本文選題：裸鼠 + 萊菔硫烷�。� 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：裸鼠是一種全身沒(méi)有毛發(fā)，缺乏胸腺的動(dòng)物。胸腺具有制造T細(xì)胞的功能。裸鼠缺乏T細(xì)胞的原因是由于其一基因的突變，這種突變基因被稱(chēng)為nu基因，屬于隱性遺傳基因，只有當(dāng)nu/nu結(jié)合時(shí)，才能出現(xiàn)表現(xiàn)型。T細(xì)胞具有免疫的功能，能夠輔助機(jī)體抑制以及殺傷進(jìn)入體內(nèi)的外源物質(zhì)。沒(méi)有胸腺，不能產(chǎn)生T細(xì)胞的裸鼠，其免疫力很低，當(dāng)外源物質(zhì)進(jìn)入體內(nèi)時(shí)，不能夠及時(shí)的被抑制或者消除�；诼闶蟮倪@些特征，其就成為了移植瘤模型的一種理想動(dòng)物。通常，裸鼠經(jīng)常被用來(lái)研究惡性腫瘤的治療。根據(jù)裸鼠的這些特征，裸鼠移植瘤模型就成為本次實(shí)驗(yàn)研究Bcl-2家族成員在萊菔硫烷抑制人涎腺腺樣囊性癌移植瘤中的作用的理想模型。 涎腺腺樣囊性癌（salivary adenoid cystic carcinoma,SACC）是一種惡性程度較高的口腔頜面部腫瘤。其臨床特征為轉(zhuǎn)移性強(qiáng)，浸潤(rùn)周?chē)窠?jīng)生長(zhǎng)等。細(xì)胞學(xué)特征主要是基底細(xì)胞樣腫瘤，由上皮細(xì)胞和肌上皮細(xì)胞組成并排列成類(lèi)管狀，類(lèi)篩網(wǎng)狀，和實(shí)性巢樣等不同的結(jié)構(gòu)。 根據(jù)我們前期實(shí)驗(yàn)，萊菔硫烷（Sulforaphane，SFN）能夠在體外抑制涎腺腺樣囊性癌ACC-M細(xì)胞的增殖并誘導(dǎo)細(xì)胞凋亡。SFN誘導(dǎo)涎腺腺樣囊性癌凋亡的過(guò)程是由Caspase介導(dǎo)的。另外，在體外萊菔硫烷誘導(dǎo)人涎腺腺樣囊細(xì)胞凋亡的過(guò)程中，Bcl-2家族成員起到了不可忽視的作用：萊菔硫烷可以引起促凋亡蛋白因子Bax、Bak的表達(dá)增加，而使抗凋亡蛋白因子Bcl-2和Bcl-xl的表達(dá)下降。我們剛剛進(jìn)行的萊菔硫烷抑制人腺樣囊性癌裸鼠移植瘤的體內(nèi)實(shí)驗(yàn)研究顯示，體內(nèi)環(huán)境中，萊菔硫烷可抑制腺樣囊性癌ACC-M裸鼠移植瘤的增殖生長(zhǎng)，并能誘導(dǎo)腺樣囊性癌ACC-M在裸鼠移植瘤中發(fā)生細(xì)胞凋亡。在這個(gè)過(guò)程中，Bcl-2蛋白家族是否也和體外實(shí)驗(yàn)一樣，起到重要作用，國(guó)內(nèi)外尚未見(jiàn)報(bào)道。本研究的目的，是利用人涎腺腺樣囊性癌細(xì)胞構(gòu)建的裸鼠移植瘤模型，通過(guò)免疫組織化學(xué)方法來(lái)驗(yàn)證Bcl-2家族蛋白的表達(dá)是否與體外實(shí)驗(yàn)中萊菔硫烷誘導(dǎo)人腺樣囊性癌ACC-M細(xì)胞凋亡的情況一致。 方法： 1細(xì)胞培養(yǎng)：復(fù)蘇凍存的人涎腺腺樣囊性癌細(xì)胞，在37度,5%CO2的恒溫培養(yǎng)箱中進(jìn)行貼壁培養(yǎng)。當(dāng)細(xì)胞達(dá)到一定濃度時(shí)，用0.25%的胰酶進(jìn)行消化傳代，收集并調(diào)整細(xì)胞密度為2×107/ml的細(xì)胞懸液。 2建立移植瘤模型：將預(yù)備好的細(xì)胞懸液注射于裸鼠的左前背部以及右后背部，并分組（給藥組、對(duì)照組）經(jīng)口腔給藥，給藥組給予萊菔硫烷（6mmol SFN/0.1ml PBS），對(duì)照組給予等量的PBS，每周給藥3次。觀(guān)察。 3解剖瘤體：三周后，采用斷頸的方法處死裸鼠，通過(guò)外科手術(shù)的方法剝離瘤體，，去除瘤體表面的壞死組織，以及小血管組織等。剝離下來(lái)的腫瘤組織經(jīng)過(guò)石蠟的包埋，處理，制成蠟塊，備用。 4免疫組織化學(xué)染色：將制成的蠟片，采用免疫組織化學(xué)染色的方法，在顯微鏡下觀(guān)察Bcl-2家族成員中Bcl-2、Bax、Bcl-xl、Bak四種蛋白的表達(dá)情況，并記分。 5統(tǒng)計(jì)學(xué)分析：采用秩和檢驗(yàn)的方法對(duì)等級(jí)資料結(jié)果進(jìn)行統(tǒng)計(jì)計(jì)算分析，并得出結(jié)論。 結(jié)果： 與對(duì)照組相比，實(shí)驗(yàn)組抑制凋亡蛋白Bcl-2、Bcl-xl表達(dá)顯著減少，有統(tǒng)計(jì)學(xué)差異(P＜0.05)；促凋亡蛋白Bax、Bak蛋白的表達(dá)略有增加，但無(wú)統(tǒng)計(jì)學(xué)差異（P＞0.05）。 結(jié)論： 在體內(nèi)用萊菔硫烷誘導(dǎo)人涎腺腺樣囊性癌細(xì)胞ACC-M移植瘤的凋亡過(guò)程中,Bcl-2家族成員起重要作用。
[Abstract]:Objective: nude mice are a kind of animals without hair and thymus. The thymus has the function of producing T cells. The reason for the lack of T cells in nude mice is due to the mutation of one gene. This mutant gene is called the Nu gene, which belongs to the recessive gene. Only when nu/nu is combined, can the expressive.T cells have immune function and can be immune. It is sufficient to assist the body to inhibit and kill the exogenous substances that enter the body. Without thymus, nude mice that do not produce T cells have low immunity and can not be suppressed or eliminated in time when exogenous substances enter the body. Based on these characteristics of nude mice, it is an ideal animal for the transplanted tumor model. To study the treatment of malignant tumors. Based on these characteristics of nude mice, the transplanted tumor model in nude mice is an ideal model for the study of the role of Bcl-2 family members in sulforaphane suppressing human salivary adenoid cystic carcinoma.
