光动力治疗与VP3基因联合治疗鼻咽癌的疗效研究

发布时间：2018-06-30 02:54

本文选题：VP3 + 基因克隆　；参考：《中南大学》2014年博士论文

【摘要】：光动力治疗(Photodynamic therapy, PDT)是采用一定波长的光照射蓄积于肿瘤细胞内的光敏剂,发生光物理化学反应,产生单态氧和自由基等其他活性物质、破坏肿瘤微血管、增强机体免疫力,促进细胞凋亡以杀伤肿瘤细胞的一种非手术治疗方法。然而PDT是对氧的依赖性及治疗深度的局限性限制其在肿瘤治疗中的广泛应用。近年来,随着基因治疗日益成为治疗肿瘤的最有前景的手段之一,PDT与基因治疗有机结合形成的PDT/基因治疗可能是一种治疗肿瘤的崭新策略。本文将分章讨论自杀基因VP3对鼻咽癌细胞的杀伤作用及体外体内实验验证PDT联合自杀基因VP3治疗鼻咽癌的疗效。第一章自杀基因VP3对鼻咽癌CNE-2细胞生长、增殖及凋亡的影响目的体外实验研究VP3基因其对鼻咽癌细胞CNE-2生长、增殖、凋亡的影响,以求证实VP3对鼻咽癌细胞的凋亡诱导效应。方法采用脂质体转染法将构建的PcDNA3.1(-)VP3-Myc(PVP3)及对照空载体质粒PcDNA3.1(-)转染鼻咽癌CNE-2细胞。通过荧光显微镜观察质粒转染效率,CCK-8法检测细胞生存率,RT-PCR、Western blot法检测VP3表达效果,通过Annexin V检测VP3对细胞凋亡的影响。结果 1.VP3mRNA在野生型及空载体质粒转染组细胞中均无明显表达,在PVP3质粒转染组中有明显表达,相对表达量约1.27±0.24。VP3蛋白(apoptin)在野生型及空载体质粒转染组细胞中均无明显表达,在PVP3质粒转染组中有明显表达,相对表达量约0.22±0.06。 2.空载体质粒转染组在24h、48h、72h细胞存活率分别为97.6±1.4%,94.2±1.7%、92.3±2.1%；PVP3质粒转染组在24h、48h、72h细胞存活率分别为79.7±3.6%,61.5±2.3%、52.4±3.8%。PVP3质粒转染与空载体质粒转染组比较有明显统计学差异(P0.001)。 3.空载体质粒转染组在24h、48h、72h细胞凋亡率分别为4.5±1.2%、5.1±0.8%、5.7±1.4%；PVP3质粒转染组在24h、48h、72h细胞凋亡率分别为11.6±2.4%、17.8±1.4%、27.3±3.3%。PVP3质粒转染与空载体质粒转染组比较有明显统计学差异(P0.001)。结论成功建立了VP3基因稳定表达的鼻咽癌CNE-2细胞株,体外实验证实了VP3能明显抑制鼻咽癌CNE-2细胞的生长增殖,并能促进CNE-2细胞的凋亡,为后续实验提供了研究依据第二章光动力治疗联合VP3基因治疗对鼻咽癌CNE-2细胞的体外实验研究目的拟对VP3联合PDT对鼻咽癌CNE-2细胞进行体外实验研究,以证实VP3联合PDT对鼻咽癌CNE-2细胞的杀伤效果。方法稳定表达VP3基因的鼻咽癌细胞CNE-2及对照组细胞分别接受不同能量密度的PDT治疗,CCK-8法检测细胞生存率,Annexin V法、Tunnel法检测细胞凋亡。结果 1.不同能量密度的PDT处理后,PVP3组细胞存活率明显低于空载体质粒转染组细胞,实验组细胞在1、3、6.25J/cm2能量密度下生存率分别为0.533±0.036、0.312±0.027、0.171±0.032；对照组细胞在1、3、6.25J/cm2能量密度下生存率分别为0.816±0.058、0.570±0.044、0.308±0.047,两组比较有明显统计学差异(P0.001)。 2.Annexin V检测显示PVP3组细胞凋亡率明显高于空载体质粒转染组细胞,实验组细胞在1、3、6.25J/cm2能量密度下凋亡率分别为0.363±0.025、0.672±0.043、0.741±0.039；对照组细胞在1、3、6.25J/cm2能量密度下凋亡率分别为0.266±0.027、0.568±0.055、0.671±0.046,两组比较有明显统计学差异(P0.001)；Tunnel法检测显示实验组细胞中大部分细胞膨胀,细胞核碎裂,对照组细胞膜较完整,部分细胞呈空泡状,细胞核浓缩,呈块状,少量细胞细胞核碎裂。结论细胞实验研究发现与单纯PDT相比,PVP3+PDT联合治疗能显著抑制鼻咽癌CNE-2细胞的生长增殖,并且能显著增强鼻咽癌CNE-2细胞的凋亡。