| Background : Osteoporosis is a systemic metabolic bone disease,mainly due to bone loss and reduction,bone tissue microstructure destruction,bone fragility increased,resulting in patients prone to fractures,because of the early lack of obvious symptoms,so it is called "silent disease",change living habits and rational medication can control the development of the disease.Osteoporosis is divided into two categories: primary and secondary,and primary osteoporosis is divided into three types: postmenopausal osteoporosis(type I.),senile osteoporosis(type II)and idiopathic osteoporosis(including juvenile type).Secondary osteoporosis is caused by certain disease or medications,and when these causes are eliminated,osteoporosis can improve significantly.The intestinal flora is the largest in the human body and plays an integral role in maintaining overall health,and several studies have shown that the intestinal flora of women with osteoporosis after menopause is altered.At present,the construction of a mouse model of osteoporosis with ovarian disease is currently the most widely used animal model of postmenopausal osteoporosis due to its single modeling factors,high success rate,and the decline of estrogen levels.Objective:In order to verify whether Fecal Microbiota Transplantation(FMT)can improve estrogen-deficient osteoporosis,this project mainly studies the following: 1.To explore the differences in weight,fat,muscle and bone density between ovary mice and normal mice;2.To explore the effect of fecal bacteria transplantation on bone tissue of ovary depleted mice;3.To explore the effect of fecal bacteria transplantation on serum bone conversion rate in mice.Methods: Before the start of the experiment,30 6-week-old C57BL/6 were randomly divided,30 mice were numbered by the toe breaking method,divided into ovarian removal mice(20 mice in the OVX group),mice in the sham group(10 mice),5 mice were placed in each cage,and 2 mice(1st and 10th)were randomly selected in the OVX+ fecal bacteria group,and fresh feces were collected by stimulation for 2-4 h,placed in a sterile EP tube and added 20% Sterile glycerol prolongs storage time and improves microbiota activity,and stores in an ultra-low temperature freezer at-80°C for subsequent microbiota transplantation(FMT).A mouse model of ovarian removal was constructed by bilateral ovary removal surgery on the back,and then the appearance and weight of ovary mice and normal mice were monitored once a week,and fat,muscle and bone density monitoring was performed with dual-energy X-ray bone densitometer(DXA)during the experiment,and the reliability and stability of the ovary mouse model were determined by monitoring these indicators.Using the method recorded in the "Microbiome Experimental Handbook"(first edition)for the preparation of fecal bacteria and oral gavage of mice,0.1ml of bacterial solution containing fresh feces was gavaged to OVX mice,gastric gavage the next day,and after 8 weeks of gavage,the mice were sacrificed,their serum was taken,and the bone resorption index(CTX-1 and TRAP)in serum was detected by ELisa,the bone formation index(ALP)was taken and the right femur was fixed,and Micro-CT was used to detect its bone density and bone microstructure(BV/TV,TB.N,Tb.Th,Tb.Sp and SMI).Results:1.Compared with sham surgical mice,the weight of ovarian osteoporosis was significantly increased,and the weight difference between the mice and the sham surgery group showed a significant increase trend with the increase of mouse age,the fat content showed a significant increase,and there was no significant difference in muscle content,the bone density(BMD)of ovary mice showed a decreasing trend compared with that in the sham surgery group,and the bone density(BMD)after gavage fecal bacteria showed an upward trend compared with that in the OVX+ saline group.2.Micro-CT results of distal femur femur after oral gavage of fecal bacteria showed that BMD increased significantly compared with the control group,Other indicators such as bone volume score(BV/TV),number of bone trabeculae(Tb.N)and trabecular thickness(Tb.Th)showed that the OVX+ fecal bacteria group had an upward trend compared with the OVX+ saline group,but there was no significant difference,and the separation degree of trabecular bone(Tb.Sp)and structural model index(SMI)showed that the OVX+ fecal bacteria group had a downward trend compared with the OVX+saline group.The difference was also not significant.3.Serum bone conversion showed that bone resorption index type I collagen C-terminal peptide(CTX-1)decreased significantly compared with the control group,bone resorption index anti-tartrate acid phosphatase(TRAP)fecal bacteria group had a downward trend compared with the control group,but the difference was not significant,bone formation index ostealine phosphatase(ALP)ovary group had a downward trend compared with the sham surgery group,while the fecal bacteria group had an upward trend compared with OVX+ normal saline group,the difference was still not significant.4.The allogeneic gavage group had better treatment effects than the autologous gavage group.Conclusion: The use of incisions on both sides of the back can successfully construct a mouse model of osteoporosis in ovary-free mice,which has the characteristics of simple implementation,high reproducibility and low casualties,fecal bacteria transplantation(FMT)can indeed improve osteoporosis caused by estrogen deficiency in mice,Micro-CT results showed that bone density(BMD)in the fecal bacteria group was significantly improved compared with the ovarian group,and serum CTX-1 was significantly increased after oophorectomy,while there was no statistical difference in ALP level.It showed that osteoporosis caused by estrogen deficiency in mice was mainly due to increased bone resorption,and bone formation did not change significantly. |
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