The Effects And Mechanisms Of Sufu Knockout On Osteoclastogenesis And Titanium Particle-induced Osteolysis

Part one Construction and verification of conditional Sufu gene knockout mice[Objective]To build the LysM-Cre+/+;Sufufx/fx mice and verify the efficiency of the Cre on knocking out Sufu gene in myeloid cells,and to explore the effect of Sufu gene knockout on Hedgehog(Hh)pathway.[Methods]Male mice of LysM-Cre+/+;Sufufx/+ that express Cre recombinant by Lysozyme promoter was mated with female mice of LysM-Cre+/+;Sufufx/+ to obtain myelin-specific Sufu conditional knockout mice(LysM-Cre+/+;sufufx/fx);Genotypes of mice were identified by PCR.Expression levels of Sufu gene in the spleen of Sufu conditional knockout mice and control mice were detected by RT-PCR.Primary mononuclear macrophages(BMMs)were isolated from the bone marrow of the mice,and mRNA levels of Sufu and target genes of the Hh pathway were detected by RT-PCR.[Results](1)The male mice of LysM-Cre+/+;Sufufx/+ were mated with female mice of LysM-Cre+/+;Sufufx/+ to obtain LysM-Cre+/+;Sufufx/fx mice;(2)The mRNA expression of Sufu gene in the spleen in SufuCKO group and control group was(0.19±0.10)and(1.00±0.35),and the difference was statistically significant(P<0.01).(3)The mRNA expression of Sufu gene in BMMs in SufuCKO group and control group was(0.18±0.01)and(1.00±0.02),and the difference between the two groups was significant(P<0.01).(4)The mRNA expression values of Gli1 gene in BMMs in SufuCKO group and Contrl group were(8.40±1.51)and(1.00±0.49),respectively,with statistically significant differences(P<0.01).(5)The mRNA expression of Ptch1 gene in BMMs in SufuCKO group and Contrl group was(1.75±0.20)and(1.00±0.03),respectively,and the difference was statistically significant(P<0.01)[Conclusion]As a negative regulation gene of Hh signaling pathway,Sufu plays a role in inhibiting Hh signaling pathway.When Sufu gene was conditionally knocked out,Sufu expression in spleen and BMMs was significantly decreased,and Hh signaling pathway was activated,and the expression of target genes Gli1 and Ptch1 in this signaling pathway was enhanced.Part two The study on the mechanism of conditional Sufu gene knockout in BMMs inhibiting osteoclast formation in vitro[Objective]To investigate the inhibitory effect and its related mechanism of conditional Sufu gene knockout on osteoclast formation in BMMs in vitro.[Method]The primary BMMs of lysm-cre+/+;Sufufx/fx mice and the control group mice were induced in vitro.TRAP staining,ghost pen cyclic peptide staining and DAPI staining were used to detect the formation of osteoclasts.RT-PCR was used to detect the expression of genes related to osteoclasts,and western-blot was used to detect the specific nuclear proteins NFATc1 and c-fos in the differentiation process of BMMs cells into osteoclasts induced by RANKL.[results]TRAP staining showed that conditional Sufu gene knockout inhibited osteoclast formation in BMMs in vitro.The number of osteoclasts in the control group and SufuCKO group was(259.00±21.74)and(72.33±9.98)respectively,and the difference of them was statistically significant(P<0.01).The percentage of area of osteoclast induced in the Contrl group and SufuCKO group was(0.64±0.09)and(0.12±0.06),respectively,and the difference was statistically significant(P<0.01).Ghost pen cyclic peptide staining and DAPI staining showed that conditional knockout Sufu inhibited the formation of osteoclast cell membrane and nucleus in BMMs in vitro.The number of osteoclasts in SufuCKO group and control group was(54.00±3.74)and(183.67±7.41),respectively,the difference was statistically significant(P<0.01).The percentage of area of osteoclasts in SufuCKO group and control group was(0.12±0.02)and(0.71±0.05),respectively,the difference was statistically significant(P<0.01).Conditional Sufu knockout inhibited mRNA expression of genes related to osteoclast development in BMMs in vitro.RT-PCR showed that NFATc1 mRNA expression in osteoclasts was(0.29±0.02)in SufuCKO group and(1.00±0.05)in Ctrl group,and the difference was statistically significant(P<0.01).Expression of C-fos mRNA in SufuCKO group and control group was(0.23±0.02)and(1.00±0.06)respectively,and the difference was statistically significant(P<0.01).Expression of Oscar mRNA in SufuCKO group and control group was(0.22±0.01)and(1.00±0.03)respectively,and the difference was statistically significant(P<0.01).Expression of Acp5 mRNA in SufuCKO group and control group was(0.25±0.02)and(1.00±0.04)respectively,and the difference was statistically significant(P<0.01).Expression of Ctsk mRNA in SufuCKO group and control group was(0.27±0.01)and(1.00±0.08)respectively,and the difference was statistically significant.Expression of Dcstamp mRNA in SufuCKO group and coltrol group was(0.21±0.02)and(1.00±0.04)respectively,and