苹果酸舒尼替尼对体外培养的恒河猴脉络膜视网膜血管内皮细胞增殖和迁移的影响

发布时间：2018-03-31 19:22

本文选题：苹果酸舒尼替尼　切入点：恒河猴脉络膜视网膜血管内皮细胞　出处：《广西医科大学》2010年硕士论文

【摘要】： 目的探讨苹果酸舒尼替尼(Sunitinib malate, SU11248)对体外培养的眼底微血管内皮细胞(恒河猴脉络膜视网膜血管内皮细胞,RF／6A)的增殖和迁移及KDRmRNA表达的影响。方法体外培养RF／6A,分为S0、S1、S2、S3、S4、S5、S6组(各组培养基中Sunitinib的浓度分别为0 mg·L-1,0.00125 mg·L-1、0.0025 mg·L-1、0.005 mg-L-1、0.01 mg·L-1、0.02 mg-L-1和0.04 mg·L-1)。分别于培养24 h和48 h后采用磺酰罗丹明染色法(SRB)法观察0 mg·L-1、0.00125mg·L-1、0.0025 mg·L-1、0.005 mg·L-1、0.01 mg·L-1、0.02 mg·L-1和0.04 mg·L-1浓度的Sunitinib对RF／6A增殖的影响；并根据吸光度值(OD570)计算Sunitinib对RF／6A增殖抑制的抑制率；采用细胞划痕修复实验观察0 mg·L-1、0.0025 mg·L-1、0.005 mg·L-1、0.01 mg·L-10.02 mg·L-1浓度的Sunitinib对RF／6A迁移的影响；并用倒置相差显微镜观察RF／6A迁移的情况；同时利用逆转录—聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)检测0 mg·L-1、0.0025mg·L-1、0.005 mg·L-1、0.01 mg·L-1、0.02 mg·L-1浓度的Sunitinib处理前后RF/6A KDR (VEGFR-2)mRNA表达水平的变化。各组RF／6A吸光度A值,RF／6A增殖抑制的抑制率及KDRmRNA相对含量均以均数±标准差(x±s)表示,数据处理采用SPSS13.0统计软件进行单因素方差分析(one-way ANOVA),并采用LSD法进行两两比较,P0.05具有统计学意义。结果1 SRB实验结果如下： Sunitinib对RF／6A的增殖有抑制作用且呈时间—剂量依赖性,0.00125 mg·L-1、0.0025 mg·L-1、0.005 mg·L-1、0.01 mg·L-1、0.02 mg·L-1和0.04 mg·L-1浓度的Sunitinib作用于RF／6A 24 h,抑制率分别为(12.009±0.038)%、(21.440±0.007)%、(35.434±0.015)%、(43.125±0.002)%、(53.700±0.001)%、(60.971±0.003)%,各组两两比较差异有统计学意义(P0.01)；Sunitinib作用于RF／6A48 h,其抑制率分别为(36.872±0.006)%、(40.673±0.013)%、(47.313±0.004)%、(55.910±0.003)%、(63.120±0.003)%、(69.975±0.014)%,各组两两比较差异均有统计学意义(P0.01)。 2细胞迁移划痕修复实验结果如下：培养基中加入0 mg·L-1、0.0025 mg·L-1、0.005 mg·L-1、0.01mg·L-1、0.02 mg·L-1浓度的Sunitinib 24 h后RF／6A的迁移距离分别为(203.3±2.2)μm、(145.4±4.4)μm、(123.9±2.6)μm、(96.1±3.1)μm、(46.6±2.9)μm。而48 h后则为(313.1±4.1)μm、(213.9±2.8)μm、(193.9±4.2)μm、(134.5±3.2)μm、(109.9±5.7)μm。在同一时段,各组两两比较差异均有统计学意义(P0.01)。 3 RT-PCR结果如下： 0 mg·L-1、0.0025 mg·L-1、0.005 mg·L-1、0.01 mg·L-1、0.02mg·L-1浓度的Sunitinib作用24 h后,RF/6A KDRmRNA的相对表达量分别为0.583±0.004、0.570±0.008、0.553±0.007、0.531±0.003、0.513±0.005。而48 h后则分别为0.628±0.005、0.610±0.002、0.588±0.002、0.564±0.005、0.525±0.004。各组两两比较差异均有统计学意义(P0.01)。结论1Sunitinib能够抑制体外培养的恒河猴脉络膜视网膜血管内皮细胞的增殖和迁移；Sunitinib能够下调恒河猴脉络膜视网膜血管内皮细胞KDRmRNA的表达； 2 Sunitinib抑制视网膜血管内皮细胞的增殖和迁移的机制可能与其下调VEGFR的表达有关；
[Abstract]:Objective to investigate the sunitinib malate (Sunitinib, malate, SU11248) on cultured retinal microvascular endothelial cells (endothelial cells, Ganges RIver monkey choroidal retinal RF / 6A) expression of proliferation and migration and KDRmRNA.
RF / 6A culture method in vitro, divided into S0, S1, S2, S3, S4, S5, S6 group (the concentration of Sunitinib in the culture medium of each group were 0 mg - L-1,0.00125 Mg - L-1,0.0025 Mg - L-1,0.005 mg-L-1,0.01 Mg - L-1,0.02 mg-L-1 and 0.04 mg L-1). The staining was cultured for 24 h and 48 h after using sulfonyl Luo Danming (SRB) Mg - L-1,0.00125mg - L-1,0.0025 0 mg - L-1,0.005 Mg - L-1,0.01 Mg - L-1,0.02 Mg - L-1 and 0.04 mg L-1 concentration of Sunitinib on RF / 6A proliferation observation; and according to the absorbance value (OD570) of RF / Sunitinib computing to inhibit 6A proliferation rate; by cell scratch repair experimental observation of 0 