| Objective:The aims of this study were to determine whether the cytoprotection of glycine was mediated via glycine receptor (GlyR) during ATP depletion and to observe the pathologic changes in cell metabolism and proliferation during ATP depletion and recovery, which were benefit to reveal the mechanism of the cytoprotection by glycine.Method:The protective effects of glycine on ATP-depleted cells.The cDNA encoding human GlyRal was constructed into eukaryotic expression vector pcDNA3.1(b). By lipofection (lipofectAMINE?2000), the reconstituted vector was co-transfected into human embryonic kidney (HEK) 293 cells, which lack native GlyR, with pEGFP-C1 vector as a selection marker. The transfection efficiency was assayed with Flow Cytometer and Confocal Microscope. The expression of GlyRal was detected by RT-PCR and Western blot. The wild type and GlyR-transfected HEK293 cells were exposed to 3 hours of ATP depletion, then the cytoprotective effects of glycine and strychnine was observed on the ATP-deprived cells by the release of lactatedehydrogenase (LDH) from the cells and the uptake of marker compounds including PI (molecular weight = 668), 3 kDa fluorescein dextrans and 70 kDa rhodamine B dextrans. The cell morphological and the ultrastructural changes after ATP depletion were assessed by light microscope and electron microscope. Trypan blue (TB) exclusion assay was performed to test the cell viability during ATP depletion. The Madin-Darby canine kidney (MDCK) cells were used as the positive control.The affects of RNA interference with native GlyRal on the cytoprotection of glycine.Fourteen different sense and antisense primers were designed based on the human GlyRal gene sequence and the homological GlyRal gene fragments were amplified in MDCK cell line by RT-PCR. According to the criteria for siRNA design, 4 pairs of siRNAs (siRNA1-4) were applied to target the identified GlyRal DNA sequence in MDCK cells. Then the siRNAs were subcloned into pSilencel.0U6 vector and transfected to MDCK cells with LipofectAMINE?2000. After transfection for 24 and 48 hours, the cells were harvested to detect the expression of GlyRal gene by RT-PCR and Western blot. At the same time, the siRNA-transfected cells were exposed to ATP depletion. The LDH release was measured to assess the suppression of glycine-mediated cytoprotection by RNAi with endogenous GlyRα1.The affects of mutation of the ligands binding site in GlyRal on the ligand cytoprotection.Tyr202, the ligands binding site in GlyRal, was mutated to Phe or Leu, respectively. And the effects of mutation on cytoprotection of glycine and strychnine were detected after the mutated GlyRal were transfected into HEK293 cells for 24 hours.The affects of glycine on cellular metabolism and proliferation during ATP depletion-recovery.The MDCK cells were suffered ATP depletion for 2 hours in the presence or absence of glycine, then normal DMEM medium was recovered to supply energy to the injured cells. The cell injuries after ATP depletion and during recovery were assessed by light microscope, Trypan Blue (TB) exclusion assay, lactate dehydrogenase (LDH) release assay, the uptake of 3H-TdR and MTT assay, to test the effects of glycine on cellular metabolism and proliferation during ATP depletion and recovery. Result:After transfection of GlyRal-pcDNA3.1 reconstituted vector, Flow Cytometer showed the transfection efficiency of HEK293 cells was about 40%. RT-PCR and Western blot showed that GlyRal gene was successfully transcribed and expressed in transfected HEK293 cells and wild type MDCK cells. After 3 h ATP depletion, the LDH release from wild type HEK293 cells, transfected 293 cells and MDCK cells were56.58±3.30%, 59.18±8.10% and 80.43±6.53%, respectively, in the absence of glycine. In the presence of glycine, the LDH release from transfected 293 cells and MDCK cells decreased to 35.15±2.61% and 4.96±1.22%. But there was no significant difference in wild type HEK293 cells with/without glycine. The protective effect by strychnine on cultural cell was similar...
|
PDF Full Text Request