The Regulation And Relative Gene Study Of Ovariectomized Female Rats Osteoblast Differentiation By NF-κB Signaling Pathway

Objectives:To investigate the osteogenic potential and the regulation gene in osteoporosis.Method:1) The establishment and identification of osteoporotic rats:20femal rats aged3mounth were divided into two groups randomly,and a group were operated ovariectomy(OP group), another was sham-operation(NT group). Serological testing、 Micro-CT testing and uterus weight/body weight were dected3mounths later.2) Isolation、culture and identification of BMSCs:BMSCs were separated and purified by using whole marrow adherence method from both sham-operated and ovariectomized rats. Cell morphous was observed by microscope and then determined by flow cytometer.3) Detection of osteogenic capability:After osteogenic induction, both expression of alkaline phosphatase (ALP) and osteocalcin were examined on day7and day14separately. The formation of calciumnodes was analyzed by alizarin red staining on day21. 4) Screening and identification of the differential expression gene:The effect of nuclear factor-κB (NF-κB) on osteoblast differentiation during osteoporosis was analyzed by gene chip and selected genes from gene chip were confirmed by RT-PCR.Results:1) Compared to the NT group, the production of serum Estradiol in OP group decreased by20.09%(p<0.05); the production of serum ALP and serum TRACP-5b respectively increased by26.03%,55.16%(p<0.05); the Uterus weight/body weight decreased by77.51%(p<0.05); the bone mineral density (BMD)、bone volume/total volume (BV/TV), trabeculae number (Tb.N) respectively decreased by45.64%.57.08%.60.88%(P<0.05); the trabecular spacing (Tb.Sp) increased by252.01%(P<0.05)2) The cell flow cytometer assay showed that CD29and CD90.1expression rate were100%and99.2%, respectively; CD45and CDllb/c were2.9%and0.2%, respectively.3) The ALP reduced46.73%and27.97%on day7and day14in OP group VS NT group (P<0.05), osteocalcin34.63%,47.45%separately (P<0.05).There were less calcium nodes in OP group compared to NT group, the alizarin red staining was obviously attenuated in OP group.4) There were1435differential expression gene (2times difference), and7important genes involving in NF-κB signaling pathway have been demonstrated by RT-PCR, and the mRNA level of TRAF3、CXCL12、CXCR4、TNF-α、Bcl2a1、 BCL-XL increased80.00%、61.71%、72.81%、85.92%、87.19%、38.07%separately (P<0.05); the mRNA level of IL-1β decreased69.62%(P<0.05). Conclusions:1) There was a significant change in the bone of ovariectomized rats, and osteoporosis models were established successfully;2) The rat bone marrow mesenchymal stem cells were extracted successfully using the whole marrow adherence method;3) The osteogenic potential of BMSCs from OP group was decreased;4) There were several differential expression genes during osteoblast differentiation in OP group compared to NT group, and NF-κB plays an inhibitory role in osteoblast differentiation during osteoporosis.