B7-H3基因免疫联合沉默Id-1基因：治疗恶性黑色素瘤的新策略

发布时间：2018-09-17 06:57

【摘要】： 目的：恶性黑色素瘤(malignant Melanoma, MM)是来源于黑色素细胞的高度恶性肿瘤,为人类第五大常见肿瘤,临床常用的治疗方法包括手术治疗,化学治疗,免疫治疗等,这些方法虽然在临床上取得了一定的疗效,但是很难彻底治愈肿瘤。近年来,随着癌基因和抑癌基因的相继发现,分子生物学手段的发展和完善,基因治疗成为肿瘤治疗研究中的热点,出现了多种基因治疗方法。这些方法虽然都取得了一定的抑瘤效果,但疗效并不理想。其主要原因是肿瘤是一个多因素、多环节、多阶段的复杂疾病,针对单一因素的治疗并不能根除肿瘤。因此,对现有治疗方法的联合成为基因治疗研究的趋势。本课题运用基因联合治疗的方法治疗小鼠恶性黑色素瘤,将活化小鼠特异性抗肿瘤免疫和抑制癌基因的表达两种治疗方法相联合,观察基因免疫治疗联合癌基因治疗对恶性黑色素瘤的治疗效果,为恶性黑色素瘤的治疗提供新的策略。方法：构建携带小鼠B7-H3基因的腺病毒表达载体(pAd-B7-H3)和携带小鼠Id-1短发夹RNA的慢病毒载体(Lent-Id 1-shRNA),通过体外实验和体内实验进行功能鉴定。将鼠B16恶性黑色素瘤细胞株接种于昆明小鼠皮下,构建恶性黑色素瘤模型,并将小鼠随机分为四组：①pAd-B7-H3+Lent-Id1-shRNA治疗组：②pAd-B7-H3治疗组；③Lent-Id1-shRNA治疗组；④未行任何治疗的小鼠作为未干预组。测量肿瘤直径,记录小鼠带瘤生存时间,TUNEL法检测肿瘤细胞凋亡情况,比较各组间肿瘤体积、小鼠生存率和肿瘤细胞凋亡率的差异。结果： 1.构建重组腺病毒表达载体pAd-B7-H3,并获得了滴度为2×1011pfu/ml的病毒。pAd-B7-H3转染组与未转染组和pAd-CMV转染组相比,B7-H3表达明显升高(p0.05)。pAd-B7-H3转染组细胞毒性T淋巴细胞(Cytotoxic T lymphocyte, CTL)杀伤活性(31.932±2.800%),与未转染组(16.027±2.847%)和pAd-CMV转染组(16.417±2.492%)相比均有统计学差异(p0.05),而未干预组与pAd-CMV转染组比较差异无统计学意义(p0.05),表明可以诱导产生特异性细胞毒性T淋巴细胞。 2.构建了三个携带Id-1短发夹RNA的慢病毒表达载体(Lent-Id1-shRNA 1#-3#), Western blot结果表明Lent-Id1-shRNA 1#对B16细胞中Id-1的抑制作用较强(p0.05)；MTT结果显示,Lent-Id1-shRNA 1#具有较强的抑瘤效果(p0.05)。 3. pAd-B7-H3治疗组、Lent-Id1-shRNA治疗组与对照组相比肿瘤生长明显减缓。接种28天后,pAd-B7-H3治疗组肿瘤体积(2662.01±1909.67mm3)和pAd-CMV治疗组(8226.89±4063.57 mm3)、未干预组(9656.65±5024.34 mm3)比较均有统计学差异(p0.05),而pAd-CMV治疗组和未干预组之间无明显差异(p0.05)。Lent-Id1-shRNA治疗组(2539.97±1564.71 mm3)和Lent-NC-GFP治疗组(8783.29±3857.61mm3)、未干预组(9656.65±5024.34 mm3)比较均有统计学差异(p0.05),而Lent-NC-GFP治疗组和未干预组之间无明显差异(p0.05)。 4. pAd-B7-H3+Lent-Id1-shRNA治疗组肿瘤体积(144.67±252.37 mm3)明显小于pAd-B7-H3治疗组、Lent-Id1-shRNA治疗组(p0.05)。结论： 1.B7-H3疫苗和Lent-Id1-shRNA单独治疗昆明小鼠皮下恶性黑色素瘤,均能显著抑制肿瘤的生长(约为未干预组肿瘤体积的2／3),但恶性黑色素瘤的治愈率为0。 2.B7-H3疫苗和Lent-Id1-shRNA联合治疗,可使恶性黑色素瘤的治愈率达到66.67%。联合治疗组中的未治愈者的带瘤生存时间较单一治疗明显延长。B7-H3疫苗联合沉默Id1基因为临床治疗恶性黑色素瘤的治疗提供了新的思路。
[Abstract]:Objective:
Malignant melanoma (MM) is a highly malignant tumor derived from melanocytes. It is the fifth most common tumor in human beings. The commonly used clinical treatment methods include surgery, chemotherapy, immunotherapy and so on. Although these methods have achieved some clinical effects, it is difficult to cure the tumor completely. The discovery of oncogenes and tumor suppressor genes successively, the development and perfection of molecular biological methods, gene therapy has become a hot spot in tumor therapy research, and a variety of gene therapy methods have emerged. Although these methods have achieved certain tumor suppressor effect, but the effect is not ideal. The main reason is that tumor is a multi-factor, multi-link, multi-stage. Therefore, the combination of current therapies has become the trend of gene therapy. In this study, we used gene therapy to treat malignant melanoma in mice, combining two therapies: activating specific anti-tumor immunity and suppressing oncogene expression. To observe the effect of gene immunotherapy combined with oncogene therapy on malignant melanoma and provide a new strategy for the treatment of malignant melanoma.
Method:
