目的 观察HGF/Met通路对肝癌细胞株MHCC97-H干细胞样表型的影响,探讨其可能的作用机制.方法 体外培养肝癌细胞株Huh7和MHCC97-H,采用细胞免疫荧光染色技术筛选高表达Met的肝癌细胞株MHCC97-H,将其分为4组:空白组(不做处理)、HGF刺激组(50 μg/L HGF)、抑制组(50 μg/LHGF+1 mol/L HGF抑制剂PHA665752)和表皮生长因子(EGF)/成纤维细胞生长因子(FGF)刺激组(50 μg/L HGF+ 20μg/L EGF+ 20 μg/L FGF).Western blot检测前3组MHCC97-H细胞中Met和磷酸化Met(p-Met)蛋白的表达.培养24 h后光镜下观察前3组细胞形态学变化.克隆球形成实验检测4组细胞克隆球形成能力.荧光实时定量PCR检测空白组和HGF刺激组不同时间点(2、4、8、16、24 h)MHCC97-H细胞中多能干细胞相关基因表达变化.计量资料采用x±s表示,多组间比较采用单因素方差分析和重复测量的方差分析,两两比较采用LSD-t检验.结果 细胞免疫荧光染色检测结果显示:肝癌细胞株MHCC97-H与Huh7细胞比较,前者高表达Met,将其作为后续研究对象.Western blot检测结果显示:空白组、HGF刺激组和抑制组细胞中Met蛋白的相对表达量分别为0.44±0.04、0.37 ±0.03和0.41 ±0.04,3组比较,差异无统计学意义(F=2.31,P＞0.05).而3组细胞中p-Met蛋白的相对表达量分别为0.020±0.010、0.070±0.020和0.010±0.000,3组比较,差异有统计学意义(F=34.11,P＜0.05),其中HGF刺激组p-Met蛋白的表达水平高于空白组和抑制组(t=3.87,5.20,P＜0.05).HGF刺激组MHCC97-H细胞形态呈现长梭状间质样改变,而抑制组和空白组细胞形态相似,呈上皮样.克隆球形成实验结果表明:空白组、HGF刺激组、抑制组和EGF/FGF刺激组克隆球数目分别为0、(35.0±6.3)个、(4.3±1.5)个和(54.3±2.5)个,4组比较,差异有统计学意义(F =511.28,P＜0.05),其中HGF刺激组克隆球数目多于空白组和抑制组(t=9.62,8.21,P＜0.05),而少于EGF/FGF刺激组(t=4.93,P＜0.05).实时定量PCR检测结果显示:空白组和HGF刺激组不同时间点(2、4、8、16、24 h)C-myc mRNA相对表达量分别为1.00±0.11、1.68±0.09、1.08 ±0.24、1.18 ±0.13、0.78 ±0.14、1.06±0.04; Klf4 mRNA分别为1.00±0.20、1.43 ±0.16、0.87±0.28、1.19 ±0.29、2.17 ±0.43、1.41 ±0.06;Oct4mRNA分别为1.00±0.12、0.54±0.12、0.69 ±0.19、1.08±0.12、1.62 ±0.30、0.97 ±0.11,空白组和HGF刺激组不同时间点上述各指标比较,差异均有统计学意义(F=13.64,8.52,13.56,P＜0.05);HGF刺激组2 h C-myc mRNA相对表达量达到峰值,与空白组比较,约升高了1.68倍(t=8.29,P＜0.05),HGF刺激组16 h Klf4 mRNA相对表达量达到峰值,与空白组比较,约升高了2.17倍(t=4.27,P＜0.05),HGF刺激组16 h Oct4 mRNA相对表达量达到峰值,与空白组比较,约升高了1.62倍(t=3.32,P＜0.05).结论 HGF/Met通路的活化能够诱导肝癌细胞MHCC97-H的干细胞样表型转化,其可能作用机制是通过增加多能干细胞相关基因的表达而实现的.