Salivary adenoid cystic carcinoma (SACC) is a high malignancy of oral and maxillofacial tumors. Its clinical features are strong metastatic and infiltrating peripheral nerve growth. The cytological features are mainly basal cell like tumors, composed of epithelial and myoepithelial cells and arranged into tubular, sieved reticulation, and solid. Different structures such as sex nests.
According to our previous experiments, Sulforaphane (SFN) can inhibit the proliferation of ACC-M cells in salivary adenoid cystic carcinoma in vitro and induce apoptosis of adenoid cystic carcinoma of salivary gland induced by.SFN. In addition, in the process of apoptosis of human salivary gland like sac cells induced by sulforaphane in vitro, the Bcl-2 family is formed. The role played an important role: sulforaphane can cause an increase in the expression of apoptotic protein factor Bax, Bak, and the decrease of the expression of anti apoptotic factor Bcl-2 and Bcl-xl. The proliferation and growth of adenoid cystic carcinoma ACC-M nude mice can induce the apoptosis of the adenoid cystic carcinoma ACC-M in nude mice. In this process, whether the Bcl-2 protein family also plays an important role as in vitro, has not been reported at home and abroad. The purpose of this study is to construct human salivary gland adenoid cystic carcinoma cells. The model of xenograft in nude mice was used to verify whether the expression of Bcl-2 family protein was consistent with the apoptosis of human adenoid cystic carcinoma ACC-M cells induced by sulforaphane in the experiment in vitro.
Method:
1 cell culture: resuscitation of frozen human salivary gland adenoid cystic carcinoma cells, adherent culture at 37 degrees, 5%CO2 constant temperature incubator. When cells reached a certain concentration, the cells were digested with 0.25% pancreatin, and the cell density of cell density of 2 x 107/ml was collected and adjusted.
2 the model of transplanted tumor was established: the prepared cell suspension was injected into the left anterior back and right back of the nude mice. The group (drug group, control group) was given oral administration by oral administration, 6mmol SFN/0.1ml PBS was given to the drug group, the control group was given the same amount of PBS, and the drug was given 3 times a week.
3 anatomic body: after three weeks, the nude mice were killed by the method of breaking the neck. The tumor body was stripped by surgical procedure, the necrotic tissue on the surface of the tumor and the small vascular tissue were removed. The dissected tumor tissue was embedded and processed by paraffin.
4 immunohistochemical staining: the prepared wax tablets, using immunohistochemical staining method, under the microscope observation of the Bcl-2 family members Bcl-2, Bax, Bcl-xl, Bak four kinds of protein expression, and score.
5 statistical analysis: rank sum test was used to analyze and calculate the results of grade data and draw conclusions.
Result:
Compared with the control group, the experimental group inhibited the apoptosis protein Bcl-2, the expression of Bcl-xl decreased significantly (P < 0.05), and the expression of apoptotic protein Bax and Bak protein increased slightly, but there was no statistical difference (P > 0.05).
Conclusion:
Bcl-2 family members play an important role in the apoptosis of human salivary adenoid cystic carcinoma ACC-M cells induced by sulforaphane in vivo.
【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】：2014
【分類(lèi)號(hào)】：R739.87

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