【摘要】：【目的】探討同源異型核基因1(Prrx1)誘導(dǎo)上皮-間質(zhì)轉(zhuǎn)化(EMT)促進(jìn)乳腺癌細(xì)胞對多西他賽(Docetaxel)耐藥的影響�！痉椒ā�1.用Real-time PCR方法從MCF-7、BT-549和MDA-MB-468三種乳腺癌細(xì)胞中篩選出Prrx1低表達(dá)乳腺癌細(xì)胞株。2.用定制的慢病毒載體感染篩選出的Prrx1低表達(dá)乳腺癌細(xì)胞株,Real-time PCR驗證感染成功后,分別于熒光顯微鏡的白光和熒光下觀察各組細(xì)胞形態(tài)變化。運(yùn)用Real-time PCR檢測波形蛋白與E-鈣黏蛋白的表達(dá)。3.用Transwell法檢測Prrx1過表達(dá)對MCF-7細(xì)胞遷移能力的影響。4.CCK-8檢測MCF-7細(xì)胞的IC50,并檢測Docetaxel作用于實驗組、空白對照組和陰性對照組24h后的IC50。5運(yùn)用流式細(xì)胞術(shù)檢測Docetaxel作用于三組細(xì)胞24h后,Prrx1過表達(dá)對乳腺癌MCF-7細(xì)胞凋亡率的影響。6.Western blot檢測三組細(xì)胞多藥耐藥相關(guān)蛋白1(ABCB1/p-gp)和Twist1的表達(dá)�！窘Y(jié)果】三種細(xì)胞中,Prrx1相對表達(dá)量最低的是MCF-7細(xì)胞(p(27)0.001)。用Prrx1過表達(dá)慢病毒感染MCF-7細(xì)胞后,細(xì)胞形態(tài)由“鋪路石樣”變成長梭形,并且熒光效率高達(dá)80%以上。Real-time PCR結(jié)果顯示Prrx1m RNA相對表達(dá)量是轉(zhuǎn)染前252.09倍(t=44.30,p(27)0.001),波形蛋白是轉(zhuǎn)染前2.65倍(t=12.05,p(27)0.001),E-鈣黏蛋白是轉(zhuǎn)染前0.64倍(t=4.91,p(27)0.001)。Transwell結(jié)果顯示Prrx1過表達(dá)的MCF-7細(xì)胞發(fā)生遷移的細(xì)胞數(shù)(71.69±6.66)明顯高于陰性對照組(40.87±4.16)(t=15.05,p(27)0.001)和空白對照組(41.2±4.62)(15.76,p(27)0.001),差異有統(tǒng)計學(xué)意義,而兩對照組之間比較差異無統(tǒng)計學(xué)意義(t=0.21,p=0.84)。CCK-8結(jié)果顯示Docetaxel作用MCF-7細(xì)胞24h和48h后的IC50值分別是(10.94±0.64)、(10.66±1.68),差異無統(tǒng)計學(xué)意義(t=0.27,p=0.80)。慢病毒感染后,Prrx1過表達(dá)的MCF-7細(xì)胞的IC50明顯高于空白對照組(13.30,p(27)0.001)和陰性對照組(t=0.91,p(27)0.001),且差異有統(tǒng)計學(xué)意義,而兩對照組之間比較差異無統(tǒng)計學(xué)意義(t=0.22,p=0.83)。流式細(xì)胞術(shù)結(jié)果顯示Docetaxel處理三組細(xì)胞24h后Prrx1過表達(dá)的MCF-7細(xì)胞的凋亡率明顯低于陰性對照組(t=9.32,p(27)0.001)和空白對照組(t=9.70,p(27)0.001),差異有統(tǒng)計學(xué)意義,而兩對照組之間比較差異無統(tǒng)計學(xué)意義(t=0.48,p=0.65)。Western blot結(jié)果顯示:Prrx1過表達(dá)MCF-7細(xì)胞的Twist1和多藥耐藥相關(guān)蛋白1(MDR1,ABCB1,P-gp)相對表達(dá)量明顯高于兩對照組,且差異有統(tǒng)計學(xué)意義(p(27)0.001),而兩對照組之間比較差異無統(tǒng)計學(xué)意義(p(29)0.05)�！窘Y(jié)論】Prrx1誘導(dǎo)EMT促進(jìn)乳腺癌細(xì)胞對Docetaxel耐藥。
[Abstract]:[objective] to investigate the effect of homologous nuclear gene 1 (Prrx1) induced epithelial-stroma transformation (EMT) on drug resistance of breast cancer cells to docetaxel (Docetaxel). [methods] 1. Prrx1 low expression breast cancer cell lines were screened out by Real-time PCR from three kinds of breast cancer cells, MCF-7,BT-549 and MDA-MB-468. Prrx1 low expression breast cancer cell lines were screened by custom lentivirus vector infection. After the infection was verified by Real-time PCR, the morphological changes of each group were observed under the white light and fluorescence of fluorescence microscope, respectively. The expression of vimentin and E-cadherin was detected by Real-time PCR. The effect of Prrx1 overexpression on the migration ability of MCF-7 cells was detected by Transwell assay. 4. IC50, of MCF-7 cells was detected by CCK-8 and Docetaxel was detected in the experimental group. IC50.5 in the blank control group and negative control group was detected by flow cytometry after 24 hours of Docetaxel treatment. The effect of Prrx1 overexpression on apoptosis rate of breast cancer MCF-7 cells. 6. Western blot detected the expression of multi-drug resistance associated protein 1 (ABCB1/p-gp) and Twist1 in three groups of cells. [results] among the three groups, the relative expression of Prrx1 was the lowest in MCF-7 cell line (p (27) 0.001). After MCF-7 cells were infected with lentivirus with Prrx1, the morphology of MCF-7 cells changed from "paving stone" to long fusiform, and the fluorescence efficiency was more than 80%. Real-time PCR results showed that the relative expression of Prrx1m RNA was 252.09 times (t 鈮，

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