异源黑色素瘤DNA疫苗结合佐剂治疗鼠黑色素瘤的研究

发布时间：2018-06-25 00:37

本文选题：gp100 + TRP-2　；参考：《浙江理工大学》2015年硕士论文

【摘要】：恶性黑色素瘤是一种具有高度转移性的恶性肿瘤，全球范围内的黑色素瘤致死率一直居高不下。近年来，随着酪氨酸激酶相关蛋白-2(tyrosinase related protein2, TRP-2)和gp100等黑色素瘤相关抗原研究的日益深入，黑色素瘤DNA疫苗得到了极大的发展。然而，尽管已经在治疗恶性黑色素瘤方面取得了可喜成绩，黑色素瘤DNA疫苗仍然存在着容易引发免疫耐受、诱导的免疫应答微弱等问题亟待解决。 TRP-2和gp100在人类黑色素瘤中高度表达，并且在同源的鼠类黑色素瘤中有类似现象。鼠源gp100(mgp100)和鼠源TRP-2(mTRP-2)在小鼠体内容易引发免疫耐受，导致小鼠难以产生有效的保护性免疫应答反应。在本实验中，我们尝试着使用异源黑色素瘤相关抗原来打破这些免疫耐受反应，提高黑色素瘤DNA疫苗的抗肿瘤效果。然而，DNA疫苗本身难以诱导强烈的免疫应答反应，还需要使用适当的佐剂加强其效果。因此，我们选择了基因佐剂Ii-PADRE(invariant Pan DR reactive epitope)和鼠源白介素12(murine interleukin-12, mIL-12)配合我们的黑色素瘤疫苗。此外，癌症患者体内的调节性T (Treg)细胞可能会抑制针对自身肿瘤抗原如gp100和TRP-2的抗肿瘤免疫应答。我们使用地尼白介素(denileukin diftitox, ONTAK)去除Tregs，以便于疫苗更好的发挥功效。我们的实验结果显示，电脉冲法免疫人源gp100(hgp100)和人源TRP-2(hTRP-2)黑色素瘤DNA疫苗(phgp100和phTRP-2)在B16F10肿瘤模型中表现出了显著的保护作用。因此，，异源gp100和TRP-2对于打破这些同源抗原引起的免疫耐受是十分必要的；ELISPOT结果显示，phgp100、phTRP-2和pIi-PADRE共同免疫使得抗TRP-2的IFN-γ分泌细胞的数量增加了近2倍，体外抗B16F10细胞的毒性细胞的数量增加了近4倍。而phTRP-2、phgp100和pIi-PADRE(肌肉免疫)结合3次注射pmIL-12(瘤内免疫)免疫的方案治疗效果最佳，该方案免疫小鼠的无瘤生存率达到了75%，且皮下建立的B16F10肿瘤得以有效消退；此外，我们发现疫苗免疫前一天腹腔注射ONTAK去除Treg细胞，结果ONTAK去除Treg细胞后phTRP-2和phgp100的抗肿瘤效果得到了进一步的提升。综上所述，2种异源黑色素瘤DNA疫苗(phgp100和phTRP-2)共同免疫可显著提高机体对B16F10黑色素瘤的保护作用；以ONTAK去除Treg细胞可以进一步增加癌症疫苗效果；而最有效的治疗策略是phgp100和phTRP-2疫苗结合基因佐剂pIi-PADRE和pmIL-12共同免疫。该方案可以在不去除Treg细胞的情况下完全消退建立的B16F10肿瘤，有效的延续小鼠的存活时间并且防止病情复发。
[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- 纬 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.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R739.5

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