| ?Objection?We showed a significant decrease in body weight compared with control group during the construction of mice with myeloid monocytes knockout adenosine kinase(ADK).Therefore,we will use myeloid monocytes cell-specific knockout ADK micefor osteoclast(OC)differentiation and bone resorption of the study,exploring myeloid monocytes knockout ADK on impact of bone metabolism.At the same time,theeffect of Zhenzhu Tiaozhi Capsule(FTZ)on osteoclast differentiation and bone resorption was evaluated under the established osteoclast culture system.?Method?1.Identification of myeloid monocytes knockout ADK mice ADKf/f?ADKf/f Lysmcre/cre?ADKf/f Lysmcre/+ knockout mice were screened out by P CR genotyping.The expression of ADK protein in primary macrophages of mice wa s detected by Western-blot.2.In vitro osteoclast differentiation and bone resorption function detection method In this study,primary bone marrow mononuclear cells(MSCs)were isolated from ADKf/f ? ADKf/f Lysmcre/cre ? ADKf/f Lysmcre/+ mice.In vitro,the primary bone marrow mononuclear cells and osteoclasts were cultured by M-CSF and RANKL.At the same time,the primary bone marrow mononuclear cells of BL/6J mice were collected and treated with ADK inhibitor(ABT-702).We evaluate the effect of myeloid monocytes knockout ADK on the differentiation and bone resorption of osteoclasts in vitro by using the tartaric acid resistant phosphatase specific Trap staining?bone resorption test?RT-PCR detected bone metabolic function-related gene,including TRAP?NFATc1?c-Fos?MMP-9?Cts K?c-Src?CTR gene.3.Methods of bone morphometry in genotype mice The body weight and femur weight of ADKf/f?ADKf/fLysmcre/cre?ADKf/fLysmcre/+mice were measured at 4 months old.The histomorphological changes of bone tissu e were observed by HE staining.Analysis bone mass changes by bone tissue static analysis system.4.Effects of FTZ on osteoclast's differentiation and bone resorption in vitro The bone marrow mononuclear cells of wild-type BL/6J mice were collected by culture system of osteoclasts and treated with drug-serum got from rat.We evaluated the effects of FTZ on the differentiation and absorption of osteoclasts by means of tartrate-resistant phosphatase-specific Trap staining,bone resorption test and RT-PCR detected on TRAP?NFATc1?c-Fos?MMP-9?Cts K?c-Src?CTR gene that were r elated to bone metabolism.?Result?1.ADK knockout efficiency of ADKf/fLysmcre/cremice higher than ADKf/fLysmcre/+mic e and ADKf/fmice(1)PCR was utilized to detect the genotype of mice tail: ADK homozygous bands were 360bp;ADKf/fmice had a clear band at 350 bp,ADKf/fLysmcre/cremice had a cl ear band at 700 bp,ADKf/fLysmcre/+mice had two bands at 350 bp and 700 bp.(2)ADK protein level in myeloid monocytes was detected by Western blot,the date show that compared with ADKf/fgroup,the expression level of ADK protein in ADKf/fLysmcre/cre group was decreased by 69.77%(P<0.01)and ADKf/f Lysmcre/+was decreased by 36.83%(P>0.05).2.In vitro,osteoclast differentiation and bone resorption function were enhanced in myeloid monocytes knockout ADK mice (1)Myeloid monocytes knockout ADK mice increased osteoclast differentiation.Osteoclast culture of different genotypes mice showed that the number of osteoclasts in the ADKf/fLysmcre/cregroup was higher than ADKf/fgroup(P<0.01),however,ADKf/f Lysmcre/+group were no significantly changes(P>0.05).Compared with ADKf/f Lysmcre/cregroup,osteoclasts number of ADKf/fLysmcre/+group was significantly different(P<0.01).ABT-702 intervention culture results showed that the number of OCs in 100 n M group was significantly different from control group and 5% Et OH group(P<0.01).Compared with ADKf/fgroup,the expression of TRAP(P<0.01),NFATc1(P<0.05)gene in ADKf/fLysmcre/cregroup was significantly higher,the expression of c-Fos gene increased but no significant difference(P>0.05).Between ADKf/f group and ADKf/fLysmcre/+group,those gene expression had no obvious change(P>0.05).However,th e expression of TRAP and NFATc1 gene in ADKf/fLysmcre/+group was significantly lower than ADKf/fLysmcre/cregroup(P<0.05)but the expression of c-Fos gene was n o significant difference(P>0.05).The RT-PCR results of group treated with ABT-702 showed that compared with 5% Et OH group,the expression of TRAP and NFATc1 m RNA in 100 n M group was significantly higher than 5% Et OH group(P<0.05)and theexpression of c-Fos gene were increased but no significant difference(P>0.05).