發(fā)布時(shí)間：2018-05-04 20:16

  本文選題：裸鼠 + 萊菔硫烷�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:涎腺腺樣囊性癌(salivary adenoid cystic carcinoma,SACC)是一種惡性腫瘤,主要來(lái)源于涎腺上皮。具有如下特點(diǎn):局部浸潤(rùn)性非常強(qiáng),極易早期侵犯神經(jīng)和血管,所以癌細(xì)胞容易沿血液擴(kuò)散到遠(yuǎn)處。采用手術(shù)方法切除腫瘤后,仍然有較高的局部復(fù)發(fā)率和遠(yuǎn)處轉(zhuǎn)移率,SACC的肺部轉(zhuǎn)移率遠(yuǎn)遠(yuǎn)高于頜面部的其它腫瘤。截止目前,人類在SACC方面的相關(guān)研究成果還不能清楚地闡述它的發(fā)病及轉(zhuǎn)移機(jī)制,所以我們要繼續(xù)研究SACC的機(jī)制,爭(zhēng)取從分子和蛋白的層面,搞清楚腺樣囊性癌的作用機(jī)理,為早日在臨床上應(yīng)用新的抗癌方法或抗癌藥物做出貢獻(xiàn)。萊菔硫烷(sulforaphane,SFN)是從十字花科蔬菜中提取出來(lái)的一種植物性物質(zhì),它屬于異硫氰酸鹽(isothiocyanates,ITCS)類,這種物質(zhì)有抗癌功效。我們前期關(guān)于萊菔硫烷的體外研究成果顯示,萊菔硫烷可以提升半胱天冬酶(Caspase)的功能,加速了腫瘤細(xì)胞的凋亡,從而抑制了腺樣囊性癌細(xì)胞系A(chǔ)CC-M,在這個(gè)過(guò)程中,Bcl-2家族成員也起到了重要作用。凋亡抑制蛋白家族(inhibitor of apoptosis protein,IAP)主要功能是調(diào)節(jié)細(xì)胞凋亡,可以通過(guò)抑制Caspase來(lái)抑制細(xì)胞凋亡,還能參與細(xì)胞周期與細(xì)胞分裂的調(diào)節(jié)等。其家族成員共有八位,分別是c-IAP1、c-IAP2、Survivin、Livin、X-IAP、NAIP、Bruce、ILP-2。c-IAP1、c-IAP2、X-IAP能夠直接抑制Caspase-3、-7、-9,而Survivin不但能夠抑制Caspase-3、-7,還能增強(qiáng)X-IAP等其它IAP家族成員的抗凋亡作用。Livin的作用是降低Caspase-3、-7、-9的活性,ILP-2可以調(diào)節(jié)Bcl-2的表達(dá),并且使其升高,降低Caspase-9的活性,達(dá)到抑制細(xì)胞凋亡的目的。NAIP的研究主要集中在神經(jīng)系統(tǒng),Bruce可抑制Caspase-9及Smac的活性。在萊菔硫烷抑制腺樣囊性癌細(xì)胞系A(chǔ)CC-M的增殖過(guò)程中,凋亡抑制蛋白家族成員是不是起到一定的作用,在經(jīng)過(guò)SFN處理的人腺樣囊性癌裸鼠移植瘤組織中,凋亡抑制蛋白是否表達(dá)異常?以往的研究成果尚不能合理的解釋這一問(wèn)題。目的:觀察移植瘤被萊菔硫烷抑制的過(guò)程中,IAP家族成員的作用如何,是否受到抑制,家族中各個(gè)成員的表現(xiàn)是否一致,有沒(méi)有其它蛋白分子干擾IAP蛋白的作用,研究其可能的分子作用機(jī)制,為日后臨床應(yīng)用萊菔硫烷抗癌提供理論依據(jù)。方法:1材料1.1人涎腺腺樣囊性癌細(xì)胞株ACC-M購(gòu)自上海交大頜面外科實(shí)驗(yàn)室,該細(xì)胞株建于1995年。1.2萊菔硫烷(SFN)購(gòu)自美國(guó)LKT實(shí)驗(yàn)室,純度≥99%。1.3裸鼠自北京軍事醫(yī)學(xué)科學(xué)院購(gòu)買,雄性10只,4~6周齡,體重為16~20克。1.4抗體從英國(guó)Abcam公司購(gòu)買的X-IAP多克隆抗體,c-IAP2多克隆抗體,c-IAP1多克隆抗體,Livin多克隆抗體,從美國(guó)AR公司購(gòu)買Survivin多克隆抗體,自無(wú)錫傲銳東源生物科技有限公司購(gòu)買二抗酶標(biāo)羊抗小鼠/兔Ig G聚合物。2實(shí)驗(yàn)方法2.1細(xì)胞培養(yǎng)將凍存的人涎腺腺樣囊性癌細(xì)胞復(fù)蘇,然后在37度恒溫培養(yǎng)箱中培養(yǎng)。細(xì)胞密度達(dá)到要求時(shí),開(kāi)始傳代,最后收集細(xì)胞懸液。2.2移植瘤的建立碘酊消毒裸鼠的左前背部以及右后背部,然后將細(xì)胞懸液分別注射于上述部位,經(jīng)口腔給藥,實(shí)驗(yàn)組予以萊菔硫烷(6 mmol SFN/0.1ml PBS)口服,而對(duì)照組口服等量的PBS,每周分三次給藥。2.3解剖瘤體三周以后開(kāi)始將裸鼠處死,然后解剖瘤體,清除掉瘤體表面的血管、壞死組織。然后用石蠟包埋腫瘤組織塊,蠟塊準(zhǔn)備完畢后行免疫組化實(shí)驗(yàn)。2.4免疫組化本實(shí)驗(yàn)采用免疫組化的方法,觀察IAP家族成員中c-IAP1、c-IAP2、Survivin、Livin、XIAP五種蛋白在裸鼠移植瘤組織中的表達(dá)情況,并統(tǒng)計(jì)染色陽(yáng)性細(xì)胞數(shù)目。2.5數(shù)據(jù)的統(tǒng)計(jì)處理本研究有實(shí)驗(yàn)、對(duì)照兩個(gè)組,每組10個(gè)樣本,統(tǒng)計(jì)每個(gè)樣本的隨機(jī)5個(gè)視野陽(yáng)性細(xì)胞總數(shù),用t檢驗(yàn)的方法進(jìn)行分析,最后得出結(jié)論。結(jié)果:1實(shí)驗(yàn)組中,五個(gè)視野中c-IAP1陽(yáng)性細(xì)胞總數(shù)為1027.2±107.88個(gè),與對(duì)照組的1210.6±108.31個(gè)相比,陽(yáng)性細(xì)胞數(shù)明顯減少,有統(tǒng)計(jì)學(xué)差異(P0.05);2實(shí)驗(yàn)組中,五個(gè)視野中c-IAP2陽(yáng)性細(xì)胞總數(shù)為995.4±113.35個(gè),與對(duì)照組的1140.30±122.35個(gè)相比,陽(yáng)性細(xì)胞數(shù)減少明顯,有統(tǒng)計(jì)學(xué)差異(P0.05);3實(shí)驗(yàn)組中,五個(gè)視野中Livin陽(yáng)性細(xì)胞總數(shù)為1038±156.10個(gè),與對(duì)照組的1163.5±85.21個(gè)相比,陽(yáng)性細(xì)胞數(shù)減少明顯,有統(tǒng)計(jì)學(xué)差異(P0.05);4實(shí)驗(yàn)組中,五個(gè)視野中Survivin陽(yáng)性細(xì)胞總數(shù)為547.80±189.77個(gè),與對(duì)照組的959.30±179.77個(gè)相比,陽(yáng)性細(xì)胞數(shù)減少非常明顯,有統(tǒng)計(jì)學(xué)差異(P0.01);5實(shí)驗(yàn)組中,五個(gè)視野中X-IAP陽(yáng)性細(xì)胞總數(shù)為1072±86.95個(gè),與對(duì)照組的1183.2±127.49個(gè)相比,陽(yáng)性細(xì)胞數(shù)減少明顯,有統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:口服萊菔硫烷能夠使人涎腺腺樣囊性癌ACC-M裸鼠移植瘤中IAP家族成員c-IAP1、c-IAP2、Survivin、Livin、XIAP的表達(dá)明顯受到抑制。提示IAP家族成員在萊菔硫烷誘導(dǎo)人涎腺腺樣囊性癌ACC-M移植瘤細(xì)胞凋亡的過(guò)程中起重要作用,這可能是萊菔硫烷誘導(dǎo)腺樣囊性癌凋亡的機(jī)制之一。
