The Inhibition Of αB-crystallin Against Activation Of Caspase-3 On Apoptotic Pathway In Cardiomyocytes Undergoing Prolonged Interval Of Hypoxia

ObjectiveαB-crystallin gene was transfected into primary cultured cardiac muscle cells ofrats in vitro. The cultured cells were incubated under hypoxic conditions forprolonged hours and placed into reoxygenational circumstance to evaluate effect ofover expressingαB-crystallin on caspase-3 in cardiomyocytes.Methods1. Cardiac muscles were obtained from the heart of 1-3d SD rats, and digested bytrypsin, finally transported into culture medium. The cells were observed underinverted microscope every day. The cultured cells were identified by staining with HEandα-sarcometric actin immunohistochemically.2. The plasmids of pEGFP-C3 and pEGFP-αB-crystallin-C3 were extracted fromE. coli DH5α. pEGFP-αB-crystallin-C3 was comfirmed by using restriction analysis.Both plasmids were transfected into cultured cardiocytes with cationic liposome(LipofectamineTM 2000). Transfection efficiency was identified by EGFP. Cellsexpressed EGFP or not were all incubated at 37℃in a humiditied atmosphere of 95ï¼…N₂ and 5ï¼…CO₂ at 37℃for 6h, 12h and 18h repectively, after that placed into theincubator of 95ï¼…air and 5ï¼…CO₂ for 12h.αB-crystallin and p17, the product ofcaspase-3, were detected by Western Blot semi-quantitied analysis.Results1. Rat cardiac muscle cells were isolated and cultured successfully in vitro andwere identified by staining with HE andα-sarcometric actin immunohistochemically.2. Plasmids ofpEGFP-αB-crystallin-C3 were transfected into culturedcardiomyocytes successfully, EGFP was observed within the cells throughfluorescence microscope.3.Western Blot semi-quantitied analysis showed that: expression of the proteinαB-crystallin was detected much higher in positive groups than control (P＜0.05) andp17 was lower. And it seemed that the difference between different hypoxia hours wasnot significant (P＞0.05). Conclusions1. Rat cardiac muscle cells were isolated and cultured successfully in vitro.2.αB-crystallin gene was transfected into cultured cardiomyocytes successfully.The expression ofαB-crystallin was increased clearly. Under the condition ofhypoxia/reoxygenation,αB-crystallin could inhibit activation of caspase-3, the markerof apoptosis. When interval ofhypoxia was prolonged, from 6h to 18h, the effectshowed no difference.