第三章VP3联合光动力治疗对鼻咽癌CNE-2细胞裸鼠移植瘤作用的实验研究目的动物实验中比较VP3联合PDT与单纯PDT或VP3基因治疗对鼻咽癌的疗效,以进一步证实VP3联合PDT对鼻咽癌的高效杀伤效果。方法构建裸鼠人鼻咽癌移植瘤模型,PDT处理后,测量并记录肿瘤的大小、重量;HE染色比较各组移植瘤病理学特点；免疫组化检测移植瘤中apoptin的表达。结果 1.空载体质粒组、PVP3质粒组、CNE2/Mock+PDT和CNE2/VP3+PDT处理后0、3、7、14天时分别比较四组肿瘤体积,经ANOVA方差分析,四组间肿瘤体积差异有显著性意义,经两样本独立t检验方法进行两两比较,结果显示CNE2/VP3+PDT处理组肿瘤体积与其他各组相比明显减小(P0.001)；且CNE2/VP3组肿瘤体积较CNE2/Mock组明显减小(P0.001)。 2.四组移植瘤重量差异有显著性意义,经LSD方法进行两两比较,CNE2/VP3+PDT处理组移植瘤重量与各对照组相比明显减小(P0.001); CNE2/VP3组移植瘤重量较CNE2/Mock组明显减小(P0.001)。 3.HE染色检查发现CNE2/Mock组移植瘤肿瘤细胞形态正常,瘤体内见毛细血管丰富,未见明显坏死组织；CNE2/VP3组移植瘤肿瘤细胞形态正常,可见少许坏死区域；CNE2/Mock+PDT组移植瘤见大量肿瘤细胞变性及核固缩,伴有部分片状坏死区域；CNE2/VP3+PDT组移植瘤见大片状坏死区域,仅少量癌细胞岛残留。 4.免疫组化检查发现CNE2/Mock组及CNE2/Mock+PDT组未见明显apoptin蛋白表达；CNE2/VP3组及CNE2/VP3+PDT组apoptin蛋白强表达,且其主要在细胞核中表达。结论利用基因治疗或者光动力治疗均能抑制鼻咽癌裸鼠移植瘤生长,而光动力治疗联合基因治疗较单纯的光动力治疗或基因治疗对鼻咽癌裸鼠移植瘤有更好的杀伤效果。光动力治疗联合基因治疗为鼻咽癌的临床治疗提供了实验基础。
[Abstract]:Photodynamic therapy (Photodynamic therapy, PDT) is a kind of non operative treatment of tumor cells by irradiation of light sensitizers accumulated in tumor cells by light irradiation of a certain wavelength, producing light physical and chemical reactions, producing other active substances such as mono oxygen and free radicals, destroying tumor microvessels, enhancing the body's immunity and promoting cell apoptosis. However, PDT is the limitation of the dependence on oxygen and the limitation of the depth of treatment. It has been widely used in cancer treatment. In recent years, as gene therapy has become one of the most promising ways to treat cancer, the PDT/ gene therapy formed by the organic combination of PDT and gene therapy may be a new strategy for the treatment of tumors. This article will be divided into this article. This chapter discusses the killing effect of suicide gene VP3 on nasopharyngeal carcinoma cells and the effect of PDT combined suicide gene VP3 on nasopharyngeal carcinoma in vivo and in vitro.
