α-干扰素联合化疗诱导黑色素瘤血管正常化中的初步研究

发布时间：2018-07-10 11:02

本文选题：黑色素瘤 + 达卡巴嗪　；参考：《华中科技大学》2010年硕士论文

【摘要】：目的:本研究拟通过动物实验探讨IFN-α联合治疗的可能机制,以及周细胞及其新标志物RGS5在联合治疗中可能所起的作用。方法:将黑色素瘤细胞株B16细胞种植在小鼠左前下肢靠背侧皮下,7天后随机分成三组:空白对照组,DTIC单药处理组和DTIC+IFN-α联合处理组,每组7只。第16天处死小鼠,测量肿瘤长短径以计算肿瘤体积;数字放射照相术分析血管形态;免疫组化检测CD31计算微血管密度(MVD),检测α-SMA分析微血管成熟周细胞覆盖度和检测肿瘤HIF-α阳性细胞的表达个数以分析缺氧区域;实时定量PCR分析RGS5 mRNA的表达;Western Blot检测RGS5和HIF-α的表达;以及免疫荧光分析RGS5在黑色素瘤中的表达部位。结果:DTIC联合IFN-α处理组相对于单用DTIC化疗组和对照组,肿瘤生长明显受到抑制,肿瘤体积显著下降(对照组5.806±1.436 cm3 vs. DTIC组2.946±1.266 cm3 vs.联合治疗组1.658±0.949 cm3; P0.05)。IFN-α联合处理组还显示肿瘤血管正常化,膨大的血管显著减少,平均血管直径下降(对照组1.0000±0.5142 mm vs. DTIC组0.8375±0.4676 mm vs.联合化疗组0.3835±0.2122mm;P0.05);微血管密度(MVD)下降(联合化疗组21±9 vessels/mm2) vs.对照组62±16 vessels/mm2 vs. DTIC组41±11 vessels/mm2;P0.05);微血管成熟周细胞覆盖度显著上升(DTIC+IFN-α联合处理组69.7±16.8% vs. DTIC处理组32±13% vs.对照49±12%;P0.05);肿瘤缺氧显著缓解(对照组145±13 HIF-α+ cells/field vs. DTIC组119±22 HIF-α+ cells/field vs. IFN-α联合化疗组66±32 HIF-α+ cells/field;P0.05)。除此之外,我们还发现黑色素瘤高表达RGS5蛋白,但是不是表达在黑色素瘤细胞,而是表达在肿瘤管腔周围。IFN-α联合处理组相对于DTIC化疗组和对照组,RGS5表达显著下降(RGS5 mRNA对照组2.996±0.197 vs. DTIC处理组2.312±0.697 vs.联合治疗组1.102±0.208, P0.05)。结论:本实验提示黑色素瘤高表达RGS5蛋白,但是不是表达在黑色素瘤细胞,而是表达在肿瘤管腔周围——周细胞。DTIC联合IFN-α处理组抗肿瘤效应优于单用DTIC化疗组和对照组部分是由于正常化肿瘤血管从而增加传统化疗的效应。并且,IFN-α显示正常化肿瘤血管效应部分是由于下调RGS5的表达及诱导周细胞成熟,揭示了RGS5是IFN-α的靶点之一,而RGS5特异性表达细胞——周细胞是IFN-α的靶细胞之一,初步证明了RGS5和周细胞在联合免疫化疗治疗黑色素瘤中对肿瘤血管正常化中能起一定的作用。
[Abstract]:Aim: to explore the possible mechanism of IFN- 伪 combined therapy and the role of pericytes and its new marker RGS5 in combined therapy by animal experiments. Methods: the melanoma cell line B16 cells were implanted in the subcutaneous side of the left anterior and lower extremity of mice for 7 days, and were randomly divided into three groups: the blank control group treated with DTIC alone and the DTIC IFN- 伪 treated group with 7 rats in each group. On the 16th day, the mice were killed, the tumor length and diameter were measured to calculate the tumor volume, and the vascular morphology was analyzed by digital radiography. CD31 was used to calculate microvessel density (MVD), 伪 -SMA was used to analyze the coverage of microvascular mature pericytes and the number of HIF- 伪 positive cells in tumor to analyze the hypoxia region, the expression of RGS5 mRNA was detected by real-time quantitative PCR and the expression of RGS5 and HIF- 伪 was detected by Western Blot, the expression of RGS5 and HIF- 伪 was detected by Western blot. The expression of RGS5 in melanoma was analyzed by immunofluorescence. Results the tumor growth was significantly inhibited and the tumor volume was significantly decreased (control group 5.806 卤1.436 cm3 vs 1.436 cm3 vs 2.946 卤1.266 cm3 vs) in the control group treated with 1: DTIC combined with IFN- 伪. The combined treatment group (1.658 卤0.949 cm ~ (3); P0.05). IFN- 伪 combined treatment group also showed that the tumor vessels normalized, the enlarged vessels decreased significantly and the mean vessel diameter decreased (control group 1.0000 卤0.5142 mm vs. DTIC group 0.8375 卤0.4676 mm vs. The microvessel density (MVD) was decreased (21 卤9 vessels/mm2) in the combined chemotherapy group (P 0.05). In the control group (62 卤16 vessels/mm2 vs .DTIC, 41 卤11 vessels / mm2, P0.05), the coverage of microvascular matured pericytes increased significantly (DTIC IFN- 伪 combined treatment group, 69.7 卤16.8% vs. DTIC treatment group, 32 卤13% vs. Tumor anoxia was significantly alleviated in the control group (49 卤12g / kg) and tumor hypoxia (145 卤13HIF- 伪 cells/field vs.DTIC group 119 卤22HIF- 伪 cells/field vs. IFN- 伪 combined chemotherapy group 66 卤32HIF- 伪 cells / 伪 cells / p0.05). In addition, we also found that melanoma overexpression RGS5 protein, but not in melanoma cells, The expression of RGS5 in the combined treatment group was significantly lower than that in the DTIC chemotherapy group and the control group (RGS5 mRNA was 2.996 卤0.197 vs 2.312 卤0.697 vs in the DTIC group). Combined treatment group 1.102 卤0.208, P0.05). Conclusion: this study suggests that RGS5 protein is highly expressed in melanoma, but not in melanoma cells. But the antitumor effect of the treated group was better than that of the single DTIC chemotherapy group and the control group, partly because of normalizing tumor blood vessels and increasing the effect of traditional chemotherapy. IFN- 伪 showed that the normal vascular effect was partly due to down-regulation of RGS5 expression and induction of pericyte maturation, which revealed that RGS5 was one of the targets of IFN- 伪, and RGS5-specific expression cell-pericyte was one of the target cells of IFN- 伪. It was preliminarily proved that RGS5 and pericytes may play a role in the normalization of tumor blood vessels in combined immunotherapy with melanoma.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R739.5

【共引文献】