IL-15基因治疗小鼠结肠癌的实验研究
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摘要
生物医学在近几十年来取得了长足的进步,但是,许多恶性肿瘤的治疗和预后仍然很不乐观。近年来基因治疗成为肿瘤治疗的重要组成部分,尤其是细胞因子相关研究,它们已经越来越受到重视。肿瘤基因治疗研究已经取得了很多成果,但是,包括基因治疗载体的安全性和有效性等很多问题,仍有待进一步研究。
我们在以前的研究中成功构建了具有双启动子的IL-15基因表达质粒载体pHi2-IL15-CMV-tat (L1),并通过转信号肽获得可以高效表达IL-15基因的质粒载体pHi2-spIL15-CMV-tat (L3),并且成功构建了具有CEA特异性IL-15表达载体pHi2-IL15-CEA-tat (L2)、pHi2-spIL15-CEA-tat (L4)。
本研究将上述构建的质粒载体和另外一种绿荧光蛋白表达载体pHi2-EGFP-CMV-tat(L6)通过电转导法,分别转染CEA阳性结肠癌细胞系SW480和CEA阴性乳腺癌细胞系MCF-7,采用倒置荧光显微镜通过细胞绿荧光表达水平评估细胞转染情况；流式细胞术(FCM)分析肿瘤细胞的转染效率；以及酶联免疫吸附法(ELISA)检测细胞转染后上清液中IL-15的浓度,分析IL-15基因表达情况。结果表明：利用pHi2-EGFP-CMV-tat(L6)电转染SW480细胞和MCF-7细胞,倒置荧光显微镜可以观察到绿荧光蛋白表达,流式细胞术分析肿瘤细胞的转染效率在10%-30%。质粒L1,L2,L3,L4转染细胞后,上清液中可见IL-15的表达；质粒pHi2-sp IL15-CMV-tat (L3)和pHi2-spIL15-CEA-tat (L4)的IL-15的表达水平高于pHi2-IL15-CMV-tat (L1)和pHi2-IL15-CEA-tat (L2),差异具有显著性(P<0.01)；转染SW480细胞后, CEA启动子正调控的质粒L2、L4与相对应的CMV启动子正调控质粒L1、L3之间的IL-15表达水平未见显著性差异(P>0.05)；转染MCF-7细胞后, L1与L2、L3与L4之间的IL-15表达水平可见显著性差异(P<0.05),质粒L1的IL-15的表达水平高于L2,L3的IL-15的表达水平高于L4。
体内试验表明携带EGFP的质粒L6右下腹腔注射小鼠,48hr后置于体外活体成像仪中,可以看到腹膜上有明显的荧光。小鼠腹腔注射小鼠源结肠肿瘤细胞CT-26建立腹腔成瘤的荷瘤小鼠模型,定期注射L3、L4质粒观察其对荷瘤小鼠的腹水生成情况和生存期影响。结果发现注射质粒载体L3、L4的小鼠生存期高于单纯注射PBS和空载的对照组。
以EGFP的表达质粒载体L6进行体内电转染实验,小鼠皮下注射CT-26细胞建立皮下成瘤模型,瘤内注射质粒后,予以电转染,体外活体成像示有荧光反应,然后取瘤体组织作冰冻切片,荧光显微镜观察。结果发现针道附近可见绿色荧光蛋白表达。进一步以高效表达的质粒载体L3进行治疗试验,结果表明质粒载体L3的小鼠瘤体生长速度明显慢于对照组。
上述结果表明电转染方法能在体外和体内有效的将IL-15表达质粒导入肿瘤细胞,同时,腹腔注射质粒也能够使腹膜有效表达目的基因。腹腔注射IL-15表达质粒能在一定程度上抑制腹膜移植瘤的生成与发展。电转染IL-15表达质粒能抑制皮下移植的小鼠结肠癌的生长。本研究为IL-15为基础的肿瘤免疫基因治疗的临床应用提供了实验依据。
Although great progress has been made in the biomedical field in recent decades, the treatment and prognosis of malignant tumors is still far from optimistic. Gene therapy has become an important component of cancer treatment in recent years, especially the cytokine-based immunogene therapy. Many achievements have been made in gene therapy of cancer; however, problems still exist, such as the safety of gene transfer vector and the efficacy of transfection which need further improved. We had constructed the plasmid vector pHi2-IL15-CMV-tat (L1) with dual-promoter amplified expressing of human IL-15. The IL-15 signal sequence was replaced by IL-2 signal sequence, resulting plasmid pHi2-spIL15-CMV-tat (L3). The CEA promoter was cloned into plasmid L1、L3 and replaced CMV promoter to construct plasmid pHi2-IL15-CEA-tat (L2)、pHi2-spIL15-CEA-tat (L4). Current study will further evaluate these vectors in vitro and in vivo.
