全反式维甲酸对食管癌细胞系Eca109 VEGF和bFGF表达的影响

发布时间：2018-06-02 05:35

本文选题：全反式维甲酸 + 食管癌细胞　；参考：《郑州大学》2015年硕士论文

【摘要】：背景食管癌是发病率较高、预后较差的实体肿瘤之一。肿瘤直径小于1mm时,可以通过自身渗透作用为之提供营养,当肿瘤直径达到1-2mm时,肿瘤内部发生了血管的新生现象,可以为肿瘤的生长提供充足的营养,新生血管可以促进肿瘤的发展、浸润和转移。多种生长因子共同参与调节着肿瘤血管的生成。其中我们研究最为广泛和深入的生长因子是血管内皮生长因子(VEGF),主要调控着肿瘤血管的生成,促进了多种恶性肿瘤的生长、转移[1-3]。另一类较大的生长因子家族是成纤维生长因子(FGF)家族,其中b FGF是第一个被鉴定的促血管生成因子[4],在血管生成中也起着重要的调节作用。诱导分化概念的提出,为肿瘤的治疗提供了一种新的、有效的治疗理念。越来越多的肿瘤专家开始关注诱导分化治疗的研究进展。全反式维甲酸(ATRA)作为一种最重要的诱导分化剂,已经在白血病及部分实体肿瘤中得到应用。本研究通过体外实验观察ATRA对食管癌细胞系Eca109中VEGF、b FGF蛋白和m RNA表达的影响,为ATRA在食管癌抗血管生成的治疗提供理论依据。研究不同浓度ATRA对食管癌细胞系Eca109 VEGF和b FGF表达的影响。方法终浓度分别为0、1、5、10μmol/L的ATRA作用于人食管癌Eca109细胞,采用噻唑蓝比色法(MTT)评价药物对细胞的增殖抑制情况;通过划痕实验测定ATRA作用于Eca109 48h前后的迁移能力;采用细胞免疫组织化学法和逆转录酶聚合链式反应(RT-PCR)分别检测不同浓度ATRA作用于Eca109 48h后VEGF、b FGF蛋白和m RNA的表达情况。结果1.MTT结果:作用于24h后,实验组,即1μmol/L、5μmol/L、10μmol/L组的增殖抑制率分别为:(44.57±1.36)%、(50.26±1.64)%、(54.24±1.39)%;48h后,上述实验组的增殖抑制率分别为:(54.29±0.15)%、(59.52±0.74)%、(65.45±4.33)%;72h后,上述实验组的增殖抑制率分别为:(66.39±0.87)%、(74.33±2.69)%、(85.24±1.49)%。而对照组,0μmol/L组三个时间点的抑制率均为0.00%。经统计学分析,各个实验组分别与对照组相比,差异均具有统计学意义(P0.05),且实验组之间进行两两比较,差异也具有统计学意义(P0.05)。2.划痕实验结果:1、5、10μmol/L ATRA作用于Eca109 48h后,细胞的迁移率分别为(50.82±1.61)%,(46.80±5.12)%,(32.31±3.25)%,对照组(68.81±2.64)%。0h各组细胞的迁移率均为0.00%。实验组数据分别与对照组相比,差异均具有统计学意义(P0.05),且实验组之间两两比较,差异具有统计学意义(P0.05)。3.免疫组化结果:VEGF蛋白阳性表达体现为细胞内出现的棕黄或棕褐色颗粒状,主要分布于细胞质,核膜亦可见阳性表达。b FGF蛋白阳性表达为细胞核内被染成棕黄色,并且胞浆、核膜可见阳性表达。0、1、5、10μmol/L ATRA作用于Eca109 48h后,细胞内VEGF蛋白相对表达量依次为:(85.43±0.53)×10-2、(77.45±0.51)×10-2、(51.43±0.34)×10-2、(21.74±0.11)×10-2;b FGF蛋白的相对表达量依次为:(71.41±0.52)×10-2、(51.63±0.46)×10-2、(38.44±0.32)×10-2、(11.24±0.21)×10-2。可以看出,随着药物浓度的增加,各个指标的相对表达量逐渐减少,经统计学分析,各个实验组分别与对照组比较,差异均具有统计学意义(P0.05),且实验组之间进行两两比较,差异也具有统计学意义(P0.05)。4.PCR结果:0、1、5、10μmol/L ATRA作用于Eca109细胞48h后,VEGF m RNA表达量依次为:(12.40±1.11)×10-2、(7.27±0.71)×10-2、(4.63±0.45)×10-2、(1.62±0.25)×10-2;b FGF m RNA表达量依次为:(17.96±0.46)×10-2、(15.32±0.33)×10-2、(12.04±0.50)×10-2、(9.13±0.30)×10-2。可以看出,随着药物浓度的增加,各个指标的表达量逐渐减少,经统计学分析,各个实验组分别与对照组比较,差异均具有统计学意义(P0.05),且实验组之间进行两两比较,差异也具有统计学意义(P0.05)。结论1、在一定浓度范围内,ATRA对食管癌Eca109细胞的增殖具有抑制作用,并且随着剂量的增大或作用时间的延长,抑制作用越明显,因此具有剂量和时间依赖性。2、在一定浓度范围内,ATRA可以抑制Eca109细胞的迁移,抑制VEGF、b FGF的m RNA和蛋白的表达,并且随着药物浓度的增加,抑制作用越明显。3、ATRA可能通过下调VEGF、b FGF基因的表达影响食管癌Eca109血管新生从而抑制其生长。
[Abstract]:Background esophageal cancer is one of the solid tumors with high incidence and poor prognosis. When the diameter of the tumor is less than 1mm, it can provide nutrition through its own osmosis. When the diameter of the tumor reaches 1-2mm, the neoplasm occurs in the tumor, which can provide sufficient nutrition for the growth of the tumor. The new blood vessels can promote the development of the tumor. Spread, infiltration and metastasis. Multiple growth factors are involved in regulating the formation of tumor vessels. We study the most extensive and deep growth factor is vascular endothelial growth factor (VEGF), which mainly regulates the formation of tumor vessels, promotes the growth of many malignant tumors, and transfers the other large growth factor family of [1-3].. The FGF family, in which B FGF is the first identified angiogenic factor [4], also plays an important regulatory role in angiogenesis. The concept of induction of differentiation provides a new and effective treatment concept for the treatment of tumors. More and more cancer experts have begun to pay attention to the study of induction of differentiation therapy. All trans retinoic acid (ATRA), as one of the most important inducer and differentiation agents, has been used in leukemia and some solid tumors. In this study, the effect of ATRA on the expression of VEGF, B FGF protein and m RNA in Eca109 of esophageal cancer cell line was observed in vitro. The study provided a theoretical basis for the treatment of anti angiogenesis in gastric cancer. The effects of different concentrations of ATRA on the expression of Eca109 VEGF and B FGF in esophageal carcinoma