本文選題：肺癌 + 紫杉醇　； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:本實(shí)驗(yàn)的研究目的是為了探究載紫杉醇(PTX)的聚己內(nèi)酯-聚乙二醇-聚己內(nèi)酯(poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone)(PCL-PEG-PCL,PCEC))聚合物納米顆粒(PTX-NPs)時(shí)辰給藥對(duì)肺癌的抗腫瘤作用及其相關(guān)機(jī)制,并初步篩選出一天中最適合給藥的時(shí)辰點(diǎn)。方法:本實(shí)驗(yàn)具體分為體外實(shí)驗(yàn)和體內(nèi)實(shí)驗(yàn)兩部分完成。在體外研究中,首先以聚乙二醇(poly(ethylene glycol),PEG)和己內(nèi)酯(ε-caprolactone)為原料,成功制備出PCEC聚合物,并將其作為載體;然后,通過薄膜分散法成功制備出PTX-NPs,用透射電子顯微鏡考察其形貌,通過動(dòng)態(tài)光散射掃描儀評(píng)估其粒徑,采用高效液相色譜儀(High performance liquid chromatography,HPLC)測(cè)得其百分包封率、載藥率及在體外的藥物釋放特性。采用噻唑藍(lán)(methylthiazoletetrazolium,MTT)試驗(yàn)研究PTX-NPs和空載體PCEC聚合物納米顆粒的細(xì)胞毒性。通過熒光顯微鏡(Fluorescence microscope)觀察PCEC聚合物納米顆粒的體外細(xì)胞攝取功能。在體內(nèi)研究中,先用常規(guī)方法培養(yǎng)A549肺癌細(xì)胞。然后,在相同的光照環(huán)境下飼養(yǎng)裸小鼠3周,建立相同的生物節(jié)律,光照時(shí)間(07:00-19:00),黑暗時(shí)間(19:00-07:00)。構(gòu)建A549肺癌皮下異種移植瘤裸小鼠動(dòng)物模型,待腫瘤體積長(zhǎng)大至100mm3左右,隨機(jī)分成三組,每組按0、5、10、15HALO(Hours After Light Onset設(shè)光照后小時(shí))(即分別對(duì)應(yīng)一天中的07:00、12:00、17:00、22:00四個(gè)時(shí)辰點(diǎn))再分成四個(gè)亞組。其中兩組作為治療組,嚴(yán)格按照晝夜24小時(shí)的四個(gè)不同時(shí)辰點(diǎn)尾靜脈注射ptx注射液和ptx-nps溶液。第三組作為對(duì)照組,尾靜脈注射體積相同的生理鹽水(ns)。ptx注射液和ptx-nps溶液均按ptx10mg/kg給藥,每隔3天一次,共給藥3次。每隔一天測(cè)量裸鼠體重、腫瘤長(zhǎng)短徑,計(jì)算腫瘤體積,繪制出腫瘤生長(zhǎng)曲線、計(jì)算出腫瘤抑制率,并用小動(dòng)物18f-fdg(fluorine-18-deoxyglucose)pet/ct(positronemissiontomography/computedtomography)顯像觀察與分析。治療12天后處死裸小鼠,剝離瘤塊,固定,采用免疫組織化學(xué)技術(shù)(immunohistochemistry,ihc)檢測(cè)各組腫瘤標(biāo)本中cd-31和ki-67的表達(dá)情況,并采用末端標(biāo)記法(tdt-mediateddutpnickendlabeling,tunel)檢測(cè)各組腫瘤標(biāo)本中細(xì)胞凋亡的情況。結(jié)果:本研究制備的ptx-nps的外觀呈球形結(jié)構(gòu),平均粒徑約168nm,粒徑分布均勻,具有初始快速釋放而隨后緩慢而持續(xù)釋放藥物的特點(diǎn)。mtt試驗(yàn)研究結(jié)果表明,空載體pcec聚合物納米顆粒沒有明顯的細(xì)胞毒性作用;ptx-nps的體外細(xì)胞毒性隨著藥物濃度的增大而增大,并且在四個(gè)不同的時(shí)辰點(diǎn)給藥ptx-nps溶液(ptx濃度相同),細(xì)胞活性呈現(xiàn)出逐漸遞增的趨勢(shì):22:0017:0012:0007:00。體內(nèi)抗腫瘤作用研究表明,ptx-nps溶液組和ptx注射液組的腫瘤抑制率均明顯高于對(duì)照組(p0.01),并且兩者均在22:00給藥時(shí)的腫瘤抑制率最大,其數(shù)值分別為84.36%和68.84%(p0.05)。此外,治療組的cd-31和ki-67的表達(dá)均呈現(xiàn)出遞減的趨勢(shì):07:0012:0017:0022:00;治療組的細(xì)胞凋亡數(shù)呈現(xiàn)出遞增的趨勢(shì):07:0012:0017:0022:00。并且,在22:00給藥時(shí),PTX-NPs溶液組的CD-31和Ki-67的表達(dá)均比PTX注射液組低(P0.05),細(xì)胞凋亡數(shù)比PTX注射液組高(P0.05)。小動(dòng)物18F-FDG PET/CT檢查結(jié)果顯示,在四個(gè)不同時(shí)辰點(diǎn)(07:00、12:00、17:00和22:00)給藥,對(duì)照組的SUVmax值分別為:2.15±0.23,2.10±0.20,2.5±0.25和2.12±0.18。PTX注射液組的SUVmax值分別為:1.44±0.44,1.26±0.11,1.15±0.06和0.83±0.06。PTX-NPs溶液組的SUVmax值分別為:1.14±0.09,0.97±0.07,0.86±0.04和0.58±0.03。結(jié)論:1.本實(shí)驗(yàn)通過開環(huán)聚合法成功制備出PCEC聚合物,并將其制備成納米顆粒,其毒性相對(duì)較低,且該納米顆粒能比較容易地被腫瘤細(xì)胞攝取,是一種比較理想的、安全的藥物載體。2.通過薄膜分散法成功制備出PTX-NPs,外觀呈均勻球形,具有良好的載藥率、包封率和藥物釋放特性;其粒徑較小,且呈單分散分布,非常適合靜脈給藥。3.PTX-NPs的體外細(xì)胞毒性隨著藥物濃度的增大而增大,并具有時(shí)辰依賴性。4.在體內(nèi)抗腫瘤作用研究中發(fā)現(xiàn),PTX-NPs時(shí)辰給藥對(duì)肺癌表現(xiàn)出顯著的抑制作用,具有協(xié)同效應(yīng),并在15HALO給藥時(shí)其抗腫瘤作用達(dá)到最佳,其機(jī)制與抑制腫瘤血管的生成、腫瘤細(xì)胞的增殖和促進(jìn)細(xì)胞凋亡有關(guān)。