Plasmid vectors expressing IL-15 or encoding enhanced green fluorescence protein (EGFP) were transfected by electroporation into SW480, a CEA-positive human colon cancer cell line, and MCF-7, a CEA-negative human breast cancer cell line. The transfection efficiency was determined by fluorescence microscope and flow cytometry. IL-15 expression was detected by ELISA. In in vivo study, plasmid L6 was injected into the peritoneum of BALB/c mice and transfected into the subcutaneously growing CT-26 tumor to evaluate the transfection efficacy by in vivo imaging techniques. BALB/c mice were inoculated with CT-26 cells by peritoneal injection, and the plasmid vectors L3, L4 were transfected by intraperitoneal injection after tumor inoculation. Empty plasmid and PBS were used as control. Weight change and life span of the mice were. In addition, the therapeutic effects of L3 were studied through electroporation to control the growth of subcutaneously inoculated CT-26 tumor.
Results showed that green fluorescent protein (GFP) could be detected in the transfected cells 24 hours after transfection. The percentage of transfection was 10%-30%. The IL-15 expression in culture supernatant of L3 and L4 transfectant was significantly higher than that of L1 and L2 (P＜0.05). In transfected SW480 cells, there is no significant deference for IL-15 expression between the CEA promoter positively controlled plasmids L2, L4 and the CMV promoter positively controlled plasmids L1, L3 respectively (P>0.05). In transfected MCF-7 cells, CEA promoter positively controlled plasmids L2, L4 gave rise to lower IL-15 expression than the CMV promoter positively controlled plasmids L1, L3, respectively (P<0.01). GFP expression was observed after intraperitoneal injection of plasmid L6 and electroporation into the subcutaneously inoculated CT-26 tumor. Comparing to the control group, and repeated injection of plasmid L3 (pHi2-spIL15-CMV-tat) and L4 (pHi2-spIL15-CEA-tat) DNA were able to inhibit tumor growth, and prolong the life of tumor-bearing mice. In addition, in vivo transfection of plasmid L3 through electroporation slowed the subcutaneously inoculated CT-26 growth.
In conclusions, electroporation can efficiently transfer IL-15 expression plasmids into tumor cells in vitro and in vivo, in addition, intraperitoneal injection results in efficient transgene expression by epithelial cells in the peritoneum. IL-15 expression plasmid can decrease the growth of inoculated tumor cells after intrapertoneal injection. In addition, electroporation of IL-15 expression plasmid can inhibit the growth of subcutaneously inoculated mice CT-16 tumor. Therefore, this study provides evidence for the clinical application of IL-15 base gene therapy.
我们在以前的研究中成功构建了具有双启动子的IL-15基因表达质粒载体pHi2-IL15-CMV-tat (L1),并通过转信号肽获得可以高效表达IL-15基因的质粒载体pHi2-spIL15-CMV-tat (L3),并且成功构建了具有CEA特异性IL-15表达载体pHi2-IL15-CEA-tat (L2)、pHi2-spIL15-CEA-tat (L4)。
本研究将上述构建的质粒载体和另外一种绿荧光蛋白表达载体pHi2-EGFP-CMV-tat(L6)通过电转导法,分别转染CEA阳性结肠癌细胞系SW480和CEA阴性乳腺癌细胞系MCF-7,采用倒置荧光显微镜通过细胞绿荧光表达水平评估细胞转染情况；流式细胞术(FCM)分析肿瘤细胞的转染效率；以及酶联免疫吸附法(ELISA)检测细胞转染后上清液中IL-15的浓度,分析IL-15基因表达情况。结果表明：利用pHi2-EGFP-CMV-tat(L6)电转染SW480细胞和MCF-7细胞,倒置荧光显微镜可以观察到绿荧光蛋白表达,流式细胞术分析肿瘤细胞的转染效率在10%-30%。质粒L1,L2,L3,L4转染细胞后,上清液中可见IL-15的表达；质粒pHi2-sp IL15-CMV-tat (L3)和pHi2-spIL15-CEA-tat (L4)的IL-15的表达水平高于pHi2-IL15-CMV-tat (L1)和pHi2-IL15-CEA-tat (L2),差异具有显著性(P<0.01)；转染SW480细胞后, CEA启动子正调控的质粒L2、L4与相对应的CMV启动子正调控质粒L1、L3之间的IL-15表达水平未见显著性差异(P>0.05)；转染MCF-7细胞后, L1与L2、L3与L4之间的IL-15表达水平可见显著性差异(P<0.05),质粒L1的IL-15的表达水平高于L2,L3的IL-15的表达水平高于L4。
体内试验表明携带EGFP的质粒L6右下腹腔注射小鼠,48hr后置于体外活体成像仪中,可以看到腹膜上有明显的荧光。小鼠腹腔注射小鼠源结肠肿瘤细胞CT-26建立腹腔成瘤的荷瘤小鼠模型,定期注射L3、L4质粒观察其对荷瘤小鼠的腹水生成情况和生存期影响。结果发现注射质粒载体L3、L4的小鼠生存期高于单纯注射PBS和空载的对照组。
以EGFP的表达质粒载体L6进行体内电转染实验,小鼠皮下注射CT-26细胞建立皮下成瘤模型,瘤内注射质粒后,予以电转染,体外活体成像示有荧光反应,然后取瘤体组织作冰冻切片,荧光显微镜观察。结果发现针道附近可见绿色荧光蛋白表达。进一步以高效表达的质粒载体L3进行治疗试验,结果表明质粒载体L3的小鼠瘤体生长速度明显慢于对照组。
