Objective:To investigate the effect and molecular mechanism of high concentration of IL-17A on osteoclast differentiation by inhibiting autophagy of osteoclast precursor cells through PI3K/Akt pathway.Methods:With RANKL (50 ng/ml) inducing osteoclast precursor cells (osteoclast we cells, OCPs), osteoclast differentiation model is set up. In osteoclast differentiation model of high levels of IL-17A (100 ng/ml), RAW264.7 cells were divided into negative control CTR-N group, CTR-R group with RANKL, IL-17A group, IL-17A+LY294002 group. BMMs were divided into negative control CTR-N group with M-CSF, CTR-R group, IL-17A group and IL-17A+LY294002 group with M-CSF and RANKL. IL-17A was applied to OCPs, and tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclast differentiation. The number of autolysosomes was observed under transmission electron microscope. RAW264.7 was treated with IL-17A. Western blot was used to detect the relative expression levels of p-Akt/Akt, p-mtor/mTOR, p-PI3K/PI3K, p-ULK1/ULK1, Cleaved-caspase3/caspase3, Beclin1/β-actin. The apoptosis rate of RAW264.7 cells treated with IL-17A was detected by flow cytometry. OCPs were treated with IL-17A and PI3K inhibitor LY294002, and TRAP staining was used to observe the number of osteoclast differentiation.Results:The TRAP staining showed that the positive ratio for RAW264.7 cells CTR-N group, CTR-R group, IL-17A group was 1.33%±0.58%, 100%±3.01%, 51.11%±4.02% with that of IL-17A significantly lower than CTR-R group ( t=16.970, P<0.05). The positive rates of BMMs in the CTR-N group, CTR-R group and IL-17A group were 1.67%±0.58%, 100%±1.01% and 50.33%±2.52%, respectively, with that of IL-17A group significantly lower than CTR-R group ( t=31.770, P<0.05). Transmission electron microscopy showed that the number of autophagosomes in RAW264.7 cells in CTR-R group and IL-17A group were 3.67±1.53 and 0.67±0.58, respectively, with significant difference between the groups ( t=3.182, P<0.05). While in BMMs cells CTR-R group and IL-17 the numbers of autophagosome were 3.00±1.00 and 0.33±0.58 with significant difference ( t=4.000, P<0.05); Western blot results showed 0.69±0.03、0.69±0.13、1.47±0.13、0.78±0.04、0.66±0.10、0.82±0.03 for RAW264.7 cells CTR-R group Akt/Akt, p-mTOR/mTOR, p-PI3K/PI3K, p-ULK1/ULK1, Cleaved caspase3/caspase3, Beclin1/β-Actin and 0.89±0.04、1.14±0.18、1.87±0.04、0.53±0.09、0.93±0.02、0.54±0.03 for RAW264.7 cells IL-17A group p-Akt/Akt, p-mTOR/mTOR, p-PI3K/PI3K, p-ULK1/ULK1, Cleaved caspase3/caspase3, Beclin1/β-Actin with significant difference ( t=6.708; t= 3.497; t=5.424; t=4.542; t=4.638; t=11.220, all P<0.05); Flow cytometry detection showed that in CTR-R group, IL-17A RAW264.7 cells apoptosis rates of group A were 6.92%±0.62%, 12.12%±0.69%, with significant difference between the two groups ( t=9.747, P<0.05); After using LY294002 TRAP staining, it showed a positive result of 9.00%±2.00%, 158.33%±3.51%, 100%±2.65% and 128.99%±4.01% for CTR-N, CTR-R, IL-17A and IL-17A+LY294002 in RAW264.7 cells respectively with significant difference between IL-17A+LY294002 group and the IL-17A in group A ( t=10.470, P<0.05). For BMMs cells CTR-N, CTR-R group, IL-17A in group, IL-17A+LY294002 group, the positive rate was 8.01%±0.99%, 151.67%±4.51%, 100%±3.61%, with significant difference between IL-17A+LY294002 group and IL-17A group ( t=6.535, P<0.05). Conclusion:High concentration of IL-17A inhibits osteoclast differentiation by inhibiting autophagy of osteoclast precursor cells through PI3K/Akt pathway.

The ultimate goal of synthetic biology is to build customized cells or organisms to meet specific industrial or medical needs. The most important part of the customized cell is a synthetic genome. Advanced genomic writing technologies are required to build such an artificial genome. Recently, the partially-completed synthetic yeast genome project represents a milestone in this field. In this mini review, we briefly introduce the techniques for de novo genome synthesis and genome editing. Furthermore, we summarize recent research progresses and highlight several applications in the synthetic genome field. Finally, we discuss current challenges and future prospects.
Animals ; CRISPR-Cas Systems ; Gene Editing ; methods ; Genetic Engineering ; methods ; Genome, Human ; High-Throughput Nucleotide Sequencing ; Humans