本文關(guān)鍵詞： 犬乳腺腫瘤細(xì)胞系 CHMp CHMm 鹽酸多柔比星 細(xì)胞凋亡　出處：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：犬乳腺腫瘤是僅次于皮膚腫瘤的常見(jiàn)腫瘤,迄今外科手術(shù)聯(lián)合化學(xué)藥物是最有效的治療方法。基于獸醫(yī)臨床尚無(wú)理想的化療藥物、復(fù)制人醫(yī)臨床的化療藥物應(yīng)用于治療犬乳腺腫瘤缺乏理論及依據(jù)、研發(fā)新藥時(shí)程長(zhǎng)且成功率低等現(xiàn)狀,本研究選用在治療人耐藥性乳腺腫瘤中取得良好效果的鹽酸多柔比星進(jìn)行體外抗腫瘤實(shí)驗(yàn)。研究該藥誘導(dǎo)犬乳腺腫瘤細(xì)胞的凋亡機(jī)制,目的在于探求其在抗犬乳腺腫瘤的作用靶點(diǎn)及機(jī)理,為該藥的化學(xué)療法在應(yīng)用于寵物臨床提供理論依據(jù),以解決寵物臨床化療藥物匱乏的燃眉之急。選用鹽酸多柔比星作為誘導(dǎo)劑及體外培養(yǎng)的犬乳腺腫瘤原發(fā)病灶及其轉(zhuǎn)移灶的細(xì)胞系CHMp株和CHMm株為研究對(duì)象,利用CCK-8法分析鹽酸多柔比星對(duì)兩株乳腺腫瘤細(xì)胞系的抑制情況,選用不同的藥物濃度誘導(dǎo)腫瘤細(xì)胞,進(jìn)行形態(tài)學(xué)觀察。同時(shí)利用AO/EB染色法考證藥物對(duì)兩株細(xì)胞系的促凋亡作用,并通過(guò)分光光度法檢測(cè)處理后胞內(nèi)凋亡相關(guān)蛋白Caspase-3、6、8、9的活性變化,Real-time PCR法檢測(cè)細(xì)胞中Bcl-2 mRNA、Bax mRNA、p53 mRNA等相關(guān)凋亡因子表達(dá)量變化,Western Blotting方法檢測(cè)胞漿蛋白中促凋亡蛋白Bax的表達(dá)情況,從而初步探究鹽酸多柔比星誘導(dǎo)犬乳腺腫瘤細(xì)胞的機(jī)制。實(shí)驗(yàn)結(jié)果顯示,不同濃度的鹽酸多柔比星均能有效的誘導(dǎo)犬乳腺腫瘤細(xì)胞凋亡,藥物濃度越高,抑制能力越強(qiáng),且能顯著提高CHMp胞內(nèi)Caspase-3、6、8、9的表達(dá)量(P≤0.05);極顯著提高CHMm胞內(nèi)Caspase-3的表達(dá)量(P≤0.001),顯著提高Caspase-6、8、9的表達(dá)量(P≤0.05)。與此同時(shí),不同的藥物濃度均使CHMp細(xì)胞內(nèi)p53 mRNA、Bax mRNA表達(dá)量極顯著提高(P≤0.001),Bcl-2 mRNA表達(dá)無(wú)明顯差異;使CHMm細(xì)胞內(nèi)p53 mRNA表達(dá)顯著提高(P≤0.05),Bax mRNA表達(dá)量極顯著提高(P≤0.001),Bcl-2 mRNA表達(dá)無(wú)明顯差異或呈極顯著降低(P≤0.001)。Western Blotting結(jié)果顯示,藥物作用誘導(dǎo)細(xì)胞以后,兩種細(xì)胞胞內(nèi)促凋亡蛋白Bax表達(dá)量提高,且表達(dá)量與藥物濃度呈正相關(guān)。該實(shí)驗(yàn)結(jié)果表明鹽酸多柔比星可通過(guò)促進(jìn)細(xì)胞周期調(diào)控相關(guān)因子p53、線粒體凋亡通路相關(guān)因子Bcl-2家族成員Bax、死亡受體通路相關(guān)成員Caspase家族,激活Caspase聯(lián)級(jí)反應(yīng)啟動(dòng)細(xì)胞凋亡程序,從而誘導(dǎo)細(xì)胞發(fā)生凋亡。本實(shí)驗(yàn)初步揭示了鹽酸多柔比星誘導(dǎo)犬乳腺腫瘤細(xì)胞凋亡的相關(guān)機(jī)制,為獸醫(yī)臨床應(yīng)用該藥物奠定科學(xué)理論基礎(chǔ),為篩選有效寵物臨床抗腫瘤用藥提供參考。
[Abstract]:The canine breast tumor is the second most common tumor after the skin tumor. So far, surgery combined with chemical drugs is the most effective treatment. The clinical chemotherapeutic drugs used in replicating human medicine for the treatment of canine breast tumors lack theory and basis, and have a long history and low success rate in the development of new drugs. In this study, doxorubicin hydrochloride, which had a good effect in the treatment of human breast cancer with drug resistance, was used to study the mechanism of apoptosis induced by doxorubicin hydrochloride. The aim of this study was to explore the target and mechanism of its antitumor effect in dogs, and to provide a theoretical basis for the application of chemotherapeutic drugs in pet clinic. In order to solve the urgent need of clinical chemotherapeutic drugs in pets, doxorubicin hydrochloride was used as inducer and CHMp and CHMm strains of canine breast tumor primary tumor and its metastatic foci were cultured in vitro. The inhibition of doxorubicin hydrochloride on two breast tumor cell lines was analyzed by CCK-8 method. The tumor cells were induced by different drug concentration and the morphological observation was carried out. Meanwhile, AO/EB staining was used to study the effect of drug on promoting apoptosis of two breast tumor cell lines. The expression of apoptosis factors such as Bcl-2 mRNA-Bax mRNA-p53 mRNA was detected by real-time PCR method. The expression of apoptosis-promoting protein Bax in cytoplasmic protein was detected by Western Blotting. The results showed that doxorubicin hydrochloride could induce the apoptosis of canine breast tumor cells effectively, and the higher the concentration of doxorubicin, the stronger the inhibition ability. In addition, the expression of Caspase-3, Caspase-3, Caspase-6, Caspase-8, Caspase-6, Caspase-6, and Caspase-6 were significantly increased in CHMp cells (P 鈮，

本文編號(hào)：1550973