cell lines. The final concentration of 0,1,5,10 mu mol/L was respectively on the Eca109 cells of human esophageal cancer. The proliferation inhibition of the cells was evaluated by thiazolium colorimetric assay (MTT), and the migration ability of ATRA before and after the Eca109 was measured by scratch test. Cell immuno histochemical method and reverse transcriptase polymerized chain reaction (RT-PCR) were used to detect the expression of VEGF, B FGF protein and m RNA after Eca109 48h, respectively. Results 1.MTT results: after 24h, the experimental group, namely, 1 micron mol/L, 5 micron, and 10 micron groups were (44.57 + 1.36)%, (50.26 + 1.64)%, (54.24). After 48h, the proliferation inhibition rate of the experimental group was: (54.29 + 0.15)%, (59.52 + 0.74)% and (65.45 + 4.33)%. After 72h, the inhibition rate of the experimental group was (66.39 + 0.87)%, (74.33 + 2.69)%, (85.24 + 1.49)%, and the control group was statistically analyzed by 0.00%. by statistical analysis, and the experimental groups were all divided by the control group. Compared with the control group, the differences were statistically significant (P0.05), and the difference between the 22 experimental groups was also statistically significant (P0.05).2. scratch test results: 1,5,10 mu mol/L ATRA after Eca109 48h, the cell migration rate was (50.82 + 1.61)%, (46.80 + 5.12)%, (32.31 + 3.25)%, and the control group (68.81 + 2.64)%.0h each. The migration rates of the group cells were both 0.00%. experimental group and the control group, the difference was statistically significant (P0.05), and the difference between the experimental groups was 22, and the difference was statistically significant (P0.05).3. immunohistochemical results: the positive expression of VEGF protein was manifested as brown or brown granules in the cell, mainly distributed in the cytoplasm. The positive expression of the positive expression of.B FGF protein was found to be brown and yellow in the nucleus, and the cytoplasm, and the positive expression of.0,1,5,10 mu mol/L ATRA in the nuclear membrane, was found to be (85.43 + 0.53) * 10-2, (77.45 + 0.51) * 10-2, (51.43 + 0.34) x 10-2, (21.74 + 0.11) x 10-2, B FGF eggs The relative expression of white was as follows: (71.41 + 0.52) x 10-2, (51.63 + 0.46) x 10-2, (38.44 + 0.32) x 10-2 and (11.24 + 0.21) x 10-2. can be seen that the relative expression of each index gradually decreased with the increase of drug concentration. The differences were statistically significant (P0.05), and the difference was statistically significant (P0.05). The 22 comparison between the groups was statistically significant (P0.05).4.PCR results: after 0,1,5,10 mu mol/L ATRA acted on Eca109 cell 48h, the VEGF m RNA expression was (12.40 + 1.11) x 10-2, (7.27 + 0.71) x 10-2, (4.63 + 0.45) * 10-2, (1.62 + 0.25) * 10-2, B FGF X 10-2, (12.04 + 0.50) x 10-2, (9.13 + 0.30) x 10-2. can see that with the increase of drug concentration, the expression of each index gradually decreased. By statistical analysis, the differences were statistically significant (P0.05) compared with the control group (P0.05). The difference was statistically significant (P0.05). 1, in a certain concentration range, ATRA can inhibit the proliferation of Eca109 cells in esophageal cancer, and the inhibition effect is more obvious with the increase of dose or the prolongation of action time. Therefore, it has a dose and time dependent.2. In a certain concentration range, ATRA can inhibit the migration of Eca109 cells and inhibit VEGF, m RNA and protein of B FGF. With the increase of drug concentration, the more obvious the inhibitory effect is.3, and ATRA may inhibit the growth of esophageal cancer Eca109 angiogenesis by down regulation of VEGF and the expression of B FGF gene.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.1

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