P2X_7受体在原代培养星形胶质细胞生长中的作用

The mechanism of growth enhancement of P2X_7 receptor on primary cultured rat cortical astrocyte

目的探讨ATP是否促进原代培养的星形胶质细胞(astrocytes,As)发生类似反应性星形胶质化的变化以及嘌呤类受体P2X7在该过程中的作用。方法原代As的分离和培养;实时照相观察As形态变化;用Br-dU掺入法及流式细胞观察As增殖变化;用荧光标记实时定量PCR(real-time RT-PCR)检测GFAP mRNA,P2X7mRNA,TGF-β1mRNA表达的变化;用Western blot检测GFAP表达;ELISA检测培养上清中TGF-β1的变化。结果成功分离并培养原代As,免疫荧光鉴定阳性率为99%。不同浓度ATP(50μmol/L,100μmol/L,500μmol/L)作用于As,均可使之表现出类似反应性胶质化的形态改变,表现为胞浆丰富,细胞突起增加且更为明显。ATP100μmol/L作用1d后,As的S期细胞数目明显增加,提示细胞增殖的活跃,5-溴脱氧尿嘧啶核苷(5-bromodeoxyuridine,5-BrdU)掺入法显示细胞核阳性率明显增加。500μmol/L浓度的ATP能促进As的GFAP mRNA,P2X7mRNA表达。用P2X7特异性的受体激动剂2′-3-′O-(4-benzoylbenzoyl)-adenosine-5′-triphosphate(BzATP)50μmol/L,75μmol/L,100μmol/L作用于原代培养的As,在培养基有Ca2+情况下可明显促进GFAP表达。在培养基有Ca2+的情况下BzATP作用2h及12h时As中TGF-β1mRNA升高。培养上清中TGF-β1蛋白的含量在8h及48h均有升高。TGF-β1中和抗体能部分抑制P2X7引起的As GFAP含量的增加。结论100μmol/L的ATP可以促进As增殖。各种浓度ATP均可促进As形态产生类似胶质化反应的变化。P2X7特异性受体激动剂BzATP可引发胶质化样反应,并且在有Ca2+的情况下促进TGF-β1的转录和释放。TGF-β1参与了P2X7受体诱导的类似反应性星形胶质化过程。

Purpose The purpose of the present study was to investigate whether ATP could promote reactive astrogliosis and the role of purine receptor P2X7 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, P2X7 mRNA, 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/Lcould 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 Ca^2＋ . After cultured with Ca^2＋ 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 Ca^2＋ plays an important role in enhancing the transcription of TGF-β1 mRNA and release of TGF-β1 protein into the supernatant.