【摘要】：目的：CD147,又名基質(zhì)金屬蛋白酶誘導(dǎo)因子,屬于免疫球蛋白超家族的細(xì)胞膜表面高糖基化蛋白,在許多惡性腫瘤中表達(dá)增高,能促進(jìn)腫瘤的存活、生長、侵襲與轉(zhuǎn)移。CD147高表達(dá)還能提高腫瘤細(xì)胞對(duì)化療藥物的耐藥性。相關(guān)研究報(bào)道,CD147在喉癌細(xì)胞株Hep2中高表達(dá),因此利用本實(shí)驗(yàn)室前期構(gòu)建的CD147ShRNA真核表達(dá)載體,利用RNAi技術(shù)高效特異地沉默喉癌細(xì)胞株Hep2中CD147的表達(dá),以分析其在喉癌侵襲、轉(zhuǎn)移及其對(duì)順鉑敏感性的作用機(jī)制。 研究方法： 1)將前期構(gòu)建的重組質(zhì)粒pSilencer/shRNA-control、pSilencer/shRNA1、pSilencer/shRNA2穩(wěn)定轉(zhuǎn)染至Hep2細(xì)胞中,采用RT-PCR法和Western法分別檢測(cè)轉(zhuǎn)染細(xì)胞中CD147mRNA和蛋白表達(dá)的變化； 2)采用MTT法檢測(cè)轉(zhuǎn)染pSilencer/shRNA-control、pSilencer/shRNA1、pSilencer/shRNA2后Hep2細(xì)胞增殖水平的變化及其對(duì)化療藥物順鉑的敏感性； 3)采用Transwell法檢測(cè)CD147shRNA對(duì)Hep2細(xì)胞的體外侵襲能力的影響； 4)采用明膠酶譜法檢測(cè)轉(zhuǎn)染pSilencer/shRNA-control、pSilencer/shRNAl、pSilencer/shRNA2后Hep2細(xì)胞培養(yǎng)液中MMP-2和MMP-9的活力； 5)采用裸鼠皮下異種移植模型觀察CD147shRNA對(duì)Hep2細(xì)胞在體內(nèi)侵襲和轉(zhuǎn)移能力的影響。 結(jié)果： 1)所構(gòu)建載體經(jīng)陽離子脂質(zhì)體介導(dǎo)轉(zhuǎn)染人喉癌細(xì)胞株Hep2,G418篩選后有限稀釋法成功獲得陽性克隆株,RT-PCR法和western法顯示兩組ShRNA均高效而特異地沉默了喉癌細(xì)胞Hep2中CD147的表達(dá)。 2)MTT結(jié)果顯示,在轉(zhuǎn)染72h、96h、120h后,Hep2/shRNA1和Hep2/shRNA2組細(xì)胞增殖能力分別下降到了77.34%(P0.01)和70.43%(P0.01),62.99%(P0.01)和73.76%(P0.01),67.49%(P0.01)和61.37%(P0.01)。Hep2/shRNA1和Hep2/shRNA2組細(xì)胞對(duì)順鉑敏感性也明顯增高。 3)細(xì)胞體外侵襲力測(cè)定結(jié)果顯示：Hep2/shRNA1和Hep2/shRNA2組細(xì)胞的穿膜細(xì)胞數(shù)低于Hep2和Hep2/shRNA-control組。 4)明膠酶譜結(jié)果顯示：Hep2/shRNA1和Hep2/shRNA2組的MMP-2和MMP-9明膠酶活性顯著降低。 5) Hep2/shRNAl和Hep2/shRNA2組細(xì)胞在裸鼠皮下的成瘤體積較Hep2組分別降低’至38.35%和36.08%。 結(jié)論： 1)RNAi技術(shù)特異性沉默Hep2細(xì)胞CD147表達(dá)后,細(xì)胞增殖能力受到明顯抑制,說明CD147能促進(jìn)喉癌細(xì)胞的生長。 2)RNAi技術(shù)特異性沉默Hep2細(xì)胞CD147表達(dá)后,細(xì)胞對(duì)化療藥物順鉑的敏感性也明顯增強(qiáng),說明CD147可以作為化療藥物的一個(gè)靶分子。 3)Hep2細(xì)胞的CD147基因經(jīng)RNAi技術(shù)特異性沉默后,細(xì)胞體外侵襲能力和體內(nèi)成瘤能力明顯降低,說明CD147與喉癌的進(jìn)展相關(guān),可能為一種新的喉癌治療靶分子。
[Abstract]:Objective: CD147, also known as matrix metalloproteinase-inducible factor, belongs to the immunoglobulin superfamily with high glycosylated protein on the surface of the cell membrane. It is highly expressed in many malignant tumors and can promote the survival and growth of the tumor. High expression of invasion and metastasis. CD 147 also increased the resistance of tumor cells to chemotherapeutic drugs. The high expression of CD147 in laryngeal carcinoma cell line Hep2 was reported. Therefore, using the eukaryotic expression vector of CD147 ShRNA constructed in our laboratory, the expression of CD147 in laryngeal carcinoma cell line Hep2 was effectively and specifically silenced by RNAi technique to analyze the invasion of CD147 in laryngeal carcinoma cell line Hep2. The mechanism of metastasis and its sensitivity to cisplatin. Methods: 1) Recombinant plasmid pSilencer / shRNA-control pSilencer-1 pSilencer / shRNA2 was stably transfected into Hep2 cells. The expression of CD147 mRNA and protein in transfected cells was detected by RT-PCR and Western methods. 2) MTT assay was used to detect the proliferation level of Hep2 cells and its sensitivity to cisplatin after transfection of pSilencer- control pSilencer- / shRNA1pSilencer- pSilencer- rshRNA2.3The effect of CD147 shRNA on the invasiveness of Hep2 cells in vitro was detected by Transwell assay. 4) the activity of MMP-2 and MMP-9 in Hep2 cell culture medium after pSilencer- rshRNA-control-pSilencer- control pSilencer- psilencerrshRNA2 transfection was detected by gelatin zymogram. 5) the effect of CD147 shRNA on invasion and metastasis of Hep2 cells in vivo was observed by subcutaneous xenotransplantation in nude mice. Results: 1) the constructed vector was transfected by cationic liposome into human laryngeal carcinoma cell line Hep2G418. After screening with limited dilution method, positive clones were successfully obtained. RT-PCR and western showed that both groups were highly efficient and specific in silencing the larynx. Expression of CD147 in Hep2 cells. 2) MTT assay showed that, The proliferative ability of Hep2 / rshRNA1 and Hep2 / rshRNA2 groups decreased to 77.34% (P0.01) and 70.43% (P0.01), respectively, 62.99% (P0.01) and 73.76% (P0.01) 67.49% (P0.01) and 61.37% (P0.01). Hep2rshRNA1 and Hep2rshRNA2 cells were significantly more sensitive to cisplatin in vitro. The results of gelatinase analysis showed that the MMP-2 and MMP-9 gelatinase activities of the two groups were significantly lower than those of the Hep2 / rshRNA1 and Hep2 / shRNA2 groups. 5) the subcutaneous tumorigenic volume of Hep2p-shRNAl and Hep2p-shRNA2 cells in nude mice was higher than that of Hep2 fractions. Don't lower'to 38.35% and 36.08%. Conclusion: 1) after silencing the expression of CD147 in Hep2 cells by RNAi, the proliferation of Hep2 cells was significantly inhibited, indicating that CD147 could promote the growth of laryngeal cancer cells. 2) the expression of CD147 in Hep2 cells was specifically silenced by RNAi. The sensitivity of CD147 cells to cisplatin was also significantly increased, indicating that CD147 could be used as a target molecule of chemotherapeutic drugs. 3) the CD147 gene of Hep2 cells was silenced by RNAi technique. The ability of invasiveness in vitro and tumorigenesis in vivo were significantly decreased, which indicated that CD147 might be a new target molecule for laryngeal cancer treatment, which was related to the progression of laryngeal carcinoma.
【學(xué)位授予單位】：南京師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R739.65

