双氢青蒿素对犬乳腺肿瘤细胞侵袭性及EMT相关因子的影响研究
发布时间：2018-08-24 08:33
【摘要】：犬乳腺肿瘤是一种严重危害机体健康的肿瘤性疾病,通过恶性增殖、转移侵袭至其他脏器导致器官衰竭。目前针对犬乳腺肿瘤的治疗多为手术及放、化疗,但结果多易复发或副作用较大。双氢青蒿素(Dihydroartemisinin,DHA)作为中药提取物,其抗疟功效已经得到了广泛的认同,随着研究的深入,其抗肿瘤的效果也逐步显现。本试验研究了双氢青蒿素对犬乳腺肿瘤细胞侵袭性及EMT相关因子是否具有影响及其影响机制。取对数增长期状态相同的犬乳腺肿瘤细胞CHMm,使用低(5μM)、中(10μM)、高(20μM)浓度DHA处理并分别培养24、48、72h。收集各组细胞使用CCK-8法检测细胞活性;细胞划痕试验检测细胞迁移性;transwell检测细胞侵袭性;ELISA检测细胞基质金属蛋白酶(MMP-9)含量及基质金属蛋白酶抑制剂1(TIMP-1)含量;实时荧光定量PCR方法检测细胞E钙粘蛋白(CDH1)、锌指结合蛋白(ZEB)、锌指转录因子(SLUG)等细胞上皮间质转化(EMT)相关因子表达量变化。1.使用CCK-8检测细胞毒性发现,相同培养时间,与空白对照组相比较,低、中、高试验组对CHMm细胞抑制性影响效果极显著(p0.01)。相同DHA作用浓度,培养48h、72h与培养24h相比较DHA对细胞抑制性影响差异极显著(p0.01)。DHA对CHMm细胞抑制率呈明显药物时间、浓度依赖性。2.细胞划痕试验检测细胞迁移速率结果表明,相同培养时间,与空白对照组相比较低、中、高试验组对CHMm细胞迁移性抑制作用效果极显著(p0.01)。相同DHA作用浓度,培养48h、72h与培养24h相比较DHA对细胞迁移性抑制影响差异极显著(p0.01)。DHA对CHMm细胞迁移性抑制呈明显的药物时间、浓度依赖性。3.细胞侵袭试验检测细胞侵袭性结果表明,相同培养时间,与空白对照组相比较低、中、高试验组穿透基底膜着色细胞个数显著增多,说明DHA对CHMm细胞侵袭性抑制效果极显著(P0.01)。相同DHA作用浓度,培养48h、72h与培养24h相比较DHA对细胞侵袭性抑制影响差异极显著(p0.01)。DHA对CHMm细胞侵袭性抑制呈明显的药物时间、浓度依赖性。4.细胞基质金属蛋白酶检测结果表明,相同培养时间,与空白对照组相比较各浓度试验组细胞MMP-9含量降低极显著(p0.01)。相同作用浓度,随培养时间的增加细胞MMP-9含量变化差异不显著(p0.05)。DHA对CHMm细胞分泌MMP-9具有显著抑制性,呈明显药物浓度依赖性。5.细胞基质金属蛋白酶抑制剂结果表明,相同培养时间,与空白对照组相比各浓度DHA试验组TIMP1含量没有显著变化(p0.05)。相同DHA作用浓度,增加培养时间,各试验组TIMP1含量也没有显著变化(p0.05)。6.EMT相关因子mRNA表达结果表明,相同培养时间,与空白对照组相比,各浓度DHA试验组目的基因CDH1的mRNA表达量上调极显著(P0.01),目的基因SLUG、ZEB1、ZEB2、Twist、HMGA2、MMP-9 及 VEGF 的 mRNA 表达量下调极显著(p0.01)。双氢青蒿素对犬乳腺肿瘤细胞具有增殖抑制作用,并通过对EMT相关因子表达的调控抑制细胞的侵袭性。
[Abstract]:Canine mammary neoplasms are a kind of tumor disease which seriously endangers the body's health. By malignant proliferation, metastasis and invasion to other organs, organ failure is caused. At present, most of the treatment for canine breast tumors are surgery, radiotherapy and chemotherapy, but the results are more likely to relapse or side effects. Dihydroartemisinin (Dihydroartemisinin,DHA), as a traditional Chinese medicine extract, has been widely recognized for its antimalarial efficacy. The effects of dihydroartemisinin on the invasiveness and EMT related factors of canine breast tumor cells were studied. Canine breast tumor cells with the same logarithmic growth state were treated with low (5 渭 M),) (10 渭 M),) high (20 渭 M) DHA for 72 h. The cell activity was detected by CCK-8 assay, the content of matrix metalloproteinase (MMP-9) and matrix metalloproteinase inhibitor 1 (TIMP-1) were detected by cell scratch assay and invasive Elisa. The expression of E-cadherin (CDH1), zinc finger binding protein (ZEB), zinc finger transcription factor (SLUG) and (EMT) related factors were detected by real-time fluorescence quantitative PCR. Using CCK-8 to detect the cytotoxicity, the same culture time, compared with the blank control group, lower, medium, high test group on the inhibitory effect of CHMm cells was very significant (p0.01). At the same concentration of DHA, the inhibitory effect of DHA on CHMm cells was significantly different between 48 h and 24 h (p0.01) .DHA was in a dose-dependent and time-dependent manner. The results of cell scratch test showed that the same culture time was lower than that of the blank control group, and the inhibitory effect of the high test group on the migration of CHMm cells was very significant (p0.01). At the same concentration of DHA, the effects of DHA on migration inhibition of CHMm cells were significantly different between 48 h and 24 h (p0.01) .DHA showed a significant dose-dependent effect on the inhibition of migration of CHMm cells in a dose-dependent manner. The results of cell invasion test showed that the number of infiltrating basement membrane stained cells in the high test group was significantly higher than that in the blank control group at the same culture time, which indicated that DHA had a significant inhibitory effect on the invasion of CHMm cells (P0.01). At the same concentration of DHA, the inhibitory effects of DHA on the invasion of CHMm cells were significantly different between 48 h and 24 h (p0.01) .DHA showed a dose-dependent effect on the inhibition of invasion of CHMm cells in a dose-dependent manner. The results of cell matrix metalloproteinase assay showed that the MMP-9 content of the cells in each concentration group was significantly lower than that in the blank control group at the same culture time (p0.01). With the increase of culture time, the change of MMP-9 content was not significant (p0.05) .DHA had significant inhibitory effect on the secretion of MMP-9 by CHMm cells in a dose-dependent manner. The results of cell matrix metalloproteinase inhibitor showed that there was no significant change in TIMP1 content in each concentration of DHA test group compared with the control group for the same culture time (p0.05). At the same concentration of DHA and increasing culture time, there was no significant change in TIMP1 content in each experimental group (p0.05) .6.The results showed that the same culture time, compared with the blank control group, had no significant change in the expression of EMT related factor mRNA. The mRNA expression of CDH1 was significantly up-regulated (P0.01), and the mRNA expression of SLUG,ZEB1,ZEB2,Twist,HMGA2,MMP-9 and VEGF was down-regulated (p0.01) in each concentration of DHA test group. Dihydroartemisinin can inhibit the proliferation of canine breast tumor cells and inhibit the invasion of canine breast tumor cells by regulating the expression of EMT related factors.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.292
