AIM: To investigate the role of human La protein in HBV mRNA expression.METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping ...AIM: To investigate the role of human La protein in HBV mRNA expression.METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen(HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR.RESULTS: SEC products containing U6+1 snRNA promoter,and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+l-hLa833 was the most efficient,which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+l-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HI3s and HBe mRNA levels were significantly decreased by 8-and 66-fold in U6+l-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs.CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease ofHBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.展开更多
AIM: To explore the changes of nuclear factor-kappa B (NF-κB) DNA-binding activity, the expression of intercellular adhesion molecule-1(ICAM-1) regulated by IMF-κB at various times and to evaluate the effects of pyr...AIM: To explore the changes of nuclear factor-kappa B (NF-κB) DNA-binding activity, the expression of intercellular adhesion molecule-1(ICAM-1) regulated by IMF-κB at various times and to evaluate the effects of pyrrolidine dithiocarbamate (PDTC) on trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. METHODS: TNBS of 0.6 mL was mixed with ethanol of 0.3 mL solution and instilled into the lumen of the rat colon. The rat models were divided into 6 groups, which were killed at 24 h, 3, 7,14, and 21 d after enema. Colonic inflammation and damage were assessed by macroscopical and histological criteria. Activity of NF-κB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA). Expression of ICAM-1 was detected by in situ hybridization (ISH) and immunohistochemistry (IH). Then various doses of PDTC were injected into rat abdomen 30 min before enema with TNBS/ethanol as pretreatment. The rats were killed 4 h after enema and the colonic inflammation, myeloperoxidase (MPO) activity, malondialdehyde (MDA) level, and DNA-binding activity of NF-κB were assessed. Finally, PDTC was injected intraperitoneally after colitis was induced. Changes of morphology were assayed. RESULTS: During the first week, hyperemia, hemorrhage, edema and ulceration of the colonic mucosa appeared with predominant infiltration of leukocytes. Neutrophils, macrophages, lymphocytes infiltrated in mucosa and submucosa 14 d later. Fibroblasts and granuloma-like structures were also obviously seen. The binding activity of NF-κB began to increase at 24 h time point and reached a peak at 14 d, then decreased but still was higher than control group at 21 d (P<0.01). Levels of ICAM-1 mRNA and protein significantly elevated at 24 h and the peak was at 21 d. Pretreatment with PDTC could attenuate the development of inflammation but not by reducing NF-KB activity. This attenuation of inflammation had a positive relationship with the dose of PDTC. PDTC at the dose of 100 mg/kg had no therapeutic effect after colitis was induced. CONCLUSION: NF-κB activation is an important event that may be involved in acute and chronic inflammation development and may contribute to self-protection against early inflammation damage. NF-κB also regulates ICAM-1 expression during colonic inflammation. Pretreatment of PDTC may attenuate the inflammation development. But PDTC has no therapeutic effect after the colitis is induced.展开更多
基金Supported by the MajorPrograms of Health Bureau of Zhejiang Province,No.2002ZD007 and the National Natural Science Foundation of China,No.30371270 and the Major Programs of Department of Science and Technology of Zhejiang Province,No.2003C13015
文摘AIM: To investigate the role of human La protein in HBV mRNA expression.METHODS: Three human La protein (hLa) specific siRNA expression cassettes (SECs) containing U6+1 promoter were prepared via one-step overlapping extension PCR. After transfection with SECs into HepG2 cells, inhibition effects on hLa expression were analyzed by semi-quantitative RT-PCR and Western blotting. Then, effective SECs were screened out and transfected into 2.2.15 cells, a stable HBV-producing cell line. HBV surface antigen(HBsAg) and e antigen (HBeAg) secretions into culture media were detected by microparticle enzyme immunoassay (MEIA) and HBs and HBe mRNA levels were analyzed by semi-quantitative RT-PCR.RESULTS: SEC products containing U6+1 snRNA promoter,and 3 sites of hLa mRNA specific siRNA were obtained successfully by one-step overlapping extension PCR and could be directly transfected into HepG2 cells, resulting in inhibition of La protein expression in both mRNA and protein levels, among which U6+l-hLa833 was the most efficient,which reduced 18.6-fold mRNA and 89% protein level respectively. In 2.2.15 cells, U6+l-hLa833 was also efficient on inhibition of hLa expression. Furthermore, semi-quantitative RT-PCR showed that HI3s and HBe mRNA levels were significantly decreased by 8-and 66-fold in U6+l-hLa833 transfected cells compared to control. Accordingly, HBsAg and HBeAg secretions were decreased partly posttransfection with SECs.CONCLUSION: PCR-based SECs can be used to mediate RNAi in mammalian cells and provide a novel approach to study the function of La protein. The inhibition of La protein expression can result in a significant decrease ofHBV mRNA, which implies that the hLa protein is also involved HBV RNA metabolism as one of the HBV RNA-stabilizing factors in human cells.
基金Supported by a Grant From Health Department Foundation of Guangdong Province, No. A2003554
文摘AIM: To explore the changes of nuclear factor-kappa B (NF-κB) DNA-binding activity, the expression of intercellular adhesion molecule-1(ICAM-1) regulated by IMF-κB at various times and to evaluate the effects of pyrrolidine dithiocarbamate (PDTC) on trinitrobenzene sulfonic acid (TNBS)-induced rat colitis. METHODS: TNBS of 0.6 mL was mixed with ethanol of 0.3 mL solution and instilled into the lumen of the rat colon. The rat models were divided into 6 groups, which were killed at 24 h, 3, 7,14, and 21 d after enema. Colonic inflammation and damage were assessed by macroscopical and histological criteria. Activity of NF-κB DNA-binding was analyzed by electrophoresis mobility shift assays (EMSA). Expression of ICAM-1 was detected by in situ hybridization (ISH) and immunohistochemistry (IH). Then various doses of PDTC were injected into rat abdomen 30 min before enema with TNBS/ethanol as pretreatment. The rats were killed 4 h after enema and the colonic inflammation, myeloperoxidase (MPO) activity, malondialdehyde (MDA) level, and DNA-binding activity of NF-κB were assessed. Finally, PDTC was injected intraperitoneally after colitis was induced. Changes of morphology were assayed. RESULTS: During the first week, hyperemia, hemorrhage, edema and ulceration of the colonic mucosa appeared with predominant infiltration of leukocytes. Neutrophils, macrophages, lymphocytes infiltrated in mucosa and submucosa 14 d later. Fibroblasts and granuloma-like structures were also obviously seen. The binding activity of NF-κB began to increase at 24 h time point and reached a peak at 14 d, then decreased but still was higher than control group at 21 d (P<0.01). Levels of ICAM-1 mRNA and protein significantly elevated at 24 h and the peak was at 21 d. Pretreatment with PDTC could attenuate the development of inflammation but not by reducing NF-KB activity. This attenuation of inflammation had a positive relationship with the dose of PDTC. PDTC at the dose of 100 mg/kg had no therapeutic effect after colitis was induced. CONCLUSION: NF-κB activation is an important event that may be involved in acute and chronic inflammation development and may contribute to self-protection against early inflammation damage. NF-κB also regulates ICAM-1 expression during colonic inflammation. Pretreatment of PDTC may attenuate the inflammation development. But PDTC has no therapeutic effect after the colitis is induced.
