抑制HES1、HEY1對涎腺腺樣囊性癌細(xì)胞功能的影響
發(fā)布時間：2018-08-28 12:51
【摘要】：涎腺腺樣囊性癌(SACC)是好發(fā)于腮腺、頜下腺及小涎腺的口腔頜面部惡性腫瘤之一,具有嗜神經(jīng)侵襲以及遠(yuǎn)處轉(zhuǎn)移兩大生物學(xué)特性,其臨床生物學(xué)特性表現(xiàn)為感覺和(或)運(yùn)動神經(jīng)功能障礙,易通過血行轉(zhuǎn)移至肺,術(shù)后易復(fù)發(fā),預(yù)后較差。本課題組通過前期大量的實(shí)驗(yàn)研究,發(fā)現(xiàn)Notch信號通路與腫瘤的發(fā)生發(fā)展密切相關(guān),通路中的受體基因Notch1能夠作為促癌基因促進(jìn)涎腺腺樣囊性癌的增殖、侵襲及遷移。國內(nèi)外眾多研究表明,HES1、HEY1基因是Notch1信號通路的下游基因,二者可能在多種腫瘤的發(fā)展演化中發(fā)揮著癌基因的作用。本研究在課題組前期研究的基礎(chǔ)上,篩選出Notch1信號通路的下游基因HES1、HEY1,研究其與涎腺腺樣囊性癌細(xì)胞增殖、侵襲、遷移的關(guān)系并對可能的機(jī)制進(jìn)行探討。本研究按照基因的篩選和細(xì)胞功能學(xué)實(shí)驗(yàn)分為兩部分,分述如下:一、在SACC細(xì)胞中利用Real-time PCR技術(shù)篩選出與Notch1變化相關(guān)的下游基因,并在組織水平驗(yàn)證其表達(dá)。目的:篩選與Notch1表達(dá)變化相關(guān)的下游靶基因。方法:根據(jù)文獻(xiàn)選取的常見的Notch1的下游基因設(shè)計(jì)引物。用Notch1的si RNA轉(zhuǎn)染SACC-83細(xì)胞,通過Real-time PCR技術(shù)檢測Notch1下游基因的表達(dá)量;構(gòu)建Notch1基因重組腺病毒表達(dá)載體,感染SACC-83細(xì)胞,應(yīng)用Real-time PCR技術(shù)研究其下游基因的表達(dá)變化。收集涎腺腺樣囊性癌組織和癌旁組織的石蠟標(biāo)本,利用篩選出的下游基因的相應(yīng)抗體進(jìn)行免疫組化,經(jīng)2名病理科醫(yī)師閱片評分后統(tǒng)計(jì)結(jié)果。結(jié)果:Real-time PCR檢測結(jié)果顯示Notch1表達(dá)下降后,HES1、HEY1的表達(dá)也相應(yīng)降低;而Notch1表達(dá)升高后,HES1、HEY1的表達(dá)也隨之上升。涎腺腺樣囊性癌癌組織標(biāo)本中HES1、HEY1的表達(dá)高于癌旁組織。結(jié)論:HES1、HEY1的表達(dá)變化與Notch1的表達(dá)變化呈正相關(guān)關(guān)系。二、利用si RNA干擾技術(shù)探討HES1、HEY1對SACC細(xì)胞功能的影響。目的:研究HES1、HEY1在SACC細(xì)胞增殖、侵襲以及遷移中的作用。方法:設(shè)計(jì)合成HES1、HEY1的si RNA,轉(zhuǎn)染SACC-83細(xì)胞,HES1、HEY1的表達(dá)量的變化通過Real-time PCR技術(shù)進(jìn)行驗(yàn)證,篩選出兩條干擾有效的si RNA,轉(zhuǎn)染SACC-83細(xì)胞使其HES1、HEY1的表達(dá)顯著下調(diào)后,采用CCK8法、平板克隆形成實(shí)驗(yàn)研究細(xì)胞增殖能力的變化;采用細(xì)胞劃痕愈合法研究細(xì)胞遷移能力的變化;采用侵襲小室法研究細(xì)胞侵襲能力的變化;采用流式細(xì)胞術(shù)研究細(xì)胞周期和凋亡的變化。結(jié)果:Real-time PCR結(jié)果顯示HES1、HEY1表達(dá)下降;CCK8和平板克隆形成實(shí)驗(yàn)證實(shí)HES1、HEY1轉(zhuǎn)染后,細(xì)胞增殖能力弱于陰性對照組;侵襲小室、劃痕愈合實(shí)驗(yàn)結(jié)果表明,HES1、HEY1表達(dá)下降后,SACC-83細(xì)胞的侵襲、遷移能力也隨之下降;流式細(xì)胞術(shù)的結(jié)果發(fā)現(xiàn)轉(zhuǎn)染HES1、HEY1后細(xì)胞的早期和晚期凋亡率升高,而細(xì)胞周期和對照組相比沒有差異。結(jié)論:抑制HES1、HEY1基因的表達(dá)后SACC細(xì)胞的增殖、侵襲和遷移能力均下降,而細(xì)胞凋亡率升高。
[Abstract]:Salivary adenoid cystic carcinoma (SACC) is one of the most common oral and maxillofacial malignancies in parotid, submandibular and small salivary glands. Its clinical biological characteristics were sensory and / or motor nerve dysfunction, which were easily metastasized to the lung by blood, and were easy to recur after operation, and the prognosis was poor. Through a large number of experimental studies, we found that the Notch signaling pathway is closely related to the occurrence and development of the tumor. The receptor gene Notch1 in the pathway can promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma (SCC) as a gene to promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma. Many studies at home and abroad have shown that the HES1 gene is the downstream gene of Notch1 signaling pathway, and both of them may play an important role in the development and evolution of many kinds of tumors. On the basis of the previous study, the downstream gene HES1,HEY1, of Notch1 signaling pathway was selected to study its relationship with salivary adenoid cystic carcinoma cell proliferation, invasion and migration, and the possible mechanism was discussed. This study was divided into two parts according to gene screening and cell function experiment. Firstly, the downstream genes associated with Notch1 changes were screened by Real-time PCR technique in SACC cells, and their expression was verified at the tissue level. Objective: to screen the downstream