采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转...采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.展开更多
Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATC...Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sup><</sup>sub>50</sub> value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.展开更多
目的观察青藤碱对高侵袭性人结肠癌细胞株SW 480移植瘤细胞周期及结肠瘤体组织中环氧合酶-2(COX-2)表达的影响,研究青藤碱防治结肠癌的机制。方法先用5只免疫功能正常的BALB/c-nu/nu裸小鼠建立高侵袭性人结肠癌瘤源,3周后取直径约2 mm...目的观察青藤碱对高侵袭性人结肠癌细胞株SW 480移植瘤细胞周期及结肠瘤体组织中环氧合酶-2(COX-2)表达的影响,研究青藤碱防治结肠癌的机制。方法先用5只免疫功能正常的BALB/c-nu/nu裸小鼠建立高侵袭性人结肠癌瘤源,3周后取直径约2 mm的瘤块移植于20只免疫缺陷的BALB/c-nu/nu2裸小鼠回盲浆膜凹龛部,1周后再随机分为结肠癌组和青藤碱组。青藤碱组按10 m L/(kg·d)灌10%青藤碱治疗30 d,结肠癌组灌0.9%氯化钠注射液对照。测量BALB/c-nu/nu裸小鼠结肠瘤体积、瘤质量并计算肿瘤抑制率;流式细胞仪检测结肠瘤体组织SW 480细胞在G1、G2、M和S期的细胞周期比例;免疫组化法检测结肠瘤体组织COX-2的表达,RT-PCR法检测COX-2 m RNA表达。结果与结肠癌组比较,青藤碱组结肠瘤体积、瘤质量及S期细胞比值明显降低、G1、G2期细胞比值提高,肿瘤抑制率为(39.75%)(P<0.05);COX-2和COX-2 m RNA低表达(P<0.05)。结论青藤碱可能在G1、G2和S期诱导SW 480细胞阻滞、抑制结肠癌细胞株SW 480细胞的有丝分裂,并通过下调COX-2表达而间接影响癌细胞的增殖,具有抗高侵袭性人结肠癌的作用。展开更多
文摘采用慢病毒载体系统构建辣椒素受体基因TRPV1过表达的人结直肠腺癌细胞Caco-2稳定重组株.将双酶切后的慢病毒空载体pCDH和TRPV1全基因PCR产物通过T4 DNA Ligase连接,构建包含TRPV1基因的过表达载体pCDH-TRPV1.将过表达载体pCDH-TRPV1转化DH 5α感受态细菌,大量扩繁后提取过表达载体pCDH-TRPV1的质粒,与psPAX2和pMD两种含有慢病毒包装所必需元件的质粒混合,再与脂质体混合制备脂质体-载体混合液.将脂质体-载体混合液转染至单层的293T细胞中,培养48h进行病毒包装.收集富含慢病毒颗粒的293T细胞上清液,超离心纯化成浓缩病毒,然后再与polybrene一起感染单层Caco-2细胞,通过GFP绿荧光信号来筛选获得TRPV1基因过表达的稳定细胞株.通过Realtime PCR方法和Western-blot检测TRPV1的mRNA表达量及蛋白表达量,结果表明,Caco-2-TRPV1重组细胞株的TRPV1的mRNA表达量及蛋白表达量均显著高于Caco-2-GFP对照细胞(P<0.05).成功构建了TRPV1基因过表达的稳定细胞株,为后续辣椒素降脂机理的研究提供了正向调控细胞模型.
文摘Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sup><</sup>sub>50</sub> value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.
文摘目的观察青藤碱对高侵袭性人结肠癌细胞株SW 480移植瘤细胞周期及结肠瘤体组织中环氧合酶-2(COX-2)表达的影响,研究青藤碱防治结肠癌的机制。方法先用5只免疫功能正常的BALB/c-nu/nu裸小鼠建立高侵袭性人结肠癌瘤源,3周后取直径约2 mm的瘤块移植于20只免疫缺陷的BALB/c-nu/nu2裸小鼠回盲浆膜凹龛部,1周后再随机分为结肠癌组和青藤碱组。青藤碱组按10 m L/(kg·d)灌10%青藤碱治疗30 d,结肠癌组灌0.9%氯化钠注射液对照。测量BALB/c-nu/nu裸小鼠结肠瘤体积、瘤质量并计算肿瘤抑制率;流式细胞仪检测结肠瘤体组织SW 480细胞在G1、G2、M和S期的细胞周期比例;免疫组化法检测结肠瘤体组织COX-2的表达,RT-PCR法检测COX-2 m RNA表达。结果与结肠癌组比较,青藤碱组结肠瘤体积、瘤质量及S期细胞比值明显降低、G1、G2期细胞比值提高,肿瘤抑制率为(39.75%)(P<0.05);COX-2和COX-2 m RNA低表达(P<0.05)。结论青藤碱可能在G1、G2和S期诱导SW 480细胞阻滞、抑制结肠癌细胞株SW 480细胞的有丝分裂,并通过下调COX-2表达而间接影响癌细胞的增殖,具有抗高侵袭性人结肠癌的作用。
