不同位点磷酸化Src酪氨酸激酶与食管鳞状细胞癌的关系

目的 探讨不同位点磷酸化Src酪氨酸激酶与食管鳞状细胞癌(ESCC)发生的关系.方法 收集30例食管鳞状细胞癌石蜡包埋组织块及3例新鲜食管鳞痛组织标本,培养人食管鳞癌TE1细胞株,分别采用免疫组织化学法、免疫印迹法和免疫荧光法分析食管鳞状细胞癌组织不同位点磷酸化Src酪氨酸激酶蛋白表达水平的变化.结果 免疫组织化学染色、免疫印迹和免疫荧光均显示食管鳞状细胞癌组织中Py416Src蛋白表达水平高于Py527Src,差异有统计学意义(P＜0.05).结论 食管鳞状细胞癌组织中Py416Src蛋白高表达,Py527Src蛋白低表达,提示Src酪氨酸激酶不同位点磷酸化水平在食管鳞状细胞癌的发生发展过程中起重要作用.

Abstract：

Objective To determine the relationship between phospholation of Src tyrosine kinase at different sites and esophageal squamous cell carcinoma (ESCC). Methods Thirty cases of ESCC paraffin-embedded tissue blocks and 3 cases of fresh ESCC tissue samples were collected, and human ESCC TE1 cells were cultured. The protein expression level of phosphorylation of Src tyrosine kinase at different sites was detected by using immunohistochemical staining, Western blotting and immunofluorescence. Results Py416Src protein expression level was higher than Py527Src in ESCC (P＜0.05). Conclusion Phosphorylation of Src tyrosine kinase at different sites plays a critical role in pathogenesis of ESCC.     

Srcasm负调控Src家族酪氨酸激酶Fyn在人食管鳞状细胞癌TE1细胞株中的表达

目的 观察人食管鳞状细胞癌TE1细胞株中Srcasm对Src家族酪氨酸激酶Fyn表达的影响.方法 分别转染0、0.25、0.5、1.0、2.0 μg人Srcasm质粒DNA入TE1细胞株中观察其对Fyn表达的影响.结果 转染Srcasm质粒DNA可以降低人食管鳞状细胞癌TE1细胞中Fyn的表达,且呈剂量依赖性.结论 人食管鳞状细胞癌中Srcasm可能是Src家族酪氨酸激酶Fyn的负调控因子,在食管鳞状细胞癌的发生发展过程中起重要作用.     

不同位点磷酸化Src酪氨酸激酶与食管鳞状细胞癌的关系

Objective To determine the relationship between phospholation of Src tyrosine kinase at different sites and esophageal squamous cell carcinoma (ESCC). Methods Thirty cases of ESCC paraffin-embedded tissue blocks and 3 cases of fresh ESCC tissue samples were collected, and human ESCC TE1 cells were cultured. The protein expression level of phosphorylation of Src tyrosine kinase at different sites was detected by using immunohistochemical staining, Western blotting and immunofluorescence. Results Py416Src protein expression level was higher than Py527Src in ESCC (P＜0.05). Conclusion Phosphorylation of Src tyrosine kinase at different sites plays a critical role in pathogenesis of ESCC.     

不同位点磷酸化Src酪氨酸激酶与食管鳞状细胞癌的关系

Objective To determine the relationship between phospholation of Src tyrosine kinase at different sites and esophageal squamous cell carcinoma (ESCC). Methods Thirty cases of ESCC paraffin-embedded tissue blocks and 3 cases of fresh ESCC tissue samples were collected, and human ESCC TE1 cells were cultured. The protein expression level of phosphorylation of Src tyrosine kinase at different sites was detected by using immunohistochemical staining, Western blotting and immunofluorescence. Results Py416Src protein expression level was higher than Py527Src in ESCC (P＜0.05). Conclusion Phosphorylation of Src tyrosine kinase at different sites plays a critical role in pathogenesis of ESCC.     

