CAJ | 학술논문

目的探讨活化血管内皮生长因子受体1(VEGFR-1)诱导肝癌细胞株MHCC97-H细胞上皮-间叶表型转化(EMT)的分子机制.方法将MHCC97-H细胞分为对照组(1％胎牛血清的DMEM培养)、PP2组(10 μmol/L PP2培养)、PBS组(10 μmol/L PBS培养)、VEGF-B组(50 μg/L的VEGF-B培养)、PP2+ VEGF-B组(10 μmol/L PP2和50 μg/L的VEGF-B培养)、PBS+ VEGF-B组(10 μmol/L PBS和50 μg/L VEGF-B培养).Westem blot法检测各组上皮标志物E-钙黏蛋白、α-连环蛋白和间叶标志物波形蛋白、N-钙黏蛋白的表达水平;细胞免疫荧光检测上述蛋白的表达部位;细胞侵袭和迁移试验检测各组MHCC 97-H细胞的侵袭和运动能力.组间比较采用t检验.结果对照组、PP2组、PBS组、VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞上皮标志物E-钙黏蛋白的表达分别为3.23±0.76、4.18±0.32、2.83 +0.65、2.06±0.15、6.12±0.08、1.36±0.54;α-连环蛋白的表达分别为3.01 +0.25、3.29+0.11、3.03±0.27、2.84+0.76、5.45±0.37、1.26±0.45;波形蛋白的表达分别为3.01±0.22、4.85±0.36、1.37±0.24、5.79±0.38、3.36±0.42、4.05±0.17;N-钙黏蛋白的表达分别为2.63±0.40、3.02±0.52、2.98±0.36、5.54±0.28、3.26±0.13、1.05±0.33.PP2组、PP2+ VEGF-B组的MHCC97-H细胞E-钙黏蛋白和α-连环蛋白表达明显上调,PP2+ VEGF-B组与VEGF-B组比较,差异有统计学意义(t=7.625,9.931,P＜0.05).PP2+ VEGF-B组的波形蛋白和N-钙黏蛋白的表达显著低于VEGF-B组(t=12.001,11.910,P＜0.05).VEGF-B处理6h后,VEGF-B组、PP2+ VEGF-B组、PBS+ VEGF-B组的MHCC97-H细胞迁移数量分别为19±l、5±2和16±1,VEGF-B组MHCC97-H细胞迁移数量显著多于PP2+ VEGF-B组(t=13.566,P＜0.05),PP2+ VEGF-B组中MHCC97-H细胞穿过Matrigel包被的改良侵袭小室的数量为4±2,显著少于VEGF-B组的16±l(t=12.350,P＜0.05).结论 VEGFR-1活化诱导MHCC97-H细胞发生EMT是由c-Src激酶信号转导通路介导的,c-Src作为该信号通路的关键因子之一可能是干预肝细胞癌侵袭和转移的有效靶点. 目的探討活化血管內皮生長因子受體1(VEGFR-1)誘導肝癌細胞株MHCC97-H細胞上皮-間葉錶型轉化(EMT)的分子機製.方法將MHCC97-H細胞分為對照組(1％胎牛血清的DMEM培養)、PP2組(10 μmol/L PP2培養)、PBS組(10 μmol/L PBS培養)、VEGF-B組(50 μg/L的VEGF-B培養)、PP2+ VEGF-B組(10 μmol/L PP2和50 μg/L的VEGF-B培養)、PBS+ VEGF-B組(10 μmol/L PBS和50 μg/L VEGF-B培養).Westem blot法檢測各組上皮標誌物E-鈣黏蛋白、α-連環蛋白和間葉標誌物波形蛋白、N-鈣黏蛋白的錶達水平;細胞免疫熒光檢測上述蛋白的錶達部位;細胞侵襲和遷移試驗檢測各組MHCC 97-H細胞的侵襲和運動能力.組間比較採用t檢驗.