目的 觀察HGF/Met通路對肝癌細胞株MHCC97-H榦細胞樣錶型的影響,探討其可能的作用機製.方法 體外培養肝癌細胞株Huh7和MHCC97-H,採用細胞免疫熒光染色技術篩選高錶達Met的肝癌細胞株MHCC97-H,將其分為4組:空白組(不做處理)、HGF刺激組(50 μg/L HGF)、抑製組(50 μg/LHGF+1 mol/L HGF抑製劑PHA665752)和錶皮生長因子(EGF)/成纖維細胞生長因子(FGF)刺激組(50 μg/L HGF+ 20μg/L EGF+ 20 μg/L FGF).Western blot檢測前3組MHCC97-H細胞中Met和燐痠化Met(p-Met)蛋白的錶達.培養24 h後光鏡下觀察前3組細胞形態學變化.剋隆毬形成實驗檢測4組細胞剋隆毬形成能力.熒光實時定量PCR檢測空白組和HGF刺激組不同時間點(2、4、8、16、24 h)MHCC97-H細胞中多能榦細胞相關基因錶達變化.計量資料採用x±s錶示,多組間比較採用單因素方差分析和重複測量的方差分析,兩兩比較採用LSD-t檢驗.結果 細胞免疫熒光染色檢測結果顯示:肝癌細胞株MHCC97-H與Huh7細胞比較,前者高錶達Met,將其作為後續研究對象.Western blot檢測結果顯示:空白組、HGF刺激組和抑製組細胞中Met蛋白的相對錶達量分彆為0.44±0.04、0.37 ±0.03和0.41 ±0.04,3組比較,差異無統計學意義(F=2.31,P＞0.05).而3組細胞中p-Met蛋白的相對錶達量分彆為0.020±0.010、0.070±0.020和0.010±0.000,3組比較,差異有統計學意義(F=34.11,P＜0.05),其中HGF刺激組p-Met蛋白的錶達水平高于空白組和抑製組(t=3.87,5.20,P＜0.05).HGF刺激組MHCC97-H細胞形態呈現長梭狀間質樣改變,而抑製組和空白組細胞形態相似,呈上皮樣.剋隆毬形成實驗結果錶明:空白組、HGF刺激組、抑製組和EGF/FGF刺激組剋隆毬數目分彆為0、(35.0±6.3)箇、(4.3±1.5)箇和(54.3±2.5)箇,4組比較,差異有統計學意義(F =511.28,P＜0.05),其中HGF刺激組剋隆毬數目多于空白組和抑製組(t=9.62,8.21,P＜0.05),而少于EGF/FGF刺激組(t=4.93,P＜0.05).實時定量PCR檢測結果顯示:空白組和HGF刺激組不同時間點(2、4、8、16、24 h)C-myc mRNA相對錶達量分彆為1.00±0.11、1.68±0.09、1.08 ±0.24、1.18 ±0.13、0.78 ±0.14、1.06±0.04; Klf4 mRNA分彆為1.00±0.20、1.43 ±0.16、0.87±0.28、1.19 ±0.29、2.17 ±0.43、1.41 ±0.06;Oct4mRNA分彆為1.00±0.12、0.54±0.12、0.69 ±0.19、1.08±0.12、1.62 ±0.30、0.97 ±0.11,空白組和HGF刺激組不同時間點上述各指標比較,差異均有統計學意義(F=13.64,8.52,13.56,P＜0.05);HGF刺激組2 h C-myc mRNA相對錶達量達到峰值,與空白組比較,約升高瞭1.68倍(t=8.29,P＜0.05),HGF刺激組16 h Klf4 mRNA相對錶達量達到峰值,與空白組比較,約升高瞭2.17倍(t=4.27,P＜0.05),HGF刺激組16 h Oct4 mRNA相對錶達量達到峰值,與空白組比較,約升高瞭1.62倍(t=3.32,P＜0.05).結論 HGF/Met通路的活化能夠誘導肝癌細胞MHCC97-H的榦細胞樣錶型轉化,其可能作用機製是通過增加多能榦細胞相關基因的錶達而實現的.