Adenovirus expression vector carrying mouse B7-H3 gene (pAd-B7-H3) and lentivirus vector carrying mouse Id-1 short hairpin RNA (Lent-Id-1-shRNA) were constructed and their functions were identified by in vitro and in vivo experiments. There were four groups: 1) pAd-B7-H3 + Lent-Id1-shRNA treatment group; 2) pAd-B7-H3 treatment group; 3) Lent-Id1-shRNA treatment group; 4) mice without any treatment as non-intervention group. Tumor diameter was measured, survival time was recorded, tumor cell apoptosis was detected by TUNEL, tumor volume, survival rate and tumor size were compared among the groups. The difference of apoptosis rate.
Result:
1. The recombinant adenovirus expression vector pAd-B7-H3 was constructed and the virus titer was 2 *1011 pfu/ml. The expression of B7-H3 in pAd-B7-H3 transfection group was significantly higher than that in non-transfection group and pAd-CMV transfection group (p0.05). Cytotoxic T lymphocyte (CTL) killing activity in pAd-B7-H3 transfection group (31.932 +2.800%) was higher than that in non-transfection group (16. There was a significant difference between the two groups (p0.05). There was no significant difference between the non-intervention group and the pAd-CMV transfection group (p0.05). It indicated that the specific cytotoxic T lymphocytes could be induced.
2. Three lentiviral expression vectors carrying Id-1 short hairpin RNA (Lent-Id1-shRNA 1#-3#) were constructed. Western blot results showed that Lent-Id1-shRNA 1# had a strong inhibitory effect on Id-1 in B16 cells (p0.05); MTT results showed that Lent-Id1-shRNA 1# had a strong inhibitory effect on tumor (p0.05).
3. The growth of tumor in pAd-B7-H3 treatment group, Lent-Id1-shRNA treatment group and control group was significantly slower than that in pAd-B7-H3 treatment group. After 28 days of inoculation, the tumor volume in pAd-B7-H3 treatment group (2662.01 +1909.67 mm3) and pAd-CMV treatment group (8226.89 +4063.57 mm3) were significantly different from that in non-intervention group (9656.65 +5024.34 mm3), but there were significant differences between pAd-CMV treatment group and non-intervention group (p0.05). There was no significant difference between Lent-Id1-shRNA treatment group (2539.97+1564.71 mm3) and Lent-NC-GFP treatment group (8783.29+3857.61 mm3) and non-intervention group (9656.65+5024.34 mm3), but there was no significant difference between Lent-NC-GFP treatment group and non-intervention group (p0.05).
4. The tumor volume of pAd-B7-H3+Lent-Id1-shRNA treatment group (144.67+252.37 mm3) was significantly smaller than that of pAd-B7-H3 treatment group and Lent-Id1-shRNA treatment group (p0.05).
Conclusion:
1. B7-H3 vaccine and Lent-Id1-shRNA alone can significantly inhibit the growth of subcutaneous malignant melanoma in Kunming mice (about 2/3 of the tumor volume of the non-intervention group), but the cure rate of malignant melanoma is 0.
B7-H3 vaccine combined with Lent-Id1-shRNA treatment can make the cure rate of malignant melanoma reach 66.67%. The survival time of incurable patients in the combined treatment group is significantly longer than that in the single treatment group. B7-H3 vaccine combined with silencing Id1 gene provides a new idea for clinical treatment of malignant melanoma.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

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