【摘要】：本文制備了荊豆凝集素介導(dǎo)的生物粘附脂質(zhì)體作為口服疫苗的載體。由于荊豆凝集素可以專(zhuān)屬性粘附并靶向于小鼠腸道派伊爾氏結(jié)上的免疫細(xì)胞—M細(xì)胞,因此,可以誘發(fā)機(jī)體同時(shí)產(chǎn)生系統(tǒng)及粘膜免疫應(yīng)答,并驗(yàn)證了該微粒體作為口服疫苗載體的可行性。 建立了考馬斯亮藍(lán)法測(cè)定模型抗原牛血清白蛋白(BSA)的含量；穩(wěn)定性考察結(jié)果表明：溫度、超聲過(guò)程和光照對(duì)BSA溶液穩(wěn)定性基本沒(méi)有影響。 采用逆向蒸發(fā)法制備了牛血清白蛋白脂質(zhì)體(LIP),以藥物包封率和脂質(zhì)體形態(tài)為指標(biāo),對(duì)影響脂質(zhì)體制備的處方及工藝因素進(jìn)行了考察,并用L9(34)正交設(shè)計(jì)實(shí)驗(yàn)對(duì)處方進(jìn)行優(yōu)化。按最優(yōu)處方制備脂質(zhì)體平均粒徑為4.796±0.416Zeta電位為-15.4±2.6 mv,包封率為69.74%±3.41%。LIP體外釋放曲線表明其具有一定緩釋作用。 建立考馬斯亮藍(lán)法和紅細(xì)胞凝集實(shí)驗(yàn)法測(cè)定荊豆凝集素(UEAI)的含量和活性。UEAI穩(wěn)定性考察結(jié)果表明：4℃-80℃范圍內(nèi),UEAI穩(wěn)定；100℃時(shí),隨著時(shí)間延長(zhǎng),UEAI活性顯著降低；pH2.0-10.0的PBS緩沖液中,UEAI溶液穩(wěn)定。安全性評(píng)價(jià)結(jié)果表明：在濃度104.6-1046.5 mg·L-1范圍內(nèi),UEAI對(duì)腸道是安全無(wú)刺激的,可以作為口服疫苗載體的修飾材料。同時(shí),細(xì)胞毒性研究結(jié)果表明UEAI對(duì)Caco-2細(xì)胞具有一定的促進(jìn)生長(zhǎng)作用。 合成了膽固醇單琥珀酸酯(CHS),對(duì)其進(jìn)行結(jié)構(gòu)表征后,采用化學(xué)偶聯(lián)技術(shù)制備了荊豆凝集素修飾的牛血清白蛋白脂質(zhì)體(UEAI-LIP),平均粒徑為5.129±1.671μm, Zeta電位為-21.4±1.5 mv；用Sepharose4B凝膠柱測(cè)定了UEAI-LIP連接率,最佳連接率為70.80%±2.28%。與LIP釋藥曲線比較可見(jiàn)：凝集素的修飾減少了藥物突釋,降低了藥物釋放速度。酶解穩(wěn)定性考察結(jié)果表明：UEAI-LIP在人工胃液(含胃蛋白酶)和人工腸液(含胰蛋白酶)中脂質(zhì)體表面有UEAI修飾會(huì)明顯增加脂質(zhì)體抗酶解能力,從而保證有更多抗原傳遞到免疫部位。 脂質(zhì)體與小鼠胃腸道體內(nèi)、外粘附性考察結(jié)果表明：與LIP相比,UEAI-LIP在含有PPs的小腸區(qū)域粘附力顯著提高,且隨表面修飾UEAI濃度的增加而增加。同時(shí),單糖抑制實(shí)驗(yàn)表明UEAI與M細(xì)胞的特異性結(jié)合可被單糖a-L-巖藻糖競(jìng)爭(zhēng)性抑制。 利用ELISA法來(lái)測(cè)定小鼠血清樣本中的IgG含量和小腸分泌液樣本中的IgA含量,從而確定誘發(fā)最佳免疫應(yīng)答的脂質(zhì)體處方。結(jié)果表明：①當(dāng)抗原以不同載體口服免疫時(shí)：high-UEAI-LIP組誘發(fā)小鼠的IgG和IgA水平最高,其次是low-UEAI-LIP組和LIP組,最低的為BSA溶液組；②當(dāng)抗原以不同粒徑脂質(zhì)體口服免疫時(shí)：平均粒徑為137±18 nm的小粒徑脂質(zhì)體可以刺激機(jī)體的免疫系統(tǒng)產(chǎn)生更強(qiáng)的系統(tǒng)免疫應(yīng)答,平均粒徑為5.657±1.924μm的大粒徑脂質(zhì)體誘發(fā)高度的粘膜免疫應(yīng)答；③當(dāng)脂質(zhì)體中包裹抗原劑量增加時(shí),可以產(chǎn)生更多的IgA,起到更好的粘膜保護(hù)作用。④當(dāng)BSA溶液以肌注方式免疫可產(chǎn)生高水平IgG；而脂質(zhì)體以口服方式給藥,則可產(chǎn)生較高IgA,即誘發(fā)充分的粘膜免疫應(yīng)答。 以市售雙價(jià)腎綜合征出血熱疫苗(HFRS)為模型抗原,對(duì)UEAI修飾脂質(zhì)體作為口服疫苗載體的可行性進(jìn)行驗(yàn)證。結(jié)果顯示：將HFRS溶液以肌肉注射方式免疫小鼠,可激發(fā)機(jī)體免疫系統(tǒng)產(chǎn)生全身免疫應(yīng)答；其所誘發(fā)的IgA水平在21天時(shí),抗體水平基本達(dá)到高峰,但隨后逐漸降低。而將抗原以UEAI修飾脂質(zhì)體形式口服免疫后,可產(chǎn)生與肌注HFRS溶液相近IgG水平和更強(qiáng)IgA水平,并隨免疫次數(shù)的增加,抗體IgA濃度明顯提高,即可誘發(fā)高水平的粘膜免疫應(yīng)答。
[Abstract]:The bioadhesive liposomes mediated by Jingdou agglutinin were prepared as the carriers of oral vaccine. Because Jingdou agglutinin can specifically adhere to and target the immune cells M cells in the intestinal Pair's node of mice, it can induce the simultaneous systemic and mucosal immune responses of the organism, and it has been proved that Jingdou agglutinin can be used as an oral vaccine carrier. Feasibility of vaccine vector.
The content of BSA was determined by Coomassie brilliant blue method. The results showed that temperature, ultrasonic process and illumination had little effect on the stability of BSA solution.
