The Study On Biological Behaviors Of Bmmscs And SHED Derived From Children With Hypophosphatasia

-7-Hypophosphatasia (HPP) is an inherited disorder characterized by defective bone and teeth mineralization and deficiency of serum and bone alkaline phosphatase activity. According to allelic heterogeneity of ALPL gene, clinical phenotypes have great variety such as early primary teeth missing, bone abnormality and hypercalcemia. Since then, more than 214 distinct ALPL mutations have been described. Tissue-nonspecific alkaline phosphatase encoded by ALPL could play a role in biological mineralization, and have effects on biological behaviors of the related stem cells resulting in hypoplasia of teeth and/or bone. In the present study, we screened the ALPL mutation from a HPP patient, and then cultured and tested the SHED and BMMSCs derived from this HPP patient. We anticipated to explore the effect of ALPL mutation on biological behaviors of SHED and BMMSCs, and to provide the experimental evidence for the HPP pathogenic mechanism.Experiment one: Mutation screening of ALPL gene from the HPP patientIn this experiment, we screened the ALPL exons of HPP patient and detected the mutation by PCR technique and gene sequencing. Examination results revealed that, a missense mutation of T→C transition was found in Exon7 of ALPL gene, which resulted in ganimalon to histidine substitution at 263rd amino acid. And the point mutation was present in heterozygous state. In addition, among the healthy members of the patient's family, as well as in 50 unrelated control individuals, this mutation was not detected. By bibliographic retrieval, this ALPL mutation (c.787T>C, p.Y263H) was never reported, which should be responsible for the phenotype of this HPP patient.Experiment two: The study on biological behaviors of SHED derived from children with hypophosphatasiaBy comparing the biological behaviors of SHED from the deciduous teeth of patients and normal children, we aimed to explore the effects of low activity of TNAP on the proliferation and differentiation of SHED. The results of our study showed that SHED from HPP patient presented the weaker proliferation and differentiation ability compared with SHED from healthy children.Real-time PCR results showed that the expression of ALP, COL1, DSPP were higher in SHED from HPP patient. But RUNX2 expression was lower compared with normal SHED. These results suggested that low activity of TNAP may play a role in the proliferation and differentiation machanism of SHED.Experiment three: The study on biological behaviors of BMMSCs derived from children with hypophosphatasiaIn this study, we isolated and cultured BMMSCs from patients and normal children. By immunocytochemistry test and flow cytometry examination, we identified the origin of these stem cells. We also did some analysis on the proliferation and differentiation abilities of BMMSCs. The results showed that the cells were presented with positive expression of CD29,CD90 and CD105, and negative expression of CD11b,CD14,CD34 and CD45. The results of our study showed that BMMSCs from HPP presented the weaker proliferation and differentiation ability compared with BMMSCs from healthy children.Real-time PCR results showed the results as same as SHED. These results suggested that the proliferation and differentiation machanism of BMMSCs are effected by low activity of TNAP .