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 劉鵬飛,唐文淵,朱政鳴;CD147和MMP-2、MMP-9與膠質(zhì)細(xì)胞瘤侵襲性的關(guān)系[J];重慶醫(yī)學(xué);2005年11期

2 周宏偉;蔣建利;郭衛(wèi)平;陳志南;張洪新;;βig-h3在肝癌細(xì)胞系中差異表達(dá)的篩選和鑒定[J];第四軍醫(yī)大學(xué)學(xué)報(bào);2006年12期

3 周筠,朱平,蔣建利,陳志南,賈俊峰,葉卉,唐浩,姚西英,樊春梅,楊勇;CD147在單核-巨噬細(xì)胞促進(jìn)成纖維細(xì)胞分泌活化基質(zhì)金屬蛋白酶中的作用及意義[J];中華風(fēng)濕病學(xué)雜志;2005年05期

4 田福亮;鮑倩;唐志鵬;王書奎;;CD147在腫瘤血管生成中的作用[J];國際檢驗(yàn)醫(yī)學(xué)雜志;2011年02期

5 李吉;CD147及其與腫瘤關(guān)系的研究進(jìn)展[J];國外醫(yī)學(xué)(腫瘤學(xué)分冊(cè));2003年04期

6 李浩,游潮;CD147/EMMPRIN與腦膠質(zhì)瘤關(guān)系的研究進(jìn)展[J];華西醫(yī)學(xué);2005年02期

7 郭衛(wèi)平;周宏偉;蔣建利;張洪新;;Differential expression of βig-h3 in human hepatoma cell lines[J];Journal of Medical Colleges of PLA;2007年01期

