中华肝脏病杂志
中華肝髒病雜誌
중화간장병잡지
CHINESE JOURNAL OF HEPATOLOGY
2015年
6期
437-442
,共6页
李传飞%陈小凤%张文广%刘佳%吕琳%梅浙川
李傳飛%陳小鳳%張文廣%劉佳%呂琳%梅浙川
리전비%진소봉%장문엄%류가%려림%매절천
慢病毒属%水通道蛋白质9%细胞增殖%细胞凋亡
慢病毒屬%水通道蛋白質9%細胞增殖%細胞凋亡
만병독속%수통도단백질9%세포증식%세포조망
Lentivirus%Aquaporin 9%Cell proliferation%Apoptosis
目的 探讨水通道蛋白9(AQP9)对人肝癌细胞系HepG2增殖、凋亡和侵袭迁移的影响.方法 构建靶向AQP9基因的慢病毒过表达载体,转染HepG2细胞并用嘌呤霉素筛选出稳定细胞株,用激光共聚焦验证转染效率,用qRT-PCR及Western blot检测AQP9表达情况.实验分组:“CC组”为无慢病毒转染的HepG2细胞、“PWPI组”为空载体慢病毒转染的HepG2细胞,“过表达AQP9组”为含过表达AQP9基因慢病毒载体转染的HepG2细胞.利用平板克隆、划痕愈合实验、Transwell小室侵袭实验以及流式细胞仪分别检测AQP9对HepG2增殖、迁移、侵袭、凋亡以及周期的影响.多组间数据比较采用单因素方差分析,两组间数据比较采用独立样本t检验. 结果 激光共聚焦结果显示,HepG2转染AQP9后细胞膜高表达绿色荧光蛋白,qRT-PCR及Western blot检测AQP9的mRNA和蛋白质表达较其他2组均上升,差异均有统计学意义(P值均＜0.01).平板克隆结果显示,CC组、PWPI组和过表达AQP9组克隆形成率分别为23.53％±2.10％、23.00％±2.022％、16.93％±3.19％,组间比较,差异有统计学意义(F=6.46,P=0.032).流式测凋亡结果显示,CC组、PWPI组和过表达AQP9组总凋亡率分别为19.70％±2.49％、24.37％±2.38％、44.96％±3.53％,组间比较,差异有统计学意义(F=66.88,P＜0.01),与PWPI组和CC组比较,差异均有统计学意义(P值均＜0.01);流式测周期结果显示,CC组、PWPI组和过表达AQP9组细胞停留在S期和G1期的细胞百分比分别为34.39％±4.33％和54.58％±0.89％;35.65％±1.39％和53.08％±2.06％;15.25％±1.81％和66.58％±0.99％,组间比较,差异均有统计学意义(F值分别为49.25和82.09,P值均＜0.01).划痕愈合实验结果显示,过表达AQP9组细胞24h的迁移率为6.38％±1.70％,与PWPI组16.74％±1.40％比较,差异有统计学意义(t=8.133,P=0.01).Transwell实验结果显示,过表达AQP9组24h穿过细胞数为(17±8)个,与PWPI组(95±11)个和CC组(109±9)个比较,差异均有统计学意义(P值均＜ 0.05). 结论 AQP9能够抑制HepG2细胞迁移和侵袭能力,诱导细胞发生凋亡并通过产生G1/S期阻滞抑制细胞增殖.
目的 探討水通道蛋白9(AQP9)對人肝癌細胞繫HepG2增殖、凋亡和侵襲遷移的影響.方法 構建靶嚮AQP9基因的慢病毒過錶達載體,轉染HepG2細胞併用嘌呤黴素篩選齣穩定細胞株,用激光共聚焦驗證轉染效率,用qRT-PCR及Western blot檢測AQP9錶達情況.實驗分組:“CC組”為無慢病毒轉染的HepG2細胞、“PWPI組”為空載體慢病毒轉染的HepG2細胞,“過錶達AQP9組”為含過錶達AQP9基因慢病毒載體轉染的HepG2細胞.利用平闆剋隆、劃痕愈閤實驗、Transwell小室侵襲實驗以及流式細胞儀分彆檢測AQP9對HepG2增殖、遷移、侵襲、凋亡以及週期的影響.多組間數據比較採用單因素方差分析,兩組間數據比較採用獨立樣本t檢驗. 結果 激光共聚焦結果顯示,HepG2轉染AQP9後細胞膜高錶達綠色熒光蛋白,qRT-PCR及Western blot檢測AQP9的mRNA和蛋白質錶達較其他2組均上升,差異均有統計學意義(P值均＜0.01).平闆剋隆結果顯示,CC組、PWPI組和過錶達AQP9組剋隆形成率分彆為23.53％±2.10％、23.00％±2.022％、16.93％±3.19％,組間比較,差異有統計學意義(F=6.46,P=0.032).流式測凋亡結果顯示,CC組、PWPI組和過錶達AQP9組總凋亡率分彆為19.70％±2.49％、24.37％±2.38％、44.96％±3.53％,組間比較,差異有統計學意義(F=66.88,P＜0.01),與PWPI組和CC組比較,差異均有統計學意義(P值均＜0.01);流式測週期結果顯示,CC組、PWPI組和過錶達AQP9組細胞停留在S期和G1期的細胞百分比分彆為34.39％±4.33％和54.58％±0.89％;35.65％±1.39％和53.08％±2.06％;15.25％±1.81％和66.58％±0.99％,組間比較,差異均有統計學意義(F值分彆為49.25和82.09,P值均＜0.01).劃痕愈閤實驗結果顯示,過錶達AQP9組細胞24h的遷移率為6.38％±1.70％,與PWPI組16.74％±1.40％比較,差異有統計學意義(t=8.133,P=0.01).Transwell實驗結果顯示,過錶達AQP9組24h穿過細胞數為(17±8)箇,與PWPI組(95±11)箇和CC組(109±9)箇比較,差異均有統計學意義(P值均＜ 0.05). 結論 AQP9能夠抑製HepG2細胞遷移和侵襲能力,誘導細胞髮生凋亡併通過產生G1/S期阻滯抑製細胞增殖.
