中华消化杂志
中華消化雜誌
중화소화잡지
Chinese Journal of Digestion
2012年
4期
256-261
,共6页
李俊峰%陈莲香%吕霞%舒建昌
李俊峰%陳蓮香%呂霞%舒建昌
리준봉%진련향%려하%서건창
神经组织蛋白质类%受体,神经生长因子类%肝%细胞分裂%细胞凋亡%细胞,培养的%基因表达
神經組織蛋白質類%受體,神經生長因子類%肝%細胞分裂%細胞凋亡%細胞,培養的%基因錶達
신경조직단백질류%수체,신경생장인자류%간%세포분렬%세포조망%세포,배양적%기인표체
Nerve tissue proteins%Receptors,nerve growth factor%Liver%Cell division%Apoptosis%Cells,cultured%Gene expression
目的 观察神经生长因子(NGF)及受体(TrkANGFR、P75NTR)在肝细胞中的表达,探讨外源性p75NTR蛋白对肝细胞的生物学作用.方法 体外培养L02肝细胞,免疫细胞化学和荧光定量PCR法分别检测NGF、TrkANGFR、P75NTR在L02细胞中的表达.XTT法检测外源性P75NTR、NGF、NGF－ P75NTR、抗TrkANGFR、抗p75NTR对L02细胞增殖的作用.流式细胞术(膜联蛋白V/碘化丙啶)检测外源性P75NTR对L02细胞凋亡的作用.流式细胞术(碘化丙啶)检测外源性p75NTR对L02细胞周期的影响.结果 P75NTR促进L02细胞增殖,呈剂量依赖性.NGF和NGF+ P75NTR组吸光度值(A)值分别为0.4916±0.0565和0.5839±0.0733,与阴性对照组比较差异有统计学意义(0.3601±0.0310,P＜0.05)；抗TrkANGFRA值为0.2689±0.0229,与阴性对照组比较差异有统计学意义(P=0.003)；抗P75 NTRA值为0.3524±0.0312,与阴性对照组比较差异无统计学意义(P=1.000).外源性P75NTR对L02细胞有抗凋亡趋势,加入100 ng/ml P75NTR抗凋亡作用最强,表达量为3.70±0.26,但与对照组比较差异无统计学意义(4.10±0.62,P=1.000).P75NTR作用于L02细胞周期的S期,呈剂量依赖性,呈倒置的U形曲线,浓度为100 ng/ml时作用最强(25.60±0.40),与对照组比较差异有统计学意义(20.10±1.00,P=0.000).外源性NGF、P75NTR、NGF+ P75NTR上调了L02细胞NGFmRNA、TrkANGFR mRNA、P75NTRmRNA的基因表达,与对照组比较差异有统计学意义(P＜0.05)；抗TrkANGFR、抗P75 NTR对NGFmRNA、TrkANGFR mRNA、P75NTR mRNA的基因表达无明显影响,与对照组比较差异无统计学意义(P＞0.05).结论 L02细胞表达NGF及受体TrkANGFR、p75NTR,合适剂量的外源性P75NTR可能通过TrkANGFR/P75NTR异源二聚体信号途径促进L02细胞增殖.
目的 觀察神經生長因子(NGF)及受體(TrkANGFR、P75NTR)在肝細胞中的錶達,探討外源性p75NTR蛋白對肝細胞的生物學作用.方法 體外培養L02肝細胞,免疫細胞化學和熒光定量PCR法分彆檢測NGF、TrkANGFR、P75NTR在L02細胞中的錶達.XTT法檢測外源性P75NTR、NGF、NGF－ P75NTR、抗TrkANGFR、抗p75NTR對L02細胞增殖的作用.流式細胞術(膜聯蛋白V/碘化丙啶)檢測外源性P75NTR對L02細胞凋亡的作用.流式細胞術(碘化丙啶)檢測外源性p75NTR對L02細胞週期的影響.結果 P75NTR促進L02細胞增殖,呈劑量依賴性.NGF和NGF+ P75NTR組吸光度值(A)值分彆為0.4916±0.0565和0.5839±0.0733,與陰性對照組比較差異有統計學意義(0.3601±0.0310,P＜0.05)；抗TrkANGFRA值為0.2689±0.0229,與陰性對照組比較差異有統計學意義(P=0.003)；抗P75 NTRA值為0.3524±0.0312,與陰性對照組比較差異無統計學意義(P=1.000).外源性P75NTR對L02細胞有抗凋亡趨勢,加入100 ng/ml P75NTR抗凋亡作用最彊,錶達量為3.70±0.26,但與對照組比較差異無統計學意義(4.10±0.62,P=1.000).P75NTR作用于L02細胞週期的S期,呈劑量依賴性,呈倒置的U形麯線,濃度為100 ng/ml時作用最彊(25.60±0.40),與對照組比較差異有統計學意義(20.10±1.00,P=0.000).外源性NGF、P75NTR、NGF+ P75NTR上調瞭L02細胞NGFmRNA、TrkANGFR mRNA、P75NTRmRNA的基因錶達,與對照組比較差異有統計學意義(P＜0.05)；抗TrkANGFR、抗P75 NTR對NGFmRNA、TrkANGFR mRNA、P75NTR mRNA的基因錶達無明顯影響,與對照組比較差異無統計學意義(P＞0.05).結論 L02細胞錶達NGF及受體TrkANGFR、p75NTR,閤適劑量的外源性P75NTR可能通過TrkANGFR/P75NTR異源二聚體信號途徑促進L02細胞增殖.
