本文關鍵詞： Stathmin siRNA 紫杉醇 潛伏膜蛋白1 微管 鼻咽癌 凋亡 增殖 侵襲 轉移　出處：《中南大學》2011年博士論文　論文類型：學位論文

【摘要】：目的：微管是構成細胞骨架的主要組成成分之一,其對于細胞的各種主動運動至關重要,這包括細胞形態(tài)的改變、細胞的移動、染色體的分離和細胞的分裂等。細胞中各種微管結構和功能的差異決定于其亞細胞定位及其與之結合的微管結合蛋白。Stathmin就是一個已經(jīng)明確的使微管失穩(wěn)定的微管結合蛋白,它通過促使微管解聚和隔離組成微管的Tubulin亞基而使微管失穩(wěn)定,多種激酶可以通過磷酸化和去磷酸化而使Stathmin蛋白的微管失穩(wěn)定活性失活和活化。鼻咽癌是中國人群特有的高發(fā)腫瘤,EB病毒的感染與鼻咽癌的發(fā)生發(fā)展密切相關,EB病毒編碼的潛伏膜蛋白LMP1是一個已經(jīng)確認的瘤致蛋白質(zhì)。前期的研究發(fā)現(xiàn)Stathmin是LMP1調(diào)控網(wǎng)絡中的一個下游分子,LMP1可以通過多條通路調(diào)節(jié)Stathmin的磷酸化,使微管失穩(wěn)定而促使細胞永生化、增強腫瘤細胞增殖、轉移和侵襲。 siRNA是人工RNAi技術中一個重要小分子,其可以激發(fā)與之互補的目標mRNA的沉默。具有高特異性、高效率、可遺傳等重要特性。本研究以期通過構建的靶向Stathmin的siRNA質(zhì)粒沉默其蛋白質(zhì)的表達,降低微管的解聚,增強微管的聚合,從而促使腫瘤細胞凋亡、抑制其增殖、轉移和侵襲。 紫杉醇是作用于細胞微管的主要抗腫瘤藥物之一,它通過促進微管聚合,抑制其解聚,保持微管穩(wěn)定,抑制細胞有絲分裂,具有顯著的放射增敏作用,有著與靶向Stathmin的siRNA類似的作用。因此,本研究擬通過二者聯(lián)合應用以期放大它們對腫瘤細胞的生物學效應。 方法：本文以鼻咽癌細胞CNE1-LMP1為研究模型,用本實驗室構建的靶向Stathmin的siRNA質(zhì)粒為策略,探討靶向Stathmin的siRNA抑制其蛋白質(zhì)的表達而對鼻咽癌細胞的凋亡和增殖、侵襲和遷移的影響,并評價Stathmin的siRNA與抗微管化療藥物聯(lián)合應用的效應。 本研究首先以RT-PCR和Western blot證實本研究所應用的靶向Stathmin的siRNA質(zhì)�？煞褚种票茄拾┘毎鸆NE1-LMP1的Stathmin的mRNA和蛋白質(zhì)的表達水平。以MTT實驗證實靶向siRNA對鼻咽癌細胞生長增殖的抑制作用。利用流式細胞術確定靶向Stathmin的siRNA誘導鼻咽癌細胞的凋亡、細胞周期和線粒體膜電位的改變,以AO/EB染色和TUNEL實驗證實其對鼻咽癌細胞凋亡的誘導作用,以Western blot檢測siRNA轉染后細胞凋亡標志性蛋白質(zhì)Caspase的變化,以期確認鼻咽癌細胞的凋亡及凋亡途徑。 其次,以Western blot檢測轉染靶向Stathmin的siRNA的鼻咽癌細胞中可溶性微管和聚合性微管比例的變化。以間接熒光染色檢測該siRNA質(zhì)粒對微管多聚化的調(diào)節(jié)作用。以劃痕實驗檢測該siRNA對鼻咽癌細胞在二維平面上遷移能力的影響。以Transwell檢測該siRNA對鼻咽癌細胞在三維基質(zhì)中遷移和侵襲能力的調(diào)節(jié)。 再次,以MTT實驗檢測轉染靶向Stathmin的siRNA和紫杉醇聯(lián)合應用對鼻咽癌細胞增殖作用的影響。以流式細胞術檢測二者聯(lián)合應用對細胞的促凋亡作用和細胞周期的改變。以間接熒光法和Western blot檢測轉染該siRNA和紫杉醇聯(lián)合應用對鼻咽癌細胞微管產(chǎn)生影響。 最后,以RT-PCR和Western blot檢測紫杉醇對鼻咽癌細胞以及其它多種高表達Stathmin細胞A375、MGC和Hela中Stathmin表達在mRNA和蛋白水平的調(diào)節(jié)作用。 結果：首先,RT-PCR和Western blot實驗證實了本研究所用的靶向Stathmin的siRNA質(zhì)粒可以明顯抑制鼻咽癌細胞Stathmin的mRNA和蛋白質(zhì)表達水平。MTT實驗檢測細胞的生長曲線顯示該siRNA的轉染對鼻咽癌細胞的增殖也有明顯的抑制作用。流式細胞術分析顯示該siRNA可明顯促進鼻咽癌細胞的凋亡(達到30.9%),使腫瘤細胞阻滯于G2/M期(16.3%)。同時,AO/EB染色、TUNEL實驗以及Western blot檢測凋亡標志性蛋白Caspase-3、8和9也進一步證實該siRNA對細胞凋亡的促進作用。并且,Caspase和線粒體膜電位的檢測也明確該siRNA是通過線粒體凋亡途徑誘導細胞凋亡的。 其次,Western blot證實向鼻咽癌細胞轉染靶向Stathmin的siRNA可明顯增加細胞的聚合性微管的量而減少可溶性微管,使得聚合性微管／可溶性微管的比值明顯提高(P/S=3.43)；間接免疫熒光也顯示轉染siRNA的細胞微管長而成束,熒光強度明顯提高。雖然二維的劃痕實驗并沒有顯示轉染了該siRNA的鼻咽癌細胞運動能力有明顯的改變,但Transwell實驗顯示轉染該siRNA的細胞遷移和侵襲能力都受到明顯抑制。 再次,將靶向Stathmin的siRNA質(zhì)粒和紫杉醇聯(lián)合應用于鼻咽癌細胞CNE1-LMP1。