Src家族酪氨酸激酶Fyn在人食管鳞状细胞癌中的表达

目的 探讨食管鳞状细胞癌组织中Fyn的表达水平及其意义.方法 收集13例食管鳞状细胞癌组织(ESCC)和10例食管正常黏膜上皮组织(UNR),采用免疫组织化学法和免疫印迹法分析食管鳞状细胞癌组织及食管正常黏膜组织Fyn蛋白表达水平的变化.结果 免疫组织化学染色显示食管鳞状细胞癌组织中Fyn蛋白表达水平(9.68±2.31)高于食管正常黏膜组织中染色指数(3.21±1.25),差异有统计学意义(P<0.01).免疫印迹结果亦显示食管鳞癌组织中Fyn蛋白表达强于食管正常黏膜组织.结论 Fyn蛋白在食管鳞状细胞癌组织中高表达,提示Fyn基因在食管鳞状细胞癌的发生发展过程中可能起重要作用.     

细胞分裂周期蛋白42mRNA和蛋白在食管鳞癌中的表达及其病理生物学意义

目的 探讨细胞分裂周期蛋白42(Cdc42)在食管鳞状细胞癌(ESCC)中的表达及其与临床病理参数间的关系.方法 应用实时荧光定量-聚合酶链反应(qRT-PCR)、蛋白免疫印迹和免疫组织化学方法,从mRNA和蛋白两个水平检测ESCC中Cdc42的表达,并分析其与临床病理参数的关系.结果 22对新鲜ESCC与癌旁正常组织中,Cdc42 mRNA在ESCC中的表达量(0.21±0.14)显著高于其在癌旁正常组织中(0.16±0.12)的表达(P＜0.05);Cdc42蛋白在ESCC中的表达量(0.83±0.35)高于其在癌旁正常组织中(0.75±0.24)的表达;在175对ESCC中,Cdc42蛋白的阳性表达率为73.7%(129/175),高于其在配对的癌旁正常组织中的表达62.9%(110/175,P＜0.05).此外,Cdc42的表达与ESCC患者的年龄、淋巴结转移及分化程度明显相关(P＜0.05).结论 Cdc42可能参与ESCC发生及转移的过程.

Abstract：

Objective To explore the expression of cell division cycle 42 (Cdc42) in human esophageal squamous cell carcinoma (ESCC) and investigate the association between Cdc42 and clinicopathological parameters. Methods The expression levels of Cdc42 mRNA and protein in ESCC and corresponding adjacent normal tissues were detected by real-time fluorescent quantitative polymerase chain reaction ( qRT-PCR), Western blotting and immunohistochemistry, respectively. The correlations between Cdc42 expression and clinicopathological parameters were analyzed. Results The expression of Cdc42 mRNA was significantly higher in ESCC tissues (0. 21 ± 0. 14 ) than that in corresponding controls (0. 16 ±0. 12) (t test,P ＜0. 05). The protein expression of Cdc42 was significantly higher in ESCC tissues (0. 83 ± 0. 35 ) than that in corresponding controls ( 0. 75 ± 0. 24). Immunohistochemistry revealed that 73.7% (129/175) of the ESCC samples had higher expression of Cdc42 protein than the corresponding controls[62. 9% (110/175) (χ2 test, P ＜ 0. 05 )]. Cdc42 expression level was correlated with age,lymphoid node metastasis and differentiation ( P all ＜ 0. 05 ), but not with the clinicopathological features,such as gender, ethnicity and macroscopical types (P ＞ 0. 05 ). Conclusion The higher expression of Cdc42 played a certain role in the carcinogenesis and metastasis of ESCC.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

