AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astr...AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.展开更多
目的 :对汉族人胶质细胞源性神经营养因子 (GDNF)前体 c DNA进行核苷酸序列分析及功能研究。 方法 :通过RT- PCR法扩增汉族人 GDNF前体 c DNA ,利用昆虫杆状病毒表达系统 (BES)表达此前体 c DNA,原代培养中脑多巴胺能神经元对表达产物...目的 :对汉族人胶质细胞源性神经营养因子 (GDNF)前体 c DNA进行核苷酸序列分析及功能研究。 方法 :通过RT- PCR法扩增汉族人 GDNF前体 c DNA ,利用昆虫杆状病毒表达系统 (BES)表达此前体 c DNA,原代培养中脑多巴胺能神经元对表达产物进行活性测定。结果 :汉族人 GDNF前体 c DNA为截短的 5 5 5 bp的转录体 ,其在昆虫细胞中的分泌表达产物能促进多巴胺能神经元的存活和分化。 结论 :汉族人 GDNF前体 c DNA序列与文献报道的 6 33 bp的序列相比缺失了 78个碱基 ,此 78个碱基的缺失并不影响人展开更多
基金Supported by the Biomedical Research Councilthe Institute of Bioengineering and Nanotechnology,the Republic of Singapore
文摘AIM： The GFAP was traditionally considered to be a biomarker for neural gila （mainly astrocytes and nonmyelinating Schwann cells）. Genetically, a 2.2-kb human GFAP promoter has been successfully used to target astrocytes in vitro and in vivo. More recently, GFAP was also established as one of the several makers for identifying hepatic stellate cells （HSC）. In this project, possible application of the same 2.2-kb human GFAP promoter for targeting HSC was investigated. METHODS： The GFAP-lacZ transgene was transfected into various cell lines （HSC, hepatocyte, and other nonHSC cell types）. The transgene expression specificity was determined by X-gal staining of the β-galactosidase activity. And the responsiveness of the transgene was tested with a typical pro-fibrotic cytokine TGF-β1. The expression of endogenous GFAP gene was assessed by real-time RT-PCR, providing a reference for the transgene expression. RESULTS： The results demonstrated for the first time that the 2.2 kb hGFAP promoter was not only capable of directing HSC-specific expression, but also responding to a known pro-fibrogenic cytokine TGF-β1 by upregulation in a doseand time-dependent manner, similar to the endogenous GFAP. CONCLUSION： In conclusion, these findings suggested novel utilities for using the GFAP promoter to specifically manipulate HSC for therapeutic purpose.