Chapter 1 the effect of suicide gene VP3 on growth, proliferation and apoptosis of nasopharyngeal carcinoma CNE-2 cells
Objective to study the effect of VP3 gene on the growth, proliferation and apoptosis of nasopharyngeal carcinoma cell CNE-2 in vitro, in order to verify the apoptosis inducing effect of real VP3 on nasopharyngeal carcinoma cells.
Methods the transfected PcDNA3.1 (-) (-) VP3-Myc (PVP3) and PcDNA3.1 (-) were transfected into the CNE-2 cells of nasopharyngeal carcinoma by liposome transfection. The transfection efficiency of the plasmid was observed by the fluorescence microscope, the cell survival rate was detected by CCK-8 method. The expression of VP3 was detected by RT-PCR, Western blot, and the effect of Annexin V on the apoptosis was detected by Annexin V.
Result
There was no obvious expression of 1.VP3mRNA in the transfected group cells of wild type and unloaded body, and there was obvious expression in the transfection group of PVP3 plasmid. The relative expression of about 1.27 + 0.24.VP3 protein (apoptin) had no obvious expression in the cells of the wild type and unloaded body transfected group, and the expression of relative expression was about 0.22 + 0 in the PVP3 plasmid transfection group. Six
The survival rate of 24h, 48h and 72h cells was 97.6 + 1.4%, 94.2 + 1.7%, 92.3 + 2.1%, respectively. The survival rate of PVP3 plasmid transfected group in 24h, 48h, 72h cells was 79.7 + 3.6%, 61.5 + and 61.5 +, respectively, and 52.4 + 3.8%.PVP3 plasmid transfection was significantly different (P0.001) compared with the empty body transfection group (P0.001).
The apoptosis rate of 24h, 48h and 72h cells was 4.5 + 1.2%, 5.1 + 0.8%, 5.7 + 1.4%, respectively. The apoptosis rate of PVP3 plasmid transfected group in 24h, 48h, 72h cells was 11.6 + 2.4%, 17.8 + and 17.8 +, respectively, and 27.3 + 3.3%.PVP3 plasmid transfection was significantly different (P0.001) compared with the empty body transfection group (P0.001).
Conclusion the CNE-2 cell line of nasopharyngeal carcinoma with stable expression of VP3 gene was successfully established. In vitro experiments confirmed that VP3 could obviously inhibit the growth and proliferation of CNE-2 cells in nasopharyngeal carcinoma and promote the apoptosis of CNE-2 cells, which provides a basis for further research.
Second Zhang Guang dynamic therapy combined with VP3 gene therapy for nasopharyngeal carcinoma CNE-2 cells in vitro
Objective to study the effect of VP3 combined with PDT on nasopharyngeal carcinoma CNE-2 cells in vitro, in order to confirm the killing effect of VP3 combined with PDT on nasopharyngeal carcinoma CNE-2 cells.
Methods the CNE-2 of nasopharyngeal carcinoma cells expressing VP3 gene and the control group cells were treated with PDT with different energy density respectively. The cell survival rate was detected by CCK-8 method, Annexin V method and Tunnel method were used to detect the apoptosis.
Result
1. after PDT treatment with different energy density, the survival rate of cells in PVP3 group was significantly lower than that of the empty body mass transfected group. The survival rate of the cells in the experimental group was 0.533 + 0.036,0.312 + 0.027,0.171 0.032 respectively under the 1,3,6.25J/cm2 energy density, and the survival rate of the cells in the control group was 0.816 + 0.058,0.570 + 0.044,0.3 under the 1,3,6.25J/cm2 energy density, respectively. 08 + 0.047, there was a significant difference between the two groups (P0.001).