[Abstract]:Objective: the purpose of this study was to explore the antitumor effect and mechanism of the drug on lung cancer by PTX (poly (ethylene glycol) -poly (ethylene glycol) -poly (PCEC) -poly (PCEC) -poly (PCL-PEG-PCL, PCEC)). Methods: the most suitable time point for drug delivery. Methods: this experiment is divided into two parts in vitro and in vivo. In the study in vitro, first of all, poly (ethylene glycol), PEG) and caprolactone (epsilon -caprolactone) are used as raw materials to prepare PCEC polymers successfully and take them as carriers; and then, the membrane dispersion method is successfully made. PTX-NPs was prepared by transmission electron microscopy, and its particle size was evaluated by dynamic light scattering scanner (High performance liquid chromatography, HPLC). The percentage of encapsulation, drug loading rate and drug release in vitro were measured by high performance liquid chromatography (chromatography, HPLC). P (methylthiazoletetrazolium, MTT) was used to study P Cytotoxicity of TX-NPs and empty carrier PCEC polymer nanoparticles. The cell uptake of PCEC polymer nanoparticles in vitro was observed by fluorescence microscopy (Fluorescence microscope). In vivo, A549 lung cancer cells were cultured in a conventional method. Then, 3 weeks of nude mice were raised in the same light environment, and the same organisms were established. Rhythm, light time (07:00-19:00), dark time (19:00-07:00). A nude mouse model of subcutaneous xenograft tumor of A549 lung cancer was constructed, and the tumor volume grew up to about 100mm3, and was randomly divided into three groups, each group was treated with 0,5,10,15HALO (Hours After Light Onset set light after light) (that is, four times of 07:00,12:00,17:00,22:00 in one day, respectively. " The two groups were divided into four subgroups, of which two groups were treated with PTX injection and ptx-nps solution strictly according to the caudal vein at 24 hours of day and night. The third group was used as the control group. The same volume of saline (NS).Ptx injection and ptx-nps solution were given by ptx10mg/kg, once every 3 days. 3 times. Every day, the body weight of the nude mice was measured, the tumor size was measured, the tumor growth curve was drawn, the tumor inhibition rate was calculated, and the 18F-FDG (fluorine-18-deoxyglucose) pet/ct (positronemissiontomography/computedtomography) imaging of the small animals was observed and analyzed. The nude mice were killed, the lump of the tumor was stripped and fixed after 12 days. Immunohistochemistry (IHC) was used to detect the expression of cd-31 and Ki-67 in all the tumor specimens, and the apoptosis of the tumor specimens was detected by tdt-mediateddutpnickendlabeling (tdt-mediateddutpnickendlabeling, TUNEL). Results: the appearance of ptx-nps in this study was spherical, with an average size of about 16 8nm, the particle size distribution is uniform, with the initial