本文選題：納米脂質(zhì)體 + MAGE3　； 參考：《第四軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 惡性腫瘤危害巨大,但惡性腫瘤的三大傳統(tǒng)療法-手術(shù)、放療和化療,治療效果不盡人意。隨著對腫瘤免疫理論的深入認(rèn)識和一些新的實驗方法的涌現(xiàn),腫瘤疫苗已成為預(yù)防和治療腫瘤轉(zhuǎn)移、復(fù)發(fā)的有力手段。但由于機體免疫網(wǎng)絡(luò)的復(fù)雜性、腫瘤異質(zhì)性、疫苗生物利用度差等問題,使得現(xiàn)有腫瘤疫苗的免疫效果并不理想。黑色素瘤抗原(Melanoma Antigen,MAGE)是第一個被發(fā)現(xiàn)的人類腫瘤特異性抗原,MAGE3是MAGE家族的重要成員之一,在多種不同類型惡性腫瘤中均有表達(dá),在正常組織細(xì)胞(除睪丸、胎盤外)不表達(dá);且MAGE3可與人類白細(xì)胞抗原(Human Leukocytes Antigen,HLA)分子結(jié)合形成抗原復(fù)合物,此抗原復(fù)合物可被細(xì)胞毒性T淋巴細(xì)胞(Cytotoxic T Lymphocyte,CTL)的T細(xì)胞受體(T cell receptor,TCR)所識別,誘導(dǎo)特異性CTL的殺傷作用,是腫瘤特異性免疫治療理想的靶分子。熱休克蛋白(Heat Shock Protein,HSP)具有分子伴侶作用,參與腫瘤抗原的加工遞呈,在誘導(dǎo)腫瘤免疫反應(yīng)中發(fā)揮著重要作用;本室研究表明,HSP70與MAGE3融合蛋白能提高M(jìn)AGE3蛋白疫苗的免疫效果。納米載藥系統(tǒng)與傳統(tǒng)載藥系統(tǒng)相比有很多優(yōu)點,可有效解決蛋白質(zhì)藥物的生物利用度差的問題,目前已經(jīng)有許多深入而廣泛的研究。 本研究以納米脂質(zhì)體包裹腫瘤特異性抗原MAGE3與熱休克蛋白HSP70的融合蛋白(MH),制備出納米脂質(zhì)體疫苗NL(MH),著重對其抗腫瘤轉(zhuǎn)移與復(fù)發(fā)作用進(jìn)行研究,為該納米疫苗臨床前研究奠定基礎(chǔ)。 一納米脂質(zhì)體疫苗制備 目的:制備無明顯毒性、高效的惡性腫瘤基因工程納米疫苗。方法:純化融合蛋白MH,薄膜分散-超聲法制備包裹MH的納米脂質(zhì)體腫瘤疫苗NL(MH),并評價其粒徑、包裹率及穩(wěn)定性;觀察NL(MH)免疫組小鼠的急性毒性反應(yīng),及重要臟器的病理學(xué)改變。結(jié)果:①成功制備出粒徑為80±25 nm的納米脂質(zhì)體,其藥物包裹率為30%,于4℃放置6個月后或3000rpm 10分鐘離心后無分層,證明其穩(wěn)定性良好;②與PBS對照組相比,免疫組小鼠未見明顯毒性反應(yīng)。結(jié)論:該惡性腫瘤基因工程納米疫苗具有良好的理化性質(zhì),未見毒性反應(yīng)。 二納米疫苗預(yù)防和治療腫瘤轉(zhuǎn)移和復(fù)發(fā)效應(yīng)研究 目的:在小鼠體內(nèi)觀察納米疫苗預(yù)防和治療腫瘤肺轉(zhuǎn)移及預(yù)防腫瘤復(fù)發(fā)的作用。方法:以PBS、空白脂質(zhì)體、MH、MH /NL、NL(MH)免疫C57BL/6小鼠3次后,IFN-γELISPOT和LDH殺傷試實驗檢測納米疫苗激活小鼠特異性細(xì)胞免疫反應(yīng)的情況;以實驗性肺轉(zhuǎn)移模型和自然肺轉(zhuǎn)移模型評價納米疫苗預(yù)防和治療腫瘤轉(zhuǎn)移作用;以腫瘤攻擊實驗和腫瘤切除后復(fù)發(fā)實驗來評價納米疫苗預(yù)防腫瘤復(fù)發(fā)作用。結(jié)果:與其它組相比,NL(MH)組小鼠脾淋巴細(xì)胞中分泌IFN-γ的T細(xì)胞數(shù)量明顯增多(P0.05),CTL對B16-MAGE3細(xì)胞具有顯著的特異性殺傷作用;在預(yù)防和治療腫瘤肺轉(zhuǎn)移實驗中,與其它組小鼠相比,NL(MH)免疫組小鼠B16-MAGE3腫瘤肺臟轉(zhuǎn)移結(jié)節(jié)數(shù)明顯減少(P0.05);在腫瘤攻擊實驗中,NL(MH)組B16-MAGE3腫瘤成瘤時間長、成瘤率低;腫瘤切除后復(fù)發(fā)實驗中,NL(MH)組B16-MAGE3腫瘤復(fù)發(fā)時間延遲,復(fù)發(fā)率明顯降低。結(jié)論: NL(MH)能夠刺激機體產(chǎn)生強烈的MAGE3特異性的細(xì)胞免疫反應(yīng),對表達(dá)MAGE3的腫瘤細(xì)胞具有顯著的殺傷作用,能有效預(yù)防和治療B16-MAGE3腫瘤轉(zhuǎn)移及預(yù)防腫瘤切除后復(fù)發(fā)。 綜上所述,本研究成功制備出粒徑為80±25nm的納米脂質(zhì)體疫苗NL(MH),其穩(wěn)定性良好;NL(MH)能誘導(dǎo)機體產(chǎn)生強烈的MAGE3特異性CTL,對表達(dá)MAGE3腫瘤轉(zhuǎn)移和復(fù)發(fā)具有良好的治療和預(yù)防作用,對表達(dá)MAGE3腫瘤復(fù)發(fā)具有良好的預(yù)防作用。本研究為研制使用安全、高效的腫瘤疫苗展示了良好的前景,為本疫苗的臨床前研究奠定了基礎(chǔ)。
[Abstract]:Malignant tumor is very harmful, but the three traditional therapies of malignant tumor - surgery, radiotherapy and chemotherapy are unsatisfactory. With the deep understanding of the theory of tumor immunity and the emergence of some new experimental methods, the tumor vaccine has become a powerful means to prevent and treat tumor metastasis and relapse. But the complexity of the immune network of the body is complicated. Melanoma Antigen (MAGE) is the first human tumor specific antigen found. MAGE3 is one of the important members of the MAGE family, which is expressed in many different types of malignant tumors and in the normal group. The cells (except the testis, the placenta) are not expressed, and MAGE3 can be combined with the Human Leukocytes Antigen (HLA) molecules to form an antigen complex, which can be identified by the T cell receptor of the cytotoxic T lymphocyte (Cytotoxic T Lymphocyte, CTL), and induces the killing effect of the specific antigen. It is an ideal target molecule for tumor specific immunotherapy. Heat Shock Protein (HSP) has the role of molecular chaperone, participates in the processing of tumor antigen and plays an important role in inducing tumor immune response. This laboratory study shows that the fusion of egg white with HSP70 and MAGE3 can improve the immune effect of the MAGE3 protein vaccine. Compared with the traditional drug carrying system, the system has many advantages, which can effectively solve the problem of poor bioavailability of protein drugs, and there have been a lot of in-depth and extensive research.