MTT實驗顯示聯(lián)合應用對細胞增殖的抑制效果明顯高于其中任何一種方式的單獨應用。而且,轉染了該siRNA的細胞,其細胞增殖的受抑制的程度與紫杉醇在一定的濃度范圍內(nèi)有劑量關系。間接免疫熒光實驗同樣顯示雖然二者單獨應用都可以一定程度上使被處理的細胞微管變粗和變長,熒光強度有所增強,但二者聯(lián)合應用的結果顯示出微管變得更粗和更長,熒光強度更強。同樣,Western blot檢測細胞的可溶性和聚合性微管的量也顯示雖然二者單獨應用都可以一定程度上使被處理細胞的聚合性微管增加,可溶性微管減少,聚合性微管／可溶性微管的比值有所增加。但二者聯(lián)合使用,細胞中的聚合性微管增加和可溶性微管減少的程度要明顯的多,聚合性微管／可溶性微管的比值顯著提高(P/S=2.05)。 最后,研究還發(fā)現(xiàn)在鼻咽癌細胞以及其它多種高表達Stathmin的腫瘤細胞中,紫杉醇對細胞中的Stathmin表達有明顯的抑制作用,而且,在鼻咽癌細胞中,這種抑制作用與紫杉醇的濃度有一定的劑量效應。 結論：靶向Stathmin的siRNA通過抑制其蛋白質(zhì)的表達而抑制鼻咽癌細胞的增殖,遷移和侵襲,并通過線粒體途徑促進細胞凋亡。這種對鼻咽癌細胞凋亡和增殖,遷移和侵襲的調(diào)控是通過增強微管的聚合而減少其降解,使微管多聚化、使微管穩(wěn)定而實現(xiàn)的。紫杉醇可下調(diào)Stathmin的表達,靶向Stathmin的siRNA與化療藥物紫杉醇對鼻咽癌細胞有聯(lián)合治療效應。這將為鼻咽癌臨床的化學治療提供了一條新的啟示。
[Abstract]:Objective: the microtubule is one of the major components of cytoskeleton, it is important for cell active movement, including the changes in cell morphology, cell migration, cell division and chromosome separation. The differences of structure and function of all cells in the microtubule depend on its subcellular localization and binding of microtubule associated protein.Stathmin is already a clear microtubule instability of microtubule binding protein, it makes microtubule instability by Tubulin subunit to microtubule depolymerization and isolation composed of microtubules, multiple kinases through phosphorylation and dephosphorylation of the Stathmin protein and microtubule instability activity inactivation and activation of nasopharyngeal carcinoma is Chinese people. The high incidence of tumors, closely related to the occurrence and development of nasopharyngeal carcinoma and infection of EB virus, EB virus encoding latent membrane protein LMP1 is a confirmed tumor Protein discovery. Previous studies found that Stathmin is a downstream molecule in LMP1 regulatory network. LMP1 can regulate phosphorylation of Stathmin through multiple pathways, causing microtubule instability, promoting cell immortalization and enhancing tumor cell proliferation, metastasis and invasion.
SiRNA is a small molecule artificial RNAi technology, which can stimulate and complementary target mRNA silencing. With high specificity, high efficiency, genetic and other important features. In this study, in order to silence the expression of siRNA protein of plasmid Stathmin by constructing the target, reduce the microtubule depolymerization of microtubules enhanced the polymerization, thereby inducing tumor cell apoptosis, inhibit the proliferation, metastasis and invasion.