8 何慧嬋;鐘惟德;;腫瘤中CD147誘導(dǎo)MMPs的分子機(jī)制[J];現(xiàn)代泌尿生殖腫瘤雜志;2009年02期

9 ;Novel Tumor-associated Antigen of Hepatocellular Carcinoma Defined by Monoclonal Antibody E4-65[J];Acta Biochimica et Biophysica Sinica;2007年05期

10 賈莉;康曉慧;張嘉寧;;CD147在腫瘤發(fā)展和組織修復(fù)中的作用[J];生命的化學(xué);2007年02期

相關(guān)博士學(xué)位論文 前10條

1 李存孝;CD147在人骨肉瘤細(xì)胞中的表達(dá)及其反義RNA對(duì)骨肉瘤侵襲和轉(zhuǎn)移的影響[D];第四軍醫(yī)大學(xué);2005年

2 郭永川;Ki67核抗原、MMP-2（基質(zhì)金屬蛋白酶-2）、TIMP-2（基質(zhì)金屬蛋白酶-2抑制因子）、CD147、LN-R（層粘連蛋白受體）的表達(dá)與侵襲性垂體腺瘤的關(guān)系[D];吉林大學(xué);2005年

3 賈莉;CD147糖基化在小鼠肝癌細(xì)胞淋巴道轉(zhuǎn)移中的作用[D];大連醫(yī)科大學(xué);2006年

4 霍鋼;侵襲性垂體腺瘤的影像學(xué)與實(shí)驗(yàn)研究[D];重慶醫(yī)科大學(xué);2006年

5 封青川;人cd147的克隆、表達(dá)、純化以其功能研究[D];中國醫(yī)科大學(xué);2006年

6 李浩;CD147、MMP-2在人腦膠質(zhì)瘤中的表達(dá)及CD147單抗抑制U251細(xì)胞侵襲性的實(shí)驗(yàn)研究[D];四川大學(xué);2007年

7 黨懿;CD147在急性心肌梗死大鼠中的表達(dá)及其與基質(zhì)金屬蛋白酶-9關(guān)系的研究[D];河北醫(yī)科大學(xué);2008年

8 徐靜;腫瘤新靶點(diǎn)HAb18G/CD147與癌—基質(zhì)的交互作用[D];第四軍醫(yī)大學(xué);2008年

9 楊向民;全人抗體高效真核表達(dá)載體的構(gòu)建與表面重塑抗體rCAb1[D];第四軍醫(yī)大學(xué);2008年

10 余曉玲;HAb18G/CD147晶體結(jié)構(gòu)—免疫球蛋白超家族新二聚化模式的發(fā)現(xiàn)[D];第四軍醫(yī)大學(xué);2008年

相關(guān)碩士學(xué)位論文 前10條

1 胡杰;EMMPRIN在人肝癌細(xì)胞SMMC-7721中對(duì)多藥耐藥的調(diào)節(jié)作用[D];山西醫(yī)科大學(xué);2011年

2 殷悅;CD147和MMP-9的表達(dá)與涎腺腫瘤侵襲性的關(guān)系[D];吉林大學(xué);2011年

3 周嬌;CD147、MMP-2在口腔鱗癌中的表達(dá)及意義[D];吉林大學(xué);2011年

4 羅美;CD147在肝癌肝移植患者組織中的表達(dá)及其預(yù)后意義[D];遼寧醫(yī)學(xué)院;2011年

5 梁宇翔;CD147和VEGF在終末期腎細(xì)胞癌中的表達(dá)及意義[D];廣州醫(yī)學(xué)院;2010年

6 郭云山;HAb18G/CD147通過調(diào)節(jié)鈣信號(hào)依賴的ERK1/2活化調(diào)控肝癌細(xì)胞周期進(jìn)程的研究[D];第四軍醫(yī)大學(xué);2011年

7 侯向華;CD147在卵巢癌中的表達(dá)調(diào)節(jié)作用及人卵巢癌原位移植裸鼠模型的構(gòu)建[D];第四軍醫(yī)大學(xué);2004年

8 鄒偉;RNAi沉默CD147基因?qū)β殉舶┘?xì)胞HO-8910pm生物學(xué)行為的影響[D];第四軍醫(yī)大學(xué);2005年

9 田加坤;MMP-2、TIMP-2及CD147與垂體腺瘤侵襲性關(guān)系的研究[D];重慶醫(yī)科大學(xué);2005年

10 楊延冬;原癌基因c-Mct和分子CD147在子宮內(nèi)膜癌中的表達(dá)及其臨床意義[D];青島大學(xué);2006年

，

本文編號(hào)：2123247