[Role of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells and its mechanism].
To investigate the role and mechanism of apoptosis in cryoinjury of cord blood hematopoietic stem/progenitor cells, apoptosis of CD34(+) cells, mitochondrial membrane potential (MMP) and Fas antigen expression were detected by flow cytometry (FCM), the Bcl-2 protein expression was detected by immunohistochemistry, caspase-3 expression was determined by Western blot and caspase-3 activity analysis, colony-forming units (CFU) was performed by semi-solid methylcellulose culture. The results showed that when cells were store at -196 degrees C for 2 weeks or 4 weeks, apoptotic cells increased, gel electrophoresis displayed typical DNA ladder, and CFU decreased by 25.2% and 30.1%. The value of MMP reduced and expression of Bcl-2 protein was down-regulated during the freeze-thaw process, but the Fas antigen expression was not effected. However, only the 32 kD inactive caspase-3 proenzyme was detected in freshly isolated CD34(+) cells. After freeze-thaw, caspase-3 was activated and a cleavage of 20 kD protein was detected. Cryopreserved cells showed a 1.2-fold and 1.5-fold increase in caspase-3 activity, respectively. It is concluded that apoptosis plays an important role in cryoinjury of cord blood hematopoietic stem/progenitor cells, which triggers a mitochondrial apoptotic pathway that is caspase-dependent but does not require death receptors, where caspase-3 is the key effector.
Xiao
Juan
J
Institute of Hematology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. juanxiao@sina.com
Zou
Ping
P
Huang
Shi-Ang
SA
Hu
Zhong-Bo
ZB
Liu
Ling-Bo
LB
You
Yong
Y
D018952:Antigens, CD34 / Q000032:analysis; D017209:Apoptosis; D001793:Blood Preservation; D053148:Caspase 3; D020169:Caspases / Q000032:analysis; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D019014:fas Receptor / Q000032:analysis
D018952:Antigens, CD34; D019253:Proto-Oncogene Proteins c-bcl-2; D019014:fas Receptor; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases