tRNA-ValAAC-5促进肝癌细胞增殖和迁移作用机制研究

目的:本研究论证肝细胞癌(hepatocellular carcinoma,HCC)细胞中tRNA-ValAAC-5表达发挥作用及机制。方法:使用Real-time PCR法检测tRNA-ValAAC-5在人正常细胞肝癌细胞(THLE2,THLE3)和HCC细胞株(Hep3B,HuH7,SNU398,Hep3G2)和中的表达。Hep3B和Hep3G2细胞分别转染tRNA-ValAAC-5-inhibitor及对照tRNA-ValAAC-5-NC即为inhibitor组和NC组。两组细胞分别通过CCK-8法检测细胞增殖,Transwell小室实验检测细胞侵袭转移能力,Western blot法检测p21、基质金属蛋白酶2(MMP2)和基质金属蛋白酶9(MMP9)的表达。结果:相较于THLE2、THLE3细胞,tRNA-ValAAC-5在Hep3B、HuH7、SNU398、Hep3G2细胞中相对表达量均有升高,差异有统计学意义(P<0.05)。与tRNAValAAC-NC相比,转染tRNA-ValAAC-5-inhibitor的Hep3B、Hep3G2细胞tRNA-ValAAC-5表达均明显降低,两组差异有统计学意义(t=36.52、27.45,P<0.001),表示转染成功。与tRNA-ValAAC-5-NC相比,Hep3B、Hep3G2细胞转染tRNA-ValAAC-5-inhibitor后24、48、72、96 h增殖能力受到抑制,差异均有统计学意义(t=5.25、8.23、7.33、14.16,P<0.001)、(t=4.25、5.11、9.39、7.59,P<0.001)。转染tRNA-ValAAC-5-inhibitor组Hep3B、Hep3G2细胞侵袭转移减少,两组差异有统计学意义(t=14.01、21.85,P<0.001)。转染tRNA-ValAAC-5-inhibitor组Hep3B和Hep3G2细胞p21表达升高、MMP2和MMP9的表达量降低,差异均有统计学意义(t=8.96、12.80、4.652,P<0.01)、(t=15.17、22.36、12.61,P<0.01)。结论:tRNA-ValAAC-5能够有效促进肝癌增殖,侵袭和转移,其可能的作用机制与调控p21、MMP2和MMP9的表达有关。

Study on the mechanism of tRNA-ValAAC-5 in promoting the proliferation and migration of hepatocellular carcinoma cells

Objective:To demonstrate the role and mechanism of tRNA-ValAAC-5 expression in hepatocellular carcinoma(HCC)cells.Methods:The expression levels of tRNA-ValAAC-5 in HCC(Hep3B,HuH7,SNU398,Hep3G2)and human hepatocellular carcinoma(THLE2,THLE3)were detected by real-time PCR.HEP3B and Hep3G2 cells were respectively transfected with tRNA-ValAAC-5-inhibitor and tRNA-ValAAC-5-NC as the inhibitor group and the NC group.Then the ability of cell proliferation was detected by CCK-8 assay and the ability of invasion and metastasis was detected by Transwell assay.The protein expression levels of p21,Matrix metalloproteinase 2(MMP2)and Matrix metalloproteinase 9(MMP9)were determined by Western blot.Results:The relative expression of tRNA-ValAAC-5 in Hep3B,HuH7,SNU398 and Hep3G2 cells were significantly higher than THLE2 and THLE3 cells,the differences were statistically significant(P<0.05).After tRNA-ValAAC-5-inhibitor transfection,the expression of tRNA-ValAAC-5 in Hep3B and Hep3G2 cells were reduced than tRNA-ValAAC-NC group.Both of the differences were statistically significant(t=36.52,27.45,P<0.001),which indicated the transfection was successful.The proliferative ability of Hep3B and Hep3G2 cells transfected with tRNA-ValAAC-5-inhibitor after 24,48,72,96 h were inhibited effectively compared with tRNA-ValAAC-5-NC group.All of the differences were statistically significant in Hep3B(t=5.25,8.23,7.33,14.16,P<0.001)and Hep3G2(t=4.25,5.11,9.39,7.59,P<0.001)cells.The number of invasion and metastasis of Hep3B and Hep3G2 cells were reduced in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,there was significant difference(t=14.01,21.85,P<0.001).The protein expression levels of P21 were lower,MMP2 and MMP9 were higher in tRNA-ValAAC-5-inhibitor group compared with tRNA-ValAAC-5-NC group,the differences were statistically significant in Hep3B(t=8.96,12.80,4.652,P<0.001)cells and Hep3G2(t=15.17,22.36,12.61,P<0.001)cells.Conclusion:tRNA-ValAAC-5 can effectively promote the proliferation,invasion and metastasis of HCC,and its possible mechanism is related to regulating the expression of p21,MMP2 and MMP9.

