Ang2-siRNA慢病毒载体干预裸鼠移植性恶性黑色素瘤的研究

发布时间：2018-02-01 14:29

本文关键词： 恶性黑色素瘤促血管生成2 RNA干扰微血管密度　出处：《福建医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：构建人恶性黑色素瘤裸鼠移植瘤模型，利用Ang2-siRNA慢病毒载体介导的RNA干扰作用，沉默Ang2基因在裸鼠移植瘤中的表达，研究在体内Ang2-siRNA慢病毒对移植瘤中Ang2基因的影响，并检测移植瘤微血管密度，肿瘤的增殖情况和体积，评价及初步明确Ang2表达水平、微血管密度与肿瘤增殖之间的关系，为肿瘤的基因治疗提供一定的基础实验研究依据。方法：1、将15雄性裸鼠随机分成空白组（PBScontrolgroup）、空载组（Vectorcontrolgroup）、实验组（RNAigroup）3组，每组5只。 2、正常培养、传代A375细胞，制成单细胞悬液，调整细胞密度至约为5×107个/ml。 3、在裸鼠右腋皮下，用100μl微量进样器接种制备好的A375单细胞悬液，，100μl/只，建立人恶性黑色素瘤裸鼠移植瘤模型。 4、空白组、空载组、实验组3组裸鼠皮下移植瘤分别多点注射PBS、pNL-EGFP-Vector和pNL-EGFP-Ang2-siRNA慢病毒液，200μl/只，并于腹腔内加强注射同种溶液500μl/只，进行干扰试验研究。 5、观察并记录裸鼠移植瘤增殖情况和体积，统计、分析各组之间体积差异，并绘制肿瘤生长曲线。 6、用实时荧光定量RT-PCR法检测移植瘤中Ang2基因的相对表达量，统计分析各组之间Ang2基因表达水平的差异和肿瘤增殖程度的关系。 7、用免疫组织化学法检测移植瘤的微血管密度，统计分析各组之间差异及其与肿瘤增殖程度的关系。结果：1、成功建立了人恶性黑色素瘤裸鼠移植瘤模型。 2、实验组移植瘤体积显著小于空白组和空载组，有统计学意义（P＜0.01），空白组和空载组移植瘤体积之间差异无统计学意义（P＞0.05）。 3、实验组移植瘤Ang2基因相对表达水平显著低于空白组和空载组，有统计学意义（P＜0.01），空白组和空载组Ang2基因相对表达水平之间差异无统计学意义（P＞0.05）。 4、实验组微血管密度显著低于空白组和空载组，有统计学意义（P＜0.01），空白组和空载组之间差异无统计学意义（P＞0.05）。结论：利用Ang2-siRNA慢病毒载体介导的RNA干扰作用，能够成功降低人恶性黑色素瘤裸鼠移植瘤中Ang2基因的表达水平，减少移植瘤中微血管的生成，从而抑制肿瘤的生长、增殖。
[Abstract]:Objective: to construct a human malignant melanoma xenograft model in nude mice, and to silence the expression of Ang2 gene in nude mice xenografts by RNA interference mediated by Ang2-siRNA lentivirus vector. To study the effect of Ang2-siRNA lentivirus on the Ang2 gene in transplanted tumor, and to detect the microvessel density, tumor proliferation and volume. The expression level of Ang2, the relationship between microvessel density and tumor proliferation were evaluated and preliminarily determined, which provided a basis for the basic experimental study of tumor gene therapy. Methods 15 male nude mice were randomly divided into control group and control group. The experimental group was treated with RNAi group (n = 5). 2. A375 cells were cultured and subcultured to form a single cell suspension, and the cell density was adjusted to about 5 脳 107 / ml. 3Subcutaneously in the right axilla of nude mice, 100 渭 l / mouse A375 single cell suspension was inoculated with 100 渭 l microsampler to establish the transplanted tumor model of human malignant melanoma in nude mice. (4) PBS was injected into nude mice subcutaneously in blank group, no-load group and experimental group respectively. PNL-EGFP-Vector and pNL-EGFP-Ang2-siRNA lentivirus were injected intraperitoneally with 500 渭 l / rat lentivirus. The interference test was carried out. 5. The proliferation and volume of xenografts in nude mice were observed and recorded. The volume difference was analyzed and the tumor growth curve was drawn. 6. The relative expression of Ang2 gene in transplanted tumor was detected by real-time fluorescence quantitative RT-PCR, and the relationship between the expression of Ang2 gene and the degree of tumor proliferation was statistically analyzed. 7. The microvessel density of transplanted tumor was detected by immunohistochemical method, and the relationship between the microvessel density and the degree of tumor proliferation was analyzed statistically. Results: 1, the transplanted tumor model of human malignant melanoma was successfully established in nude mice. 2. The volume of transplanted tumor in the experimental group was significantly smaller than that in the blank group and the no-load group (P < 0.01), but there was no significant difference between the blank group and the no-load group (P > 0.05). 3The relative expression level of Ang2 gene in experimental group was significantly lower than that in blank group and no-load group (P < 0.01). There was no significant difference in relative expression of Ang2 gene between blank group and no-load group (P > 0.05). 4. The microvessel density in the experimental group was significantly lower than that in the blank group and the no-load group (P < 0.01), but there was no significant difference between the blank group and the no-load group (P > 0.05). Conclusion: RNA interference mediated by Ang2-siRNA lentivirus vector can successfully reduce the expression of Ang2 gene in human malignant melanoma xenografts in nude mice. Reduce the formation of microvessels in the tumor, thereby inhibiting the growth and proliferation of the tumor.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.5

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