1.Establishment of a Stable Cell Line Expressing"Toxic" Transient Receptor Potential A1 Channel
Shaopeng WANG ; Lianghui MA ; Jixian LI ; Zhigang PENG ; Jing CAO
Progress in Biochemistry and Biophysics 2008;35(12):1378-1386
Transient receptor potential A1 (TRPAl) is a cold sensitive cation channel, which could also be activated by various pungent compounds. As a transduction channel in a number of sensory modalities, TRPAl expressing in heterogonous systems serves to provide great convenience in pharmacological analysis and functional investigation. Due to cellular toxicity, establishment of stable TRPAl cell line has always been challenging. Nevertheless, the first stable human embryonic kidney (HEK-293) cell line with un-controlled expression of TRPAl was successfully established. It was also confirmed that this stable cell line retained TRPAl expression for more than 25 passages in culture. The functional analysis of the cell response verified the stability and specificity of this novel recombinant TRPAl cell line. Altogether, the data indicated this TRPAl-HEK cell line would be a useful tool for functional analysis of TRPAl and for the development of high throughput screening (HTS) compatible assay in the effort to identify TRPAl modulators.
2.Comparative Proteome Analysis of Plasma Membrane from Different Differential Nasopharyngeal Carcinoma Cell Lines
Tingting SHENG ; Lijun ZHANG ; Xiaohui LIU ; Xiaofang JIA ; Jixian XIONG ; Quanyuan HE ; Rui CAO ; Xia PENG ; Ni SHI ; Songping LIANG
Chinese Journal of Biochemistry and Molecular Biology 2008;24(8):712-718
A subcellular proteomic method was applied to investigate the protein expression profiles of nasopharyngeal carcinoma (NPC) cell lines,CNE1 and CNE2,at various differentiation levels.Plasma membrane (PM) proteins were obtained by Percoll density grade centrifugation and subjected to twodimensional electrophoresis (2DE) followed by PDQuest software analysis.Nine proteins expressed with more than two folds difference were identified by MALDI-TOF-TOF,of which functions involved in cell differentiation,signal transduction,and metabolism.Half of these proteins,such as galectin-1 and annexin Ⅱ,were analyzed with real-time quantitative PCR or Westem blotting.We have tested a proteomic method to study differentiated NPCs at different levels and found several proteins that might be related to their biological characteristics.
3.Establishment of a cell based high throughput screening model for transient receptor potential V3 modulators.
Shaopeng WANG ; Jixian LI ; Zhigang PENG ; Jing CAO
Chinese Journal of Biotechnology 2008;24(11):1895-1901
We established a cell based high throughput screening model by calcium assay on fluorometric imaging plate reader for finding modulators of TRPV3. The TRPV3 expression vector was transfected into HEK-293 and stable cell line expressing TRPV3 was selected with antibiotics. Upon TRPV3 specific modulators stimulated, pharmacological characteristics of TRPV3 over expression cell line were detected by calcium assay on fluorometric imaging plate reader. Assay conditions were optimized and stability of the model was observed. The reliability and accuracy of application to 96 and 384 well format high throughput screening were also evaluated. A stable HEK-293 cell line highly expressing TRPV3 was established. TRPV3 specific modulators could modulate calcium signal through TRPV3 in dose dependent manner. Optimized screening condition was established by assay development. This model is stable and sensitive, and meets the requirement of high throughput screening by Z'factor validation and Spiking test. This cell model can be applied to screening TRPV3 modulators by calcium assay.
Calcium
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metabolism
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Cell Line
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Humans
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Ion Transport
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Kidney
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cytology
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Membrane Transport Modulators
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metabolism
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Models, Biological
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TRPV Cation Channels
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biosynthesis
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genetics
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Transfection
4.Drug screening model of treating pigmentation disorders in tyrp1a transgenic zebrafish
Li LIU ; Siran PEI ; Huali WU ; Qingshun ZHAO ; Jixian PENG ; Jing SHANG
Journal of China Pharmaceutical University 2016;47(6):740-743
To effectively screen treating pigmentation disorders drug, a new transgenic zebrafish drug screening model was constructed. Green fluorescent protein(GFP)were drived by tyrosinase-related protein 1a (tyrp1a)promoter specific expression in melanocyte. Effect of N-Phenylthiourea(PTU)and alpha-melanaocyte stimulating hormone(α-MSH)on the GFP expression and melanogenic of tyrp1a: eGFP zebrafish were photographed under the steromicroscope. Results showed that PTU could significantly inhibit the melanogenic and expression of GFP of zebrafish. As compared with control group, α-MSH treatment resulted in marked stimulation of body pigmentation and expression of GFP. In conclusion, a new transgenic zebrafish drug screening model was successfully established, which can be used for treating pigmentation disorders.