本文選題：阿霉素脂質(zhì)體 + GCS�。� 參考：《第二軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：多藥耐藥(MDR)，，是腫瘤細胞對多種化療藥物耐藥的種常見的耐藥形式，也是導(dǎo)致腫瘤化療失敗和腫瘤復(fù)發(fā)的重要原因，成為當(dāng)前研究腫瘤治療的熱點話題。納米技術(shù)應(yīng)用于臨床制藥是化療研究的項重大突破，而且利用納米技術(shù)克服腫瘤細胞耐藥引起了廣泛關(guān)注。本文結(jié)合了腫瘤耐藥細胞的自身代謝特點，采用乙醇注入法制備PEG-mCeramide修飾的脂質(zhì)體，以硫酸銨梯度法包裹阿霉素，得到PEG-mCeramide修飾的阿霉素脂質(zhì)體（Lipo-ADR-Cer-）。本項研究展示了，Lipo-ADR-Cer-能夠降低腫瘤耐藥細胞對阿霉素的IC50，體外明顯抑制腫瘤耐藥細胞MCF-7-ADR和HL-60-ADR的細胞活性，體內(nèi)具備強的抗腫瘤效果。 通過檢測糖基化ceramide合酶（GCS）在乳腺癌和白血病細胞株中的表達發(fā)現(xiàn)，在MCF-7-ADR和HL-60-ADR細胞株上GCS的表達均比MCF-7和HL-60敏感腫瘤細胞株增多。我們考慮GCS的過表達與腫瘤細胞的耐藥相關(guān)，這些實驗結(jié)果為利用外源性的ceramide來克服腫瘤耐藥增加可能。同時，體外游離藥物細胞殺傷實驗表明PEG-mCeramide比對照PEG-DSPE的細胞毒性明顯增強，不同濃度的游離的PEG-mCeramide與游離阿霉素聯(lián)合給藥有增敏效果，增敏指數(shù)隨著PEG-mCeramide濃度的增高而不斷增加，而且我們發(fā)現(xiàn)在耐藥細胞株上，低劑量的PEG-mCeramide聯(lián)合阿霉素的增敏效果比敏感細胞強。 本課題中我們采用乙醇注入法制備PEG-mCeramide修飾的空白脂質(zhì)體和無修飾的對照空白脂質(zhì)體，以硫酸銨梯度法包裹化療藥物阿霉素，制備PEG-mCeramide修飾的阿霉素脂質(zhì)體（Lipo-ADR-Cer-）和對照阿霉素脂質(zhì)體（Lipo-ADR-）。對納米脂質(zhì)體的粒徑、Zeta電位、載藥量，包封率等基本表征進行檢測，結(jié)果表明兩種阿霉素脂質(zhì)體的基本表征無明顯差異，而且兩種阿霉素脂質(zhì)體的不同PH條件下的體外釋放實驗結(jié)果亦無明顯差異。我們通過cck8法檢測Lipo-ADR-Cer-、Lipo-ADR、游離ADR和游離ADR+PEG-mCeramide在不同給藥時間點對MCF-7、MCF-7-ADR、HL-60和HL-60-ADR細胞株的體外殺傷效果，結(jié)果展示了在兩株敏感腫瘤細胞株（MCF-7和HL-60）上,Lipo-ADR-Cer-和Lipo-ADR-的殺傷效果無統(tǒng)計學(xué)差異，而在兩株耐藥的腫瘤細胞株（MCF-7-ADR和HL-60-ADR）上, Lipo-ADR-Cer-的殺傷效果明顯強于Lipo-ADR-。為探討Lipo-ADR-Cer-殺傷效果增強的機制，我們利用流式細胞術(shù)和共聚焦顯微鏡技術(shù)對比了兩種脂質(zhì)體在細胞轉(zhuǎn)染和內(nèi)吞的差異，結(jié)果顯示兩者均無統(tǒng)計學(xué)差異。故我們考慮Lipo-ADR-Cer-殺傷效果增強的機制在于耐藥細胞自身的代謝特點。 在體內(nèi)實驗中，我們構(gòu)建了乳腺癌腋下負荷裸鼠的動物模型和白血病裸鼠動物模型，通過系統(tǒng)觀察了腫瘤小鼠的體重變化和腫瘤大小，定期定量對小鼠尾靜脈注射Lipo-ADR-Cer-、Lipo-ADR-、游離ADR和游離ADR+PEG-mCeramide以及PBS后，評價乳腺癌小鼠的體內(nèi)腫瘤抑制效果和白血病小鼠的生存狀況。體內(nèi)抗腫瘤實驗表明，在兩種腫瘤細胞的動物模型上，PEG-mCeramide修飾的阿霉素脂質(zhì)體組的體內(nèi)抗腫瘤效果比對照阿霉素脂質(zhì)體組的效果強，游離的阿霉素聯(lián)合PEG-mCeramide體內(nèi)抗腫瘤效果比游離阿霉素組的效果強，但兩組游離藥物組的治療效果均不及兩組納米藥物組，而且游離阿霉素組與PBS組的效果相近。 結(jié)論：與敏感的腫瘤細胞株相比，GCS在耐藥腫瘤細胞株上的表達增多；對比對照阿霉素脂質(zhì)體，PEG-mCeramide修飾的阿霉素脂質(zhì)體的基本表征、體外釋放過程、體外轉(zhuǎn)染效率以及細胞的內(nèi)吞過程均無統(tǒng)計學(xué)差異；在耐藥細胞株上，對比對照阿霉素脂質(zhì)體，PEG-mCeramide修飾的阿霉素脂質(zhì)體的體外殺傷效果和體內(nèi)抗腫瘤效果增強。本課題結(jié)合腫瘤耐藥細胞的自身特點，利用具有殺傷效應(yīng)PEG-mCeramide修飾納米脂質(zhì)體載體，制備PEG-mCeramide修飾的阿霉素脂質(zhì)體，為探討克服腫瘤細胞耐藥提供了新的證據(jù)，為治療MDR提供了條新的途徑。
[Abstract]:Multidrug resistance (MDR) is a common form of drug resistance to a variety of chemotherapeutic drugs. It is also an important cause of tumor chemotherapy failure and tumor recurrence. It has become a hot topic in the study of tumor treatment. The application of nanotechnology to clinical pharmacy is a major breakthrough in the study of chemotherapy and the use of nanotechnology to overcome cancer. In this paper, PEG-mCeramide modified liposomes were prepared by ethanol injection, and adriamycin (Lipo-ADR-Cer-) modified by PEG-mCeramide was obtained by the ammonium sulfate gradient method. This study shows that Lipo-ADR-Cer- can reduce the swelling. Tumor resistant cells to adriamycin IC50 significantly inhibited the cell activity of tumor resistant cells MCF-7-ADR and HL-60-ADR in vitro, and had strong anti-tumor effect in vivo.
The expression of glycosylated ceramide synthase (GCS) in breast cancer and leukemia cells showed that the expression of GCS on MCF-7-ADR and HL-60-ADR cell lines increased more than that of MCF-7 and HL-60 sensitive tumor cells. We consider that the overexpression of GCS is associated with the drug resistance of the tumor cells. These experimental results are the use of exogenous ceramide to a gram. At the same time, the cytotoxicity test of free drug cells in vitro showed that the cytotoxicity of PEG-mCeramide was significantly higher than that of the control PEG-DSPE. The combination of free PEG-mCeramide and free adriamycin in different concentrations had a sensitizing effect, and the sensitization index increased with the increase of PEG-mCeramide concentration, and we found that In drug-resistant cell lines, low dose of PEG-mCeramide combined with adriamycin has stronger sensitivity than sensitive cells.