上述结果表明电转染方法能在体外和体内有效的将IL-15表达质粒导入肿瘤细胞,同时,腹腔注射质粒也能够使腹膜有效表达目的基因。腹腔注射IL-15表达质粒能在一定程度上抑制腹膜移植瘤的生成与发展。电转染IL-15表达质粒能抑制皮下移植的小鼠结肠癌的生长。本研究为IL-15为基础的肿瘤免疫基因治疗的临床应用提供了实验依据。
Although great progress has been made in the biomedical field in recent decades, the treatment and prognosis of malignant tumors is still far from optimistic. Gene therapy has become an important component of cancer treatment in recent years, especially the cytokine-based immunogene therapy. Many achievements have been made in gene therapy of cancer; however, problems still exist, such as the safety of gene transfer vector and the efficacy of transfection which need further improved. We had constructed the plasmid vector pHi2-IL15-CMV-tat (L1) with dual-promoter amplified expressing of human IL-15. The IL-15 signal sequence was replaced by IL-2 signal sequence, resulting plasmid pHi2-spIL15-CMV-tat (L3). The CEA promoter was cloned into plasmid L1、L3 and replaced CMV promoter to construct plasmid pHi2-IL15-CEA-tat (L2)、pHi2-spIL15-CEA-tat (L4). Current study will further evaluate these vectors in vitro and in vivo.
Plasmid vectors expressing IL-15 or encoding enhanced green fluorescence protein (EGFP) were transfected by electroporation into SW480, a CEA-positive human colon cancer cell line, and MCF-7, a CEA-negative human breast cancer cell line. The transfection efficiency was determined by fluorescence microscope and flow cytometry. IL-15 expression was detected by ELISA. In in vivo study, plasmid L6 was injected into the peritoneum of BALB/c mice and transfected into the subcutaneously growing CT-26 tumor to evaluate the transfection efficacy by in vivo imaging techniques. BALB/c mice were inoculated with CT-26 cells by peritoneal injection, and the plasmid vectors L3, L4 were transfected by intraperitoneal injection after tumor inoculation. Empty plasmid and PBS were used as control. Weight change and life span of the mice were. In addition, the therapeutic effects of L3 were studied through electroporation to control the growth of subcutaneously inoculated CT-26 tumor.
Results showed that green fluorescent protein (GFP) could be detected in the transfected cells 24 hours after transfection. The percentage of transfection was 10%-30%. The IL-15 expression in culture supernatant of L3 and L4 transfectant was significantly higher than that of L1 and L2 (P＜0.05). In transfected SW480 cells, there is no significant deference for IL-15 expression between the CEA promoter positively controlled plasmids L2, L4 and the CMV promoter positively controlled plasmids L1, L3 respectively (P>0.05). In transfected MCF-7 cells, CEA promoter positively controlled plasmids L2, L4 gave rise to lower IL-15 expression than the CMV promoter positively controlled plasmids L1, L3, respectively (P<0.01). GFP expression was observed after intraperitoneal injection of plasmid L6 and electroporation into the subcutaneously inoculated CT-26 tumor. Comparing to the control group, and repeated injection of plasmid L3 (pHi2-spIL15-CMV-tat) and L4 (pHi2-spIL15-CEA-tat) DNA were able to inhibit tumor growth, and prolong the life of tumor-bearing mice. In addition, in vivo transfection of plasmid L3 through electroporation slowed the subcutaneously inoculated CT-26 growth.
In conclusions, electroporation can efficiently transfer IL-15 expression plasmids into tumor cells in vitro and in vivo, in addition, intraperitoneal injection results in efficient transgene expression by epithelial cells in the peritoneum. IL-15 expression plasmid can decrease the growth of inoculated tumor cells after intrapertoneal injection. In addition, electroporation of IL-15 expression plasmid can inhibit the growth of subcutaneously inoculated mice CT-16 tumor. Therefore, this study provides evidence for the clinical application of IL-15 base gene therapy.
引文
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