Method for inducing deposition and maturation of bone comprising a co-therapeutic regimen of LMP-1 and BMP-2
a bone and co-therapeutic technology, applied in the direction of osteogenic factors, drug compositions, peptide/protein ingredients, etc., can solve the problems of high cost, low efficacy, and low efficiency of bone deposition and maturation, and achieve the effects of reducing the risk of aging, and improving the quality of li
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example 1
LMP-1 Dramatically and Synergistically Increases the Responsiveness of Mesenchymal Stem Cells (MSCs) to BMP-2
[0156] It is known that LMP-1 induces bone but produces very small amounts of BMPs. In an effort to elucidate the mechanism behind this phenomena, the hypothesis that LMP-1 increases the responsiveness of cells allowing them to respond to lower levels of BMPs with respect to osteoblastice differentiation, was tested. MSC cultures transfected with the chimeric Ad5F35 vector overexpressiong LMP-1 were treated with either LMP-1 (0, 1, 5, 10 pfu / cell) or BMP-2 (100 ng / mL) alone or in combination. Neither BMP-2 nor LMP-1 alone induce any bone nodule mineralization in human MSCs on day 21, but treating MSCs overexpressing LMP-1 (5-10 pfu / cell) with rhBMP-2 (100 ng / mL) induced dramatic bone nodule mineralization as shown by alizarin red staining. Thus, unexpectedly, concurrent exposure to LMP-1 enabled an ineffective dose of BMP-2 to facilitate bone formation suggesting that LMP-1 ...
example 2
LMP Variant (LMP-2) Does not Induce Nodule Formation in Rat Calvarial Osteoblast Cultures
[0158] Secondary rat calvarial osteoblasts do not spontaneously differentiate without exposure to a stimulus such as glucocorticoid (GC). Cells were transfected with plasmids containing three LMP (hLMP-1; hLMP-2 and hLMP-3) variants and assessed for multilayer mineralized nodule formation 14 days after treatment. Control cells received no treatment and all data were normalized to the control group. Overexpression of hLMP-1 resulted in nodule formation (225 nodules) comparable to that seen with GC (275 nodules). hLMP-3, which is a truncated version of LMP-1 also induced nodule formation (290 nodules). However, hLMP-2 which lacks a 45aa region failed to induce nodule formation (20 nodules), suggesting that the missing 45aa are required for LMP's osteoinductive properties.
example 3
Detection of Smad1 and Phosphorvlated Smad1 (P-Smad1)
[0159] Measuring activation of the BMP signaling pathway requires the ability to measure Smad1 and Smad5. SDS-PAGE separated cytoplasmic and nuclear protein blots were probed with Smad-1 specific antibody. A comparison of cytoplasmic and nuclear protein extracts from untreated pleuripotent cells demonstrated that most of the Smad1 (54 kDa) was detected in the cytoplasmic fraction.
[0160] For detection of P-Smad1 (phosphorylated Smad1) in the nucleus, SDS-PAGE blots of nuclear proteins were prepared from MSCs overexpressing LMP-1 at 4 and 8 hours following infection with Ad5F35-LMP-1. The blots were probed with primary antibody specific to P-Smad1. The binding of primary antibody was detected using the HRP-labeled second antibody after signal development by enhanced chemi-luminescence (ECL). A single band at the apparent size of 54 kDa showed an increase of phosphorylated Smad1 in the nuclear fraction as early as 4 hours in cells ...
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