The Preliminary Study Of The Effects Of Krüppel-like Factor4on The Differentiation And Functions Of Osteoclasts

Objective1. To construct pCGF5IEGZ-KLF4ER eukaryotic expression vector containing enhanced green fluorescent protein (EGFP)and zeocin resistance gene and to screen and identification the recombinant transfected murine RAW264.7macrophages.2. To probe the effect of KLF4on murine RAW264.7macrophages differentiating into osteoclast and its function, and to investigate the mechanism.3. To construct transgenic mice model by the copulation of CD11b-Cre mouse with the KLF41oxp mouse to achieve the selective-deletion of KLF4gene in osteoclast and its precursor cells. To observe the osteoclast differentiation and its function after KLF4gene knock-out.Methods1. The full length of KLF4was obtained, a modified ligand-binding domain construct of murine oestrogen receptor (MOR G525R) was fused to KLF4, and then inserted into the pCGF5-IEGZ vector. The recombinant transfected into AW264.7cells and to screen the transfected cells steadily expressing KLF4. Transfection efficiency was determined by using flow cytometry. The expression of KLF4was detected by Western-blot and Q-PCR; localization of the recombinant in cells was determined after DPAI staining.2. Dynamic expression of the KLF4at specific points during differentiation was detected by Realtime-PCR and Western blot. The multinuclear cells (MNCs) after the overexprssion of KLF4in induced cells were examined using tartrate resistant acid phosphatase(TRAP) staining and bone cell culture test was conducted to detect the bone resorbing activity; Expression of the osteoclast specific gene makers, cathepsin K, matrix metallopeptidase-9(MMP-9) and RANK, were analyzed using real-time PCR or Western-blot. 3. KLF41oxp (The loxp sites in introns1and3) mouse mated with CDllb-cre (Cre recombinase under the control of the CD11b promotor) mouse to produce targeted homozygous mice (KLF4fl/fl;CD11b-Cre); The osteoclast precursor cells without KLF4gene were isolated from targeted mouse, and observe their ability of differentiation and bone resorption.Results1. Both the lengths of KLF4PCR product and the KLF4-ER gene fragment form recombinant were consistent with expected, expression vector construction was achieved. The transfection efficiency was95.31%at19d after transfection, the expression of both KLF4protein and mRNA were greater and higher than pCGF5IEGZ groups. The biological effects of KLF4induced by Tamoxifen were better.2. There was no significantly different among the expressions of KLF4at1,3and5day after differentiation in RAW264.7, but the expression of KLF4during the differentiation decreased; The activity of osteoclast, the expression of osteoelast specific genes (cathepsin K, MMP-9, TRAP) and RANK were suppressed or decreased in transfected group. The expression of RANK in transfected group after differentiation.3. Heterozygous F1mice(genotype:KLF4Floxp/+, X/YCre) which obtained by the copulation of CDllb-Cre mouse with the KLF41oxp mouse was identified by PCR; the heterozygous F1mouse mated with KLF41oxp mouse to produce targeted heterozygous mice (KLF4fl/fl;CDllb-Cre) which was also identified by PCR. The osteoclast precursor cells isolated from targeted mouse showed no difference in its differentiation into osteoclast and bone resorbing activity.Conclusion1. The recombinant expression vector pCGF5IEGZ-KLF4ER has been successfully established, it was the first domestic application of pCGF5IEGZ vector containing inducible HBD of ER for RAW264.7transfection.2. KLF4inhibits differentiation and function of osteoclast, and the these effects may be associated with the down-regulation of RANK.3. The transgenic mice which characterized by selective-deletion of KLF4gene in osteoclast and its precursor cells could achieve by the copulation of CDllb-Cre mouse with KLF41oxp mouse; the induced differentiation into osteoclast and the bone resorption function exhibited no abnormal in targeted mice.