曲尼司特对环孢素A诱导的人肾小管上皮细胞向间充质转变的抑制作用

Objective To study the effect of tranilast on cyclosporine A (CsA)-induced epithelial-to-mesenchymal transition in human renal tubular epithelial cells, and investigate the mechanism of its antifibrotic effect. Methods Cultured HK-2 cells were divided into four groups: (1)In the control group, cells were treated without any medicine; (2) The cell were treated with CsA (4. 2μmol/L) for 72 h; (3) The cells were treated with a combination of CsA (4. 2 μmol/L) and tranilast (100μmol/L); (4) The cells were treated with tranilast (100 μmol/L) alone for 72 h.Morphological changes of the cells were assessed by phase-contrast microscopy. The immunofluorescence and Western blotting were adopted to detect the expression of E-cadherin, α-SMA and OPN mRNA and proteins respectively. Results Tranilast could markedly ameliorate the morphological changes of HK-2 cells stimulated by CsA. The irmmunofluorescence staining revealed the expression of E-cadherin was markedly decreased in HK-2 cells stimulated with CsA for 72 as compared with the control group, while the expression of α-SMA and OPN was significantly higher in CsA group than the control group. The expression of E-cadherin in the CsA + Tranilast group was higher than the CsA group, while the expression of α-SMA and OPN in the CsA + Tranilast group was lower than the CsA group. Western blotting showed that protein expression level of E-cadherin in CsA group was dramatically lower than that in the control group (P＜0. 05), while that of α-SMA and OPN in CsA group was significantly higher than in the control group (P＜0.05). The protein expression level of E-cadherin in HK-2 cells in the CsA + Tranilast group was markedly higher than in the CsA group (P＜0.05), and that of α-SMA and OPN in CsA + Tranilast group was significantly lower than in the CsA group (P＜0. 05). Conclusion Tranilast can block the CsA-induced epithelialto-mesenchymal transition in HK-2 cells probably by suppressing the expression of OPN.     

XB130 as an Independent Prognostic Factor in Human Esophageal Squamous Cell Carcinoma

Background

Adaptor proteins, with multimodular structures, can participate in the regulation of various cellular functions. A novel adaptor protein XB130 has been implicated as a substrate and regulator of tyrosine kinase-mediated signaling and in controlling cell proliferation and apoptosis in thyroid and lung cancer cells. However, its expression and role in gastrointestinal cancer have not been investigated. We sought to determine the role of XB130 in cell cycle progression of esophageal squamous cell carcinoma (ESCC) cells and to examine its expression and effects on the prognosis of patients with ESCC.

Methods

Expression of XB130 in human ESCC cell lines was analyzed by Western blot testing and immunofluorescent staining. Knockdown experiments with XB130 small interfering RNA (siRNA) were conducted, and the effect on cell cycle progression was analyzed. Immunohistochemistry of XB130 for 52 primary tumor samples obtained from patients with ESCC undergoing esophagectomy was performed.

Results

XB130 was highly expressed in TE2, TE5, and TE9 cells. In these cells, knockdown of XB130 with siRNA inhibited G₁–S phase progression and increased the expression of p21, the cyclin-dependent kinase inhibitor. Immunohistochemistry showed that 71.2 % of the patients expressed XB130 in the nuclei and/or cytoplasm of the ESCC cells. Further, nuclear expression of XB130 was an independent prognostic factor of postoperative survival.

Conclusions

These observations suggest that the expression of XB130 in ESCC cells may affect cell cycle progression and impact prognosis of patients with ESCC. A deeper understanding of XB130 as a mediator and/or biomarker in ESCC is needed.     