mg - L-1,0.0025 Mg - L-1,0.005 Mg - L-1,0.01 Mg - L-10.02 Mg - L-1 concentration effect of Sunitinib on RF / 6A migration; and observed by phase contrast RF / 6A migration of the microscope; at the same time by using reverse transcription polymerase chain reaction (reverse, tra Nscription-polymerase chain reaction, RT-PCR Sunitinib) before and after treatment was detected in 0 mg - L-1,0.0025mg - L-1,0.005 Mg - L-1,0.01 Mg - L-1,0.02 Mg - L-1 concentration of RF/6A KDR (VEGFR-2) expression of mRNA. Each RF / 6A value A, the relative content of KDRmRNA RF / 6A and the inhibition rate of proliferation are mean standard the difference (x + s), data processing using SPSS13.0 statistical software for single factor analysis of variance (one-way ANOVA), and LSD method was used to compare the 22, P0.05 was statistically significant.
Results 1 SRB experimental results are as follows:
Sunitinib on RF / 6A inhibited the proliferation and showed time and dose dependent, 0.00125 Mg - L-1,0.0025 Mg - L-1,0.005 Mg - L-1,0.01 Mg - L-1,0.02 Mg - L-1 and 0.04 mg L-1 concentration of Sunitinib in RF / 6A 24 h, the inhibition rate of (12.009 + 0.038)%, (21.440. 0.007)% and (35.434 + 0.015)% and (43.125 + 0.002)% and (53.700 + 0.001)% and (60.971 + 0.003)%, 22 groups was statistically significant difference (P0.01); the effect of Sunitinib on RF / 6A48 h, and the inhibition rates were (36.872 + 0.006)%, (40.673 + 0.013)% and (47.313 + 0.004)% and (55.910 + 0.003)% and (63.120 + 0.003)% and (69.975 + 0.014)%, 22 groups were statistically significant (P0.01).
2 cell migration scratch repair experimental results are as follows:
Adding 0 mg - L-1,0.0025 culture mg L-1,0.005 mg L-1,0.01mg L-1,0.02 Mg - L-1 concentration in medium Sunitinib 24 h after the migration distance of RF / 6A respectively (203.3 + 2.2) m, (145.4 + 4.4) m, (123.9 + 2.6) m, (96.1 + 3.1) m, (46.6 + 2.9) M. and 48 h for (313.1 + 4.1) m, (213.9 + 2.8) m, (193.9 + 4.2) m, (134.5 + 3.2) m, (109.9 + 5.7) M. at the same time, the 22 groups had statistical difference meaning (P0.01).
3 RT-PCR results are as follows:
Sunitinib 0 mg - L-1,0.0025 Mg - L-1,0.005 Mg - L-1,0.01 Mg - L-1,0.02mg - L-1 concentration of 24 h, the relative expression of RF/6A KDRmRNA were 0.583 + 0.004,0.570 + 0.008,0.553 + 0.007,0.531 + 0.003,0.513 + 0.005. and 48 h respectively for 0.628 + 0.005,0.610 + 0.002,0.588 + 0.002,0.564 + 0.005,0.525 + 0.004. were 22 difference there was statistically significant (P0.01).
The proliferation and migration of Ganges RIver monkey choroid retinal endothelial cells in vitro. Conclusion 1Sunitinib can inhibit the expression of Sunitinib can down regulate endothelial cells; KDRmRNA Ganges RIver monkey choroidal retinal vessels;
The mechanism of 2 Sunitinib inhibit the proliferation and migration of retinal vascular endothelial cells may be associated with down-regulation of VEGFR;

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774

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