【摘要】：背景與目的近年來,惡性腫瘤的發(fā)病率及死亡率逐年增加,而傳統(tǒng)腫瘤治療手段的臨床療效十分有限,促使惡性腫瘤成為威脅人類健康的大隱患。作為胃腸外科領(lǐng)域常見惡性腫瘤的結(jié)直腸癌,這種趨勢更加凸顯。因此,研制開發(fā)具有新的治療機(jī)制的治療方案對于目前晚期診斷和有限療效的結(jié)直腸癌現(xiàn)狀十分必要和緊急。生物治療作為第四種腫瘤治療模式,在惡性腫瘤的新興療法中別樹一幟,現(xiàn)已逐漸登上腫瘤治療的主要舞臺。作為生物治療家族中一員的溶瘤病毒是天然或經(jīng)人工改造的能特異性殺傷腫瘤細(xì)胞的一類病毒,在多種實(shí)體瘤的治療中具有廣闊的應(yīng)用前景。oHSV2是一種人工改造的重組2型單純皰疹溶瘤病毒,具有腫瘤特異性和條件復(fù)制性。病毒基因組中插入了粒細(xì)胞-巨噬細(xì)胞集落刺激因子(Granulocyte Colony-stimulating,GM-CSF)基因,使得病毒在復(fù)制時(shí)表達(dá)GM-CSF細(xì)胞因子。GM-CSF具有多功能性,能募集并誘導(dǎo)活化抗原遞呈細(xì)胞(Antigen presenting cell,APC),產(chǎn)生特異性的抗腫瘤免疫。在本研究中,我們首次評估oHSV2在結(jié)直腸癌中的抗腫瘤效果,并初步探討其可能的體內(nèi)外殺傷腫瘤細(xì)胞的機(jī)制。方法在體外,oHSV2感染CT26、LoVo、HT-29、HCT116等多種結(jié)直腸癌細(xì)胞,倒置顯微鏡觀察各細(xì)胞感染病毒后的形態(tài)變化及病變情況;MTT法分析oHSV2對LoVo和CT26細(xì)胞生長及活性的影響;流式細(xì)胞術(shù)分析oHSV2對Lo Vo和CT26細(xì)胞周期及凋亡的影響。在體內(nèi),BALB/c小鼠皮下接種小鼠結(jié)腸癌細(xì)胞CT26,構(gòu)建相應(yīng)的小鼠結(jié)腸癌移植瘤模型。1)oHSV2體內(nèi)復(fù)制表達(dá)GM-CSF檢測:荷瘤小鼠瘤內(nèi)注射oHSV2,于2,4,6,8,10,15,20天采用酶聯(lián)免疫吸附法(ELISA)測定血清中GM-CSF濃度,繪制濃度時(shí)間變化曲線。2)oHSV2治療結(jié)直腸癌療效評價(jià):荷瘤小鼠分為oHSV2組,5-氟尿嘧啶(5-FU)組(陽性對照組),磷酸鹽緩沖液(PBS)組(陰性對照組)。給藥后,記錄小鼠體重、腫瘤體積、生存期、生活狀態(tài)以及病毒注射區(qū)有無紅腫、破潰等變化以評估oHSV2抗腫瘤效果及副反應(yīng)。3)流式細(xì)胞術(shù)定量檢測小鼠脾臟內(nèi)骨髓來源抑制細(xì)胞(Myeloid-derived suppressor cell,MDSC)和調(diào)節(jié)性T細(xì)胞(Tregulatory T cells,Tregs),腫瘤引流淋巴結(jié)(Tumor-draining lymphode,TDLN)內(nèi)樹突狀細(xì)胞(Dendritic cell,DC)及瘤體內(nèi)CD4+T與CD8+T細(xì)胞的比例變化以探究oHSV2表達(dá)GM-CSF對荷瘤小鼠體內(nèi)免疫的影響。結(jié)果體外實(shí)驗(yàn):1)倒置顯微鏡觀察到多種人源和鼠源的結(jié)直腸癌細(xì)胞在感染病毒后均出現(xiàn)細(xì)胞離壁懸浮,變圓,細(xì)胞間隙變大。相鄰被感染的瘤細(xì)胞相互融合形成相當(dāng)數(shù)量的合胞體(Syncytia)。