목적 관찰HGF/Met통로대간암세포주MHCC97-H간세포양표형적영향,탐토기가능적작용궤제.방법 체외배양간암세포주Huh7화MHCC97-H,채용세포면역형광염색기술사선고표체Met적간암세포주MHCC97-H,장기분위4조:공백조(불주처리)、HGF자격조(50 μg/L HGF)、억제조(50 μg/LHGF+1 mol/L HGF억제제PHA665752)화표피생장인자(EGF)/성섬유세포생장인자(FGF)자격조(50 μg/L HGF+ 20μg/L EGF+ 20 μg/L FGF).Western blot검측전3조MHCC97-H세포중Met화린산화Met(p-Met)단백적표체.배양24 h후광경하관찰전3조세포형태학변화.극륭구형성실험검측4조세포극륭구형성능력.형광실시정량PCR검측공백조화HGF자격조불동시간점(2、4、8、16、24 h)MHCC97-H세포중다능간세포상관기인표체변화.계량자료채용x±s표시,다조간비교채용단인소방차분석화중복측량적방차분석,량량비교채용LSD-t검험.결과 세포면역형광염색검측결과현시:간암세포주MHCC97-H여Huh7세포비교,전자고표체Met,장기작위후속연구대상.Western blot검측결과현시:공백조、HGF자격조화억제조세포중Met단백적상대표체량분별위0.44±0.04、0.37 ±0.03화0.41 ±0.04,3조비교,차이무통계학의의(F=2.31,P＞0.05).이3조세포중p-Met단백적상대표체량분별위0.020±0.010、0.070±0.020화0.010±0.000,3조비교,차이유통계학의의(F=34.11,P＜0.05),기중HGF자격조p-Met단백적표체수평고우공백조화억제조(t=3.87,5.20,P＜0.05).HGF자격조MHCC97-H세포형태정현장사상간질양개변,이억제조화공백조세포형태상사,정상피양.극륭구형성실험결과표명:공백조、HGF자격조、억제조화EGF/FGF자격조극륭구수목분별위0、(35.0±6.3)개、(4.3±1.5)개화(54.3±2.5)개,4조비교,차이유통계학의의(F =511.28,P＜0.05),기중HGF자격조극륭구수목다우공백조화억제조(t=9.62,8.21,P＜0.05),이소우EGF/FGF자격조(t=4.93,P＜0.05).실시정량PCR검측결과현시:공백조화HGF자격조불동시간점(2、4、8、16、24 h)C-myc mRNA상대표체량분별위1.00±0.11、1.68±0.09、1.08 ±0.24、1.18 ±0.13、0.78 ±0.14、1.06±0.04; Klf4 mRNA분별위1.00±0.20、1.43 ±0.16、0.87±0.28、1.19 ±0.29、2.17 ±0.43、1.41 ±0.06;Oct4mRNA분별위1.00±0.12、0.54±0.12、0.69 ±0.19、1.08±0.12、1.62 ±0.30、0.97 ±0.11,공백조화HGF자격조불동시간점상술각지표비교,차이균유통계학의의(F=13.64,8.52,13.56,P＜0.05);HGF자격조2 h C-myc mRNA상대표체량체도봉치,여공백조비교,약승고료1.68배(t=8.29,P＜0.05),HGF자격조16 h Klf4 mRNA상대표체량체도봉치,여공백조비교,약승고료2.17배(t=4.27,P＜0.05),HGF자격조16 h Oct4 mRNA상대표체량체도봉치,여공백조비교,약승고료1.62배(t=3.32,P＜0.05).결론 HGF/Met통로적활화능구유도간암세포MHCC97-H적간세포양표형전화,기가능작용궤제시통과증가다능간세포상관기인적표체이실현적.