Taxol is one of the main antitumor drugs to cells, it is by promoting microtubule polymerization, inhibit the depolymerization of microtubules remain stable, inhibition of cell mitosis, and has remarkable radiosensitizing effect, with Stathmin targeting siRNA similar role. Therefore, this study proposed by the combination of the two methods in order to enlarge their biological effect on tumor cells.
Methods: the nasopharyngeal carcinoma cell line CNE1-LMP1 as a model constructed by our laboratory siRNA plasmid targeting Stathmin strategy, to investigate the expression of Stathmin targeting siRNA inhibits the protein on nasopharyngeal carcinoma cell apoptosis and proliferation, invasion and migration in vitro, and assess the effect of Stathmin and siRNA price antimicrotubule chemotherapy application.
This study firstly by RT-PCR and Western blot confirmed that the expression level of mRNA protein and the research on Application of the siRNA plasmid targeting Stathmin could inhibit CNE1-LMP1 nasopharyngeal carcinoma cell line Stathmin. MTT experiment confirmed that targeting inhibition of siRNA on proliferation of nasopharyngeal carcinoma cells. Using flow cytometry to determine apoptosis of nasopharyngeal carcinoma cell targeting Stathmin siRNA, cell cycle and mitochondrial membrane potential changes with AO/EB staining and TUNEL experiments showed that the induction of apoptosis of nasopharyngeal carcinoma cells, to change Western blot detection siRNA transfected cells apoptosis marker protein Caspase, in order to confirm the apoptosis and apoptosis of nasopharyngeal carcinoma cells.
Secondly, the changes of soluble microtubule with Western detected by blot targeting Stathmin siRNA in nasopharyngeal carcinoma cells and the proportion of polymeric microtubules by indirect fluorescent staining. The siRNA plasmid multimerization of microtubule regulation. In order to influence the scratch assay siRNA on nasopharyngeal carcinoma cell migration in the two-dimensional plane. Detected by Transwell the regulation of siRNA on nasopharyngeal carcinoma cells in three-dimensional matrix in migration and invasion.