迷走神经电刺激抑制NF-κB信号通路对结肠炎大鼠血管内皮功能的损伤

[目的]观察迷走神经电刺激(vagus nerve stimulation,VNS)对结肠炎大鼠胸主动脉形态、功能的影响,探讨其对结肠炎大鼠血管内皮损伤的作用。[方法]将健康SD大鼠随机分为对照组(15只)、葡聚糖硫酸钠(DSS)组(45只),对照组不作任何处理,DSS组通过饮用5%DSS诱导构建结肠炎模型。将DSS组大鼠随机选择30只又分为DSS+VNS组和DSS+Sham组,每组15只;DSS+VNS组给予大鼠VNS,而DSS+Sham组进行假手术。HE染色检测各组大鼠结肠和胸主动脉组织病理学变化,酶联免疫吸附实验检测大鼠血清中肿瘤坏死因子-α(TNF-α),白细胞介素-1β(IL-1β),白细胞介素-6(IL-6)和内皮素-1(ET-1)的含量,比色法检测胸主动脉中亚硝酸盐/硝酸盐的浓度,实时荧光定量PCR检测胸主动脉中细胞间黏附分子-1(ICAM-1)mRNA和血管内皮细胞黏附分子-1(VCAM-1)mRNA的表达水平,免疫组织化学染色检测胸主动脉中NF-κB p65阳性细胞表达情况,Western blot检测胸主动脉中NF-κB p65、κBα以及p-lκBα蛋白表达水平。[结果](1)与对照组比较,DSS组大鼠血清中TNF-α、IL-1β、IL-6和ET-1的含量升高,血管形态发生病变,内皮细胞脱落,胸主动脉中亚硝酸盐/硝酸盐浓度降低,ICAM-1和VCAM-1m RNA表达升高,NF-κB p65阳性表达率高,NF-κB p65和p-lκBα的蛋白表达水平升高而WBα蛋白表达水平降低,变化均显著(P<0.01)。(2)与DSS组和DSS+Sham组比较.DSS+VNS组大鼠血清中TNF-α、IL-1β、IL-6和ET-1的含量下降,大鼠血管形态明显改善并抑制了内皮细胞脱落的现象,胸主动脉中亚硝酸盐/硝酸盐浓度上升,ICAM-1和VCAM-1 mRNA表达降低,NF-κB p65阳性表达率下降,同时抑制了NF-κB p65和p-lκBα的蛋白表达水平升高以及lκBα蛋白表达水平降低,变化均显著(P<0.01)。而DSS+Sham组与DSS组比较,这些变化差异不显著(P>0.05)。[结论]VNS能在DSS诱导的结肠炎大鼠中起到抗炎作用.可以减轻炎症过程中的血管内皮损伤,这一过程可能是通过抑制NF-κB信号通路实现的。

胃十二指肠疾病与幽门螺杆菌血清抗体分型的关系探讨

[目的]探讨抗体分型与慢性浅表性胃炎(chronic superficial gastritis,CSG)、慢性萎缩性胃炎(chronic atrophic gastritis,CAG)、消化性溃疡(peptic ulcer,PU)和胃癌(gastriccancer,GC)的关系。[方法]选取常见胃十二指肠疾病患者113例,分为CSG组、CAG组、PU组、GC组,所有患者行胃镜检查,取静脉血,采用Western blot法测血清幽门螺杆菌(Hp)抗体:细胞毒素相关蛋白A(CagA)、细胞空泡毒素A(Vac)、尿素酶A(UreA)、尿素酶B(UreB)抗体,进行Hp血清分型。CagA和(或)VacA抗体阳性为Ⅰ型(HpⅠ),CagA和VacA抗体阴性,UreA和(或)UreB抗体阳性为Ⅱ型(HpⅡ)。[结果]113例患者Hp感染率为84.1%,Ⅰ型感染比例高于Ⅱ型感染。CAG组、PU组、GC组患者的Ⅰ型感染比例高于CSG组。[结论]单中心临床调查显示胃十二指肠疾病患者人群中感染Hp主要以毒力强的Ⅰ型为主,Ⅰ型菌株与GC、CAG、PU的发生有相关性;Hp血清抗体分型检测有助于临床医师对Hp相关的胃十二指肠疾病的预后风险评价及指导Hp感染的治疗策略。