發(fā)布時(shí)間：2018-05-17 22:17

  本文選題：乳腺癌 + 藥物靶向��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：背景:乳腺癌已經(jīng)成為中國女性最常見的惡性腫瘤之一,在世界范圍內(nèi)也是如此。紫杉醇(Paclitaxel,PTX)是乳腺癌的一線化療藥物。但其水溶性差,且會(huì)引起較嚴(yán)重的不良反應(yīng),如過敏反應(yīng)等,使其臨床應(yīng)用受到了一定限制[4]。易溶于水、毒性更低的新型紫杉醇藥物傳送系統(tǒng)就成為研究的熱點(diǎn)。中國科學(xué)院應(yīng)用化學(xué)研究所合成了一系列功能性聚乙二醇-聚(乳酸-碳酸酯)[poly(ethylene glycol)-b-poly(L-lactide-co-carbonate)]及其與紫杉醇和靶向基團(tuán)的鍵合物。但這種藥物傳遞系統(tǒng)對(duì)乳腺癌在體內(nèi)及體外的作用情況仍未知曉。本研究中,我們選擇葉酸作為靶向基團(tuán),并制成紫杉醇膠束(PTX micelles,M(PTX))和葉酸靶向紫杉醇膠束(folic acid-bearing polymer-PTX micelles,(M(FA/PTX))。我們期待這些膠束能擁有多種功能,包括水溶性、緩釋作用、生物降解能力、葉酸受體靶向能力等,與目前臨床應(yīng)用的藥物劑型相比較,療效相近或更優(yōu),但副作用更低。目的:探索紫杉醇膠束和葉酸靶向紫杉醇膠束作用于EMT-6小鼠乳腺癌細(xì)胞株和動(dòng)物模型的效果,檢測(cè)兩種膠束的腫瘤抑制率、腫瘤細(xì)胞的凋亡效果和對(duì)生存率的影響。材料與方法:制備含25%PTX的(M(PTX))和含22.5%PTX的(M(FA/PTX))兩種PTX鍵合物膠束。(1)對(duì)EMT-6小鼠乳腺癌細(xì)胞進(jìn)行常規(guī)培養(yǎng)、傳代。應(yīng)用MTT法檢測(cè)PTX、M(PTX)和(M(FA/PTX))以等量PTX的不同濃度梯度(10ug/ml、1ug/ml、0.1ug/ml、0.01ug/ml、1ng/ml)作用EMT-6細(xì)胞24、48、72小時(shí)的細(xì)胞抑制率。應(yīng)用流式細(xì)胞儀檢測(cè)含等量PTX濃度為1ug/ml時(shí),PTX、M(PTX)和(M(FA/PTX))作用于EMT-6細(xì)胞24、48、72小時(shí)的細(xì)胞凋亡率。應(yīng)用倒置顯微鏡觀察含等量PTX 1μg/ml濃度的PTX、M(PTX)、M(PTX)作用于EMT-6細(xì)胞24和72小時(shí)的細(xì)胞形態(tài)變化。應(yīng)用Hoechst 33258熒光染色檢測(cè)含等量PTX 1μg/ml濃度的PTX、M(PTX)、M(PTX)作用于EMT-6細(xì)胞72小時(shí)的凋亡指數(shù)。(2)制作EMT-6小鼠乳腺癌動(dòng)物模型。在抑瘤實(shí)驗(yàn)中,分為空白對(duì)照、PTX、M(PTX)和M(FA/PTX)4個(gè)組,每組12只小鼠,給藥總劑量為20mg/kg為標(biāo)準(zhǔn)(以等量PTX計(jì)算),第5天處死動(dòng)物。測(cè)量瘤重,計(jì)算腫瘤抑制率。瘤組織一部分做流式細(xì)胞檢測(cè),其余的迅速用4%多聚甲醛固定,石蠟包埋,進(jìn)行HE染色、Bax和Bcl-2的免疫組化檢測(cè)。生存實(shí)驗(yàn)中,分為空白對(duì)照、PTX、M(PTX)和M(FA/PTX)4個(gè)組,每組18只小鼠,給藥總劑量為26.7mg/kg為標(biāo)準(zhǔn)(以等量PTX計(jì)算),每日觀察記錄小鼠生存狀態(tài)。(3)應(yīng)用SPSS18.0統(tǒng)計(jì)軟件,計(jì)量資料用均數(shù)±標(biāo)準(zhǔn)差描述,組間差異應(yīng)用單項(xiàng)方差分析比較,以p0.05為有統(tǒng)計(jì)學(xué)意義。計(jì)數(shù)資料應(yīng)用χ2檢驗(yàn)進(jìn)行樣本率間比較,以p0.05為有統(tǒng)計(jì)學(xué)意義。應(yīng)用Kaplan-Meier法計(jì)算生存率,繪制生存曲線。各組間生存差異比較采用Log-rank檢驗(yàn)。結(jié)果:(1)在體外實(shí)驗(yàn)中,MTT試驗(yàn)結(jié)果顯示在72h時(shí),在1ug/ml的濃度下M(PTX)和M(FA/PTX)的抑制效果達(dá)到最佳。隨著作用時(shí)間的延長,各組藥物對(duì)EMT-6乳腺癌細(xì)胞生長的抑制率也隨之增加。流式細(xì)胞結(jié)果顯示在藥物作用72h時(shí),各用藥組的細(xì)胞凋亡率均高于對(duì)照組,對(duì)照組、PTX組、M(PTX)組和M(FA/PTX)組的細(xì)胞凋亡率分別為14.4%、20.7%、19.7%和25.7%,M(FA/PTX)組的細(xì)胞凋亡率最高,與其他兩用藥組比較其差異有統(tǒng)計(jì)學(xué)意義(p0.05)。倒置顯微鏡下,PTX、M(PTX)和M(FA/PTX)三種藥物作用于EMT-6小鼠乳腺癌細(xì)胞后可以觀察到細(xì)胞形態(tài)較對(duì)照組細(xì)胞發(fā)生明顯的改變。Hoechst 33258染色結(jié)果顯示對(duì)照組、PTX組、M(PTX)組和M(FA/PTX)組的細(xì)胞凋亡率分別為5.8%、15.8%、15.2%和21%。(2)在體內(nèi)抑瘤實(shí)驗(yàn)中,PTX組的動(dòng)物在通過尾靜脈給藥時(shí),均煩躁不安,持續(xù)尖叫,而M(PTX)和M(FA/PTX)兩組的小鼠在給藥時(shí)均無明顯的異常反應(yīng)出現(xiàn)。在給藥5天后,PTX、M(PT X)和M(FA/PTX)組的瘤重抑制率分別為29.0%、46.5%和54.9%。在組織學(xué)觀察中,三個(gè)用藥組均可觀察到壞死區(qū)域,M(FA/PTX)組的壞死區(qū)域的面積要比PTX和M(PTX)組明顯增大。對(duì)照組、PTX、M(PTX)和M(FA/PTX)組的Bax蛋白陽性表達(dá)率分別為8.3%、50%、60%和75%,Bcl-2蛋白陽性表達(dá)率分別為91.7%%、40%、60%和50%。流式細(xì)胞檢測(cè)結(jié)果顯示,對(duì)照組、PTX、M(PTX)和M(FA/PTX)組的細(xì)胞凋亡率分別為1.0%、36.6%、55.9%和66.1%。生存時(shí)間結(jié)果顯示,對(duì)照組、PTX組、M(PTX)組和M(FA/PTX)組在用藥30天時(shí)的生存率分別為50%、55.6%、72.2%和77.8%,中位生存時(shí)間分別為33天、31天、34天和42天(log-rank test:c2=9.789,P=0.02)。結(jié)論:在體外細(xì)胞試驗(yàn)中,M(PTX)和M(FA/PTX)兩種藥物可以獲得與PTX近似的對(duì)EMT-6小鼠乳腺癌細(xì)胞的腫瘤抑制效果,且M(FA/PTX)表現(xiàn)出比M(PTX)更好的腫瘤抑制效果。在體內(nèi)動(dòng)物實(shí)驗(yàn)中,與PTX和M(PTX)相比,M(FA/PTX)顯示出更好地對(duì)小鼠皮下移植瘤的抑制效果,能夠更有效誘導(dǎo)腫瘤細(xì)胞凋亡。M(FA/PTX)能更好的延長荷瘤小鼠的生存時(shí)間。M(FA/PTX)在人乳腺癌中治療效果值得期待。
