摘要
目的明确甘氨酸对缺氧肾细胞保护作用的分子靶点。方法分别定点突变甘氨酸受体（glycine receptor α1，ClyR α1）配基结合位点202位酪氨酸（Thr）为苯丙氨酸（Phi）和亮氨酸（Leu），野生型及突变型GlyR α1转染入人胚胎肾细胞（HEK293细胞）细胞，观察GlyR α1突变是否影响甘氨酸和士的宁对缺氧肾细胞的保护作用；RT—PCR扩增犬肾细胞（MDCK细胞）内人源性GlyR α1的同源基因，设计4对短链寡核苷酸（siRNA），观察RNAi抑制内源性GlyR α1表达对甘氨酸细胞保护作用的影响。结果细胞ATP耗竭3h后，甘氨酸处理组中，转染了正常GlyR α1、苯丙氨酸突变的GlyR α1及亮氨酸突变的GlyR α1的HEK293细胞的LDH释放率分别为（41．17±2．61）％、（62．67±4．53）％及（41．33±6．02）％。士的宁处理组中，分别有（44．50±5．97）％、（45．33±6．60）％及（59．17±6．97）％的LDH自上述3种转染细胞中释放到培养介质。4对siRNA对靶基因表达的抑制不完全相同，最强抑制作用达70％以上，细胞LDH释放率随GlyR α1表达水平下降而升高。结论GlyR α1配基结合位点的不同突变，影响了不同配基的保护作用。抑制内源性GlyR的表达减弱了甘氨酸对ATP耗竭细胞的保护作用。甘氨酸发挥细胞保护作用时，其靶分子为GlyR。
Objective This study is aimed to clarify whether GIyR （glycine receptor） acts as a molecular target in eytoprotection of glycine. Methods The vector GlyR α1-pcDNA3.1 （b） was constructed. Tyr202, the ligand binding site in GlyR α1, was mutated to Phe or Leu, respectively. The peDNA3. 1 （b） vectors containing wild-type or mutated GlyR α1 were respectively transfected into HEK293 cells to observe whether the mutation of GlyR α1 affects cytoprotection on anoxic renal cells by glycine and strychnine. The human homological GlyR α1 gene was amplified in MDCK cell line by RT-PCR. And four GlyRα1 siRNAs duplexes designed to target the identified sequences were transfected into MDCK cells, respectively. The cytoprotection effects by glycine were surveyed after inhibition of endogenous GlyR α1 under the condition of cellular ATP depletion. Results In the presence of glycine, 3 h after ATP depletion, the release rate of LDH from HEK293 cells transfected with wild GlyR α1, T202F mutative GlyR ctl or T202L mutative GlyR α1, was 41.17 ± 2.61%, 62. 67 ±4. 53 % and 41.33 ±6. 02 %, respectively. Similarly, in addition of strychnine to the above cells with 3 h ATP depletion, the percentage of LDH release was 44. 50±5.97%, 45.33 ±6. 60% and 59. 17 ±6.97%. Differently, when knocked down by 4 GlyRctl siRNAs, the transcription and protein expression of GlyR α1 was interfered and reduced to various degrees. The most inhibitive effect on GlyR α1 expression was more than 90 %. The suppression of GlyR α1 exression attenuated the cytoprotection of glycine on ATP-depleted cells. Conclusion Mutation of ligand-binding site in GlyR α1 reduces the ligand-mediated cytoprotection, which results from the lowered affinities ligand binding to its receptor. Suppression of endogenous GlyR expression attenuates the eytoprotection of glycine on ATP-depleted cells. Our results demonstrated that GlyR is a key molecular target in cytoprotection of glycine.
出处
《医学分子生物学杂志》
CAS
CSCD
2009年第2期124-129,共6页
Journal of Medical Molecular Biology
关键词
甘氨酸受体
ATP耗竭
定点突变
RNA干涉
glycine receptor
ATP depletion
cytoprotection
mutation in site
RNA interference
