重组溶瘤单纯疱疹病毒oHSV2治疗结直肠癌的体内外研究
发布时间：2018-09-19 14:30
【摘要】：背景与目的近年来,恶性肿瘤的发病率及死亡率逐年增加,而传统肿瘤治疗手段的临床疗效十分有限,促使恶性肿瘤成为威胁人类健康的大隐患。作为胃肠外科领域常见恶性肿瘤的结直肠癌,这种趋势更加凸显。因此,研制开发具有新的治疗机制的治疗方案对于目前晚期诊断和有限疗效的结直肠癌现状十分必要和紧急。生物治疗作为第四种肿瘤治疗模式,在恶性肿瘤的新兴疗法中别树一帜,现已逐渐登上肿瘤治疗的主要舞台。作为生物治疗家族中一员的溶瘤病毒是天然或经人工改造的能特异性杀伤肿瘤细胞的一类病毒,在多种实体瘤的治疗中具有广阔的应用前景。oHSV2是一种人工改造的重组2型单纯疱疹溶瘤病毒,具有肿瘤特异性和条件复制性。病毒基因组中插入了粒细胞-巨噬细胞集落刺激因子(Granulocyte Colony-stimulating,GM-CSF)基因,使得病毒在复制时表达GM-CSF细胞因子。GM-CSF具有多功能性,能募集并诱导活化抗原递呈细胞(Antigen presenting cell,APC),产生特异性的抗肿瘤免疫。在本研究中,我们首次评估oHSV2在结直肠癌中的抗肿瘤效果,并初步探讨其可能的体内外杀伤肿瘤细胞的机制。方法在体外,oHSV2感染CT26、LoVo、HT-29、HCT116等多种结直肠癌细胞,倒置显微镜观察各细胞感染病毒后的形态变化及病变情况;MTT法分析oHSV2对LoVo和CT26细胞生长及活性的影响;流式细胞术分析oHSV2对Lo Vo和CT26细胞周期及凋亡的影响。在体内,BALB/c小鼠皮下接种小鼠结肠癌细胞CT26,构建相应的小鼠结肠癌移植瘤模型。1)oHSV2体内复制表达GM-CSF检测:荷瘤小鼠瘤内注射oHSV2,于2,4,6,8,10,15,20天采用酶联免疫吸附法(ELISA)测定血清中GM-CSF浓度,绘制浓度时间变化曲线。2)oHSV2治疗结直肠癌疗效评价:荷瘤小鼠分为oHSV2组,5-氟尿嘧啶(5-FU)组(阳性对照组),磷酸盐缓冲液(PBS)组(阴性对照组)。给药后,记录小鼠体重、肿瘤体积、生存期、生活状态以及病毒注射区有无红肿、破溃等变化以评估oHSV2抗肿瘤效果及副反应。3)流式细胞术定量检测小鼠脾脏内骨髓来源抑制细胞(Myeloid-derived suppressor cell,MDSC)和调节性T细胞(Tregulatory T cells,Tregs),肿瘤引流淋巴结(Tumor-draining lymphode,TDLN)内树突状细胞(Dendritic cell,DC)及瘤体内CD4+T与CD8+T细胞的比例变化以探究oHSV2表达GM-CSF对荷瘤小鼠体内免疫的影响。结果体外实验:1)倒置显微镜观察到多种人源和鼠源的结直肠癌细胞在感染病毒后均出现细胞离壁悬浮,变圆,细胞间隙变大。相邻被感染的瘤细胞相互融合形成相当数量的合胞体(Syncytia)。2)MTT结果显示oHSV2对肿瘤细胞的生长有极强的抑制作用,其杀伤效应呈现出剂量依耐性和时间依耐性。3)细胞周期结果显示相比较于阴性对照组,oHSV2杀伤大肠癌细胞不依赖细胞周期,可均一的抑制处于各个细胞周期的肿瘤细胞(P0.05)。这与传统化疗药物5-FU不同,它主要是将癌细胞阻断在S期,杀伤肿瘤细胞呈现周期特异性(P0.05)。4)细胞凋亡结果显示相对于PBS组,oHSV2主要引起LoVo和CT26细胞发生坏死(P0.05),并呈剂量依耐性(P0.05),而非致癌细胞发生凋亡(P0.05)。体内实验:1)瘤内注射oHSV2后,血清中GM-CSF浓度不断升高,并在首次给药后的第8天出现高峰(3150±327.1 pg/mL),之后缓慢下降。2)oHSV2和5-FU治疗荷瘤小鼠均表现出显著的抗肿瘤效果,小鼠中位生存期都显著延长(50d vs 36d,p0.01;51d vs 36d,p0.01)。但oHSV2治疗未引起小鼠体重下降。在治疗起始第28天,5-FU组平均体重较阴性对照组有显著差异(16.61g vs 22.07g,P0.01),oHSV2组与PBS组无统计学差异(P0.05),且小鼠病毒注射区皮肤未见坏死、溃疡。3)流式分析结果显示相对于阴性对照组(14.60%),5-FU治疗组和病毒治疗组的MDSC比例均下降(7.84%和2.50%,P0.01),但oHSV2更能有效的降低脾内MDSC比例。相比较于PBS组,Tregs比例在5-FU组显著升高(14.50%vs 8.94%,P0.01),但oHSV2组的平均比例有所下降(4.60%,P0.05)。在TDLN及瘤体内,相对于阴性对照组,oHSV2治疗组的DC(6.49%vs 3.73%,P0.01),CD4+T(15%vs 8.57%,P0.01),CD8+T(8.19%vs 5.15%,P0.01)比例均升高,差异明显。而5-FU组各免疫细胞比例较PBS组则明显降低(P0.05)。结论1)本实验研究首次表明oHSV2在体内外均有明显的杀伤结直肠癌细胞的作用。病毒抑制肿瘤细胞不依赖于细胞周期,主要以细胞坏死的方式杀伤瘤细胞。2)oHSV2瘤内注射可在荷瘤小鼠体内复制产生具有生物活性的GM-CSF以增强抗肿瘤免疫。3)相对于化疗药物,病毒治疗不伴有明显的毒副作用。因此,oHSV2可通过直接细胞毒作用(溶瘤效应)以及增强抗肿瘤免疫的双重机制来抑制肿瘤细胞生长。这为结直肠癌提供了一个有效的潜在治疗新策略。
[Abstract]:BACKGROUND & OBJECTIVE In recent years, the incidence and mortality of malignant tumors have been increasing year by year. However, the clinical efficacy of traditional tumor therapy is very limited, which makes malignant tumors become a great hidden danger to human health. Biotherapy, as the fourth mode of cancer treatment, has become a new method in the treatment of malignant tumors, and has been gradually on the main stage of cancer treatment. Oncolytic viruses, as a member of the family of biotherapy, are known to be oncolytic viruses. OHSV2 is an artificially modified recombinant herpes simplex oncolytic virus type 2 with tumor specificity and conditional replication. Granulocyte-macrophage colony stimulating factor is inserted into the virus genome. Granulocyte Colony-stimulating (GM-CSF) gene enables the virus to express GM-CSF cytokines when replicating. GM-CSF is multifunctional and can recruit and induce activated antigen presenting cells (APCs) to produce specific antitumor immunity. In this study, we first evaluated the antitumor effect of oHSV2 in colorectal cancer. Methods