target genes associated with the change of Notch1 expression. Methods: primers were designed according to the common downstream genes of Notch1. SACC-83 cells were transfected with si RNA of Notch1 and the expression of downstream gene of Notch1 was detected by Real-time PCR. The recombinant adenovirus expression vector of Notch1 gene was constructed and infected with SACC-83 cells. The expression of downstream gene was studied by Real-time PCR technique. Paraffin specimens of salivary adenoid cystic carcinoma and paracancerous tissues were collected. The corresponding antibodies of the downstream genes were used for immunohistochemistry. The results were evaluated by two pathologists. Results the expression of HES1 and HEY1 was also decreased after the decrease of Notch1 expression, and the expression of HES 1 + HEY1 was also increased after the increase of Notch1 expression. The expression of HES1,HEY1 in salivary adenoid cystic carcinoma tissues was higher than that in paracancerous tissues. Conclusion there is a positive correlation between the expression of Notch1 and the changes of the expression of HeS1 and HEY1. Secondly, the effect of HES1,HEY1 on the function of SACC cells was studied by si RNA interference technique. Aim: to investigate the role of HES1,HEY1 in the proliferation, invasion and migration of SACC cells. Methods: the changes of the expression level of HES1,HEY1 si RNA, transfected SACC-83 cells were verified by Real-time PCR technique. Two interfering si RNA, transfected SACC-83 cells were screened out and their HES1,HEY1 expression was significantly down-regulated. CCK8 assay was used. The changes of cell proliferation ability were studied by plate clone formation assay, cell migration ability was studied by cell scratch healing method, and cell invasion ability was studied by invasive chamber method. The changes of cell cycle and apoptosis were studied by flow cytometry. Results the cell proliferation ability after transfection of HES1,HEY1 was weaker than that of negative control group, the cell proliferation ability was weaker after transfection of HES1,HEY1, and the invasion of SACC-83 cells after HES1,HEY1 transfection and scratch healing test showed that the expression of HES 1 and HEY1 decreased. Flow cytometry showed that the early and late apoptosis rate of HES1,HEY1 transfected cells increased, but the cell cycle had no difference compared with the control group. Conclusion: after inhibiting the expression of HES1,HEY1 gene, the proliferation, invasion and migration of SACC cells decreased, while the rate of apoptosis increased.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R739.87
[Abstract]:Salivary adenoid cystic carcinoma (SACC) is one of the most common oral and maxillofacial malignancies in parotid, submandibular and small salivary glands. Its clinical biological characteristics were sensory and / or motor nerve dysfunction, which were easily metastasized to the lung by blood, and were easy to recur after operation, and the prognosis was poor. Through a large number of experimental studies, we found that the Notch signaling pathway is closely related to the occurrence and development of the tumor. The receptor gene Notch1 in the pathway can promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma (SCC) as a gene to promote the proliferation, invasion and migration of salivary adenoid cystic carcinoma. Many studies at home and abroad have shown that the HES1 gene is the downstream gene