[Abstract]:Background: salivary adenoid cystic carcinoma (SACC) is a malignant tumor, mainly from the salivary epithelium. It has the following characteristics: the local invasion is very strong, and it is very vulnerable to the early invasion of the nerve and blood vessels, so the cancer cells are easy to spread along the blood. After surgical resection of the tumor, there is still a higher position. The lung metastasis rate of SACC is far higher than that of other tumors in the maxillofacial region. So far, the related research results in SACC can not clearly explain its pathogenesis and metastasis mechanism, so we should continue to study the mechanism of SACC and strive for the understanding of adenoid cystic carcinoma from the molecular and protein level. Sulforaphane (SFN) is a plant substance extracted from the cruciferous vegetables, and it belongs to the isothiocyanates (ITCS) class. This substance has anti-cancer effect. The results show that sulforaphane can enhance the function of caspase (Caspase), accelerate the apoptosis of tumor cells and inhibit the adenoid cystic carcinoma cell line ACC-M. In this process, the members of the Bcl-2 family also play an important role. The main function of the apoptosis inhibitory protein family (inhibitor of apoptosis protein, IAP) is to regulate the main function. Apoptosis can be inhibited by inhibiting Caspase to inhibit cell apoptosis, and can also participate in the regulation of cell cycle and cell division. The family members have eight members, which are c-IAP1, c-IAP2, Survivin, Livin, X-IAP, NAIP, Bruce, ILP-2.c-IAP1, c-IAP2. The anti apoptotic effect of.Livin, such as X-IAP and other IAP family members, is to reduce the activity of Caspase-3, -7, -9. ILP-2 can regulate the expression of Bcl-2, and make it increase, reduce the activity of Caspase-9, and achieve the purpose of inhibiting the apoptosis of.NAIP mainly concentrated in the divine system, Bruce can inhibit Caspase-9 and activity. Does sulforaphane inhibit the proliferation of adenoid cystic carcinoma cell line ACC-M, does the member of the apoptosis inhibitory protein family play a certain role. Is the apoptosis inhibitory protein expression abnormal in the transplanted tumor tissue of human adenoid cystic carcinoma treated with SFN? The previous research results can not explain this problem reasonably. In the process of inhibition of sulforaphane, how is the role of IAP family members, whether it is inhibited, the expression of each member of the family is consistent, the role of other protein molecules interfering with IAP protein, and the possible molecular mechanism of its molecular action to provide a theoretical basis for the future clinical application of sulforaphane anticancer. Method: 1 material 1.1 The human salivary adenoid cystic carcinoma cell line ACC-M was purchased from the Shanghai Jiaotong maxillofacial surgery laboratory. The cell line was built in 1995.1.2 sulforaphane (SFN) purchased from the American LKT laboratory. The purity of the cell line was purchased from the Military Medical Science Academy of the PLA, the purity of the nude mice from the Military Medical Science Academy of the PLA, Beijing, and the male 10, 4~6 weeks old, and the weight of 16 ~20 grams of.1.4 antibody purchased from the UK Abcam company. C-IAP2 polyclonal antibody, c-IAP1 polyclonal antibody, Livin polyclonal antibody, Livin polyclonal antibody, purchased Survivin polyclonal