Objective To investigate the effects of HGF/Met signaling pathway on stem-like phenotype of hepatocellular carcinoma cell line MHCC97-H.Methods The hepatocellular carcinoma cell lines Huh7 and MHCC97-H were cultured in vitro.Cell lines with high expression of Met were selected and divided into the blank group (cells untreated),HGF stimulation group (cells treated with 50 μg/L of HGF),HGF inhibition group (cells treated with 50 μg/L of HGF + 1 mol/L of PHA665752) and epidermal growth factor/fibroblast growth factor (EGF/FGF) stimulation group (cells treated with 50 μg/L of HGF + 20 μg/L of EGF + 20 μg/L of FGF).The protein expressions of Met and phospho-Met (p-Met) in the blank group,the HGF stimulation group and the HGF inhibition group were detected by Western blot.The cell morphological alteration of the blank group,the HGF stimulation group and the HGF inhibition group was observed under the light microscope at 24 hours after the treatment.Different sphere-formation ability was detected by sphere-formation assay under serum-free condition.The expression change of induced pluripotent stem cells related genes was analyzed by Real-Time polymerase chain reaction when MHCC97-H cells had been treated with HGF for different hours (2,4,8,16,24 hours).The measurement data were presented as.x ± s.All data were analyzed using the one-way analysis of variance,repeated measurement and LSD-t test.Results The results of immunofluorescence showed that MHCC97-H cells possessed high expression of Met when compared with Huh7 cells.In the blank group,HGF stimulation group and the HGF inhibition group,the protein expressions of Met were 0.44 ± 0.04,0.37 ± 0.03,0.41 ± 0.04,with no significant difference between the 3 groups (F =2.31,P ＞ 0.05),and the protein expressions of p-Met in the 3 groups were 0.020 ±0.010,0.070 ± 0.020,0.010 ± 0.000,with significant difference between the 3 groups (F =34.11,P ＜ 0.05).The protein expression of p-Met in the HGF stimulation group was significantly higher than that in the blank group and the HGF inhibition group (t =3.87,5.20,P ＜ 0.05).MHCC97-H cells in the HGF stimulation group were partly spindle-shaped which were similar to mesenchymal phenotype when cells had been treated with HGF for 24 hours,and PHA665752 could prevent this morphological alteration.MHCC97-H cells in the HGF inhibition group were like epithelium cells,which were similar to those in the blank group.The numbers of the spheres of the blank group,HGF stimulation group,HGF inhibition group and the EGF/FGF stimulation group were 0,35.0 ± 6.3,4.3 ± 1.5 and 54.3 ± 2.5,with significant difference between the 4 groups (F =511.28,P ＜ 0.05).The number of spheres of the HGF stimulation group was greater than that of the blank group and the HGF inhibition group (t =9.62,8.21,P ＜0.05),while lesser than that of the EGF/FGF stimulation group (t =4.93,P ＜ 0.05).In the blank group and the HGF stimulation groups (2,4,8,16,24 h),the expressions of C-myc mRNA were 1.00 ±0.11,1.68 ±0.09,1.08 ±0.24,1.18 ±0.13,0.78 ±0.14,1.06 ±0.04; the expressions of Klf4 mRNA were 1.00±0.20,1.43 ±0.16,0.87 ±0.28,1.19 ±0.29,2.17 ±0.43,1.41 ± 0.06; the expressions of Oct4 mRNA were 1.00 ±0.12,0.54 ±0.12,0.69 ±0.19,1.08 ±0.12,1.62 ±0.30,0.97 ± 0.1 1,with significant difference (F =13.64,8.52,13.56,P ＜ 0.05).The expression of C-myc mRNA in the HGF stimulation group (HGF stimulation for 2 hours) was 1.68 times higher than that of the blank group (t =8.29,P＜0.05).The expression of K1f4 mRNA of the HGF stimulation group (HGF stimulation for 16 hours) was 2.17 times higher than that of the blank group (t =4.27,P ＜ 0.05).The expression of Oct4 mRNA of the HGF stimulation group (HGF stimulation for 16 hours) was 1.62 times higher than that of the blank group (t =3.32,P ＜ 0.05).Conclusion HGF/Met signaling pathway might prompt the stem-like phenotype of MHCC97-H liver cancer cells through upregulation of pluripotent stem cells related genes.