of Notch1 signaling pathway, and both of them may play an important role in the development and evolution of many kinds of tumors. On the basis of the previous study, the downstream gene HES1,HEY1, of Notch1 signaling pathway was selected to study its relationship with salivary adenoid cystic carcinoma cell proliferation, invasion and migration, and the possible mechanism was discussed. This study was divided into two parts according to gene screening and cell function experiment. Firstly, the downstream genes associated with Notch1 changes were screened by Real-time PCR technique in SACC cells, and their expression was verified at the tissue level. Objective: to screen the downstream target genes associated with the change of Notch1 expression. Methods: primers were designed according to the common downstream genes of Notch1. SACC-83 cells were transfected with si RNA of Notch1 and the expression of downstream gene of Notch1 was detected by Real-time PCR. The recombinant adenovirus expression vector of Notch1 gene was constructed and infected with SACC-83 cells. The expression of downstream gene was studied by Real-time PCR technique. Paraffin specimens of salivary adenoid cystic carcinoma and paracancerous tissues were collected. The corresponding antibodies of the downstream genes were used for immunohistochemistry. The results were evaluated by two pathologists. Results the expression of HES1 and HEY1 was also decreased after the decrease of Notch1 expression, and the expression of HES 1 + HEY1 was also increased after the increase of Notch1 expression. The expression of HES1,HEY1 in salivary adenoid cystic carcinoma tissues was higher than that in paracancerous tissues. Conclusion there is a positive correlation between the expression of Notch1 and the changes of the expression of HeS1 and HEY1. Secondly, the effect of HES1,HEY1 on the function of SACC cells was studied by si RNA interference technique. Aim: to investigate the role of HES1,HEY1 in the proliferation, invasion and migration of SACC cells. Methods: the changes of the expression level of HES1,HEY1 si RNA, transfected SACC-83 cells were verified by Real-time PCR technique. Two interfering si RNA, transfected SACC-83 cells were screened out and their HES1,HEY1 expression was significantly down-regulated. CCK8 assay was used. The changes of cell proliferation ability were studied by plate clone formation assay, cell migration ability was studied by cell scratch healing method, and cell invasion ability was studied by invasive chamber method. The changes of cell cycle and apoptosis were studied by flow cytometry. Results the cell proliferation ability after transfection of HES1,HEY1 was weaker than that of negative control group, the cell proliferation ability was weaker after transfection of HES1,HEY1, and the invasion of SACC-83 cells after HES1,HEY1 transfection and scratch healing test showed that the expression of HES 1 and HEY1 decreased. Flow cytometry showed that the early and late apoptosis rate of HES1,HEY1 transfected cells increased, but the cell cycle had no difference compared with the control group. Conclusion: after inhibiting the expression of HES1,HEY1 gene, the proliferation, invasion and migration of SACC cells decreased, while the rate of apoptosis increased.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R739.87
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 王春華;鄭志z，
本文編號：2209447
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