，
本文编号：2200223
[Abstract]:Canine mammary neoplasms are a kind of tumor disease which seriously endangers the body's health. By malignant proliferation, metastasis and invasion to other organs, organ failure is caused. At present, most of the treatment for canine breast tumors are surgery, radiotherapy and chemotherapy, but the results are more likely to relapse or side effects. Dihydroartemisinin (Dihydroartemisinin,DHA), as a traditional Chinese medicine extract, has been widely recognized for its antimalarial efficacy. The effects of dihydroartemisinin on the invasiveness and EMT related factors of canine breast tumor cells were studied. Canine breast tumor cells with the same logarithmic growth state were treated with low (5 渭 M),) (10 渭 M),) high (20 渭 M) DHA for 72 h. The cell activity was detected by CCK-8 assay, the content of matrix metalloproteinase (MMP-9) and matrix metalloproteinase inhibitor 1 (TIMP-1) were detected by cell scratch assay and invasive Elisa. The expression of E-cadherin (CDH1), zinc finger binding protein (ZEB), zinc finger transcription factor (SLUG) and (EMT) related factors were detected by real-time fluorescence quantitative PCR. Using CCK-8 to detect the cytotoxicity, the same culture time, compared with the blank control group, lower, medium, high test group on the inhibitory effect of CHMm cells was very significant (p0.01). At the same concentration of DHA, the inhibitory effect of DHA on CHMm cells was significantly different between 48 h and 24 h (p0.01) .DHA was in a dose-dependent and time-dependent manner. The results of cell scratch test showed that the same culture time was lower than that of the blank control group, and the inhibitory effect of the high test group on the migration of CHMm cells was very significant (p0.01). At the same concentration of DHA, the effects of DHA on migration inhibition of CHMm cells were significantly different between 48 h and 24 h (p0.01) .DHA showed a significant dose-dependent effect on the inhibition of migration of CHMm cells in a dose-dependent manner. The results of cell invasion test showed that the number of infiltrating basement membrane stained cells in the high test group was significantly higher than that in the blank control group at the same culture time, which indicated that DHA had a significant inhibitory effect on the invasion of CHMm cells (P0.01). At the same concentration of DHA, the inhibitory effects of DHA on the invasion of CHMm cells were significantly different between 48 h and 24 h (p0.01) .DHA showed a dose-dependent effect on the inhibition of invasion of CHMm cells in a dose-dependent manner. The results of cell matrix metalloproteinase assay showed that the MMP-9 content of the cells in each concentration group was significantly lower than that in the blank control group at the same culture time (p0.01). With the increase of culture time, the change of MMP-9 content was not significant (p0.05) .DHA had significant inhibitory effect on the secretion of MMP-9 by CHMm cells in a dose-dependent manner. The results of cell matrix metalloproteinase inhibitor showed that there was no significant change in TIMP1 content in each concentration of DHA test group compared with the control group for the same culture time (p0.05). At the same concentration of DHA and increasing culture time, there was no significant change in TIMP1 content in each experimental group (p0.05) .6.The results showed that the same culture time, compared with the blank control group, had no significant change in the expression of EMT related factor mRNA. The mRNA expression of CDH1 was significantly up-regulated (P0.01), and the mRNA expression of SLUG,ZEB1,ZEB2,Twist,HMGA2,MMP-9 and VEGF was down-regulated (p0.01) in each concentration of DHA test group. Dihydroartemisinin can inhibit the proliferation of canine breast tumor cells and inhibit the invasion of canine breast tumor cells by regulating the expression of EMT related factors.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.292
，
本文编号：2200223
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