rapid release and the subsequent slow and sustained release of the drug.Mtt test results show that the no-load pCEC polymer nanoparticles have no obvious cytotoxicity, and the cytotoxicity of ptx-nps in vitro increases with the increase of drug concentration, and is given to ptx-np at four different hour points. The s solution (PTX concentration was the same), the cell activity showed a gradual increasing trend: the tumor inhibition rate in the ptx-nps solution group and PTX injection group was significantly higher than that of the control group (P0.01), and the tumor inhibition rates were the highest at 22:00, and the values were 84.36% and 68.84% respectively (P 0.05). In addition, the expression of cd-31 and Ki-67 in the treatment group showed a decreasing trend: 07:0012:0017:0022:00; the number of apoptotic cells in the treatment group showed an increasing trend: 07:0012:0017:0022:00. and the expression of CD-31 and Ki-67 in PTX-NPs solution group was lower than that of PTX injection group at 22:00 (P0.05), and the number of apoptotic cells was higher than that of PTX injection. Group high (P0.05). The results of 18F-FDG PET/CT examination of small animals showed that at four different hour points (07:00,12:00,17:00 and 22:00), the SUVmax values of the control group were respectively: 2.15 + 0.23,2.10 + 0.20,2.5 + 0.25 and 2.12 + 0.18.PTX injection groups, respectively: 1.44 + 0.44,1.26 +, 0.11,1.15 + 0.06 and 0.83 + 0.06.PTX-NPs solution groups The values are as follows: 1.14 + 0.09,0.97 + 0.07,0.86 + 0.04 and 0.58 + 0.03. conclusion: 1. this experiment successfully prepared PCEC polymer by open ring polymerization, and prepared it into nano particles, its toxicity is relatively low, and the nanoparticles can be easily absorbed by tumor cells. It is an ideal, safe drug carrier.2. through thin film. The dispersion method successfully prepared PTX-NPs with a uniform appearance and spherical appearance, with good drug loading rate, encapsulation efficiency and drug release characteristics. Its particle size is small, and it is monodisperse distribution. The cytotoxicity of.3.PTX-NPs in vitro is very suitable for the increase of drug concentration, and the antitumor effect of time dependent.4. in the body is studied. It is found that PTX-NPs time administration has a significant inhibitory effect on lung cancer and has a synergistic effect, and its anti-tumor effect is best when 15HALO is administered. The mechanism is related to inhibiting the formation of tumor vessels, proliferation of tumor cells and promoting cell apoptosis.

【學(xué)位授予單位】：西南醫(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)】：R734.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 李海東;官元;辛賀;程鳳梅;;PCL及PCL-PEG-PCL共聚物的合成與表征[J];合成樹脂及塑料;2012年03期

2 張琳華;何穎娜;馬桂蕾;宋存先;;葉酸靶向紫杉醇聚合物納米囊泡的制備及其抗腫瘤活性研究[J];中國(guó)藥學(xué)雜志;2010年22期

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本文編號(hào)：1887198