肝窦内皮细胞的一氧化氮合成酶促进肝细胞增殖  

Endothelial nitric oxide synthase derived from hepatic sinusoidal endothelial cells promotes hepatocyte proliferation

作　　者：黄小龙[1] 杨青壮 韩霖[1] 范伟强[1] 呼增吉 符誉[1] 翁杰[1] 陈有科 尹秋实[1] 林师佈 杨彦[1] Huang Xiaolong;Yang Qingzhuang;Han Lin;Fan Weiqiang;Hu Zengji;Fu Yu;Weng Jie;Chen Youke;Yin Qiushi;Lin Shibu;Yang Yan(Department of Hepatobiliary Surgery,the First Affiliated Hospital of Hainan Medical College,Haikou 570102,China)

机构地区：[1]海南医学院附属第一医院肝胆外科,海口570102

出　　处：《中华实验外科杂志》2020年第10期1823-1825,共3页Chinese Journal of Experimental Surgery

基　　金：海南省自然科学基金(817331);国家自然科学基金地区基金项目(81860514)。

摘　　要：目的:探讨肝窦内皮细胞(LSECs)的内皮型一氧化氮合成酶(eNOS)对肝细胞(HC)增殖的影响及其机制。方法:培养细胞并分组为HC组,HC+LSECs组,HC+N-硝基-L-精氨酸甲酯(L-NAME)组,HC+LSECs+L-NAME组,HC+LSECs+肝细胞生长因子(HGF)组,HC+LSECs+HGF+L-NAME组。5-乙炔基-2’-脱氧尿苷(EDU)方法检测HC增殖率,酶联免疫吸附实验(ELISA)方法检测各组HC的蛋白激酶B(Akt)、细胞间充质-上皮转化因子(c-Met)表达。蛋白质印迹法(Western blot)法及即时聚合酶链锁反应(RT-PCR)法检测LSECs、LSECs+L-NAME组中细胞的eNOS、血管生成素2(Ang2)、转化生长因子-β1(TGF-β1)的表达。应用SPSS 19.0统计软件进行分析,多组间比较采用单因素方差分析,两组间均数比较采用t检验。结果:HC+LSECs组与HC+LSECs+L-NAME组比较,HC增殖率高[(42.30±4.43)%比(32.80±3.18)%];HC+LSECs+HGF组与HC+LSECs+HGF+L-NAME组比较,HC增殖率高[(96.34±2.48)%比(59.53±2.88)%],差异均有统计学意义(F=246.325,P<0.01)。Akt、c-Met的表达:HC+LSECs组与HC+LSECs+L-NAME组比较表达升高(4.71±0.02、9.18±0.09比2.66±0.01、6.78±0.08);HC+LSECs+HGF组与HC+LSECs+HGF+L-NAME组比较表达升高(10.55±0.02、17.53±0.16比5.82±0.02、10.72±0.05,F=103175.549、8465.544,P<0.01)。Ang2、TGF-β1、eNOS的表达:相对于LSECs细胞组,LSECs+L-NAME组中eNOS的表达下降(0.372838±0.019876比0.117049±0.008135,t=20.732,P<0.01);(0.02793±0.00092比0.01057±0.00015,t=32.126,P<0.01),而Ang2、TGF-β1的表达升高(0.216319±0.018801比0.557341±0.034118,t=-84.663,P<0.01),(0.00499±0.00006比0.01700±0.00010,t=-181.747,P<0.01);(0.137316±0.012621比0.628727±0.045014,t=-75.773,P<0.01),(0.010930±0.000351比0.034670±0.000830,t=-45.488,P<0.01)。结论:来源于LSECs的eNOS,可以通过抑制LSECs中Ang2/TGF-β1信号通路表达,达到促进HC增殖作用。Objective To explore the effect of liver sinusoidal endo-thelial cells(LSECs)-derived endothelial nitric oxide synthase(eNOS)on the proliferation of hepatocytes(HC).Methods The cells were cultured and divide into the following groups:HC,HC+LSECs,HC+n-nitro-l-arginine methyl esters(L-NAME),HC+LSECs+L-NAME,HC+LSECs+hepatocyte growth factor(HGF),HC+LSECs+HGF+L-NAME.The 5-acetylenyl-2’-deo-xyuridine(EDU)method was used to detect the proliferation of HC in each group.The expression of protein kinase B(Akt)and cellular-mesenchymal to epithelial transition factor(c-Met)in HC of each group was detected by enzyme linked immunosorbent assay(ELISA).Western blotting and real-time polymerase chain reaction were used to detect the expression of eNOS,Angiopoietin-2(Ang2),transforming growth factor-β1(TGF-β1)in LSECs group and LSECs+L-NAME group.SPSS 19.0 statistical software was used to analyze the data.Results HC+LSECs group had higher proliferation rate than HC+LSECs+L-NAME group[(42.30±4.43)%vs.(32.80±3.18)%].The proliferation rate in HC+LSECs+HGF group was higher than in HC+LSECs+HGF+L-NAME group[(96.34±2.48)%vs.(59.53±2.88)%,F=246.325,P<0.01].The expression of Akt and c-Met in HC+LSECs group was higher than in HC+LSECs+L-NAME group(4.71±0.02,9.18±0.09 vs.2.66±0.01,6.78±0.08),higher in HC+LSECs+HGF group than in HC+LSECs+HGF+L-NAME group(10.55±0.02,17.53±0.16 vs.5.82±0.02,10.72±0.05,F=103175.549,8465.544,P<0.01).As compared with the LSECs group,the expression of eNOS in the LSECs+L-NAME group was decreased(0.372838±0.019876 vs.0.117049±0.008135,t=20.732,P<0.01);(0.02793±0.00092 vs.0.01057±0.00015,t=32.126,P<0.01),while the expression of Ang2 and TGF-β1 was increased(0.216319±0.018801 vs.0.557341±0.034118,t=-84.663,P<0.01),(0.00499±0.00006 vs.0.01700±0.00010,t=-181.747,P<0.01);(0.137316±0.012621 vs.0.628727±0.045014,t=-75.773,P<0.01),(0.010930±0.000351 vs.0.034670±0.000830,t=-45.488,P<0.01).Conclusion eNOS derived from LSECs can promote HC proliferation by inhibiting the expression of Ang2/TGF-β

关 键 词：肝窦内皮细胞 内皮型一氧化氮合成酶 血管生成素2 肝细胞 转化生长因子-Β1 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]