摘要
背景与目的：胃癌是最常见的恶性肿瘤之一,发病率逐年增高。由于临床症状隐匿,多数患者就诊时已进展至中晚期,传统治疗复发率高,预后较差。如何进一步提高疗效、降低死亡率一直是研究的重要课题。随着分子生物学的发展,肿瘤基因治疗已成为目前研究热点。然而,临床实验提示单基因治疗恶性肿瘤效果有限,主要原因可能在于肿瘤的发生、发展涉及多个基因的变化,功能失常的基因互相配合,促进肿瘤进展。因此,向肿瘤细胞导入多种相关基因以提高抗肿瘤效果是基因治疗的发展方向。基因疗法中,自杀基因以其强大的杀伤肿瘤细胞作用及“旁观者效应”备受关注。目前应用最广泛的自杀基因/前药治疗系统是胞嘧啶脱氨酶基因/5-氟胞嘧啶(cytosine deaminase/5-Fluorocytosine, CD/5-FC)和单纯疱疹病毒胸苷激酶基因/环氧鸟苷(herps simplex virus thymidine kinase/ganciclovir, HSV-TK/GCV)。这两种自杀基因/前药系统各有特点：HSV-TK/GCV直接杀伤肿瘤细胞的作用较强,而CD/5-FC旁观者效应更为显著；此外,不同类型的肿瘤对这两种自杀基因/前药系统的敏感性不同,推测联合应用CD/5-FC和HSV-TK/GCV可增加抗肿瘤效应。实体瘤的生长、浸润及转移都离不开瘤内新生血管形成,抑制新生血管形成已成为治疗肿瘤的重要靶点之一。血管内皮生长因子(vascular endothelial growth factor, VEGF)是最重要的促血管生成因子之一,在多数肿瘤组织中高表达,与肿瘤的预后及复发密切相关。RNA干扰(RNA interference, RNAi)是在mRNA水平上的基因阻断技术,可高效、特异抑制靶基因表达。既往研究证实利用RNAi可有效下调VEGF在鼻咽癌、结肠癌、胃癌等多种肿瘤细胞中表达,降低微血管密度(micro vessel density, MVD),有效抑制肿瘤生长。因此,本课题通过构建表达融合自杀基因yCDglyTK及VEGF-shRNA的联合基因表达载体,通过体内外实验研究其抗胃癌作用,探讨联合应用自杀基因治疗与抗血管生成基因疗法治疗胃癌的可行性。
方法：(1)选择有效靶序列并构建靶向VEGF的干扰质粒pGenesil-shVEGF;采用PCR法从pGenesil-shVEGF中扩增shRNA表达框架,亚克隆至融合自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)shVEGF-yCDglyTK;酶切、测序等鉴定重组质粒；(2)以磷酸钙纳米颗粒(calcium phosphate nanoparticles, CPNPs)为载体,介导空白质粒、靶向VEGF的干扰质粒、融合自杀基因质粒及联合基因质粒分别转染SGC7901细胞,G418筛选,建立稳定转染的细胞株,RT-PCR、Western-blot及免疫荧光检测目的基因表达；(3)MTT法检测shVEGF-yCDglyTK/5-FC联合基因/前药治疗体系对胃癌SGC7901细胞的杀伤作用及该体系的“旁观者效应”；平板克隆形成实验检测联合基因/5-FC对胃癌细胞克隆形成率的影响；高效液相色谱仪(high-performance liquid chromatography, HPLC)测定细胞上清中5-FC和5-FU的含量；Hoechst 33258染色及流式细胞仪检测细胞凋亡；(4)以SGC7901细胞建立裸鼠胃癌皮下移植瘤模型,实验分5组：A组为空白对照组,未施加任何处理；B组：瘤内注射CPNPs-空白质粒,腹腔注射5-FC；C组：瘤内注射CPNPs-干扰质粒；D组：瘤内注射CPNPs-融合自杀基因质粒,腹腔注射5-FC；E组：瘤内注射CPNPs-联合基因质粒,腹腔注射5-FC。观察各组移植瘤生长情况；待观察期满,瘤组织行HE染色鉴定组织病理类型；免疫组化染色鉴定肿瘤组织中yCDglyTK和VEGF的表达并测定微血管密度(MVD)。
结果：(1)成功构建干扰质粒pGenesil-shVEGF及联合基因质粒pcDNA3.1(-)shVEGF-yCDglyTK;(2)成功建立4种稳定转染不同质粒的胃癌细胞株：SGC/null, SGC/shVEGF, SGC/CDTK及SGC/shVEGF-CDTK;在SGC/shVEGF-CDTK中,检测到yCDglyTK基因表达,VEGF基因表达较野生型SGC7901明显下调；(3)体外细胞实验：加入前体药物5-FC后,SGC/shVEGF, SGC/CDTK、SGC/shVEGF-CDTK与野生型SGC7901细胞及SGC/null相比较,细胞存活率下降,且凋亡比例增高,以SGC/shVEGF-CDTK组差异最明显(P< 0.01); HPLC检测该组细胞上清中5-FC向5-FU转化的效率为92.00%；该组细胞具有强大的旁观者效应,当阳性细胞比例达40%时,细胞相对存活率仅为(19.00±2.44)%。(4)体内动物实验：与A、B对照组相比,实验组C、D组移植瘤生长受到不同程度抑制,E组抑制效应最明显；至观察期满,与空白对照组A组相比,E组抑瘤率为72.91%。E组瘤组织常规病理检查显示大量肿瘤细胞坏死；免疫组化可检测到yCDglyTK的表达；与对照组A组或B组相比,E组瘤组织VEGF表达下调,MVD明显减少。
结论：(1)成功构建联合基因载体pcDNA3.1(-)shVEGF-yCDglyTK,在该载体中,shVEGF及yCDglyTK都可正常表达。(2)shVEGF-yCDglyTK/5-FC联合基因治疗体系可有效杀伤胃癌SGC7901细胞,并具有强大的“旁观者效应”；诱导细胞凋亡可能是该联合基因治疗体系发挥作用的重要机制。(3)瘤内直接注射CPNPs-pcDNA3.1(-)shVEGF-yCDglyTK复合物,联合腹腔注射5-FC,可有效抑制胃癌生长,减少肿瘤组织新生血管形成,是一种具有良好应用前景的治疗策略,为胃癌的临床治疗提供新思路。
