本文選題：乳鐵蛋白 + 白藜蘆醇��； 參考：《遵義醫(yī)學院》2017年碩士論文

【摘要】：目的:制備乳鐵蛋白修飾的白藜蘆醇納米粒(Lactoferrin modified Resveratrol Nano Particles,Lf-Res-NPs),考察其在小鼠體內藥物代謝動力學,觀察其對阿爾茨海默病(Alzheimer’s disease,AD)模型大鼠的干預作用,初步評價其腦靶向作用。方法:(1)界面聚合法制備白藜蘆醇納米粒(Resveratrol Nano Particles,Res-NPs),星點設計優(yōu)化處方和工藝,采用羥基聚乙二醇馬來酰亞胺(Hydroxyl-PEG-Maleimide,OH-PEG-MAL)的-OH基團鏈接Res-NPs,MAL基團與活化后的乳鐵蛋白巰基結合,制備Lf-Res-NPs,激光粒度分析儀檢測其粒徑,透射電鏡觀察其形態(tài)。(2)SPF級昆明小鼠270只,雄性,隨機分為3組:Res溶液組、Res-NPs組和Lf-Res-NPs組。常規(guī)飼養(yǎng)1周后腹腔注射給藥,給藥后0.17、0.5、0.75、1、2、6、10、12、24h采血,每個時間點10只小鼠。采用HPLC法分析血漿及組織中藥物濃度,計算藥代動力學參數(shù)和各組織靶向指數(shù)。(3)SPF級SD大鼠50只,雌雄各半,隨機分為正常組、AD模型組、Res溶液組、Res-NPs組和Lf-Res-NPs組,每組10只。正常組每天腹腔注射生理鹽水和灌胃蒸餾水,其余各組腹腔注射D-半乳糖150mg·kg-1·d-1和灌胃三氯化鋁10mg·kg-1·d-1,連續(xù)65d。第31d正常組和AD模型組腹腔注射生理鹽水,藥物干預組腹腔注射不同劑型Res,Res給藥劑量為30mg·kg-1·d-1。造模65d后,Morris水迷宮檢測大鼠認知功能;比色法檢測血清及腦組織中谷胱甘肽過氧化物酶(Glutathione peroxidase,GSH-PX)、超氧化物歧化酶(Superoxide dismutase,SOD)活力及丙二醛(Malondialdehyde,MDA)含量;免疫組織化學染色觀察海馬β-淀粉樣蛋白1-42(βeta amyloid peptides1-42,Aβ1-42)及半胱氨酸天冬氨酸蛋白酶3(Cysteinecontaining aspartate-specific protease3,Casepase-3)表達;ELISA法檢測海馬組織Aβ1-42的表達;Western Bolt檢測海馬β-淀粉樣前體蛋白(βeta-Amyloid precursor protein,APP)、Caspase-3及核轉錄因子-κB(Nuclear factor-κB,NF-κB)蛋白的表達。結果:(1)(1)Res-NPs制備最佳處方:油水體積比4.0m L:8.0m L,水相p H 2.0,右旋糖酐-70 50mg,Res 48mg,BCA 52μL。其粒徑(229.02±4.30)nm,載藥量(10.13±0.96)%,包封率(80.36±2.70)%;(2)在p H7.4 PBS釋放介質中,Res溶液2h累積釋放Res(80.26±1.61)%,8h累積釋放Res(86.16±0.10)%;Res-NPs 2h累積釋放Res(56.77±2.47)%,8h累積釋放Res(73.11±7.14)%,72h累積釋放Res(80.24±2.78)%;Res-NPs中加入血漿時,2h累積釋放Res(56.62±0.30)%,8h時累積釋放Res(82.22±3.22)%,72h累積釋放Res(87.96±0.72)%;(3)Lf-Res-NPs粒徑(240±4.652)nm,透射電鏡鑒定Lf與Res-NPs成功連接。(2)(1)藥代動力學結果顯示:與Res溶液組藥時曲線下面積(AUC(0-∞))、生物半衰期(t1/2z)、達峰時間(Tmax)、達峰濃度(Cmax)和清除率(CLz/F)比較,Res-NPs組和Lf-Res-NPs組AUC(0-∞)、t1/2z、Tmax、Cmax增加,CLz/F降低(P0.05);與Res-NPs組比較,AUC(0-∞)、t1/2z、Tmax、Cmax增加,CLz/F降低(P0.05);(2)Res溶液組和Res-NPs組藥物主要集中在肝和腎組織,Lf-Res-NPs組藥物主要集中在肝、脾和腦組織,給藥120min后Res溶液組腦靶向指數(shù)最大值為14.263%,Res-NPs組腦靶向指數(shù)最大值為20.980%,Lf-Res-NPs組腦靶向指數(shù)最大值為26.879%,其Lf-Res-NPs組比Res-NPs組增加1.3倍,比Res溶液組增加1.9倍;且給藥后10、12、24h Lf-Res-NPs組腦靶向指數(shù)顯著高于其余兩組,差異有統(tǒng)計學意義(P0.05)。