목적 탐토수통도단백9(AQP9)대인간암세포계HepG2증식、조망화침습천이적영향.방법 구건파향AQP9기인적만병독과표체재체,전염HepG2세포병용표령매소사선출은정세포주,용격광공취초험증전염효솔,용qRT-PCR급Western blot검측AQP9표체정황.실험분조:“CC조”위무만병독전염적HepG2세포、“PWPI조”위공재체만병독전염적HepG2세포,“과표체AQP9조”위함과표체AQP9기인만병독재체전염적HepG2세포.이용평판극륭、화흔유합실험、Transwell소실침습실험이급류식세포의분별검측AQP9대HepG2증식、천이、침습、조망이급주기적영향.다조간수거비교채용단인소방차분석,량조간수거비교채용독립양본t검험. 결과 격광공취초결과현시,HepG2전염AQP9후세포막고표체록색형광단백,qRT-PCR급Western blot검측AQP9적mRNA화단백질표체교기타2조균상승,차이균유통계학의의(P치균＜0.01).평판극륭결과현시,CC조、PWPI조화과표체AQP9조극륭형성솔분별위23.53％±2.10％、23.00％±2.022％、16.93％±3.19％,조간비교,차이유통계학의의(F=6.46,P=0.032).류식측조망결과현시,CC조、PWPI조화과표체AQP9조총조망솔분별위19.70％±2.49％、24.37％±2.38％、44.96％±3.53％,조간비교,차이유통계학의의(F=66.88,P＜0.01),여PWPI조화CC조비교,차이균유통계학의의(P치균＜0.01);류식측주기결과현시,CC조、PWPI조화과표체AQP9조세포정류재S기화G1기적세포백분비분별위34.39％±4.33％화54.58％±0.89％;35.65％±1.39％화53.08％±2.06％;15.25％±1.81％화66.58％±0.99％,조간비교,차이균유통계학의의(F치분별위49.25화82.09,P치균＜0.01).화흔유합실험결과현시,과표체AQP9조세포24h적천이솔위6.38％±1.70％,여PWPI조16.74％±1.40％비교,차이유통계학의의(t=8.133,P=0.01).Transwell실험결과현시,과표체AQP9조24h천과세포수위(17±8)개,여PWPI조(95±11)개화CC조(109±9)개비교,차이균유통계학의의(P치균＜ 0.05). 결론 AQP9능구억제HepG2세포천이화침습능력,유도세포발생조망병통과산생G1/S기조체억제세포증식.
Objective To investigate the impact of aquaporin 9 (AQP9) on the proliferation,apoptosis,invasiveness and migration of hepatocellular carcinoma cells using the HepG2 cell line.Methods A lentiviral vector targeting the coding region of human AQP9 was constructed.The recombinant lentiviral vector was harvested from the 293T cell line and transfected into the HepG2 cell line; resistant cell clones were selected with puromycin.Three groups of cells were established,including the CC group (control without lentiviral vector),the PWPI group (control with empty carrier virus),and the AQP9 overexpression group (experimental with the AQP9 recombinant virus).Transfection efficiency was validated by laser confocal microscopy.Expression of AQP9 was detected in the transfected HepG2 cells by westem blotting (protein) and real-time qPCR (mRNA).AQP9 effects on proliferation,migration,invasion and apoptosis of the HepG2 cell line were assessed by plate colony formation assay,woumd healing assay,transwell assay and flow cytometry.Results The green fluorescent protein of the recombinant lentiviral vector was appropriately distributed in the cell membrane.The AQP9 overexpression group showed significantly higher AQP9 mRNA and protein levels than the PWPI group and the CC group (both P ＜ 0.01).Cells with AQP9 overexpression showed a lower colony formation rate (16.93±3.19％ vs.CC group:23.53±2.10％ and PWPI group:23.00±2.02％; F=6.46,P =0.032) and a lower overall apoptosis rate (44.96±3.53％ vs.CC group:19.7±2.49％ and PWPI group:24.37±2.38％; F =66.88,P ＜ 0.01).The AQP9 overexpression group also showed significantly higher number of cells in the G1 stage and significantly lower number of cells in the S stage (G1:66.58±0.99％ and S:15.25±1.81％),significantly smaller cell migration distance (P =0.01 ＜ 0.05),and significantly suppressed invasiveness (17±8 vs.CC group:109±9 and PWPI group:95±11;P =0.01 ＜ 0.05).Conclusion In HepG2 cells,AQP9 significantly reduces the migrative and invasive capabilities,induces cell apoptosis,and inhibits cell proliferation via cell cycle arrest at the G1/S phases.