[Abstract]:Background: breast cancer has become one of the most common malignant tumors in Chinese women, and is also the case in the world. Paclitaxel (PTX) is a first-line chemotherapy drug for breast cancer. But its water solubility is poor, and it can cause serious adverse reactions, such as allergic reaction, so that its clinical application is limited to [4]. soluble in water and toxicity. A lower new taxol drug delivery system has become a hot spot of research. A series of functional polyethylene glycol poly (lactic acid carbonate) [poly (ethylene glycol) -b-poly (L-lactide-co-carbonate)) and the bonds with taxol and target groups have been synthesized by the Institute of Applied Chemistry of the Chinese Academy of Sciences. In this study, we chose folic acid as a target group and made paclitaxel micelles (PTX micelles, M (PTX)) and folic acid targeted paclitaxel micelles (folic acid-bearing polymer-PTX micelles, (M (FA/PTX)) in this study. We expect these micelles to have a variety of functions, including water solubility and sustained release. The effect, biodegradability, and the targeting ability of folic acid receptor, compared with the current clinical drug formulations, are similar or better, but have lower side effects. Objective: To explore the effect of paclitaxel micelles and folic acid targeted paclitaxel micelles on EMT-6 mouse breast cancer cell lines and animal models, and to detect the tumor inhibition rate of two kinds of micelles. The effect of apoptosis and the survival rate of tumor cells. Materials and methods: two kinds of PTX binding micelles containing 25%PTX (M (PTX)) and 22.5%PTX (M (FA/PTX)) were prepared. (1) conventional culture of EMT-6 mice breast cancer cells was carried out. MTT method was used to detect PTX, M (PTX) and (0.1) concentration gradient (0.1) Ug/ml, 0.01ug/ml, 1ng/ml) acts on the cell inhibition rate of EMT-6 cells at 24,48,72 hours. Using flow cytometry to detect the cell apoptosis rate of PTX, M (PTX) and (M (FA/PTX)) when the concentration of PTX is 1ug/ml. Cell morphology changes at 24 and 72 hours. Hoechst 33258 fluorescence staining was used to detect PTX, M (PTX), M (PTX), M (PTX), and M (PTX) for 72 hours of apoptosis index of EMT-6 cells. (2) the mammary carcinoma animal model of EMT-6 mice was made. In the tumor suppressor experiment, 4 groups, 12 mice in each group, were divided into 4 groups, 12 mice in each group. The total dose was 20mg/kg as the standard (calculated by PTX), the animals were killed fifth days. The tumor weight was measured and the tumor inhibition rate was calculated. A part of the tumor tissue was detected by flow cytometry. The rest were fixed with 4% polyformaldehyde, paraffin embedded, HE staining, Bax and Bcl-2 immunohistochemistry. The survival experiments were divided into blank control, PTX, M (PTX) and M (FA/PT). X) 4 groups, each group of 18 mice, the total dose of 26.7mg/kg as the standard (with equal amount of PTX), the daily observation of the survival state of mice. (3) the application of SPSS18.0 statistical software, the measurement data were described