Again, to transfect the target detection of MTT experiment to Stathmin siRNA and paclitaxel on the proliferation of nasopharyngeal carcinoma cells. The change was measured by flow cytometry two combined application of cell apoptosis and cell cycle. By indirect immunofluorescence and Western blot detection combined with the transfection of siRNA and paclitaxel the influence on NPC cell microtubules.
Finally, we used RT-PCR and Western blot to detect paclitaxel regulating the expression of Stathmin in A375, MGC and Hela of nasopharyngeal carcinoma cells and many other high expression Stathmin cells at mRNA and protein level.
Results: first, RT-PCR and Western blot experiments confirmed the growth curve of mRNA.MTT and protein expression assay were used in this study targeted siRNA plasmid Stathmin could inhibit the proliferation of nasopharyngeal carcinoma cell line Stathmin showed that transfection of the siRNA in nasopharyngeal carcinoma cells also significantly inhibited. The analysis shows that the siRNA significantly to promote the apoptosis of nasopharyngeal carcinoma cells by flow cytometry (up to 30.9%), the tumor cells in G2/M phase (16.3%). At the same time, AO/EB staining, TUNEL assay and Western blot apoptosis protein marker Caspase-3,8 and 9 confirmed the role of siRNA on cell apoptosis. And the detection of Caspase and mitochondrial membrane potential the siRNA is also clearly induced apoptosis through the mitochondrial apoptotic pathway.
Secondly, Western blot confirmed to nasopharyngeal carcinoma cells transfected with Stathmin targeting siRNA could significantly increase the amount of polymerization of microtubule cells and reduce the ratio of soluble tubulin, microtubule polymerization / soluble microtubules increased significantly (P/S=3.43); indirect immunofluorescence also showed that transfection of siRNA cells with long microtubule bundles, the fluorescence intensity was obviously improved. Although the two-dimensional scratch test did not show the movement ability of the transfected nasopharyngeal carcinoma cell siRNA has obvious change, but Transwell test showed that cell migration and invasion of the transfected siRNA were inhibited.
Again, the siRNA plasmid targeting Stathmin and paclitaxel in nasopharyngeal carcinoma cell CNE1-LMP1.MTT assay showed that the inhibitory effect of combined application of cell proliferation was significantly higher than that of the single application in any way. Moreover, the transfected siRNA cells, the cell proliferation was inhibited and the degree of paclitaxel dose-dependent in concentration within a certain range. Indirect immunofluorescence assay also showed that although the two separate applications can make the processed microtubule thicker and longer to a certain extent, the fluorescence intensity increased, but the combination of the two results show that microtubules become thicker and longer, higher fluorescence intensity. Similarly, soluble Western blot detection cells and aggregation of microtubules also show that although the two separate applications, can to some extent make polymerization of MTS treatment increased soluble microtubule The ratio of aggregated microtubules / soluble microtubules increased. However, the combination of two components increased the number of aggregated microtubules and the decrease of soluble microtubules in cells. The ratio of polymerized microtubules / soluble microtubules increased significantly (P/S=2.05).
Finally, the study also found in NPC cells and other higher expression of Stathmin in tumor cells, and paclitaxel inhibited significantly in the cells Stathmin expression in nasopharyngeal carcinoma cells, and the inhibitory effect of paclitaxel concentration and the dose effect.
Conclusion: the Stathmin targeting siRNA can inhibit the expression of the protein and the inhibition of nasopharyngeal carcinoma cell proliferation, migration and invasion, and promote cell apoptosis through the mitochondrial pathway. The apoptosis and proliferation of nasopharyngeal carcinoma cells, the regulation of migration and invasion and reduce its degradation by enhancing the polymerization of microtubules, microtubule polymerization,. Microtubule stabilizing and implementation. The expression of paclitaxel can downregulate Stathmin, Stathmin targeting siRNA and paclitaxel in nasopharyngeal carcinoma cells with combined treatment effect. This will provide a new inspiration for the chemical treatment of nasopharyngeal carcinoma.

【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R739.63

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