목적 관찰신경생장인자(NGF)급수체(TrkANGFR、P75NTR)재간세포중적표체,탐토외원성p75NTR단백대간세포적생물학작용.방법 체외배양L02간세포,면역세포화학화형광정량PCR법분별검측NGF、TrkANGFR、P75NTR재L02세포중적표체.XTT법검측외원성P75NTR、NGF、NGF－ P75NTR、항TrkANGFR、항p75NTR대L02세포증식적작용.류식세포술(막련단백V/전화병정)검측외원성P75NTR대L02세포조망적작용.류식세포술(전화병정)검측외원성p75NTR대L02세포주기적영향.결과 P75NTR촉진L02세포증식,정제량의뢰성.NGF화NGF+ P75NTR조흡광도치(A)치분별위0.4916±0.0565화0.5839±0.0733,여음성대조조비교차이유통계학의의(0.3601±0.0310,P＜0.05)；항TrkANGFRA치위0.2689±0.0229,여음성대조조비교차이유통계학의의(P=0.003)；항P75 NTRA치위0.3524±0.0312,여음성대조조비교차이무통계학의의(P=1.000).외원성P75NTR대L02세포유항조망추세,가입100 ng/ml P75NTR항조망작용최강,표체량위3.70±0.26,단여대조조비교차이무통계학의의(4.10±0.62,P=1.000).P75NTR작용우L02세포주기적S기,정제량의뢰성,정도치적U형곡선,농도위100 ng/ml시작용최강(25.60±0.40),여대조조비교차이유통계학의의(20.10±1.00,P=0.000).외원성NGF、P75NTR、NGF+ P75NTR상조료L02세포NGFmRNA、TrkANGFR mRNA、P75NTRmRNA적기인표체,여대조조비교차이유통계학의의(P＜0.05)；항TrkANGFR、항P75 NTR대NGFmRNA、TrkANGFR mRNA、P75NTR mRNA적기인표체무명현영향,여대조조비교차이무통계학의의(P＞0.05).결론 L02세포표체NGF급수체TrkANGFR、p75NTR,합괄제량적외원성P75NTR가능통과TrkANGFR/P75NTR이원이취체신호도경촉진L02세포증식.
Objective To observe the expression of nerve growth factor(NGF)and its receptors (TrkANGFRand P75NTR) in hepatocytes and to explore the biological effects of exogenous P75NTR protein on hepatocytes.Methods L02 hepatocytes were cultured in vitro.The expression of NGF,TrkANNGFR and P75NTR in L02 cells were assessed by immunocytochemistry and fluorescent quantitation polymerase chain reaction (PCR). The effects on L02 cell proliferation by exogenous P75NTR,NGF,NGF+ P75NTR,anti-TrkANGFR and anti-P75NTR were detected by XTT assay.The effect of exogenous P75NTR on L02 cell apoptosis was measured by flow cytometry (Annexin V/PI) and the effect of exogenous P75NTR on L02 cell cycle was determined by flow cytometry (PI).Results L02 cell proliferation was promoted by P75NTR and in dose-dependent manner.The A value of NGF group and NGF+ P75NTR group was 0.4916±0.0565 and 0.5839 ± 0.0733,respectively,and there was statistical significance compared with control group (0.3601 ± 0.0310,P＜0.05).The A value ot anti-TrkANGFR group was 0.2689±0.0229,and there was statistical significance compared with control group (P=0.003).The A value of anti P75NTR was 0.3524 ± 0.0312,and there was no statistical significance compared with control group (P=1.000). Exogenous P75NTR had anti-apoptosis effect on L02 cells,the antiapoptosis effect was strongest when 100 ng/ml P75NTR was added and the expression quantity was 3.70 ±0.26.However there was no statistical significant compared with control group (4.10 ± 0.62,P=1.000).P75NTR affected the cell cycle S phase of L02 cells and in dose-dependent manner,which was inverted U shaped curve.The effect was strongest when the concentration was 100 ng/ml (25.60 ±0.40) and there was statistical significance compared with control group (20.10 ±1.00,P=0.000).Exogenous NGF,P75NTR and NGF + P75NTR up regulated the gene expression of NGFmRNA,TrkANGFRmRNA and P75NTR mRNA in L02 cells and there was statistical significance compared with control group (P＜0.05).There was no significant difference in the gene expression of NGFmRNA,TrkANGFFR mRNA and P75NTR mRNA between anti-TrkANGFR,anti-P75NTR groUp and control group (P＞0.05).Conclusion NGF and its receptors TrkANGFR and P75NTR were expressed in L02 cells.The appropriate dose of exogenous P75NTP protein promoted L02 cells proliferation via TrkANGFR/P75NTR heterodimer signal pathway.
