穩(wěn)定抑制PAK2蛋白表達(dá)的HUH—7細(xì)胞株的建立

樸蓮淑　王福光　于慶功等

[摘要] 目的 為研究細(xì)胞周期相關(guān)因子PAK2在原發(fā)性肝癌中的作用，擬用shRNA干擾技術(shù)建立穩(wěn)定抑制PAK2蛋白表達(dá)的HUH-7肝癌細(xì)胞系。 方法 用基因轉(zhuǎn)染技術(shù)將人PAK2的3個(gè)shRNA片段分別克隆至慢病毒載體pHBLV-U6-ZsGreen-Puro，包裝成病毒后利用脂質(zhì)體將載體病毒轉(zhuǎn)染至HUH-7細(xì)胞，利用G418篩選穩(wěn)定表達(dá)shRNA的細(xì)胞。利用Real-time PCR和Western blotting方法分別鑒定轉(zhuǎn)染細(xì)胞內(nèi)PAK2的RNA及蛋白表達(dá)水平。 結(jié)果 sh1-PAK2 HUH-7、sh2-PAK2 HUH-7和sh3-PAK2病毒載體均可轉(zhuǎn)染至HUH-7細(xì)胞系，且轉(zhuǎn)染效率較高（>80%）。Real time-PCR結(jié)果顯示，與對(duì)照組sh-cont比較，轉(zhuǎn)染sh1-PAK2、sh2-PAK2和sh3-PAK可明顯抑制HUH-7細(xì)胞內(nèi)PAK2-mRNA的表達(dá)，差異均有統(tǒng)計(jì)學(xué)意義（均P < 0.05），其中sh2-PAK2和sh3-PAK2的干擾效果尤為明顯，下調(diào)效率分別達(dá)68%和89%。Western blotting結(jié)果顯示，轉(zhuǎn)染sh3-PKA2細(xì)胞內(nèi)PAK2的蛋白表達(dá)量較對(duì)照組sh-cont明顯下調(diào)，其表達(dá)量為對(duì)照組sh-cont的14.4%。 結(jié)論 穩(wěn)定抑制PAK2基因表達(dá)的肝癌細(xì)胞系shPAK2 HUH-7的建立為PAK2在肝癌細(xì)胞中的作用機(jī)制研究奠定細(xì)胞基礎(chǔ)。

[關(guān)鍵詞] PAK2；原發(fā)性肝癌；RNA干擾；HUH-7

[中圖分類號(hào)] R735.7 [文獻(xiàn)標(biāo)識(shí)碼] A [文章編號(hào)] 1673-7210（2015）09（c）-0014-04

[Abstract] Objective To study the role of cell cycle related factor PAK2 in primary hepatic carcinoma， shRNA interference technology was used to establish HUH-7 cell lines that could inhibit PAK2 protein expression stably. Methods Three shRNA fragments of human PAK2 were cloned into lentiviral vector pHBLV-U6-ZsGreen-Puro respectively by gene transfection technique， after they were transfected to virus， the carrier virus was transfected into HUH-7 cells by lipidosome， and G418 was used to screen cells that could express the shRNA stably. Real-time PCR and Western blotting were used to identify the expression levels of RNA and protein of PAK2 in transfection cells. Results All virus vectors of sh1-PAK2 HUH-7， sh2-PAK2 HUH-7 and sh3-PAK2 could be transfected into HUH-7 cell lines， and the transfection efficiency was high （>80%）. The results of Real time-PCR showed that， compared with control group sh-cont， transfection sh1-PAK2， sh2-PAK2 and sh3-PAK could inhibit the expression of PAK2-mRNA in HUH-7 cells， the differences were all statistically significant （all P < 0.05）， among which， the interference effectiveness of sh2-PAK2 and sh3-PAK2 was especially obvious， the down-regulated efficiency reached to 68% and 89% respectively. The results of Western blotting showed that， the protein expression level of PAK2 in sh3-PKA2 cells was lower than that of control group sh-cont， the expression level of which was 14.4% of control group sh-cont. Conclusion The establishment of cell line shRNA-PAK2 HUH-7 that can inhibit PAK2 genetic expression stably provides a cell model for mechanism study of PAK2 in hepatocellular carcinoma cells.

[Key words] PAK2； Primary hepatic carcinoma； RNAi； HUH-7

p21激活激酶（p21-activated kinases，PAKs）是一類絲氨酸/ 蘇氨酸激酶，是小G蛋白R(shí)ho家族Rac和Cdc42重要的下游效應(yīng)分子，可分為2個(gè)亞類：PAK2屬于Ⅰ類范疇[1]。隨著研究的不斷深入，PAK2被證明作為Caspase酶的效應(yīng)底物，在凋亡的過程中被激活，發(fā)現(xiàn)PAK2可以被Caspase裂解從而顯示出催化活性[2]。近年來，有很多學(xué)者指出PAK2的高表達(dá)在乳腺癌、頭頸部腫瘤、胃癌等多種腫瘤中，對(duì)其發(fā)生、發(fā)展中起促細(xì)胞增殖、抗凋亡作用，且在放射治療的抵抗中起著重要作用，有望成為腫瘤靶向治療的新靶點(diǎn)[3-7]。