In vitro, oHSV2 was infected with CT26, LoVo, HT-29, HCT116 and other colorectal cancer cells. The morphological changes and pathological changes were observed by inverted microscope. MTT assay was used to analyze the effect of oHSV2 on the growth and activity of LoVo and CT26 cells. To study the effect of oHSV2 on cell cycle and apoptosis of Lo Vo and CT26 cells.In vivo, BALB/c mice were subcutaneously inoculated with mouse colon cancer cells CT26 to construct the corresponding transplanted tumor model.1) Detection of GM-CSF expression by oHSV2 replication in vivo: OHSV2 was injected into tumor-bearing mice, and blood samples were determined by enzyme-linked immunosorbent assay (ELISA) at 2,4,6,8,10,15 and 20 days. The mice were divided into oHSV2 group, 5-fluorouracil (5-FU) group (positive control group) and phosphate buffer (PBS) group (negative control group). To evaluate the anti-tumor effect and side effects of oHSV2. 3. Quantitative detection of Myeloid-derived suppressor cell (MDSC) and Tregulatory T cells (Tregs), dendritic cells (DC) in tumor-draining lymph nodes (TDLN) by flow cytometry in spleen of mice. The ratio of CD4+T cells to CD8+T cells in tumor was measured to investigate the effect of GM-CSF expression by oHSV2 on the immunity of tumor-bearing mice in vivo.Results In vitro experiment: 1) Inverted microscope showed that many kinds of human and mouse colorectal cancer cells suspended off the wall, became round and the cell gap became larger after infection. Syncytia. 2) MTT results showed that oHSV2 had a strong inhibitory effect on the growth of tumor cells, and its killing effect showed dose-tolerance and time-tolerance. 3) Compared with the negative control group, oHSV2 did not depend on cell cycle, but inhibited the growth of colorectal cancer cells uniformly. Tumor cells in each cell cycle (P 0.05). This is different from the traditional chemotherapy drug 5-FU, which mainly blocked the cancer cells in S phase, killing tumor cells showed cycle-specific (P 0.05). The results of apoptosis showed that compared with PBS group, oHSV2 mainly caused necrosis of LoVo and CT26 cells (P 0.05), and showed dose-tolerance (P 0.05), but not dose-dependent (P 0.05). Carcinogenic cells apoptosis (P 0.05). In vivo experiments: 1) After intratumoral injection of oHSV2, the concentration of GM-CSF in serum increased continuously, and peaked at the 8th day after the first administration (3150 (+327.1 pg/mL), then slowly decreased. 2) OHSV2 and 5-FU showed significant anti-tumor effects in tumor-bearing mice, and the median survival time of mice was significantly prolonged (50d vs 36). On the 28th day of treatment, the average body weight of the 5-FU group was significantly higher than that of the negative control group (16.61 g vs 22.07 g, P 0.01). There was no significant difference between the oHSV2 group and the PBS group (P 0.05). There was no necrosis of the skin in the injection area of the mouse virus and the ulcer. 