S期。采用Traswell小室检测nestin干扰后细胞体外迁移运动能力的改变,结果发现三组细胞运动能力的差异具有显著性(F=298.278,P0.001)。与5-8F细胞相比,5-8FNESi细胞穿过膜的细胞数显著减少(P0.001),其运动能力明显降低。采用流式细胞术检测nestin干扰后对鼻咽癌细胞凋亡的影响,结果发现三组细胞凋亡细胞比例的差异具有显著性(F=603.612,P0.001)。与5-8F和5-8FNC细胞相比,5-8FNESi细胞凋亡细胞比例明显升高(P=0.001)。裸鼠皮下成瘤接种后连续观察,于第21天处理5-8F组(接种5-8F细胞)、5-8FNC组(接种5-8FNC细胞)和5-8FNESi组(接种5-8FNESi细胞)各6只裸鼠。析因设计的方差分析结果表明,三组裸鼠移植瘤的体积增长有显著差异(F=14.670,P0.001),5-8F细胞慢病毒干扰nestin后,移植瘤的体积增长明显减慢。单因素方差分析(One-way ANOVA)结果表明,三组移植瘤的平均瘤重量也有显著差异(F=19.089,P0.001),与5-8F细胞相比,5-8FNESi细胞形成移植瘤的重量显著降低(P0.001)。 4.Nestin表达沉默后影响了鼻咽癌细胞5-8F的干性利用平板克隆形成实验、肿瘤球形成实验及流式细胞术分选SP细胞法检测nestin沉默后对鼻咽癌细胞干性的影响,统计结果采用SPSS13.0统计软件分析,结果发现,三组细胞的集落形成能力具有显著性差异(F=150.845,P0.001),SP比例有显著差异(F=205.400,P0.001)及肿瘤球的形成数量也有显著差异(F=830.622,P0.001)。5-8F细胞在nestin干扰后,平板克隆集落形成率明显低于干扰前(P0.001),SP细胞的比例明显少于干扰前(P0.001),而5-8FNESi细胞肿瘤球的形成数量也比5-8F细胞显著减少(P0.001)。结论 1.相对于永生化的鼻咽上皮细胞NP69和鼻咽慢性炎组织,nestin在鼻咽癌细胞株和鼻咽癌组织中的表达上调。 2.成功构建特异性干扰nestin的慢病毒载体pGCL-GFP/shnestin,建立稳定干扰nestin的单克隆细胞5-8FNESi及空载对照5-8FNC细胞。 3.Nestin基因沉默后显著抑制了鼻咽癌细胞的体、内外增殖和迁移能力,显著增加了鼻咽癌细胞中凋亡细胞的比例,这些结果提示nestin可能是一种与鼻咽癌相关基因。 4.Nestin基因沉默后显著减弱了鼻咽癌细胞5-8F的干性,表明nestin可能在维持鼻咽癌细胞干性中发挥重要作用。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R739.63

【共引文献】