結果對照組、PP2組、PBS組、VEGF-B組、PP2+ VEGF-B組、PBS+ VEGF-B組的MHCC97-H細胞上皮標誌物E-鈣黏蛋白的錶達分彆為3.23±0.76、4.18±0.32、2.83 +0.65、2.06±0.15、6.12±0.08、1.36±0.54;α-連環蛋白的錶達分彆為3.01 +0.25、3.29+0.11、3.03±0.27、2.84+0.76、5.45±0.37、1.26±0.45;波形蛋白的錶達分彆為3.01±0.22、4.85±0.36、1.37±0.24、5.79±0.38、3.36±0.42、4.05±0.17;N-鈣黏蛋白的錶達分彆為2.63±0.40、3.02±0.52、2.98±0.36、5.54±0.28、3.26±0.13、1.05±0.33.PP2組、PP2+ VEGF-B組的MHCC97-H細胞E-鈣黏蛋白和α-連環蛋白錶達明顯上調,PP2+ VEGF-B組與VEGF-B組比較,差異有統計學意義(t=7.625,9.931,P＜0.05).PP2+ VEGF-B組的波形蛋白和N-鈣黏蛋白的錶達顯著低于VEGF-B組(t=12.001,11.910,P＜0.05).VEGF-B處理6h後,VEGF-B組、PP2+ VEGF-B組、PBS+ VEGF-B組的MHCC97-H細胞遷移數量分彆為19±l、5±2和16±1,VEGF-B組MHCC97-H細胞遷移數量顯著多于PP2+ VEGF-B組(t=13.566,P＜0.05),PP2+ VEGF-B組中MHCC97-H細胞穿過Matrigel包被的改良侵襲小室的數量為4±2,顯著少于VEGF-B組的16±l(t=12.350,P＜0.05).結論 VEGFR-1活化誘導MHCC97-H細胞髮生EMT是由c-Src激酶信號轉導通路介導的,c-Src作為該信號通路的關鍵因子之一可能是榦預肝細胞癌侵襲和轉移的有效靶點.
목적 탐토활화혈관내피생장인자수체1(VEGFR-1)유도간암세포주MHCC97-H세포상피-간협표형전화(EMT)적분자궤제.방법 장MHCC97-H세포분위대조조(1％태우혈청적DMEM배양)、PP2조(10 μmol/L PP2배양)、PBS조(10 μmol/L PBS배양)、VEGF-B조(50 μg/L적VEGF-B배양)、PP2+ VEGF-B조(10 μmol/L PP2화50 μg/L적VEGF-B배양)、PBS+ VEGF-B조(10 μmol/L PBS화50 μg/L VEGF-B배양).Westem blot법검측각조상피표지물E-개점단백、α-련배단백화간협표지물파형단백、N-개점단백적표체수평;세포면역형광검측상술단백적표체부위;세포침습화천이시험검측각조MHCC 97-H세포적침습화운동능력.조간비교채용t검험.결과 대조조、PP2조、PBS조、VEGF-B조、PP2+ VEGF-B조、PBS+ VEGF-B조적MHCC97-H세포상피표지물E-개점단백적표체분별위3.23±0.76、4.18±0.32、2.83 +0.65、2.06±0.15、6.12±0.08、1.36±0.54;α-련배단백적표체분별위3.01 +0.25、3.29+0.11、3.03±0.27、2.84+0.76、5.45±0.37、1.26±0.45;파형단백적표체분별위3.01±0.22、4.85±0.36、1.37±0.24、5.79±0.38、3.36±0.42、4.05±0.17;N-개점단백적표체분별위2.63±0.40、3.02±0.52、2.98±0.36、5.54±0.28、3.26±0.13、1.05±0.33.PP2조、PP2+ VEGF-B조적MHCC97-H세포E-개점단백화α-련배단백표체명현상조,PP2+ VEGF-B조여VEGF-B조비교,차이유통계학의의(t=7.625,9.931,P＜0.05).PP2+ VEGF-B조적파형단백화N-개점단백적표체현저저우VEGF-B조(t=12.001,11.910,P＜0.05).VEGF-B처리6h후,VEGF-B조、PP2+ VEGF-B조、PBS+ VEGF-B조적MHCC97-H세포천이수량분별위19±l、5±2화16±1,VEGF-B조MHCC97-H세포천이수량현저다우PP2+ VEGF-B조(t=13.566,P＜0.05),PP2+ VEGF-B조중MHCC97-H세포천과Matrigel포피적개량침습소실적수량위4±2,현저소우VEGF-B조적16±l(t=12.350,P＜0.05).결론 VEGFR-1활화유도MHCC97-H세포발생EMT시유c-Src격매신호전도통로개도적,c-Src작위해신호통로적관건인자지일가능시간예간세포암침습화전이적유효파점.
