The mechanism of growth enhancement of P2X7 receptor on primary cultured rat cortical astrocyte

Ying Liu, Hua Zheng Liang, Jin Jun Sun, Zhu Rong Ye

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Purpose The purpose of the present study was to investigate whether ATP could promote reactive astrogliosis and the role of purine'receptor P2X 7 in the process. Methods Separation and culture of primary astrocytes (As) from cortices of Sparague-Dawley rats were conducted,and the purity was checked by immuno- fluorescence. 5-BrdU. incorporation and flow cytometer were used to detect the proliferation of As after treatment with ATP of different concentrations. Real-time RT-PCR was used to measure the changes of GFAP mRNA, P2X7mRNA, TGF-β1mRNA expression after ATP treatment. The expression of GFAP protein was analyzed by Western blot. ELISA was used to detect the amount of TGF-β1 protein in the supernatant. Results Separation and culture of primary As were successful, the purity of As reached 99%. The proportion of As at s-phase was significantly enhanced after 100 μmol/L ATP treatment. 5-BrdU incorporation showed enhanced positive mitosis ratio after 100 μmol/L ATP treatment. These results indicated that ATP at concentration of 100 μmol/L might induce the proliferation and hypertrophy of As in vitro. ATP at concentration of 500 μmol/L could promote the expression GFAP mRNA and P2X7 mRNA. The content of GFAP was elevated in 2 days with 2′-3′-O-(4-benzoylbenzoyl)-adenosine-5′ -triphosphate(BzATP), a selective agonist of P2X7 receptor, at concentration of 50 μmol/L, 75 μmol/L and 100 μmol/L respectively in the culture media with Ca2+. After cultured with Ca2+ and BzATP for 2 hours, TGF-β1 mRNA reached the first peak, and then reached the second peak at 12 h. TGF-β1 protein in the supernatant reached the first peak at 8 h, and another peak at 48 h. TGF-β1 neutralizing antibody with concentration of 6 μg/mL can partly inhibit GFAP expression induced by P2X7 receptor. Conclusions ATP promotes proliferation and hypertrophy of primary cultured of As,mimicking the change as reactive astrogliosis in vivo. P2X7 activated by BzATP may also induced the same morphological As. BzATP combined with Ca2+ plays an important role in enhancing the transcription of TGF-β1 mRNA and release of TGF-β1 protein into the supernatant.

Original languageEnglish
Pages (from-to)491-497
Number of pages7
JournalFudan University Journal of Medical Sciences
Issue number4
Publication statusPublished - Jul 2007
Externally publishedYes


  • ATP
  • P2X receptor
  • Primary cultured astrocytes
  • Reactive astrogliosis
  • TGF-β

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