雙氫青蒿素對犬乳腺腫瘤細胞侵襲性及EMT相關因子的影響研究
發(fā)布時間：2018-08-24 08:33
【摘要】：犬乳腺腫瘤是一種嚴重危害機體健康的腫瘤性疾病,通過惡性增殖、轉(zhuǎn)移侵襲至其他臟器導致器官衰竭。目前針對犬乳腺腫瘤的治療多為手術及放、化療,但結果多易復發(fā)或副作用較大。雙氫青蒿素(Dihydroartemisinin,DHA)作為中藥提取物,其抗瘧功效已經(jīng)得到了廣泛的認同,隨著研究的深入,其抗腫瘤的效果也逐步顯現(xiàn)。本試驗研究了雙氫青蒿素對犬乳腺腫瘤細胞侵襲性及EMT相關因子是否具有影響及其影響機制。取對數(shù)增長期狀態(tài)相同的犬乳腺腫瘤細胞CHMm,使用低(5μM)、中(10μM)、高(20μM)濃度DHA處理并分別培養(yǎng)24、48、72h。收集各組細胞使用CCK-8法檢測細胞活性;細胞劃痕試驗檢測細胞遷移性;transwell檢測細胞侵襲性;ELISA檢測細胞基質(zhì)金屬蛋白酶(MMP-9)含量及基質(zhì)金屬蛋白酶抑制劑1(TIMP-1)含量;實時熒光定量PCR方法檢測細胞E鈣粘蛋白(CDH1)、鋅指結合蛋白(ZEB)、鋅指轉(zhuǎn)錄因子(SLUG)等細胞上皮間質(zhì)轉(zhuǎn)化(EMT)相關因子表達量變化。1.使用CCK-8檢測細胞毒性發(fā)現(xiàn),相同培養(yǎng)時間,與空白對照組相比較,低、中、高試驗組對CHMm細胞抑制性影響效果極顯著(p0.01)。相同DHA作用濃度,培養(yǎng)48h、72h與培養(yǎng)24h相比較DHA對細胞抑制性影響差異極顯著(p0.01)。DHA對CHMm細胞抑制率呈明顯藥物時間、濃度依賴性。2.細胞劃痕試驗檢測細胞遷移速率結果表明,相同培養(yǎng)時間,與空白對照組相比較低、中、高試驗組對CHMm細胞遷移性抑制作用效果極顯著(p0.01)。相同DHA作用濃度,培養(yǎng)48h、72h與培養(yǎng)24h相比較DHA對細胞遷移性抑制影響差異極顯著(p0.01)。DHA對CHMm細胞遷移性抑制呈明顯的藥物時間、濃度依賴性。3.細胞侵襲試驗檢測細胞侵襲性結果表明,相同培養(yǎng)時間,與空白對照組相比較低、中、高試驗組穿透基底膜著色細胞個數(shù)顯著增多,說明DHA對CHMm細胞侵襲性抑制效果極顯著(P0.01)。相同DHA作用濃度,培養(yǎng)48h、72h與培養(yǎng)24h相比較DHA對細胞侵襲性抑制影響差異極顯著(p0.01)。DHA對CHMm細胞侵襲性抑制呈明顯的藥物時間、濃度依賴性。4.細胞基質(zhì)金屬蛋白酶檢測結果表明,相同培養(yǎng)時間,與空白對照組相比較各濃度試驗組細胞MMP-9含量降低極顯著(p0.01)。相同作用濃度,隨培養(yǎng)時間的增加細胞MMP-9含量變化差異不顯著(p0.05)。DHA對CHMm細胞分泌MMP-9具有顯著抑制性,呈明顯藥物濃度依賴性。5.細胞基質(zhì)金屬蛋白酶抑制劑結果表明,相同培養(yǎng)時間,與空白對照組相比各濃度DHA試驗組TIMP1含量沒有顯著變化(p0.05)。相同DHA作用濃度,增加培養(yǎng)時間,各試驗組TIMP1含量也沒有顯著變化(p0.05)。6.EMT相關因子mRNA表達結果表明,相同培養(yǎng)時間,與空白對照組相比,各濃度DHA試驗組目的基因CDH1的mRNA表達量上調(diào)極顯著(P0.01),目的基因SLUG、ZEB1、ZEB2、Twist、HMGA2、MMP-9 及 VEGF 的 mRNA 表達量下調(diào)極顯著(p0.01)。雙氫青蒿素對犬乳腺腫瘤細胞具有增殖抑制作用,并通過對EMT相關因子表達的調(diào)控抑制細胞的侵襲性。
[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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.292
，
本文編號：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.
【學位授予單位】：沈陽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S858.292
，
本文編號：2200223
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