【摘要】：目的：前期工作中我們首次篩選和鑒定出卵巢癌特異性結(jié)合肽（Ovariancancerspecific targeting peptide, OSTP），本實(shí)驗(yàn)進(jìn)一步研究OSTP聯(lián)合紫杉醇對(duì)卵巢癌A2780細(xì)胞生長(zhǎng)和凋亡的影響及其機(jī)制。將對(duì)探討和開發(fā)OSTP作為靶向化療增敏劑治療卵巢癌奠定實(shí)驗(yàn)基礎(chǔ)。 方法：體外培養(yǎng)卵巢癌A2780細(xì)胞，隨機(jī)分為溶劑對(duì)照組（1640和DMSO組）、OSTP組、紫杉醇組和OSTP聯(lián)合紫杉醇組；采用四甲基偶氮唑鹽（MTT法）檢測(cè)OSTP對(duì)A2780細(xì)胞生長(zhǎng)的影響及其IC50值；用Annexin-V-FITC和PI雙染法標(biāo)記A2780細(xì)胞，應(yīng)用流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡情況；通過(guò)磷酸化蛋白芯片檢測(cè)信號(hào)通路中蛋白的磷酸化水平變化情況；并用Western Blot實(shí)驗(yàn)進(jìn)一步研究信號(hào)通路中磷酸化蛋白的表達(dá)情況。 結(jié)果：MTT法檢測(cè)結(jié)果顯示：不同濃度(0.01、0.1、1、10、100umol/L)的OSTP作用于卵巢癌A2780細(xì)胞時(shí)，其抑制率分別是27.36%、32.68%、36.08%、40.77%、57.32%。與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.01)， OSTP的IC50值為78.18umol/L。應(yīng)用流式細(xì)胞技術(shù)檢測(cè)細(xì)胞凋亡發(fā)現(xiàn)：不同濃度的OSTP（20、40、80、160、320umol/L）對(duì)A2780細(xì)胞的凋亡率分別為7.88%、8.35%、8.73%、9.39%、10.68%，與對(duì)照組相比差異有統(tǒng)計(jì)學(xué)意義（P0.01），提示OSTP可誘導(dǎo)細(xì)胞凋亡，凋亡作用不是很強(qiáng)。OSTP和紫杉醇聯(lián)合應(yīng)用，即分別在OSTP80umol/L0h、3h、6h、12h后再加紫杉醇10umol/L作用于卵巢癌A2780細(xì)胞48h，結(jié)果發(fā)現(xiàn)細(xì)胞凋亡率分別是39.40%、53.09%、48.18%和45.62%，均高于OSTP和紫杉醇單獨(dú)用藥及兩者的凋亡率相加值，且差異有統(tǒng)計(jì)學(xué)意義(P0.001)，提示OSTP能增強(qiáng)紫杉醇誘導(dǎo)卵巢癌A2780細(xì)胞凋亡的作用。磷酸化蛋白芯片檢測(cè)OSTP聯(lián)合紫杉醇誘導(dǎo)卵巢癌A2780細(xì)胞凋亡中蛋白磷酸化水平的情況。共篩選出42個(gè)差異磷酸化位點(diǎn)，31個(gè)差異蛋白。其中有11個(gè)蛋白的磷酸化水平上調(diào)，23個(gè)蛋白的磷酸化水平下調(diào)。Western Blot進(jìn)一步研究了其中PI3K/Akt信號(hào)通路的兩個(gè)差異磷酸化蛋白(FoxO1/3/4、14-3-3zeta/delta)的表達(dá)情況，結(jié)果顯示，與對(duì)照組相比，，OSTP組、紫杉醇組FoxO1/3/4磷酸化蛋白表達(dá)水平降低（P0.01）；與OSTP組、紫杉醇組相比，聯(lián)合用藥組中的FoxO1/3/4磷酸化蛋白表達(dá)水平降低（P0.01），差異均具有統(tǒng)計(jì)學(xué)意義。與對(duì)照組相比，OSTP組、紫杉醇組14-3-3zeta/delta磷酸化蛋白表達(dá)水平降低（P0.01）；與OSTP組、紫杉醇組相比，聯(lián)合用藥組中的14-3-3zeta/delta磷酸化蛋白表達(dá)水平降低（P0.01）；差異均具有統(tǒng)計(jì)學(xué)意義。 結(jié)論： 1. OSTP能增強(qiáng)紫杉醇誘導(dǎo)卵巢癌A2780細(xì)胞凋亡的作用。 2. OSTP增強(qiáng)紫杉醇誘導(dǎo)卵巢癌A2780細(xì)胞凋亡作用，其機(jī)制可能與PI3K/Akt信號(hào)通路中的關(guān)鍵蛋白FoxO1/3/4和14-3-3zeta/delta磷酸化蛋白表達(dá)降低有關(guān)。
文內(nèi)圖片：
圖片說(shuō)明：OSTP作用于卵巢癌A2780細(xì)胞后的凋亡流式檢測(cè)圖
[Abstract]:Aim: in the previous work, we screened and identified the specific binding peptide (Ovariancancerspecific targeting peptide, OSTP), of ovarian cancer for the first time. The effect of OSTP combined with paclitaxel on the growth and apoptosis of ovarian cancer A2780 cells and its mechanism were further studied. It will lay an experimental foundation for the discussion and development of OSTP as a targeted chemotherapy sensitizer in the treatment of ovarian cancer. Methods: ovarian cancer A2780 cells were randomly divided into solvent control group (1640 and DMSO), OSTP group, taxol group and OSTP combined with paclitaxel group), the effect of OSTP on the growth of A2780 cells was detected by MTT assay, and the apoptosis of A2780 cells was detected by Annexin-V-FITC and PI double staining, and the apoptosis of A2780 cells was detected by flow cytometry. The phosphorylation level of protein in signal pathway was detected by phosphorylation protein chip, and the expression of phosphorylation protein in signal pathway was further studied by Western Blot assay. Results: the results of MTT assay showed that the inhibition rates of different concentrations of OSTP (0.01, 0.1, 1, 10100umol 鈮

本文編號(hào)：2510061