Objective To investigate the molecular mechanism of epithelial-mesenchymal transition (EMT) induced by activated vascular endothelial growth factor receptor-1 ( VEGFR-I ) in cell line MHCC97-H.Methods MHCC97-H cells were cultured in DMEM with 1％ fetal bovine serum (control group),10 μmol/L PP2 (PP2 group),10 μmol/L PBS (PBS group),50 μmol/L VEGF-B (VEGF-B group),l0μmol/L PP2 and 50 μmol/L VEGF-B (PP2 +VEGF group),10 μmol/L PBS and 50 μmol/L VEGF-B (PBS + VEGF-B group),respectively.Protein expressions of epithelial marker E-cadherin,α-catenin and mesenchymal marker vimentin and N-cadherin were detected by Western blot.The expression sites of E-cadherin,α-catenin and mesenchymal marker vimentin and N-cadherin were detected by cell immunofluorescence.The ability of invasion and migration of cell line MHCC97-H were assessed by cell invasion and migration test.All data were analyzed by the t test.Results The expressions of E-cadherin,α-catenin,vimentin and N-cadherin were 3.23 +0.76,3.01 ±0.25,3.01 +0.22 and 2.63 +0.40 in the control group,4.18 +0.32,3.29 +0.11,4.85 +0.36 and 3.02 +0.52 in the PP2 group,2.83 +0.65,3.03 +0.27,1.37 ±0.24 and 2.98 ±0.36 in the PBS group,2.06 ±0.15,2.84 ±0.76,5.79 ± 0.38 and 5.54 ± 0.28 in the VEGF-B group,6.12 ± 0.08,5.45 ± 0.37,3.36 ± 0.42 and 3.26 ±0.13 in the PP2 + VEGF-B group and 1.36 ±0.54,1.26 ±0.45,4.05 ±0.17 and 1.05 ±0.33 in the PBS +VEGF-B group.There was a significant difference in the expressions of E-cadherin and α-catenin between the PP2 +VEGF-B group and the VEGF-B group (t =7.625,9.931,P ＜ 0.05 ).The expressions of vimentin and N-cadherin in the PP2 + VEGF-B group were significantly lower than those in the VEGF-B group (t =12.001,11.910,P ＜ 0.05).Six hours after the treatment with VEGF-B,the numbers of MHCC97-H migrated were 19 ± 1,5 ± 2and 16 ± 1 in the VEGF-B group,PP2 + VEGF-B group and PBS + VEGF-B group,respectively.The number of MHCC97-H cells migrated in the VEGF-B group was greater than that in the PP2 ± VEGF-B group ( t =13.566,P ＜ 0.05 ).The number of MHCC97-H cells passed through the Boyden chamber was 4 + 2,which was significantly less than 16 ± 1 of the VEGF-B group (t =12.350,P ＜0.05).Conclusion EMT induced by activated VEGFR-1 was mediated via c-Src kinase signal transduction in MHCC91-H cell line,and c-Src may be a potential target to interfere the invasion and migration of hepatic cancer cells.