In this study, nano liposome specific antigen MAGE3 and heat shock protein HSP70 fusion protein (MH) was used to prepare nano liposome vaccine NL (MH), focusing on its anti-tumor metastasis and recurrence effect, which lay the foundation for the pre clinical study of the nano vaccine.
Preparation of a nano liposome vaccine
Objective: to prepare the nano vaccine of malignant tumor gene engineering without obvious toxicity and high efficiency. Methods: to purify the fusion protein MH and to prepare the nano liposome tumor vaccine NL (MH) coated with MH by thin film dispersion ultrasonic method and evaluate its size, encapsulation rate and stability. The acute toxicity of NL (MH) immunized mice and the pathological changes of important organs were observed. Results: (1) the nano liposomes with a diameter of 80 + 25 nm were successfully prepared. The drug encapsulation rate was 30%. After 6 months at 4 C or after 3000rpm 10 minutes without stratification, it proved that the stability was good. 2. Compared with the PBS control group, the immune group had no obvious toxic reaction. No toxic reaction was found in physical and chemical properties.
Two nano vaccine for prevention and treatment of tumor metastasis and recurrence
Objective: To observe the effect of nano vaccine in preventing and treating tumor lung metastasis and preventing tumor recurrence in mice. Methods: after 3 doses of PBS, blank liposomes, MH, MH /NL, NL (MH) immunized C57BL/6 mice, IFN- gamma ELISPOT and LDH killing test test was used to detect the specific cell immune response of mice activated by nano vaccine; the experimental lung transfer model was used. The effect of nano vaccine on the prevention and treatment of tumor metastasis was evaluated by the model and natural lung metastasis model. The effect of nano vaccine on tumor recurrence was evaluated by tumor attack experiment and recurrence experiment after tumor resection. Results: compared with other groups, the number of T cells secreted IFN- gamma in spleen lymphocyte of NL (MH) mice increased significantly (P0.05), CTL to B16-MAGE3 In the prevention and treatment of tumor lung metastasis, the number of metastatic nodules of lung in the NL (MH) immune group was significantly reduced in the NL (MH) immunization group than in the other mice (P0.05). In the tumor attack experiment, the NL (MH) group of B16-MAGE3 tumors had a long tumorigenesis and a low tumor rate; NL in the recurrence experiment after tumor resection, NL (MH) group B16-MAGE3 tumor recurrence time delay, recurrence rate obviously decreased. Conclusion: NL (MH) can stimulate the body to produce a strong MAGE3 specific cellular immune response, the expression of MAGE3 tumor cells have a significant killing effect, the effective prevention and treatment of B16-MAGE3 swollen tumor metastasis and the prevention of recurrence after tumor resection.
To sum up, the nano liposome vaccine NL (MH) with a particle size of 80 + 25nm was successfully prepared, and its stability was good. NL (MH) could induce a strong MAGE3 specific CTL in the body, and had a good therapeutic and preventive effect on the metastasis and recurrence of MAGE3 tumor, and had a good preventive effect on the recurrence of the MAGE3 tumor. The use of a safe and highly effective tumor vaccine shows good prospects and lays the foundation for preclinical research of this vaccine.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R392;R73-36

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