3) flow analysis showed that the average body weight of the 5-FU group was significantly lower than that of the negative control group (P 0.01). Compared with PBS group, Tregs ratio in 5-FU group was significantly higher (14.50% vs 8.94%, P 0.01), but the average ratio in oHSV2 group was lower (4.60%, P 0.05). In TDLN and tumor, the ratio of Tregs was significantly higher (14.50% vs 8.94%, P 0.01). In the negative control group, the proportion of DC (6.49% vs 3.73%, P 0.01), CD4 + T (15% vs 8.57%, P 0.01), CD8 + T (8.19% vs 5.15%, P 0.01) and CD8 + T (8.19% vs 5.15%, P 0.01) in the oHSV2 treatment group were significantly higher than those in the PBS treatment group, but the proportion of immune cells in the 5-FU treatment group was significantly lower than that in the PBS group (P 0.05). Conclusion 1. This study is the first time to show that oHSV2 can kill colorectal cancer cells in vitro and in vivo Viral inhibition of tumor cells is independent of cell cycle and kills tumor cells mainly by cell necrosis. 2) OHSV2 intratumoral injection can replicate GM-CSF with biological activity in tumor-bearing mice to enhance anti-tumor immunity. 3) Compared with chemotherapy drugs, viral treatment has no obvious toxic side effects. Therefore, oHSV2 can pass through Direct cytotoxicity (tumor lysis) and enhanced antitumor immunity inhibit tumor cell growth. This provides an effective potential therapeutic strategy for colorectal cancer.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
本文编号：2250406
[Abstract]:BACKGROUND & OBJECTIVE In recent years, the incidence and mortality of malignant tumors have been increasing year by year. However, the clinical efficacy of traditional tumor therapy is very limited, which makes malignant tumors become a great hidden danger to human health. Biotherapy, as the fourth mode of cancer treatment, has become a new method in the treatment of malignant tumors, and has been gradually on the main stage of cancer treatment. Oncolytic viruses, as a member of the family of biotherapy, are known to be oncolytic viruses. OHSV2 is an artificially modified recombinant herpes simplex oncolytic virus type 2 with tumor specificity and conditional replication. Granulocyte-macrophage colony stimulating factor is inserted into the virus genome. Granulocyte Colony-stimulating (GM-CSF) gene enables the virus to express GM-CSF cytokines when replicating. GM-CSF is multifunctional and can recruit and induce activated antigen presenting cells (APCs) to produce specific antitumor immunity. In this study, we first evaluated the antitumor effect of oHSV2 in colorectal cancer. Methods In vitro, oHSV2 was infected with CT26, LoVo, HT-29, HCT116 and other colorectal cancer cells. The morphological changes and pathological changes were observed by inverted microscope. MTT assay was used to analyze the effect of oHSV2 on the growth and activity of LoVo and CT26 cells. To study the effect of oHSV2 on cell cycle and apoptosis of Lo Vo and CT26 cells.In vivo, BALB/c mice were subcutaneously inoculated with mouse colon cancer cells CT26 to construct the corresponding transplanted tumor model.1) Detection of GM-CSF expression by oHSV2 replication in vivo: OHSV2 was injected into tumor-bearing mice, and blood samples were determined by enzyme-linked immunosorbent assay (ELISA) at 2,4,6,8,10,15 and 20 days. The mice were divided into oHSV2 group, 5-fluorouracil (5-FU) group (positive control group) and phosphate buffer (PBS) group (negative control group). To evaluate the anti-tumor effect and side effects of oHSV2. 3. Quantitative detection of Myeloid-derived suppressor cell (MDSC) and Tregulatory T cells (Tregs), dendritic cells (DC) in tumor-draining lymph nodes (TDLN) by flow cytometry in spleen of mice. The ratio of CD4+T cells to CD8+T cells in tumor was measured to investigate the effect of GM-CSF expression by oHSV2 on the immunity of tumor-bearing mice in vivo.Results In vitro experiment: 1) Inverted microscope showed that many kinds of human and mouse colorectal cancer cells suspended off the wall, became round and the cell gap became larger after infection. Syncytia. 