(3)(1)水迷宮定位航行結果顯示:與正常組比較,AD模型組1-4d逃避潛伏期延長(P0.05);與AD模型組比較,不同劑型Res干預的各組2-4d逃避潛伏期縮短(P0.05);與Res溶液組比較,Res-NPs組和Lf-ResNPs組在第3-4d逃避潛伏期縮短(P0.05);與Res-NPs組比較,Lf-Res-NPs組在第4d逃避潛伏期縮短(P0.05);空間探索結果顯示:與正常組比較,AD模型組穿環(huán)次數(shù)和滯留時間減少(P0.05);與AD模型組比較,不同劑型Res干預的各組穿環(huán)次數(shù)與滯留時間增加(P0.05);不同劑型Res組間比較,Lf-Res-NPs組穿環(huán)次數(shù)與滯留時間增加(P0.05)。(2)與正常組比較,AD模型組血清及腦組織內SOD和GXH-PX活力降低,MDA含量升高(P0.05);與AD模型組比較,Res-NPs組和Lf-Res-NPs組血清及腦組織SOD和GSH-PX活力升高,MDA含量降低(P0.05);不同劑型Res組間比較,Lf-Res-NPs組血清及腦組織SOD和GSH-PX活力增加,MDA含量降低(P0.05)。(3)免疫組化結果顯示:與正常組比較,AD模型組大鼠海馬Casepase-3和Aβ1-42蛋白表達增加(P0.05);與AD模型組比較,不同劑型Res干預的各組大鼠海馬Casepase-3和Aβ1-42蛋白表達降低(P0.05);不同劑型Res組間比較,Lf-Res-NPs組大鼠海馬Casepase-3和Aβ1-42蛋白表達降低(P0.05)。(4)ELISA檢測結果顯示:與正常組比較,AD模型組大鼠海馬Aβ1-42蛋白表達增加(P0.05);與AD模型組比較,不同劑型Res干預的各組大鼠海馬Aβ1-42蛋白表達降低(P0.05);不同劑型Res組間比較,Lf-Res-NPs組大鼠海馬Aβ1-42蛋白表達降低(P0.05)。(5)Western bolt結果顯示:與正常組比較,AD模型組大鼠海馬Casepase-3、NF-κB和APP蛋白表達增加(P0.05);與AD模型組比較,不同劑型Res干預的各組大鼠海馬Casepase-3、NF-κB、APP蛋白表達降低(P0.05);不同劑型Res組間比較,Lf-Res-NPs組大鼠海馬Casepase-3、NF-κB、APP蛋白表達降低(P0.05)。結論:(1)制備得到粒徑為(240±4.652)nm Lf-Res-NPs。(2)Lf-Res-NPs可延長Res在小鼠體內的作用時間,增加Res在小鼠體內的生物利用度,提高Res的腦靶向作用,減少其他組織對Res的攝取。(3)Lf-Res-NPs可遞送更多Res進入腦內,增加腦內Res濃度,提高抗氧化能力,抑制海馬神經(jīng)元APP和Aβ1-42蛋白表達,減輕神經(jīng)元凋亡和炎癥反應,效果較Res溶液和Res-NPs好。
[Abstract]:Objective: Resveratrol nanoparticles modified lactoferrin (Lactoferrin modified Resveratrol Nano Particles, Lf-Res-NPs), to investigate the in vivo drug metabolism in mice to observe the dynamics of Alzheimer's disease (Alzheimer 's disease, AD) intervention model rats, evaluate its brain targeting effect. Methods: (1) preparation of resveratrol nanoparticles by interfacial polymerization (Resveratrol Nano, Particles, Res-NPs), optimized design, using hydroxyl polyethylene glycol maleimide (Hydroxyl-PEG-Maleimide, OH-PEG-MAL) -OH groups link Res-NPs, MAL group combined with activated lactoferrin thiol, preparation of Lf-Res-NPs, laser particle size analyzer and particle size, were observed morphology of TEM. (2) SPF 270 Kunming mice, male, were randomly divided into 3 groups: Res solution group, Res-NPs group and Lf-Res-NPs group. After 1 weeks of feeding abdominal routine Injection after administration of blood 0.17,0.5,0.75,1,2,6,10,12,24h, 10 rats in each time point were analyzed