Construction of lentivirus vector containing human homeobox gene HOXB4 and its expression in human umbilical cord mesenchymal stem cells.
This study was purposed to construct lentivirus vector containing human homeobox gene HOXB4 and explore changes of human umbilical cord mesenchymal stem cells (HUCMSC) after infected with HOXB4 mediated by lentivirus. PCR amplification was performed to obtain HOXB4, which was cloned in lenti-shuttle vector. Four-plasmid lentivirus packaging system was used to transfect HEK293T cells. After 48 h, lentivirus Lenti-HOXB4 was harvested and lentivirus titer was determined. Lenti-HOXB4 was used to infect HUCMSC. The infected cells were observed under inverted fluorescence microscope to determine the optimal multiplicity of infection (MOI). Meanwhile, RT-PCR, immune fluorescence staining, CCK-8 and flow cytometry (FCM) were used to determine the expression of HOXB4 and its effect on cell growth. The results indicated that lenti-HOXB4 was successfully obtained by co-transfecting the 293T cells with four plasmids. The determined virus titer was 3×10(8) TU/ml; when MOI was 20. Lenti-HOXB4 had a high transfection rate in HUCMSC, over 80%. In HUCMSC infected with lenti-HOXB4, the expression of target gene could be detected both at mRNA and protein levels. It could promote the proliferation of HUCMSC. FCM results indicated HOXB4 gene did not significantly influence the surface marker of HUCMSC. It is concluded that HOXB4 gene can promote the high proliferation of HUCMSC and does not significantly influence the expression of the surface marker of HUCMSC.
Department of Hematology, Affiliated Hospital of Qingdao University Medical School, Qingdao, Shandong Province, China.
Yan
Y
Qiao
Chun-Ting
CT
Zhao
Zhu-Zhen
ZZ
Liu
Xian-Qi
XQ
Feng
Li
L
Wang
Shi-Hai
SH
Liu
D049109:Cell Proliferation; D002478:Cells, Cultured; D005434:Flow Cytometry; D005801:Genes, Homeobox; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000166:cytology; D010957:Plasmids; D014157:Transcription Factors / Q000235:genetics*; D014470:Umbilical Cord / Q000166:cytology
C090460:HOXB4 protein, human; D018398:Homeodomain Proteins; D014157:Transcription Factors