目的:观察人剪切修复基因人类着色性干皮病D组基因(xeroderma pigmentosum group D,XPD)转染至人肝癌细胞株SMMC-7721细胞后XPD、DNp73和GADD45β基因的表达变化以及对肝癌细胞生长的影响.方法:实验分4组:重组质粒SMMC-7721-pEGFP-N2-XP...目的:观察人剪切修复基因人类着色性干皮病D组基因(xeroderma pigmentosum group D,XPD)转染至人肝癌细胞株SMMC-7721细胞后XPD、DNp73和GADD45β基因的表达变化以及对肝癌细胞生长的影响.方法:实验分4组:重组质粒SMMC-7721-pEGFP-N2-XPD(XPD组)、空载质粒SMMC-7721-pEGFP-N2组(N2组),脂质体组和SMMC-7721细胞空白对照组.应用Lipofectamine2000脂质体瞬时转染,逆转录聚合酶链反应(RT-PCR)和蛋白印迹(Western blot)法检测转XPD基因后,人肝癌细胞株SMMC-7721细胞中DNp73以及GADD45β的mRNA和蛋白质的表达量变化,并用四甲基偶氮唑盐(MTT)法检测细胞增殖的活力,流式细胞仪检测细胞凋亡的变化.结果:荧光显微镜下,XPD组和N2组细胞中观察到绿色荧光蛋白表达,说明转染成功;RT-PCR检测显示:XPD组中DNp73 mRNA相对表达量较其他3组显著下调,XPD和GADD45βmRNA相对表达量较其他3组明显上调(均P<0.01);Western blot检测显示:XPD、DNp73以及GADD45β蛋白相对表达量在各组间的差异与其mRNA各组间差异一致;MTT检测示:SMMC-7721细胞空白对照组、脂质体组、N2组、XPD组的吸光度(A)值分别为0.633±0.012,0.623±0.009,0.628±0.016,0.384±0.011,XPD组低于其他3组,差异均有统计学意义(均P<0.01),表明转染XPD后SMMC-7721细胞的增殖能力减弱.流式细胞仪检测SMMC-7721肝癌细胞凋亡:转染XPD的SMMC-7721细胞凋亡显著,凋亡率达56.53%,而其他3组均未见明显凋亡.结论:XPD基因在肝癌的发生发展中起抑制作用,癌基因DNp73的表达随XPD表达增加而降低,抑癌基因GADD45β则随XPD表达增加而增加,提示两者可能在XPD抑制肝癌细胞的生长机制中起重要作用.展开更多
生长阻滞和DNA损伤诱生蛋白45β(growth arrest and DNA damage 45β,GADD45β)参与多种细胞信号通路,在病毒感染过程中发挥重要作用,但在禽白血病病毒(avian leukosis virus,ALV)感染中研究较少。本研究中,用J亚群禽白血病病毒(ALV-J)...生长阻滞和DNA损伤诱生蛋白45β(growth arrest and DNA damage 45β,GADD45β)参与多种细胞信号通路,在病毒感染过程中发挥重要作用,但在禽白血病病毒(avian leukosis virus,ALV)感染中研究较少。本研究中,用J亚群禽白血病病毒(ALV-J)感染DF-1细胞,通过荧光定量PCR和Western blot检测GADD45β表达水平。此外,在过表达GADD45β和干扰GADD45β的情况下,通过Western blot、间接免疫荧光试验和ELISA检测GADD45β对ALV-J复制的影响。结果表明,ALV-J感染DF-1细胞能显著上调GADD45β表达水平(P<0.05)。过表达GADD45β后,ALV-J的蛋白表达水平显著降低(P<0.05),荧光信号强度也明显低于对照组。然而,干扰GADD45β后,ALV-J的复制水平显著上调(P<0.05),说明GADD45β可以抑制ALV-J病毒复制。本研究首次发现GADD45β具有抑制ALV-J病毒复制的功能,为抗ALV-J的研究提供了新的思路和理论基础。展开更多
Immunization with inactivated autoreactive T cells may induce idiotype anti-idiotypic reactions to deplete autoreac-tire T cells,which are involved in autoimmune diseases.However,it is unknown whether attenuated activ...Immunization with inactivated autoreactive T cells may induce idiotype anti-idiotypic reactions to deplete autoreac-tire T cells,which are involved in autoimmune diseases.However,it is unknown whether attenuated activated healthyautologous T-cell immunization could increase anti-tumor immune responses.To this end,C57B1/6 mice were immunizedwith attenuated activated autologous T cells.The splenocytes from immunized mice showed a higher proliferative abil-ity than that from naive mice.The special phenotype analysis showed that there were more CDS+T cells and CD62L+T cells in immunized mice after 24 h of culture with 10% fetal calf serum complete medium in vitro(P<0.01).Theseresults demonstrated that this immunization may activate T cells in vivo.Furthermore,the splenocytes from immunizedmice revealed resistance to activation-induced cell death(AICD)in vitro.To further study the relative genes that areresponsible for the higher proliferation and resistance to AICD,the expression of Fas/Fas ligand(FasL)and GADD45βwas measured by real-time PCR.The results indicated that GADD45β transcription was higher in the splenocytes fromimmunized mice than that in the naive mice.In addition,the Fas expression showed a parallel higher,but FasL did notchange obviously.To investigate the biologic functions induced by immunization in vivo,a tumor model was establishedby EL-4 tumor cell inoculation in C57/B1 mice.Mice receiving autologous T-cell immunization had significantly inhibitedtumor growth in vivo(P<0.01).This study implicated that immunization with attenuated activated autologous T cellsenhances anti-tumor immune responses that participate in tumor growth inhibition.展开更多
Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and ...Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.展开更多
目的研究肝泡型包虫病患者肝脏细胞Gadd45β(growth arrest and DNA damage gene 45β)及细胞周期调控相关基因的表达及意义。方法采集24例肝泡型包虫病患者肝脏标本,分为病灶旁组织(Close)和远端组织(Distance)。用重组TGF-β1(1ng/ml...目的研究肝泡型包虫病患者肝脏细胞Gadd45β(growth arrest and DNA damage gene 45β)及细胞周期调控相关基因的表达及意义。方法采集24例肝泡型包虫病患者肝脏标本,分为病灶旁组织(Close)和远端组织(Distance)。用重组TGF-β1(1ng/ml)及泡球蚴匀浆蛋白(1mg/ml)刺激体外培养的HL-7702人肝细胞,分别于30min、1h、6h、12h、24h、48h收集细胞。患者肝脏标本及刺激后收集的细胞均采用Trizol法提取RNA,反转录cDNA,通过实时荧光定量PCR检测目的基因表达并进行分析。结果近旁组织PCNA,TGF-β1,cyclinA1,cyclinE基因相对表达量分别为1.934±1.337、1.155±0.5138、1.367±0.8087、1.384±1.116、1.580±1.005、1.899±1.173和1.478±1.189,显著高于远端组织(1.00±0.00)且差异有统计学意义(P<0.05),而Gadd45β,cyclinB1,cyclinD差异无统计学意义(P>0.05)。用重组TGF-β1(1ng/ml)及泡球蚴匀浆蛋白(1mg/ml)刺激体外培养的HL-7702人肝细胞,引起Gadd45β,cyclinA1,cyclinE表达上调。结论泡球蚴匀浆蛋白刺激,可引起肝细胞Gadd45β及细胞周期相关蛋白表达上调,参与细胞抗凋亡过程。泡球蚴感染晚期,肝脏中仍存在细胞周期素(cyclins)参与的细胞增殖,但同时TGF-β1高表达,并通过Smads信号通路促进肝纤维化相关蛋白表达,促成肝脏泡型包虫病晚期病理变化。展开更多
文摘生长阻滞和DNA损伤诱生蛋白45β(growth arrest and DNA damage 45β,GADD45β)参与多种细胞信号通路,在病毒感染过程中发挥重要作用,但在禽白血病病毒(avian leukosis virus,ALV)感染中研究较少。本研究中,用J亚群禽白血病病毒(ALV-J)感染DF-1细胞,通过荧光定量PCR和Western blot检测GADD45β表达水平。此外,在过表达GADD45β和干扰GADD45β的情况下,通过Western blot、间接免疫荧光试验和ELISA检测GADD45β对ALV-J复制的影响。结果表明,ALV-J感染DF-1细胞能显著上调GADD45β表达水平(P<0.05)。过表达GADD45β后,ALV-J的蛋白表达水平显著降低(P<0.05),荧光信号强度也明显低于对照组。然而,干扰GADD45β后,ALV-J的复制水平显著上调(P<0.05),说明GADD45β可以抑制ALV-J病毒复制。本研究首次发现GADD45β具有抑制ALV-J病毒复制的功能,为抗ALV-J的研究提供了新的思路和理论基础。
文摘Immunization with inactivated autoreactive T cells may induce idiotype anti-idiotypic reactions to deplete autoreac-tire T cells,which are involved in autoimmune diseases.However,it is unknown whether attenuated activated healthyautologous T-cell immunization could increase anti-tumor immune responses.To this end,C57B1/6 mice were immunizedwith attenuated activated autologous T cells.The splenocytes from immunized mice showed a higher proliferative abil-ity than that from naive mice.The special phenotype analysis showed that there were more CDS+T cells and CD62L+T cells in immunized mice after 24 h of culture with 10% fetal calf serum complete medium in vitro(P<0.01).Theseresults demonstrated that this immunization may activate T cells in vivo.Furthermore,the splenocytes from immunizedmice revealed resistance to activation-induced cell death(AICD)in vitro.To further study the relative genes that areresponsible for the higher proliferation and resistance to AICD,the expression of Fas/Fas ligand(FasL)and GADD45βwas measured by real-time PCR.The results indicated that GADD45β transcription was higher in the splenocytes fromimmunized mice than that in the naive mice.In addition,the Fas expression showed a parallel higher,but FasL did notchange obviously.To investigate the biologic functions induced by immunization in vivo,a tumor model was establishedby EL-4 tumor cell inoculation in C57/B1 mice.Mice receiving autologous T-cell immunization had significantly inhibitedtumor growth in vivo(P<0.01).This study implicated that immunization with attenuated activated autologous T cellsenhances anti-tumor immune responses that participate in tumor growth inhibition.