by HPLC assay. The drug concentration in plasma and tissues, pharmacokinetic parameters and the target to calculate the index. (3) SPF 50 SD rats, male and female, were randomly divided into normal group, AD model group. The solution of Res group, Res-NPs group and Lf-Res-NPs group, 10 rats in each group. The normal group received intraperitoneal injection of saline and distilled water, the other groups were intraperitoneally injected with D- galactose 150mg - kg-1 - D-1 and 10mg - kg-1 - alchlor intragastric administration of D-1, the continuous 65d. 31d normal group and AD model group by intraperitoneal injection of saline. The drug intervention group by intraperitoneal injection of different dosage of Res, Res dosage was 30mg - kg-1 - d-1. model 65D, water maze Morris; ratio of serum and brain tissue glutathione peroxidase assay enzyme (Glutathione peroxidase, GSH-PX), super Superoxide dismutase (Superoxide dismutase, SOD) activity and malondialdehyde (Malondialdehyde, MDA) content of hippocampus were observed; amyloid beta protein 1-42 immunohistochemical staining (ETA amyloid beta peptides1-42, A beta 1-42) and caspase 3 (Cysteinecontaining aspartate-specific, protease3, Casepase-3) to detect the expression of A beta expression; hippocampus ELISA 1-42; Western Bolt was detected in the beta amyloid precursor protein (beta eta-Amyloid precursor protein, APP, Caspase-3) and nuclear factor kappa B (factor- K Nuclear B NF- K B) protein expression. Results: (1) Res-NPs (1) prepared by the best prescription: the volume ratio of oil to water 4.0m L:8.0m L, aqueous P H 2, -70 50mg Res 48mg, dextran, BCA 52 L. diameter (229.02 + 4.30) nm, drug loading (10.13 + 0.96)%, the encapsulation rate (80.36 + 2.70)%; (2) in the P H7.4 PBS release medium, Res solution of 2H accumulation the release of Res (80. 26鹵1.61)%,8h绱Н閲婃斁Res(86.16鹵0.10)%;Res-NPs 2h绱Н閲婃斁Res(56.77鹵2.47)%,8h绱Н閲婃斁Res(73.11鹵7.14)%,72h绱Н閲婃斁Res(80.24鹵2.78)%;Res-NPs涓姞鍏ヨ嫻嗘椂,2h绱Н閲婃斁Res(56.62鹵0.30)%,8h鏃剁瘡縐噴鏀綬es(82.22鹵3.22)%,72h绱Н閲婃斁Res(87.96鹵0.72)%;(3)Lf-Res-NPs綺掑緞(240鹵4.652)nm,閫忓皠鐢?shù)闀滈壌瀹歀f涓嶳es-NPs鎴愬姛榪炴帴.(2)(1)鑽唬鍔ㄥ姏瀛︾粨鏋滄樉紺，

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