食管鳞癌与细胞因子信号转导负调控因子3及其DNA甲基化的关系

目的 检测食管鳞癌(ESCC)组织中细胞因子信号转导负调控因子3(SOCS3)的DNA甲基化、mRNA及蛋白表达水平,探讨其在食管鳞癌发生、发展、浸润和转移中的作用.方法 采用甲基化特异性聚合酶链反应(MSP)、Real-Time聚合酶链反应(PCR)和Western blot法分别检测43例食管鳞癌组织中SOCS3的DNA甲基化、mRNA和蛋白表达水平,并与相应的癌旁正常食管组织进行对照研究,分析其与临床病理参数的关系.结果 (1)食管鳞癌组织SOCS3 DNA甲基化的阳性率(79.1%)明显高于癌旁组织(14.0%,P＜0.01);(2)食管鳞癌组织SOCS3 mRNA相对表达强度比值(0.53±0.30)明显低于癌旁组织(1.15±0.44,P＜0.01),食管鳞癌组织中甲基化组的SOCS3 mRNA表达(0.45±0.24)显著低于非甲基化组(0.86±0.29,P＜0.05);(3)食管鳞癌组织SOCS3蛋白表达(1.66±0.22)显著低于癌旁组织(1.83±0.15,P＜0.01),食管鳞癌组织中甲基化组SOCS3蛋白表达(1.61±0.21)显著低于非甲基化组(1.87±0.15,P＜0.01);(4)在TNM分期中Ⅲ期组表达均低于Ⅰ～Ⅱ期组(P＜0.05),伴有淋巴结转移组表达也都低于无淋巴结转移组(P＜0.05),未发现其在性别、年龄、家族史、吸烟史中有明显差异(P＞0.05);(5)食管鳞癌组织中SOCS3mRNA表达及其蛋白表达水平与肿瘤分化级别呈正相关(0.301＜r＜1,P＜0.05),与TNM分期、淋巴结转移呈负相关(-1＜r＜-0.301,P＜0.05).结论 食管鳞癌组织中SOCS3 DNA甲基化阳性率高,导致SOCS3基因表达下调,与食管鳞癌的分化、浸润和转移密切相关.     

Effect of liver X receptor on the thrombomodulin expression in human glomerular endothelial cells

ObjectiveTo explore the role of liver X receptor (LXR) agonist T0901317 onthrombomodulin (TM) expression in human glomerular endothelial cells and the possible mechanisms. Methods Different concentrations of T0901317 were used to stimulate human glomerular endothelial cells for different time, then LXRα, LXRβ expression were detected by using Western blotting analysis; the roles of T0901317 on TM mRNA and TM protein expression were observed by using real-time PCR, Western blotting and immunofluorescence assay. LXRα, LXRβ gene interference segment Si-hLXRα, Si-hLXRβ were transfected into human glomerular endothelial cells with the concentration of 100 nmol/L respectively, then the roles of Si-hLXRα, Si-hLXRβ on the TM protein and TM mRNA expression were assayed by Western blotting and real time PCR. Results Human glomerular endothelial cells expressed LXRα and LXRβ. Compared to the normal cells and DMSO group, T0901317 could significantly promote TM expression in human glomerular endothelial cells (P＜0.05) and showed a time- and dose-dependent manner. TM expression in Si-hLXRα transfected group was significantly lower than that in the control group (P＜0.05), while TM expression in Si-hLXRβ transfected group had no significant difference compared to the control group. Conclusions Human glomerular endothelial cells express LXRα and LXRβ. LXR agonist T0901317 promotes TM expression in human glomerular endothelial cells, which may be mainly through activating LXRa.     

短发卡RNA对人胃癌细胞STAT3基因的沉默作用

目的:探讨STAT3基因小发夹RNA（shRNA）表达质粒对胃癌MKN-45细胞STAT3基因的干扰作用。方法:根据STAT3 mRNA 编码序列，设计RNA干扰靶点，构建STAT3基因的特异性小RNA干扰质粒（psiRNA-H1/STAT3），使用脂质体转染人胃癌细胞系（MKN-45细胞）。实验分为对照（A）组，psiRNA-H1转染（B）组和psiRNA-H1/STAT3转染（C）组。通过RT-PCR和Western Blot检测STAT3特异性小RNA干扰基因对胃癌细胞STAT3基因mRNA和蛋白表达的影响。结果:psiRNA-H1/STAT3经限制性酶切及部分序列分析证明基因插入正确，并经测序证实。将其成功转染MKN-45细胞后，该细胞的STAT3 mRNA和蛋白表达均明显下降(P<0.05)。 结论:将成功构建的针对STAT3基因的shRNA表达载体转染MKN-45细胞，能有效抑制该细胞的STAT3 mRNA和蛋白表达，为STAT3基因靶向治疗提供一定的实验依据。     

