Background: Low level laser irradiation (LLLI) stimulates bone regeneration. However, the molecular mechanisms leading to this is not yet understood. The stepwise subtractive cDNA cloning technology has been developed, coupled with DNA homology searched in DNA database is useful to identify the certain gene.
Aim: In order to understand the mechanism, we attempted to identify genes whose expressions are enhanced by LLLI. MC3T3-E1 osteoblastic cells were irradiated with an 830 nm Ga-Al-As diode laser, and a cDNA library was constructed using subtractive gene cloning.
Material and methods: The cDNA library of osteoblasts which was treated by LLLI was constructed. Nucleotide sequences were analyzed and homology searched in a DNA database using BLASTN program to identify the gene with altered expression. Altered mRNA levels by LLLI were confirmed by reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR.
Results: The DNA sequence of a subtracted gene clone MCL129 indicated high homology (99%) with the microtubule-associated protein 1A (MAP1A) gene. Increase in MAP-1A mRNA level by LLLI was successfully confirmed by RT-PCR and real-time PCR.
Discussion: MAP1A has been shown to promote microtubule assembly and its functional expression. Microtubules play an important role in cell division, cell shape and polarity, cell movement, intracellular transport, signal transduction, and synthesis and secretion of collagen. Thus, enhancement of MAP1A gene expression by LLLI may be one of the molecular mechanisms involved in accelerating bone formation by LLLI.
Conclusion: LLLI irradiation enhances MAP1A gene expression and modulates microtubule assembly and the functional structure of microtubules, in turn, stimulates osteoblastic proliferation and differentiation.