發(fā)布時(shí)間：2017-12-27 23:26

  本文關(guān)鍵詞：Dickkopf-1基因在人胰腺癌生長(zhǎng)侵襲中的作用機(jī)制探討　出處：《蚌埠醫(yī)學(xué)院》2015年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 胰腺癌 DKK-1 c-Myc cyclinD1 TGF-β1 RNAi 生長(zhǎng)侵襲

【摘要】：目的:1.檢測(cè)人胰腺癌細(xì)胞系PANC-1中DKK-1的表達(dá)情況,觀察DKK-1 siRNA靶向抑制PANC-1中DKK-1表達(dá)前后癌細(xì)胞生物學(xué)特性的變化;2.檢測(cè)DKK-1 siRNA干擾PANC-1細(xì)胞中DKK-1表達(dá)前后癌細(xì)胞c-Myc、cyclinD1及TGF-β1基因表達(dá)情況變化,并對(duì)DKK-1與c-Myc、cyclinD1及TGF-β1的相關(guān)性進(jìn)行分析,探討DKK-1基因在PANC-1細(xì)胞生長(zhǎng)侵襲作用中與c-Myc、cyclinD1及TGF-β1基因的關(guān)系。方法:1.設(shè)計(jì)合成3對(duì)DKK-1干擾片段(siDKK-1-1、siDKK-1-2、siDKK-1-3),應(yīng)用RT-PCR方法檢測(cè)siDKK-1-1組、siDKK-1-2組、siDKK-1-3組PANC-1細(xì)胞中DKK-1 mRNA的表達(dá),從siDKK-1-1、siDKK-1-2、siDKK-1-3中篩選出干擾效果最顯著的目標(biāo)片段;并從如下三個(gè)方面檢測(cè)干擾DKK-1表達(dá)后PANC-1細(xì)胞生物學(xué)特性的變化:MTT法檢測(cè)轉(zhuǎn)染目標(biāo)片段前后PANC-1細(xì)胞增殖能力變化,流式細(xì)胞儀檢測(cè)轉(zhuǎn)染目標(biāo)片段前后PANC-1細(xì)胞凋亡能力變化,劃痕實(shí)驗(yàn)檢測(cè)轉(zhuǎn)染目標(biāo)片段前后PANC-1細(xì)胞運(yùn)動(dòng)侵襲能力變化,通過(guò)比較三個(gè)指標(biāo)在各組之間的差異,進(jìn)一步探討抑制DKK-1表達(dá)對(duì)PANC-1細(xì)胞生物學(xué)特性的影響。2.應(yīng)用RT-PCR法檢測(cè)DKK-1 siRNA轉(zhuǎn)染PANC-1細(xì)胞前后細(xì)胞中DKK-1、c-Myc、cyclinD1及TGF-β1基因m RNA的表達(dá);Western-blot法檢測(cè)DKK-1 siRNA轉(zhuǎn)染PANC-1細(xì)胞前后細(xì)胞中DKK-1、c-Myc、cyclinD1及TGF-β1蛋白的表達(dá),利用統(tǒng)計(jì)軟件對(duì)轉(zhuǎn)染前后各基因表達(dá)的變化進(jìn)行相關(guān)性分析,進(jìn)一步探討DKK-1在促進(jìn)人胰腺癌生長(zhǎng)侵襲中與c-Myc、cyclinD1及TGF-β1的關(guān)系。結(jié)果:1.RT-PCR結(jié)果顯示轉(zhuǎn)染siDKK-1-1后PANC-1細(xì)胞內(nèi)DKK-1 mRNA表達(dá)降低最明顯。2.轉(zhuǎn)染si DKK-1-1后PANC-1細(xì)胞增殖能力下降、凋亡增加及遷移能力降低,以上實(shí)驗(yàn)結(jié)果與空白組及NC組比較,差異有統(tǒng)計(jì)學(xué)意義(P均0.05)。3.RT-PCR檢測(cè)結(jié)果顯示轉(zhuǎn)染siDKK-1-1后PANC-1細(xì)胞內(nèi)c-Myc、cyclinD1及TGF-β1 mRNA表達(dá)明顯降低(P均0.05);Western-blot檢測(cè)結(jié)果顯示轉(zhuǎn)染si DKK-1-1后PANC-1細(xì)胞內(nèi)c-Myc、cyclinD1及TGF-β1蛋白表達(dá)明顯降低(P均0.05)。對(duì)上述實(shí)驗(yàn)結(jié)果進(jìn)行相關(guān)性分析發(fā)現(xiàn)DKK-1、c-Myc、cyclinD1及TGF-β1表達(dá)兩兩之間均呈正相關(guān)。結(jié)論:1.靶向DKK-1 siRNA阻斷人胰腺癌細(xì)胞PANC-1中DKK-1的表達(dá),可有效抑制PANC-1細(xì)胞的增殖、侵襲能力,并誘導(dǎo)細(xì)胞凋亡。2.利用DKK-1 siRNA抑制胰腺癌細(xì)胞PANC-1中DKK-1的表達(dá),可使c-Myc、cyclinD1及TGF-β1表達(dá)下調(diào)。3.DKK-1通過(guò)影響TGF-β1的表達(dá)來(lái)調(diào)控下游效應(yīng)基因c-Myc、cyclinD1,從而促進(jìn)胰腺癌細(xì)胞的增殖與遷移,并抑制其凋亡。
[Abstract]:Objective: to detect expression of DKK-1 in 1. human pancreatic cancer cell line PANC-1, observe DKK-1 siRNA targeted inhibition of DKK-1 expression and biological characteristics of cancer cells in PANC-1; 2. DKK-1 siRNA interference in PANC-1 cells to detect expression of DKK-1, cyclinD1 and c-Myc before and after cancer cell TGF- beta 1 gene expression changes, and the correlation between DKK-1 and c-Myc, cyclinD1 and TGF- beta 1 were analyzed to investigate the relationship between growth and c-Myc, cyclinD1 and DKK-1 in the invasion TGF- beta 1 gene expression in PANC-1 cells. Methods: 1. design and synthesis of 3 DKK-1 interference fragment (siDKK-1-1, siDKK-1-2, siDKK-1-3), the expression of RT-PCR was used to detect the siDKK-1-1 group, siDKK-1-2 group, siDKK-1-3 group, PANC-1 cells DKK-1 mRNA, screened out the interference effect from siDKK-1-1, siDKK-1-2, siDKK-1-3 