本文選題：gp100 + TRP-2　； 參考：《浙江理工大學(xué)》2015年碩士論文

【摘要】：惡性黑色素瘤是一種具有高度轉(zhuǎn)移性的惡性腫瘤，全球范圍內(nèi)的黑色素瘤致死率一直居高不下。近年來，隨著酪氨酸激酶相關(guān)蛋白-2(tyrosinase related protein2, TRP-2)和gp100等黑色素瘤相關(guān)抗原研究的日益深入，黑色素瘤DNA疫苗得到了極大的發(fā)展。然而，盡管已經(jīng)在治療惡性黑色素瘤方面取得了可喜成績，黑色素瘤DNA疫苗仍然存在著容易引發(fā)免疫耐受、誘導(dǎo)的免疫應(yīng)答微弱等問題亟待解決。 TRP-2和gp100在人類黑色素瘤中高度表達(dá)，并且在同源的鼠類黑色素瘤中有類似現(xiàn)象。鼠源gp100(mgp100)和鼠源TRP-2(mTRP-2)在小鼠體內(nèi)容易引發(fā)免疫耐受，導(dǎo)致小鼠難以產(chǎn)生有效的保護(hù)性免疫應(yīng)答反應(yīng)。在本實(shí)驗(yàn)中，我們嘗試著使用異源黑色素瘤相關(guān)抗原來打破這些免疫耐受反應(yīng)，提高黑色素瘤DNA疫苗的抗腫瘤效果。然而，DNA疫苗本身難以誘導(dǎo)強(qiáng)烈的免疫應(yīng)答反應(yīng)，還需要使用適當(dāng)?shù)淖魟┘訌?qiáng)其效果。因此，我們選擇了基因佐劑Ii-PADRE(invariant Pan DR reactive epitope)和鼠源白介素12(murine interleukin-12, mIL-12)配合我們的黑色素瘤疫苗。此外，癌癥患者體內(nèi)的調(diào)節(jié)性T (Treg)細(xì)胞可能會抑制針對自身腫瘤抗原如gp100和TRP-2的抗腫瘤免疫應(yīng)答。我們使用地尼白介素(denileukin diftitox, ONTAK)去除Tregs，以便于疫苗更好的發(fā)揮功效。 我們的實(shí)驗(yàn)結(jié)果顯示，電脈沖法免疫人源gp100(hgp100)和人源TRP-2(hTRP-2)黑色素瘤DNA疫苗(phgp100和phTRP-2)在B16F10腫瘤模型中表現(xiàn)出了顯著的保護(hù)作用。因此，，異源gp100和TRP-2對于打破這些同源抗原引起的免疫耐受是十分必要的；ELISPOT結(jié)果顯示，phgp100、phTRP-2和pIi-PADRE共同免疫使得抗TRP-2的IFN-γ分泌細(xì)胞的數(shù)量增加了近2倍，體外抗B16F10細(xì)胞的毒性細(xì)胞的數(shù)量增加了近4倍。而phTRP-2、phgp100和pIi-PADRE(肌肉免疫)結(jié)合3次注射pmIL-12(瘤內(nèi)免疫)免疫的方案治療效果最佳，該方案免疫小鼠的無瘤生存率達(dá)到了75%，且皮下建立的B16F10腫瘤得以有效消退；此外，我們發(fā)現(xiàn)疫苗免疫前一天腹腔注射ONTAK去除Treg細(xì)胞，結(jié)果ONTAK去除Treg細(xì)胞后phTRP-2和phgp100的抗腫瘤效果得到了進(jìn)一步的提升。 綜上所述，2種異源黑色素瘤DNA疫苗(phgp100和phTRP-2)共同免疫可顯著提高機(jī)體對B16F10黑色素瘤的保護(hù)作用；以O(shè)NTAK去除Treg細(xì)胞可以進(jìn)一步增加癌癥疫苗效果；而最有效的治療策略是phgp100和phTRP-2疫苗結(jié)合基因佐劑pIi-PADRE和pmIL-12共同免疫。該方案可以在不去除Treg細(xì)胞的情況下完全消退建立的B16F10腫瘤，有效的延續(xù)小鼠的存活時(shí)間并且防止病情復(fù)發(fā)。
[Abstract]:Malignant melanoma is a highly metastatic malignant tumor. In recent years, with the development of melanoma associated antigens such as tyrosine kinase associated protein 2 (tyrosinase related protein 2 (TRP-2) and gp100, melanoma DNA vaccines have been greatly developed. However, despite the gratifying achievements in the treatment of malignant melanoma, melanoma DNA vaccines are still prone to induce immune tolerance. TRP-2 and gp100 are highly expressed in human melanoma and have similar phenomena in homologous rat melanoma. Mouse gp100 (mgp100) and murine TRP-2 (mTRP-2) can easily induce immune tolerance in mice, which makes it difficult for mice to produce an effective protective immune response. In this study, we tried to break these immune tolerance responses by using heterogenous melanoma associated antigens to improve the antitumor effect of melanoma DNA vaccine. However, DNA vaccine itself is difficult to induce a strong immune response, and need to use appropriate adjuvant to enhance its effect. Therefore, invariant Pan Dr reactive epitope) and murine interleukin-12 (mIL-12) were selected to cooperate with our melanoma vaccine. In addition, regulatory T (Treg) cells in cancer patients may inhibit the antitumor immune response to their own tumor antigens such as gp100 and TRP-2. We use Denil (denileukin diftitox, Ontak) to remove Tregs in order to make the vaccine more effective. Our results showed that the immunized human gp100 (hgp100) and human TRP-2 (hTRP-2) melanoma DNA vaccines (phgp100 and phTRP-2) showed significant protective effects in B16F10 tumor model. Therefore, allogenic gp100 and TRP-2 are necessary to break the immune tolerance induced by these homologous antigens. The results of ELISPOT showed that co-immunization of phgp100 phTRP-2 and pIi-PADRE increased the number of IFN- 緯 secreting cells against TRP-2 by nearly 2 times. The number of anti-B 16 F 10 cells increased nearly 4 times in vitro. However, the combination of phTRP-2 phgp100 and pIi-PADRE combined with three injections of pmIL-12 (intratumoral immunity) was the best in the treatment. The tumor-free survival rate of mice immunized with phTRP-2 and pIi-PADRE was 75 and the subcutaneous B16F10 tumor subsided effectively. We found that the antitumor effect of phTRP-2 and phgp100 was further improved one day before vaccination by intraperitoneal injection of ONTAK to remove Treg cells. In conclusion, the co-immunization of two heterogenous melanoma DNA vaccines (phgp100 and phTRP-2) can significantly improve the protective effect of B16F10 melanoma, and the removal of Treg cells by ONTAK can further increase the effect of cancer vaccine. The most effective treatment strategy is the co-immunization of pIi-PADRE and pmIL-12 with phgp100 and phTRP-2 vaccine adjuvants. This scheme can completely subside the established B16F10 tumor without removing Treg cells, effectively prolong the survival time of mice and prevent the recurrence of the disease.
【學(xué)位授予單位】：浙江理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R739.5

【共引文獻(xiàn)】

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