2)MTT結(jié)果顯示oHSV2對腫瘤細(xì)胞的生長有極強(qiáng)的抑制作用,其殺傷效應(yīng)呈現(xiàn)出劑量依耐性和時(shí)間依耐性。3)細(xì)胞周期結(jié)果顯示相比較于陰性對照組,oHSV2殺傷大腸癌細(xì)胞不依賴細(xì)胞周期,可均一的抑制處于各個(gè)細(xì)胞周期的腫瘤細(xì)胞(P0.05)。這與傳統(tǒng)化療藥物5-FU不同,它主要是將癌細(xì)胞阻斷在S期,殺傷腫瘤細(xì)胞呈現(xiàn)周期特異性(P0.05)。4)細(xì)胞凋亡結(jié)果顯示相對于PBS組,oHSV2主要引起LoVo和CT26細(xì)胞發(fā)生壞死(P0.05),并呈劑量依耐性(P0.05),而非致癌細(xì)胞發(fā)生凋亡(P0.05)。體內(nèi)實(shí)驗(yàn):1)瘤內(nèi)注射oHSV2后,血清中GM-CSF濃度不斷升高,并在首次給藥后的第8天出現(xiàn)高峰(3150±327.1 pg/mL),之后緩慢下降。2)oHSV2和5-FU治療荷瘤小鼠均表現(xiàn)出顯著的抗腫瘤效果,小鼠中位生存期都顯著延長(50d vs 36d,p0.01;51d vs 36d,p0.01)。但oHSV2治療未引起小鼠體重下降。在治療起始第28天,5-FU組平均體重較陰性對照組有顯著差異(16.61g vs 22.07g,P0.01),oHSV2組與PBS組無統(tǒng)計(jì)學(xué)差異(P0.05),且小鼠病毒注射區(qū)皮膚未見壞死、潰瘍。3)流式分析結(jié)果顯示相對于陰性對照組(14.60%),5-FU治療組和病毒治療組的MDSC比例均下降(7.84%和2.50%,P0.01),但oHSV2更能有效的降低脾內(nèi)MDSC比例。相比較于PBS組,Tregs比例在5-FU組顯著升高(14.50%vs 8.94%,P0.01),但oHSV2組的平均比例有所下降(4.60%,P0.05)。在TDLN及瘤體內(nèi),相對于陰性對照組,oHSV2治療組的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,差異明顯。而5-FU組各免疫細(xì)胞比例較PBS組則明顯降低(P0.05)。結(jié)論1)本實(shí)驗(yàn)研究首次表明oHSV2在體內(nèi)外均有明顯的殺傷結(jié)直腸癌細(xì)胞的作用。病毒抑制腫瘤細(xì)胞不依賴于細(xì)胞周期,主要以細(xì)胞壞死的方式殺傷瘤細(xì)胞。2)oHSV2瘤內(nèi)注射可在荷瘤小鼠體內(nèi)復(fù)制產(chǎn)生具有生物活性的GM-CSF以增強(qiáng)抗腫瘤免疫。3)相對于化療藥物,病毒治療不伴有明顯的毒副作用。因此,oHSV2可通過直接細(xì)胞毒作用(溶瘤效應(yīng))以及增強(qiáng)抗腫瘤免疫的雙重機(jī)制來抑制腫瘤細(xì)胞生長。這為結(jié)直腸癌提供了一個(gè)有效的潛在治療新策略。
[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.
【學(xué)位授予單位】：濟(jì)南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34

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1 李延飛;毛澤勇;石曉太;鄒建文;董玉婷;孫美玲;劉濱磊;方志正;;人用重組溶瘤單純皰疹病毒Ⅱ型(OH2)生物學(xué)特性的穩(wěn)定性[J];中國生物制品學(xué)雜志;2016年05期

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