2.Annexin V detection showed that the apoptosis rate of the PVP3 group was significantly higher than that of the empty body mass transfected group. The apoptosis rate of the experimental group was 0.363 + 0.025,0.672 + 0.039 respectively under the 1,3,6.25J/cm2 energy density, and the apoptosis rate of the control group was 0.266 + 0.027,0.568 + 0.055,0.671 + 0.046, two, under the 1,3,6.25J/cm2 energy density, two There was significant difference between the groups (P0.001), and the Tunnel test showed that most of the cells in the experimental group were expanded, the nuclei were broken, the cell membrane of the control group was more complete, some cells were vacuolated, the nuclei were concentrated, and the cell nuclei were fragmented.
Conclusion compared with simple PDT, PVP3+PDT combined therapy can significantly inhibit the growth and proliferation of nasopharyngeal carcinoma CNE-2 cells, and can significantly enhance the apoptosis of nasopharyngeal carcinoma CNE-2 cells.
Third chapter VP3 combined photodynamic therapy on nasopharyngeal carcinoma CNE-2 cell xenograft in nude mice
Objective to compare the therapeutic effect of VP3 combined with PDT and simple PDT or VP3 gene therapy on nasopharyngeal carcinoma in order to further confirm the effect of VP3 combined with PDT on nasopharyngeal carcinoma.
Methods the model of human nasopharyngeal carcinoma transplanted in nude mice was constructed. After PDT treatment, the size and weight of the tumor were measured and recorded. The pathological features of the tumor were compared with HE staining, and the expression of apoptin in the transplanted tumor was detected by immunohistochemistry.
Result
1. empty body mass group, PVP3 plasmid group, CNE2/Mock+PDT and CNE2/VP3+PDT after 0,3,7,14 days were compared with four groups of tumor volume respectively. After ANOVA variance analysis, the difference of tumor volume between the four groups was significant, and the two samples were compared with the independent t test method for 22 comparison. The results showed that the volume of the tumor in the CNE2/VP3+PDT treatment group and the other groups were shown in the other groups. The ratio was significantly reduced (P0.001), and the volume of tumor in group CNE2/VP3 was significantly lower than that in group CNE2/Mock (P0.001).
2. the weight difference between the four groups was significant. Compared with the 22 comparison by the LSD method, the weight of the transplanted tumor in the CNE2/VP3+PDT treatment group was significantly lower than that of the control group (P0.001), and the weight of the transplanted tumor in the group CNE2/VP3 was significantly lower than that in the CNE2/Mock group (P0.001).
3.HE staining showed that the tumor cells of group CNE2/Mock were normal, and the blood capillary was rich in the tumor and no obvious necrotic tissue was found in the tumor. The tumor cells in group CNE2/VP3 were normal, and a little necrotic area was found. In group CNE2/Mock+PDT, a large number of tumor cell degeneration and nuclear fixation, accompanied by partial necrosis area, were found in group CNE; CNE In group 2/VP3+PDT, there were large necrotic areas and only a small number of cancer cell islands remained.
4. immunohistochemical examination showed that no obvious expression of apoptin protein was found in group CNE2/Mock and CNE2/Mock+PDT group, and the expression of apoptin protein in group CNE2/VP3 and CNE2/VP3+PDT group was strongly expressed, and it was mainly expressed in the nucleus.
Conclusion both gene therapy and photodynamic therapy can inhibit the growth of nasopharyngeal carcinoma transplanted tumor in nude mice. Photodynamic therapy combined with gene therapy has better killing effect on nude mice transplanted tumor of nasopharyngeal carcinoma than simple photodynamic therapy or gene therapy. The combination of photodynamic therapy and gene therapy provides the experimental basis for the clinical treatment of nasopharyngeal carcinoma.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R739.63

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