Bovine serum albumin liposomes (LIP) were prepared by reverse evaporation method. The formulation and technological factors affecting the preparation of liposomes were investigated by using drug encapsulation efficiency and liposome morphology as indexes. The prescription was optimized by L9(34) orthogonal design experiment. The average particle size of the liposomes prepared by the optimal prescription was 4.796+0.416 Zeta potential of - 15. 4 + 2.6 MV, the entrapment efficiency was 69.74% + 3.41%.LIP, the in vitro release curve showed that it had a certain sustained release effect.
The content and activity of UEAI were determined by Coomassie brilliant blue method and erythrocyte agglutination assay. The results of UEAI stability test showed that UEAI was stable in the range of 4 80 C, UEAI activity decreased significantly with time at 100 C, and UEAI solution was stable in PBS buffer with pH 2.0 10.0. In the range of concentration 104.6-1046.5 mg/L-1, UEAI is safe and non-irritating to the intestine, and can be used as a modified material for oral vaccine carrier.
Cholesterol MONOSUCCINATE (CHS) was synthesized and characterized. UAI-LIP modified by Jingdou agglutinin was prepared by chemical coupling technique. The average diameter of UEAI-LIP was 5.129+1.671 micron and the Zeta potential was -21.4+1.5 mv. 2.28%. Compared with the LIP release curve, the lectin modification reduced the drug burst release and the drug release rate. The results of enzymatic hydrolysis stability test showed that UEAI-LIP in artificial gastric juice (including pepsin) and artificial intestinal juice (including trypsin) could significantly increase the liposome anti-enzymatic hydrolysis ability, thus ensuring the stability of UEAI-LIP. More antigens are passed to the immune site.
Compared with LIP, the adhesion of UEAI-LIP to the small intestine containing PPs was significantly enhanced and increased with the concentration of surface modified UEAI. Simultaneously, the specific binding of UEAI to M cells was inhibited by monosaccharide a-L-fucose.
ELISA was used to determine the content of IgG in serum and the content of IgA in intestinal secretion of mice to determine the optimal liposome formulation for inducing immune response. When the antigen was orally immunized with liposomes of different sizes, the small size liposomes with an average diameter of 137 18 nm could stimulate the immune system to produce stronger systemic immune response, and the large size liposomes with an average diameter of 5.657 65 Increasing the dosage of encapsulated antigen can produce more IgA and play a better role in mucosal protection.
The feasibility of UEAI modified liposomes as oral vaccine carriers was validated by using commercially available bivalent hemorrhagic fever with renal syndrome vaccine (HFRS) as model antigen. The results showed that the immune system of mice immunized with HFRS solution by intramuscular injection could induce systemic immune response, and the level of IgA induced by HFRS could be detected at 21 days. After oral immunization in the form of UEAI modified liposomes, the levels of IgG and stronger IgA were similar to those in HFRS solution. With the increase of immunization times, the concentration of IgA increased significantly, which could induce a high level of mucosal immune response.
【學(xué)位授予單位】：沈陽(yáng)藥科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392.1

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