the difference was statistically significant(P<0.01).Expression of Atp6v0a3 mRNA in SufuCKOgroup and control group was(0.26±0.02)and(1.00±0.02)respectively,and the difference was statistically significant(P<0.01).Expression of Atp6v0d2 mRNA in SufuCKO group and control group was(0.28±0.02)and(1.00±0.05)respectively,and the difference was statistically significant(P<0.01).Conditional knockout Sufu inhibited the expression of osteoclast specific genes NFATc1 and C-fos protein in vitro.After 1 day of induction,the values of C-fos protein in the control group and SufuCKO group were(1.88±0.07)and(1.38±0.12)respectively,and the difference was statistically significant(P<0.01);The values of NFATc1 in control group and SufuCKO group were(2.90±0.35)and(1.99±0.10)respectively,and the difference was statistically significant(P<0.05).After 3 day of induction,the values of C-fos protein in the control group and SufuCKO group were(2.38±0.26)and(1.52±0.17)respectively,and the difference was statistically significant(P<0.05);The values of NFATc1 in control group and SufuCKO group were(3.74±0.42)and(2.56±0.34)respectively,and the difference was statistically significant(P<0.05).[Conclusion]M-CSF and RANKL can induce osteoclast formation and related gene expression and specific protein formation.Conditional Sufu gene knockout in BMMs inhibited osteoclast formation induced by M-CSF and RANKL,as well as related gene expression and specific protein formation.Part three Sufu gene knockout in BMMs inhibited titanium particle-induced osteolysis of the skull in vivo[Objective]To establish a mouse skull osteolysis model and investigate whether conditional Sufu gene knockout in BMMs in vivo can inhibit osteolysis induced by titanium particles.[Methods]The Micro-CT and 3D reconstruction were used to observe the skull dissolution defects of mice,and software was used to analyze the number of pores and void area values in skull BV/TV and ROI in each group.HE staining was performed on bone tissue sections to observe the formation of holes in the bone tissue of the skull of mice.Image pro-plus 6.0 software was used to analyze the erosion area of holes in the skull of mice in each group.The formation of osteoclasts in the skull was observed by TRAP staining,and the number of osteoclasts in each group was analyzed by Image pro-plus 6.0[Results]The image of Micro-CT reconstruction showed that in the control group,bone loss induced by titanium particles was clearly observed at the sagittal line of the cranium.In the Sham group,the BV/TV?number of pores and void area in ROI were respectively[(79.15±1.28)%,(20.67±3.40)and(3.68±0.58)%].In the control group,the BV/TV,number of pores and void area in ROI were respectively[(31.30±3.61)%,(94.33±6.55),and(36.10±3.68)%],and the differences were statistically significant compared with Sham group(P<0.01).In the SufuCKO group,the BV/TV?number of pores and void area in ROI were respectively[(70.91±1.77)%,(36.33±3.68),(10.29±0.93)%],and the differences were statistically significant compared with control group(P<0.01).HE staining showed that there were significantly more bone erosion holes on the skull surface of the control group,with different shapes and sizes of holes.Compared with the control group,the formation of bone erosion holes on the skull surface of Sham group mice and SufuCKO group mice was significantly reduced,and the number and shape of holes were smaller.The erosion area values of the mouse skull holes in Sham group and control group were respectively(0.90±0.27)%and(43.48±4.93)%,and the difference was statistically significant(P<0.01).The erosion area of skull cavity was(4.81±1.28)%and(43.48±4.93)%respectively in SufuCKO group and control group,and the difference was statistically significant(P<0.01).TRAP staining showed that there were more TRAP positive cells in the bone erosion area of the control group,with different sizes and irregular shapes,which were osteoclasts.The number of TRAP positive cells in SufuCKO group decreased compared with control group,as well as cell morphology.The number of osteoclasts in control group and Sham group was(44.17±4.37)and(4.00±1.41)respectively,and the difference was statistically significant(P<0.01).The number of osteoclasts in control group and SufuCKO group was(44.17±4.37)and(9.50±1.98),respectively,and the difference was statistically significant(P<0.01).[Conclusion]The conditional Sufu gene knockout in BMMs in vivo can reduce the formation of holes induced by titanium particles and the occurrence of osteolysis,which playing a role in preventing bone erosion.In the mouse skull osteolysis model,conditional Sufu gene knockout in BMMs in vivo can inhibit the formation of osteoclasts and achieve the inhibition of titanium particle induced skull osteolysis.