We used ethanol injection to prepare PEG-mCeramide modified blank liposomes and unmodified control blank liposomes, encapsulated adriamycin by ammonium sulfate gradient method, and prepared PEG-mCeramide modified adriamycin liposomes (Lipo-ADR-Cer-) and control adriamycin liposomes (Lipo-ADR-). The particle size of nano liposomes, Zet The basic characterization of a potential, drug loading and encapsulation efficiency showed that there was no significant difference in the basic characterization of two adriamycin liposomes, and there was no significant difference in the experimental results of two kinds of adriamycin liposomes under different PH conditions. We detected Lipo-ADR-Cer-, Lipo-ADR, free ADR and free ADR+PEG-mCerami by CCK8 method. The killing effect of De on MCF-7, MCF-7-ADR, HL-60 and HL-60-ADR cell lines at different time points showed that there was no significant difference in the killing effect of Lipo-ADR-Cer- and Lipo-ADR- on two sensitive tumor cell lines (MCF-7 and HL-60), and the killing of Lipo-ADR-Cer- was on the two drug-resistant swollen cell lines (MCF-7-ADR and HL-60-ADR). The effect of the injury was significantly stronger than that of Lipo-ADR-. as a mechanism for enhancing the effect of Lipo-ADR-Cer- killing. We compared the difference in cell transfection and endocytosis between two liposomes by flow cytometry and confocal microscopy. The results showed that there was no statistical difference between the two. Therefore, we consider that the mechanism of the enhancement of Lipo-ADR-Cer- killing effect is tolerance. The metabolic characteristics of the drug cell itself.
In the experiment in vivo, we constructed the animal model of breast cancer axillary load nude mice and the nude mouse model of leukemic nude mice. The weight change and tumor size of the tumor mice were observed by the system. Lipo-ADR-Cer-, Lipo-ADR-, free ADR, free ADR+PEG-mCeramide and PBS were regularly injected into the tail vein of mice to evaluate the small breast cancer. The tumor inhibition effect in vivo and the survival status of leukemia mice in vivo. In vivo antitumor experiments showed that in the animal models of two tumor cells, the anti tumor effect of PEG-mCeramide modified adriamycin liposome group was better than that of the adriamycin liposome group. Free adriamycin combined with the anti tumor effect of PEG-mCeramide in vivo. The results were better than that in the free adriamycin group, but the effect of the two groups of free drug groups was less than that of the two groups of nanoscale drugs, and the effect of the free adriamycin group was similar to that in the PBS group.
Conclusion: compared with the sensitive tumor cell lines, the expression of GCS in the resistant tumor cell lines increased; compared with the control of adriamycin liposomes, the basic characterization of the adriamycin liposomes modified by PEG-mCeramide, the release process in vitro, the transfection efficiency in vitro and the endocytosis process of the cells were not statistically different. The effects of PEG-mCeramide modified adriamycin liposomes in vitro and in vivo anti-tumor effect are enhanced according to the liposomes of adriamycin. This subject combines the characteristics of the tumor resistant cells and uses the PEG-mCeramide modified nano liposome to prepare the adriamycin liposomes modified by PEG-mCeramide. Cell resistance provides new evidence, which provides a new way for the treatment of MDR.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943;R96

【參考文獻】

相關(guān)期刊論文 前1條

1 Yoh Takuwa;Yasuo Okamoto;Noriko Takuwa;Kazuaki Yoshioka;;Roles of sphingosine-1-phosphate signaling in angiogenesis[J];World Journal of Biological Chemistry;2010年10期

本文編號：1939111