with mean standard deviation, the difference between groups was compared with the single variance analysis, and P0.05 was statistically significant. The count data were carried out by the x 2 test. The sample rate was compared with P0.05. The survival rate was calculated by Kaplan-Meier method and the survival curve was drawn. The survival difference between each group was compared with the Log-rank test. (1) in the experiment in vitro, the results of MTT test showed that the inhibition effect of M (PTX) and M (FA/PTX) was best at the concentration of 1ug/ml at the concentration of 72h. With the time of action. The inhibition rate of EMT-6 breast cancer cell growth was also increased in each group. Flow cytometry showed that the apoptosis rate of each drug group was higher than that of the control group when the drug action was 72h. The apoptosis rate of the control group, the PTX group, the M (PTX) group and the M (FA/PTX) group were 14.4%, 20.7%, 19.7% and 25.7%, and the apoptosis rate of M (FA/PTX) group. The difference was statistically significant (P0.05). Under the inverted microscope, three drugs of PTX, M (PTX) and M (FA/PTX) were used to observe the cell morphology of the breast cancer cells in EMT-6 mice. The results showed that the cell morphology of the cells in the control group was obviously changed by.Hoechst 33258 staining, and the control group, PTX, M (PTX) and M. The rate of apoptosis was 5.8%, 15.8%, 15.2% and 21%. (2) in the tumor suppressor experiment in vivo. The animals in the PTX group were restless and screaming, while the mice in the M (PTX) and M (FA/PTX) two groups had no obvious abnormal response to the drug delivery. After 5 days of administration, the tumor repressor rate of PTX, M (PT X) and M (FA/PTX) groups was respectively For 29%, 46.5%, and 54.9%. in histological observation, the necrotic area was observed in three groups, and the area of the necrotic region in the M (FA/PTX) group was significantly higher than that in the PTX and M (PTX) group. The positive rates of the Bax protein expression in the PTX, M (PTX) and M (FA/PTX) groups were 8.3%, 50%, 60% and 75% in the control group, respectively, and 40%, 60% respectively. The results of 50%. flow cytometry showed that the apoptosis rates of PTX, M (PTX) and M (FA/PTX) groups were 1%, 36.6%, 55.9% and 66.1%., respectively, and the survival time of control group, PTX group, M (PTX) group and M (FA/PTX) group were 50%, 55.6%, 72.2% and 77.8%, and the median survival time was 33 days, 31 days, 34 days, respectively. And 42 days (log-rank test:c2=9.789, P=0.02). Conclusion: in vitro cell test, M (PTX) and M (FA/PTX) drugs can obtain the tumor inhibition effect on EMT-6 mouse breast cancer cells similar to PTX, and M (FA/PTX) shows a better tumor inhibition effect than M. It is better to induce tumor cell apoptosis.M (FA/PTX) more effectively to prolong the survival time of tumor bearing mice (.M (FA/PTX) in human breast cancer.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.9;R-332

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1 吳迪;葉酸靶向紫杉醇膠束對(duì)EMT-6乳腺癌細(xì)胞株及動(dòng)物模型的抗腫瘤作用[D];吉林大學(xué);2016年

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本文編號(hào)：1903107