Bcl-XL 反义寡核苷酸对食管癌 EC9706细胞和裸鼠移植瘤组织的诱凋亡作用

目的:探讨Bcl-XL反义寡核苷酸(Bcl-XL ASODN)对食管癌EC9706细胞和人食管癌裸鼠移植瘤组织的诱凋亡作用。方法:将合成的Bcl-XL ASODN通过阳离子脂质体介导转染EC9706细胞后,以吖啶橙荧光染色、流式细胞术和TUNEL技术检测ASODN组、细胞对照组、空白对照组和N-ODN组的凋亡率;用TUNEL技术检测ASODN组、N-ODN组和细胞对照组裸鼠移植瘤组织的细胞凋亡率。结果:吖啶橙荧光染色、流式细胞术和TUNEL检测结果显示,ASODN组、N-ODN组、空白对照组和细胞对照组的凋亡率比较,差异均有统计学意义(F=26.853、38.537、28.541,P均<0.001);且ASODN组的凋亡率均高于细胞对照组、空白对照组和N-ODN组(P均<0.05);TUNEL法检测移植瘤组织的细胞凋亡率结果显示,细胞对照组、N-ODN组及ASODN组的凋亡率比较,差异有统计学意义(F=56.375,P<0.001);且ASODN组高于细胞对照组及N-ODN组(P均<0.05)。结论:Bcl-XL ASODN对食管癌EC9706细胞及食管癌裸鼠移植瘤组织均有诱凋亡作用。

咳喘落口服液对哮喘模型小鼠肺组织中嗜酸性粒细胞凋亡及调控因素的影响

目的:观察咳喘落口服液对哮喘小鼠嗜酸性粒细胞(eosinophil,EOS)凋亡及调控因素的影响,旨在进一步阐明咳喘落抑制气道炎症的机制。方法:56只BALB/c小鼠分为正常组、模型组、咳喘落组和西药(强的松龙)组。除正常对照组外,其余各组小鼠用卵白蛋白和硫酸铝钾致敏激发BALB/c小鼠,复制哮喘模型。在不同时相(末次激发后0h,第3、7和14天)分别采用改良的Giemsa染色法检测各组小鼠肺组织中EOS数量;免疫组化结合图像分析方法检测各组小鼠肺组织中Fas、Fas配体(Fas ligand,FasL)和Bcl-XL表达的情况;采用TdT介导的带生物素dUTP缺口末端标记技术(terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling,TUNEL)检测EOS凋亡率。结果:末次激发后第3、7和14天,哮喘模型组小鼠气道炎症明显,以末次激发后第3天最为严重,而咳喘落组、西药组各时相气道组织炎症均明显减轻,EOS数量低于模型组(P<0.05)。咳喘落组末次激发后第3天,其肺组织内FasL、EOSFas阳性面积和凋亡率较模型组升高,差异有统计学意义(P<0.01),且凋亡率达顶峰,而Bcl-XL的阳性面积及EOS数量低于模型组,差异有统计学意义(P<0.05);末次激发后第7和14天,中药组EOS中Fas阳性面积和Bcl-XL的阳性面积较模型组明显下降(P<0.01);且第14天,其肺组织中FasL表达和EOS凋亡率均明显下降(P<0.01)。西药阳性对照组的作用与中药治疗组类似。结论:哮喘小鼠存在以EOS浸润为主的气道炎症,同时伴有凋亡受抑和凋亡延迟。咳喘落可以提高炎症早期肺组织EOS凋亡率,可能是通过减少EOS,提高FasL、Fas表达和降低Bcl-XL表达实现的。

Down-regulation of Bcl-X_L by RNA interference suppresses cell growth and induces apoptosis in human esophageal cancer cells

AIM: To determine the inhibitory effect of the vector- generated small interfering RNAs (siRNAs) on the expression of the Bcl-XL gene in established human esophageal cancer cells, and to investigate the effect of the Bcl-XL siRNAs on cell growth and apoptosis in esophageal cancer cells. METHODS: Three siRNA-expressing vectors targeting different sites of the Bcl-XL gene were constructed from pTZ-U6+1 vector. Cultured esophageal cancer cells were transfected with the siRNA-expressing vector (or the control vector) using lipofectamine 2000. Bcl-XL gene expression was determined with semiquantitative RT- PCR assay and Western blotting. Among the three siRNA- expressing vectors, the most highly functional vector and its effect on cell growth and apoptosis in esophageal cancer cells was further analyzed. RESULTS: Of the three siRNA-expressing vectors, siRNA- expressing vector No.1 was the most potent one which suppressed Bcl-XL mRNA production to 32.5% of that in the untreated esophageal cancer cells. Western blotting analysis showed that siRNA-expressing vector No.1 markedly down-regulated the expression of Bcl-XL in human esophageal cancer cells. Treatment of esophageal cancer cells with siRNA-expressing vector No.1 resulted in inhibition of cell growth and induction of apoptosis. CONCLUSION: Down-regulation of Bcl-XL by vector- generated small interfering RNAs can suppress cell growth and induce apoptosis in human esophageal cancer cells.

Novel rapid tissue lysis method to evaluate cancer proteins: Correlation between elevated Bcl-X_L expression and colorectal cancer cell proliferation

AIM: We optimized a rapid and efficient tissue lysis method using the MagNA Lyser (Roche, Germany). Using this novel method combined with immunoblot analysis, we investigated the correlation between abnormal Bcl-XL expression and clinicopathological characteristics in colorectal cancer. METHODS: Tissue samples from Sprague-Dawley rats were tested to determine optimal lysis conditions for use with MagNA Lyser. We next used the new method to extract tissue proteins from the tumor tissue of a colorectal cancer patient. The availability of extractable tissue proteins for proteomic study was demonstrated by two-dimensional (2D) gel electrophoresis and subsequent matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In addition, we prepared tissue lysates from paired tumor tissues and adjacent nontumor tissues of 50 colorectal carcinoma patients. Ensuing immunoblot analyses were performed to detect the level of Bcl-XL expression. RESULTS: The optimal sample sizes processed were found to be around 200 mg, with oscillation frequency of 6 500 r/min for 80 s. Test of the first human tissue lysate confirmed that the MagNA Lyser method was adequate for protein extraction and subsequent identification by current proteomic protocols. The method was also applicable to immunoblot analysis. Thirty of 50 (60%) colorectal patients exhibited higher level of Bcl-XL expression in their tumor tissues. Raised level of Bcl-XL expression correlated with patients' gender and tumor cell proliferation index (P= 0.037 and P<0.001, respectively), but was independent of clinicopathological characteristics and overall survival. CONCLUSION: We report a novel tissue lysis method applicable to proteomic and immunoblot analyses, which can facilitate the discovery and detection of cancer protein alterations.

Bcl-XL反义寡核苷酸增强食管癌细胞对5-氟尿嘧啶的敏感性

目的探讨Bcl -XL反义寡核苷酸(ASODN)在下调Bcl- XL表达、增加食管癌细胞株EC9706对5氟尿嘧啶(5Fu)敏感性中的作用。方法设细胞对照组、空白对照组、无关序列寡核苷酸(N ODN)组、ASODN组、5Fu组及ASODN联合5Fu组,应用四甲基偶氮唑蓝(MTT)法检测EC9706细胞的增殖抑制率;RT PCR和Westernblot检测Bcl- XLmRNA和蛋白表达水平的改变;吖啶橙荧光染色和流式细胞技术定量检测EC9706细胞的凋亡率。结果Bcl -XLASODN联合5Fu治疗组对食管癌细胞增殖的抑制率为71.58%,对Bcl XLmRNA表达抑制率为81.25%,并可显著下调Bcl XL的蛋白表达;凋亡指数为69.5%,凋亡率为(63.32±9.23)%,与细胞对照组、空白对照组、N -ODN组、ASODN组及5Fu组比较,差异均有统计学意义(P<0.05)。结论ASODN联合5Fu可有效抑制食管癌细胞EC9706的增殖,通过ASODN下调Bcl XL表达,可显著增强食管癌细胞对5Fu的敏感性。