WWOX表达对胆管癌QBC939细胞凋亡的影响及其机制

目的 探讨含有WW结构域的氧化还原酶基因(WWOX)表达对胆管癌QBC939细胞凋亡的影响及其作用机制.方法 用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞;采用荧光定量逆转录－聚合酶链反应(RT-PCR)和Westem blot法鉴定WWOX在QBC939细胞中的表达;流式细胞仪(FCM)法检测转染前后各细胞凋亡率的变化;JC-l染色法检测细胞线粒体膜电位(△Ψm)变化;荧光定量RT-PCR和Western blot法检测胆管癌细胞bcl-2表达的变化;将未转染和转染空质粒的细胞作为对照组接种到裸鼠皮下以检测荷瘤,TUNEL方法原位检测移植瘤的凋亡.结果 建立了稳定表达WWOX基因的QBC939/WWOX细胞株,mRNA及蛋白表达明显增加.FCM显示QBC939/WWOX组的细胞凋亡率明显增高[(1.24±0.35)％比(1.73±0.48)％比(21.40±2.35)％,P＜O.01],JC-l显示转染组的线粒体膜电位下降[(4.27±0.64)％比(4.96±0.52)％比(28.60±3.94)％,P＜O.01],bcl-2 mRNA及蛋白的表达均显著降低(P＜0.05).转染组的皮下肿瘤较对照组生长速度明显减慢(P＜0.05),TUNEL实验证实转染组的皮下肿瘤凋亡指数为(13.6±1.5)％,较对照组明显增高,差异有统计学意义(P＜0.O1).结论 WWOX基因能促进胆管癌细胞的凋亡,其机制可能与下调bcl-2的表达,激活线粒体凋亡通路有关.     

苦参碱调控Snail2表达水平抑制转化生长因子β1诱导的人腹膜间皮细胞上皮间充质转分化

目的 研究苦参碱对转化生长因子β1(TGF-β1)诱导腹膜间皮细胞上皮间充质转分化(EMT)后转录因子Snail2的影响.方法 采用TGF-β1刺激人腹膜间皮细胞并同时予不同浓度苦参碱干预处理,实验分为空白对照组、TGF-β1(5 ng/ml)诱导组、TGF-β1+0.4 mg/ml苦参碱干预组、TGF-β1+0.6 mg/ml苦参碱干预组、TGF-β1+0.8 mg/ml苦参碱干预组和TGF-β1+1.0 mg/ml苦参碱干预组.实时荧光定量PCR和Western印迹检测Snail2、上皮标志分子E钙黏蛋白(E-cadherin)和间质标志分子α平滑肌肌动蛋白(α-SMA)、纤维连接蛋白(FN)、胶原Ⅲ(ColⅢ)的表达,Western印迹检测Smad2、Smad3和细胞外调节蛋白激酶1/2(ERK1/2)的蛋白磷酸化水平.结果 TGF-β1(5 ng/ml)刺激能上调人腹膜间皮细胞Snail2、α-SMA、FN和ColⅢmRNA和蛋白的表达水平,上调Smad2、Smad3和ERK1/2的蛋白磷酸化水平,下调E-cadherinmRNA和蛋白的表达水平;苦参碱(0.4、0.6、0.8、1.0 mg/ml)干预处理后能下调Snail2和α-SMA、FN和ColⅢmRNA和蛋白的表达水平以及ERK1/2的蛋白磷酸化水平,上调E-cadherin mRNA和蛋白的表达水平.结论 TGF-β1能诱导人腹膜间皮细胞EMT,苦参碱可能通过ERK1/2信号通路下调Snail2的表达水平来抑制TGF-β1诱导的人腹膜间皮细胞EMT.