Backgroud and Objective:Gastric cancer (GC) is one of the most common malignancies, the incidence rate has been increasing year by year. Most patients with GC are diagnosed at an advanced stage because of non-specific clinical symptoms. Despite a variety of traditional treatments, the prognosis for patients with advanced GC remains poor, and the recurrence rate is still high. New therapeutic approaches with more efficacy are urgently needed. With the development in molecular biology, gene therapy has been researched extensively. However, clinical trials have revealed that single-gene therapy has limited effects. The main reason might be that multiple-gene mutations are involved in the occurrence and development of tumors. These genes could co-ordinate with each other, and promote tumor progression. Thus, combination gene therapy using two or more related therapeutic genes might enhance anti-tumor effect. Suicide gene therapy has received much attention due to its direct killing mechanism and "by-stander effect". Two of the best characterized suicide gene therapy systems are herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) and cytosine deaminase/5-fluorocytosine (CD/5-FC). Each suicide gene/prodrug system has is own features, for example, HSV-TK/GCV has a stronger killing effect, while CD/5-FC shows a more powerful "by-stander effect". In addition, cell type dependency might exist with one suicide gene therapy system. Therefore, combination of HSV-TK/GCV and CD/5-FC is expected to have synergistic effects. Angiogenesis plays an essential role in tumor growth, infiltration and metastasis, and inhibition of angiogenesis is a new target for cancer therapy. Vascular endothelial growth factor (VEGF) is a most important pro-angiogenic factor, and is overexpressed in the majority of human cancers. VEGF expression level has a close relationship with prognosis and recurrence rate of malignant cancers. RNA interference (RNAi) technology could inhibit gene expression at mRNA level efficiently and specificly. RNAi could downregulate VEGF expression, resulting in a decreased micro vessel density (MVD) and tumor growth delay in several cancer cell lines, such as nasopharyngeal caner cells, colon cancer cells, and gastric cancer cells. To explore the feasibility of combination gene therapy for gastric cancer using suicide gene and anti-angiogenic genem therapy, a combination gene vector, co-expressing VEGF-shRNA and fusion suicide gene yCDglyTK was constructed in this research, and its inhibiting effect on gastric cancer in vivo and in vitro was also studied.
