ABSTRACT: To investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways.
We amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell phenotype, cell differentiation capacity into lineages of the 3 germ layers and expression levels of pluripotency-associated genes were investigated.
Smad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages maintained the phenotype of stem cells with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to differentiate into cell lineages of the 3 germ layers.
BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 10/2012; 32(10):1383-8.
ABSTRACT: To study the characteristics of lymphocyte nuclear factor kappa B (NF-κB) signal transduction kinase-related molecular mRNA differential expressions at various month age segments in aging process and the intervening effect of Epimedium flavonoids (EF) on it.
Sixty SD rats were divided into six groups, according to animals' age, i.e., the 3 days (d) group, the 4 months (m) group, the 10 m group, the 18 m group, the 27 m group, and the 27 m+EF group. RNA was extracted from separated splenic lymphocytes. Adopting NF-κB signal path functional genome oligonucleotide gene-chip (128 related genes), the integral characteristics and differences of NF-κB signal transduction kinase-related mRNA expressions were determined, and the intervening effect of EF was examined.
The mean level of the NF-κB signal transduction kinase-related mRNA expressions in rats' splenic lymphocytes lowered with aging; the highest expression was presented at 3 d after birth, and then, it lowered gradually, with the lowest level at 18 m or 27 m. After EF intervention, the expression level was raised to the 10-18 m level in the aged rats.
The changing rules of lymphocyte NF-κB-signal-transduction-kinase-related mRNA expressions in various stages of aging are helpful for selecting the well time for preventing and intervening aging, and will also give a hint to the molecular index for assessment of senility retarding researches.
Chinese Journal of Integrative Medicine 09/2011; 17(9):704-9. · 0.80 Impact Factor
ABSTRACT: Astragaloside IV (ASI), a pure compound derived from Radix Astragali, is commonly used in degenerative bone diseases such as osteoporosis. Our previous study identified in vivo the osteogenetic effect of Fu Fang Qi She Pills (FFQSP), a Chinese herbal formula containing Radix Astragali from which ASI was extracted. In this study, we investigated the osteogenetic effects of ASI under the conditions of centrifugating pressure on OCT-1 cells. These preosteoblasts were grown in 3D-culture, and treated with ASI at 50 micromol/l with centrifugation at 200 rpm, 500 rpm for 3 and 5 days. Morphocytological examination, morphometry of alkaline phosphatases (ALP) staining was performed. Expression of type I collagen (Col I) was detected by immunocytochemistry assays. ALP, Col1a2, Osteocalcin (OC), and runt-related transcription factor-2 (Runx2) mRNA expression were determined via real-time PCR. The results showed ASI plus 500 rpm for 3 days and ASI plus 200 rpm for 5 days significantly induced osteogenesis related protein and gene expression. We concluded that ASI would promote osteogenesis when cells of preosteoblast OCT-1 were subjected to proper centrifugating pressure and a pertinent period of time.
Pharmazie 01/2011; 66(1):63-8. · 1.01 Impact Factor