文摘Histone deacetylase (HDAC) inhibitors are considered as promising therapeutic agents against several malignant diseases because they inhibit cancer cell proliferation. The stress sensor genes of the growth arrest and DNA damage-inducible protein (gadd45) family exhibit disordered expression in several types of malignant diseases and are thus a novel target for cancer therapy. However, there have been only few investigations of whether HDAC inhibitors affect the expression of gadd45 genes. We examined the effects of a HDAC inhibitor, trichostatin A (TSA), on the time-dependent expression of gadd45 genes in the human colon cancer cell line LS174T. Addition of TSA to LS174T cells induced inhibition of cell proliferation by arresting the cell cycle. We found that TSA treatment of LS174T cells induced rapid upregulation of gadd45β mRNA expression within 15 min, reaching a peak level at 3 h. Although the time-dependent expression pattern of gadd45β mRNA was similar to that of gadd45β mRNA, the peak level of gadd45β was lower than that of gadd45β. TSA treatment also upregulated the mRNA level of p21Waf1/Cip1, a prolif- eration inhibitor, after 3 h, but downregulated the mRNA levels of cyclin D1, a proliferation inducer, after 3 h, and of c-Myc after 1 h. TSA treatment induced a certain level of apoptosis, but the mRNA level of p53, a potent apoptosis inducer, was down-regulated after 3 h. These results suggest that the up-regulation of p21Waf1/Cip1 and apoptosis was independent of p53 and that the early upregulation of gadd45β gene, which precedes the upregulation of p21Waf1/Cip1 and the downregulation of cyclin D1, are important in TSA-treated LS174T cells.
文摘目的研究肝泡型包虫病患者肝脏细胞Gadd45β(growth arrest and DNA damage gene 45β)及细胞周期调控相关基因的表达及意义。方法采集24例肝泡型包虫病患者肝脏标本,分为病灶旁组织(Close)和远端组织(Distance)。用重组TGF-β1(1ng/ml)及泡球蚴匀浆蛋白(1mg/ml)刺激体外培养的HL-7702人肝细胞,分别于30min、1h、6h、12h、24h、48h收集细胞。患者肝脏标本及刺激后收集的细胞均采用Trizol法提取RNA,反转录cDNA,通过实时荧光定量PCR检测目的基因表达并进行分析。结果近旁组织PCNA,TGF-β1,cyclinA1,cyclinE基因相对表达量分别为1.934±1.337、1.155±0.5138、1.367±0.8087、1.384±1.116、1.580±1.005、1.899±1.173和1.478±1.189,显著高于远端组织(1.00±0.00)且差异有统计学意义(P<0.05),而Gadd45β,cyclinB1,cyclinD差异无统计学意义(P>0.05)。用重组TGF-β1(1ng/ml)及泡球蚴匀浆蛋白(1mg/ml)刺激体外培养的HL-7702人肝细胞,引起Gadd45β,cyclinA1,cyclinE表达上调。结论泡球蚴匀浆蛋白刺激,可引起肝细胞Gadd45β及细胞周期相关蛋白表达上调,参与细胞抗凋亡过程。泡球蚴感染晚期,肝脏中仍存在细胞周期素(cyclins)参与的细胞增殖,但同时TGF-β1高表达,并通过Smads信号通路促进肝纤维化相关蛋白表达,促成肝脏泡型包虫病晚期病理变化。