Methods:(1) RNAi targeting VEGF was applied to construct an interfering plasmid pGenesil-shVEGF. Then, the VEGF-shRNA expression cassette (including the U6 promoter) was amplified by PCR and subcloned into pcDNA3.1(-)CV-yCDglyTK to build a novel combination gene plasmid pcDNA3.1(-)shVEGF-yCDglyTK. The recombinant plasmid was identified by restriction enzyme digestion and sequencing. (2) A blank plasmid pcDNA3.1(-)null, pGenesil-shVEGF, pcDNA3.1(-)CV-yCDglyTK and pcDNA3.1(-)shVEGF-yCDglyTK were transfected into SGC7901 cells using calcium phosphate nanoparticles (CPNPs), and stable transfected SGC7901 cell lines were established by G418 selection. Expressions of yCDglyTK and VEGF were detected by RT-PCR, Western-blot and immunofluorescence assays. (3) The killing efficacy and "by-stander effecf"of shVEGF-yCDglyTK/5-FC were checked using MTT assays. Plate colony forming assays were carried out to study the impact of combination gene/prodrug on SGC7901 colony forming rate. Levels of 5-FC and 5-FU in cell supernatant were determined by high-performance liquid chromatography (HPLC). Cell apoptosis were detected by Hoechst 33258 staining and flow cytometry. (4) Nude mice bearing gastric cancer were established by transplantation of SGC7901 cells. There were five groups:group A was the blank control and received no treatment; group B received intratumoral injection of CPNPs-pcDNA3.1(-)null complex and intraperitoneal injection of 5-FC; group C received intratumoral injection of CPNPs-pGenesil-shVEGF complex; group D received intratumoral injection of CPNPs-pcDNA3.1(-) CV-yCDglyTK complex and intraperitoneal injection of 5-FC; and group E received intratumoral injection of CPNPs-pcDNA3.1(-)shVEGF-yCDglyTK complex and intraperitoneal injection of 5-FC. The tumor growth was observed. When the test was terminated, tumor tissue was subjected to hematoxylin and eosin staining for histological examination. Immunohistochemistry was performed to study the expression of yCDglyTK and VEGF, and MVD was detected also.
Results:(1) Both of the interfering plasmid pGenesil-shVEGF and combination gene plasmid pcDNA3.1(-)shVEGF-yCDglyTK were successfully constructed. (2) Four stable transfectants were established. They were named SGC/null, SGC/shVEGF, SGC/CDTK and SGC/shVEGF-CDTK. In SGC/shVEGF-CDTK cells, yCDglyTK gene expression could be detected, and VEGF gene expression was significantly decreased compared with wild-type SGC7901; (3) In the presence of 5-FC, cell survival rate of SGC/shVEGF, SGC/CDTK and SGC/shVEGF-CDTK was reduced, and the proportion of apoptotic cells increased. SGC/shVEGF-CDTK were the most sensitive to 5-FC (P< 0.01); Conversion efficiency of 5-FC to 5-FU was 92.00% in SGC7901/ shVEGF-CDTK cell supernatant; When SGC/shVEGF-CDTK accounted for 40%, cell relative viability was only (19.00±2.44)%, exhibiting a powerful "by-stander effect". (4) in vivo experiments:Compared with group A and group B, tumor growth in group C, group D and group E was inhibited. CPNPs/shVEGF-CDTK+5-FC system was the most efficient in suppressing tumor growth compared with CPNPs/shVEGF therapy or CPNPs/CDTK+5-FC threatment. At the end of observation, the growth inhibition ratio in group E was 72.91%. There were obvious cell necrosis in tumor tissue of group E. Immunohistochemistry revealed yCDglyTK expression, decreased VEGF and MVD.
Conclusion:(1) The combination gene plasmid pcDNA3.1(-) shVEGF-yCDglyTK was successfully constructed, and in this novel vector, both VEGF-shRNA and yCDglyTK could expressed normally. (2) shVEGF-yCDglyTK/5-FC treatment system could kill SGC7901 cells effectively and exhibit powerful "by-stander effect"; Inducing cell apoptosis might be an important mechanism. (3) Intratumoral injection of CPNPs-pcDNA3.1(-)shVEGF-yCDglyTK, in combination with intraperi-toneal injection of 5-FC could inhibit tumor growth effectively, and reduce neovascularization. Our research suggested a promising strategy for clinical gastric cancer therapy.
引文
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