(2)Myeloid monocytes knockout ADK enhances osteoclast absorption.The area(P<0.01)and numbers(P<0.05)of pits were significantly change in ADKf/fLysmcre/cregroup,which compared with ADKf/fgroup.Comparison of ADKf/fgroup and ADKf/fLysmcre/+group,there were no obvious change in bone resorption test(P>0.05).But compared with ADKf/fLysmcre/cregroup,ADKf/fLysmcre/+group had significantly change in area and number of pits(P<0.01).ABT-702 intervention bone resorption test's results showed that not only pits area of 5%Et OH group and 100 n M were significant increased but also the numbers significant increased(P<0.01).The expression of MMP-9?Cts K?and CTR gene in ADKf/fLysmcre/cregroup was significantly higher than that in ADKf/fgroup(P<0.01),but the expression of c-Src g ene was no significantly increased(P>0.05).There was statistical significance between ADKf/fgroup and ADKf/fLysmcre/+group in the expression of Cts K gene(P<0.05).Co mpared with ADKf/fLysmcre/cregroup,ADKf/fLysmcre/+group's MMP-9,ctsk,CTR gene expression was statistically significant(P<0.05).Bone resorption related genes showed that compared to 5%Etoh group,100 nm group's MMP-9(P<0.01),ctsk(P<0.05)g ene expression increased,but c-Src,CTR expression had no significant change(P>0.05).3.Changes of bone mass in myeloid monocytes knockout ADK mice Compared to ADKf/fmice,ADKf/fLysmcre/cremice's weight of body and femur we re lightest,with significant difference(P<0.01),ADKf/f Lysmcre/+ mice had no signific ant change(P>0.05).Compared with ADKf/f Lysmcre/cre mice,ADKf/f Lysmcre/+ mice bod yand femur weight were significantly different(P<0.01).ADKf/f group's trabecular bo ne was pink and abundant with compact solid,good continuity of reticulate bone tr abecula.ADKf/fLysmcre/cregroup's trabecular bone was significantly reduced,sparse,fra cture shape.ADKf/fLysmcre/+group between the two groups with trabecular bone slend er,partial fracture.The static parameters of bone tissue showed that ADKf/fLysmcre/+mice and ADKf/fmice were respectively compared with ADKf/fLysmcre/cre,%Tb.Ar?T b.Th?Tb.N?%Ob.S/BS?Ob.N/BS?Tb.Sp?%Oc.S/BS?Oc.N/BS were statistically d ifferent(P<0.01;P<0.05).Similarly,the tibial midline analysis showed compare to th e other two groups,%Ct.Ar and %Ma.Ar of ADKf/fLysmcre/cregroup parameters chan gesignificantly(P<0.01).4.FTZ inhibits osteoclast differentiation and absorption Different concentrations performanced different inhibitory effects on osteoclasts.C ompared with the control group,the number of OCs and pits(P<0.01),the area of o steoclastic bone resorption(P<0.05)in the Alendronate group?FTZ-H group and FTZ-M group were decreased significantly,which lower than FTZ-L group(P <0.01;P<0.05).Compared with Alendronate group,the number of bone lacunae in FTZ-L group was significantly different(P<0.01).OCs' differentiation gene PT-PCR showed that compared with control group,the expression of Trap?NFATc1 gene in FTZ-H group was significantly reduced(P<0.05),the expression of c-Fos gene were not statistically significant(P>0.05).OCs' resorption gene PT-PCR showed that the expression of MMP-9 in FTZ-H group was significantly ruduced than that in control group and th ere was no significant difference in the expression of Cts K,c-Src and CTR gene be tween the two groups.?Conclusion?1.Myeloid monocytes knockout ADK mice enhanced osteoclast cell differentiation function.2.Myeloid monocytes knockout ADK mice enhanced osteoclast absorption function.3.Myeloid monocytes cell knockout ADK mice showed bone mass loss,abnormal bone structure.4.FTZ could improve osteoporosis by inhibiting osteoclast differentiation and absorp tion function. |
PDF Full Text Request