[關(guān)鍵詞]
[摘要]
目的 獲取溫郁金凝集素(CWL)重組蛋白,并研究其對結(jié)腸癌細(xì)胞凋亡、遷移和侵襲的影響。方法 根據(jù)CWL基因全長編碼序列,采用大腸桿菌原核表達(dá)系統(tǒng)重組表達(dá)及純化CWL;觀察CWL對兔紅細(xì)胞的凝血作用;CCK-8法評價CWL(10、20、40、80 μg·mL-1)對結(jié)腸癌HCT-116細(xì)胞的毒性;流式細(xì)胞儀檢測CWL(40、80 μg·mL-1)對HCT-116細(xì)胞凋亡的影響;劃痕實驗檢測細(xì)胞遷移能力;Transwell小室法檢測細(xì)胞侵襲能力;實時熒光定量PCR(qRT-PCR)實驗分析Caspase-3、Caspase-9、BAX、PARP mRNA表達(dá)水平; Western blotting檢測cleaved-Caspase-3、cleaved-Caspase-9、BAX、cleaved-PARP蛋白表達(dá)水平。結(jié)果 重組純化獲得相對分子質(zhì)量為3×104的CWL;高質(zhì)量濃度的CWL導(dǎo)致紅細(xì)胞出現(xiàn)明顯的凝血情況,隨著CWL質(zhì)量濃度的降低,其凝血活性逐漸下降,直至消失。與對照組比較,CWL對HCT-116細(xì)胞活性具有顯著抑制作用(P<0.001);40、80 μg·mL-1 CWL可顯著降低HCT-116細(xì)胞的遷移和侵襲能力(P<0.001),顯著升高HCT-116細(xì)胞凋亡率(P<0.01、0.001);顯著升高Caspase-3、Caspase-9、BAX、PARP的mRNA表達(dá)水平(P<0.05、0.01、0.001),同時顯著增加cleaved-Caspase-3、cleaved-Caspase-9、BAX、cleaved-PARP蛋白表達(dá)水平(P<0.05、0.01)。結(jié)論 CWL可通過線粒體凋亡途徑促進(jìn)HCT-116細(xì)胞凋亡并抑制其遷移和侵襲,從而發(fā)揮抗腫瘤作用。
[Key word]
[Abstract]
Objective Curcuma wenyujin lectin (CWL) recombinant protein was obtained, and its effect on the apoptosis, migration and invasion of colon cancer cells were invesitgated. Methods According to the coding sequence of CWL gene, CWL was heterogeneously expressed and purified using Escherichia coli prokaryotic expression system. CCK-8 method was used to evaluate the toxicity of CWL to colon cancer HCT-116 cells, apoptosis was detected by flow cytometry, and cells were detected by scratch test. Migration ability, cell invasion ability was assessed using transwell chamber method. the expression levels of genes in signal pathways such as Caspase-3, Caspase-9, BAX and PARP were performed using real-time fluorescence quantitative PCR (qRT-PCR) analysis. Western blotting was used to detect the expression levels of cleaved-Casase-3, cleaved-Casase-9, BAX, and cleaved-PARP proteins. Results Recombinant purification yielded CWL of relative molecular weight of 3×104. High quality concentration of CWL caused to significant coagulation of red blood cells, and as the concentration of CWL decreases, its coagulation activity gradually decreases until it disappears. Compared with control group, CWL had a significant inhibitory effect on the activity of HCT-116 cells (P < 0.001), CWL of 40, 80 μg·mL-1 could significantly reduce the migration and invasion ability of HCT-116 cells (P < 0.001), and significantly increased the apoptosis rate of HCT-116 cells (P < 0.01, 0.001). CWL of 40, 80 μg·mL-1 significantly increased mRNA expression levels of Caspase-3, Caspase-9, BAX, and PARP (P < 0.05, 0.01, 0.001), while significantly increased protein expression levels of cleaved- Caspase-3, cleaved-Caspase-9, BAX, and cleaved-PAP (P < 0.05, 0.01). Conclusion CWL can promote HCT-116 cell apoptosis through the mitochondrial apoptosis pathway, inhibit HCT-116 cell migration and invasion, thereby exerting anti-tumor effects and providing a new source of drugs for the development of clinical tumor drugs.
[中圖分類號]
R285.5
[基金項目]
安徽濟(jì)人藥業(yè)有限公司課題(KJHX2009);合肥未來藥物開發(fā)有限公司橫向課題(KJHX2008)