2) MTT results showed that oHSV2 had a strong inhibitory effect on the growth of tumor cells, and its killing effect showed dose-tolerance and time-tolerance. 3) Compared with the negative control group, oHSV2 did not depend on cell cycle, but inhibited the growth of colorectal cancer cells uniformly. Tumor cells in each cell cycle (P 0.05). This is different from the traditional chemotherapy drug 5-FU, which mainly blocked the cancer cells in S phase, killing tumor cells showed cycle-specific (P 0.05). The results of apoptosis showed that compared with PBS group, oHSV2 mainly caused necrosis of LoVo and CT26 cells (P 0.05), and showed dose-tolerance (P 0.05), but not dose-dependent (P 0.05). Carcinogenic cells apoptosis (P 0.05). In vivo experiments: 1) After intratumoral injection of oHSV2, the concentration of GM-CSF in serum increased continuously, and peaked at the 8th day after the first administration (3150 (+327.1 pg/mL), then slowly decreased. 2) OHSV2 and 5-FU showed significant anti-tumor effects in tumor-bearing mice, and the median survival time of mice was significantly prolonged (50d vs 36). On the 28th day of treatment, the average body weight of the 5-FU group was significantly higher than that of the negative control group (16.61 g vs 22.07 g, P 0.01). There was no significant difference between the oHSV2 group and the PBS group (P 0.05). There was no necrosis of the skin in the injection area of the mouse virus and the ulcer. 3) flow analysis showed that the average body weight of the 5-FU group was significantly lower than that of the negative control group (P 0.01). Compared with PBS group, Tregs ratio in 5-FU group was significantly higher (14.50% vs 8.94%, P 0.01), but the average ratio in oHSV2 group was lower (4.60%, P 0.05). In TDLN and tumor, the ratio of Tregs was significantly higher (14.50% vs 8.94%, P 0.01). In the negative control group, the proportion of DC (6.49% vs 3.73%, P 0.01), CD4 + T (15% vs 8.57%, P 0.01), CD8 + T (8.19% vs 5.15%, P 0.01) and CD8 + T (8.19% vs 5.15%, P 0.01) in the oHSV2 treatment group were significantly higher than those in the PBS treatment group, but the proportion of immune cells in the 5-FU treatment group was significantly lower than that in the PBS group (P 0.05). Conclusion 1. This study is the first time to show that oHSV2 can kill colorectal cancer cells in vitro and in vivo Viral inhibition of tumor cells is independent of cell cycle and kills tumor cells mainly by cell necrosis. 2) OHSV2 intratumoral injection can replicate GM-CSF with biological activity in tumor-bearing mice to enhance anti-tumor immunity. 3) Compared with chemotherapy drugs, viral treatment has no obvious toxic side effects. Therefore, oHSV2 can pass through Direct cytotoxicity (tumor lysis) and enhanced antitumor immunity inhibit tumor cell growth. This provides an effective potential therapeutic strategy for colorectal cancer.
【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.34
【参考文献】
相关期刊论文 前1条
1 李延飞;毛泽勇;石晓太;邹建文;董玉婷;孙美玲;刘滨磊;方志正;;人用重组溶瘤单纯疱疹病毒Ⅱ型(OH2)生物学特性的稳定性[J];中国生物制品学杂志;2016年05期
，本文编号：2250406
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