of the most significant target fragment; and from the biological characteristics of PANC-1 cell three the detection of interference DKK-1 expression changes after changes in proliferation ability of PANC-1 cells before and after transfection were detected by MTT method to detect the changes of target fragments, the apoptosis of PANC-1 cells before and after transfection ability of target fragments by flow cytometry, ability of invasion of PANC-1 cells before and after transfection of target motion fragment detection scratch test, by comparing the three indicators among the groups, further to investigate the inhibitory effect of DKK-1 expression on the biological characteristics of PANC-1 cell. Expression of DKK-1, c-Myc, cyclinD1 and TGF- beta 1 gene m RNA DKK-1 siRNA detected before and after transfection of PANC-1 cells 2. by RT-PCR; expression of DKK-1 in cells, c-Myc, cyclinD1 and TGF- beta 1 protein were detected before and after DKK-1 siRNA was transfected into PANC-1 cells by Western-blot method, to analyze the correlation between the changes of expression of each gene transfected by the statistical software, further explore the relationship of DKK-1 in invasion and promote the growth of human pancreatic cancer with c-Myc, cyclinD1 and TGF- beta 1. Results: the results of 1.RT-PCR showed that the expression of DKK-1 mRNA in PANC-1 cells decreased most obviously after siDKK-1-1 transfection. 2. after transfection of Si DKK-1-1, the proliferation ability of PANC-1 cells decreased, the apoptosis increased and the migration ability decreased. The difference between the above experimental results and the blank group and NC group was statistically significant (P 0.05). 3.RT-PCR detection showed that the expression of c-Myc, cyclinD1 and TGF- beta 1 mRNA in PANC-1 cells decreased significantly after transfection of siDKK-1-1 (P 0.05), and Western-blot test showed that the expression of PANC-1 and PANC-1 in the cells of the Si cells decreased significantly after transfection Si DKK-1-1 (all 0.05). The correlation analysis of the experimental results showed that there was a positive correlation between DKK-1, c-Myc, cyclinD1 and TGF- beta 1 expression 22. Conclusion: 1. targeting DKK-1 siRNA can inhibit the expression of DKK-1 in human pancreatic cancer cell PANC-1, which can effectively inhibit the proliferation and invasion ability of PANC-1 cells, and induce cell apoptosis. 2. the expression of DKK-1 in pancreatic cancer cell PANC-1 was inhibited by DKK-1 siRNA, and the expression of c-Myc, cyclinD1 and TGF- beta 1 was down regulated. 3.DKK-1 regulates the downstream effect gene c-Myc and cyclinD1 by affecting the expression of TGF- beta 1, which promotes the proliferation and migration of pancreatic cancer cells and inhibits its apoptosis.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R735.9

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 王雪劍;前列腺癌生物力學(xué)性質(zhì)的改變與其細(xì)胞惡性表型相關(guān)性的實(shí)驗(yàn)研究[D];大連醫(yī)科大學(xué);2014年

相關(guān)碩士學(xué)位論文 前1條

1 肖城生;前列腺癌中多重信號(hào)通路的表達(dá)檢測(cè)[D];中南大學(xué);2013年

，

本文編號(hào)：1343647