antibody from American AR company, purchased two anti enzyme labelled Sheep anti mouse / rabbit Ig G polymer.2 experiment method and 2.1 cell culture, and then resuscitated the cryopreserved human salivary gland adenoid cystic carcinoma cells, and then, then the human salivary gland adenoid cystic carcinoma cells are resuscitated. In the 37 degree constant temperature incubator, the cell density reached the requirement, and the cell suspension.2.2 transplanted tumor was finally collected to establish iodine tincture to disinfect the left anterior back and right back of the nude mice. Then the cell suspension was injected into the above part, and the oral administration was given by the oral administration, and the experimental group was given oral sulforaphane (6 mmol SFN/0.1ml PBS), while the experimental group was given oral administration. After taking oral equal amount of PBS, three times a week, the nude mice were dissected three times a week for three weeks. Then the nude mice were executed, then the tumor bodies were dissected and the blood vessels and necrotic tissues were removed. Then the tumor tissue was embedded with paraffin. After the wax was prepared, the immuno histochemical method was used to observe the immunohistochemical method and observe the IA. The expression of five kinds of protein in P family members c-IAP1, c-IAP2, Survivin, Livin, XIAP in the transplanted tumor tissues of nude mice, and statistical processing of the number of.2.5 data for the number of positive cells were statistically analyzed. Compared with the two groups of 10 samples in each group, the total number of positive 5 visual field cells in each sample was statistically analyzed by t test. In the 1 experimental group, the total number of c-IAP1 positive cells in the five field of vision was 1027.2 + 107.88, and the number of positive cells decreased significantly compared with the control group. The number of positive cells decreased significantly (P0.05). In the 2 experimental group, the total number of c-IAP2 positive cells in five fields was 995.4 + 113.35, and 1140.30 + 122.3 of the control group. The number of positive cells decreased significantly in the 5 Comparison (P0.05); in the 3 experimental group, the total number of Livin positive cells in five fields of vision was 1038 + 156.10, compared with 1163.5 + 85.21 of the control group, the number of positive cells decreased significantly (P0.05); in the 4 experimental group, the total number of Survivin positive cells in five fields was 547.80 + 189.7. 7, compared with the 959.30 + 179.77 control group, the number of positive cells decreased very obviously (P0.01). In the 5 experimental group, the total number of X-IAP positive cells in five fields was 1072 + 86.95, compared with the 1183.2 + 127.49 control group, the number of positive cells decreased significantly (P0.05). Conclusion: oral sulforaphane is able to be taken orally. The expression of c-IAP1, c-IAP2, Survivin, Livin, XIAP in human salivary adenoid cystic carcinoma (ACC-M) xenografts in nude mice is obviously inhibited. It suggests that the members of IAP family play an important role in the apoptosis of adenoid cystic carcinoma of human salivary gland induced by sulforaphane, which may be the apoptosis of adenoid cystic carcinoma induced by sulforaphane. One of the mechanisms.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R739.8

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