| Objective To explore the effect of ATF7ip expression level of Th17 cells on osteoclast differentiation and the regulation of Qianggu Yin on ATF7ip expression.Method 1.Th17 cell extraction and fluorescence identification The enrolled people were divided into three groups:normal group(without medication),osteoporosis group(oral calcium carbonate D3 tablets and calcitriol capsules)and treatment group(oral calcium carbonate D3 tablets,calcitriol capsules and Qianggu Yin).Six months later,fasting venous blood 5mL was taken in the morning,centrifuged and cultured,and Th17 cells were obtained from the three groups,and identified by CD3 and CD8 immunofluorescence.2.Transfection and PCR detection of ATF7ip overexpression plasmid vector Three groups of Th17 cells were transfected with ATF7ip overexpression,and each group was divided into three groups:control,NC and ATF7ip.Total RNA(YEASEA,19201ES60)was extracted and detected by PCR.The reaction procedure was 94℃ 5min---94℃ 30s---6℃ 40s---72℃ 50s Murray-annealing 4℃.A total of 30 cycles for an hour and a half(PCR Amplifier,Bio-Rad).3.Primary osteoclast extraction and co-culture SD rats aged 5-8 weeks were disinfected with alcohol.The long bones of the limbs were aseptically separated,and the soft tissues on the surface were removed.The bone marrow cavity was washed with aseptic PBS with a 5mL syringe until the color was whitened.The flushing fluid was collected into the centrifuge tube for osteoclast culture.Then 3 μ m Transwell plate was selected and 4 osteoclast precursors were planted in the lower chamber.Six groups of cells were inoculated in the upper chamber.Th17 cells in normal group,ATF7ip overexpression in Th17 cells in normal group,Th17 cells in osteoporosis group,ATF7ip overexpression in Th17 cells in osteoporosis group,Th17 cells in treatment group,overexpression of ATF7ip in Th17 cells in treatment group.4.Cell TRAP staining and WB detection Six groups of osteoclasts were stained and counted by TRAP respectively.Six groups of osteoclast progenitor cells were extracted and WB was used to detect the expression of CSK,RANK and MMP9 of osteoclast progenitor cells in the lower ventricle.Result After the treatment,3 cases in the normal group dropped out,4 cases in the osteoporosis group dropped out,and 6 cases in the treatment group dropped out.A total of 77 specimens were collected.1.Th17 cells were identified by CD3 and CD8,and photographed by fluorescence microscope,the treatment group,osteoporosis group and normal group had strong CD3 and CD8 fluorescence;When Th17 cells were transfected,NC used pEX-1 with fluorescent protein label,and fluorescence appeared 24 hours after transfection,which indicated that genes could be transferred into cells under the same operation,and the number of Th17 cells in the three groups was more than that in the Control group after transfection with ATF7ip overexpression plasmid vector,which indicated that Th17 cells extraction and ATF7ip overexpression transfection had better effects.2.PCR results showed that the expression of ATF7ip in the three groups transfected with ATF7ip was higher than that in the Control group,and the difference was statistically significant(P<0.05),which indicated that plasmid vector could be transfected and expressed normally in the three groups of cells.Among the three groups of ATF7ip transfected cells,the expression of ATF7ip in osteoporosis group was the highest,the expression of atf7ip in treatment group was lower than that in osteoporosis group,and the expression of atf7ip in normal group was the lowest,and the difference between the two groups was statistically significant(P<0.05).3.When the osteoclast precursor cells in the lower chamber were detected by TRAP staining,it was found that the number of burgundy positive Th17 cells+ATF7ip in the three groups was more than that in the Control group.For the Control group,the number of burgundy positives in the osteoporosis group was higher than that in both the treatment and normal groups.4.The expression of CSK in Th17 cells+ATF7ip in the three groups was higher than that in the corresponding control group,and the difference was statistically significant(P<0.05).For the control group,the expression of CSK in the osteoporosis group was higher than that in the treatment group,with a statistically significant difference(P<0.05);the expression of RANK in the normal group+ATF7ip was higher than that in the normal group+Control,and the difference was statistically significant(P<0.05).The expression of RANK in the osteoporosis group and the treatment group+ATF7ip was slightly higher than that in the osteoporosis group+Control,the difference was not statistically significant(P>0.05),for the Control group,the expression of RANK in the osteoporosis group was higher than that in the treatment group,and the difference was statistically significant(P<0.05);the expression of MMP9 in the normal group and the treatment group+ATF7ip Compared with the corresponding Control group,the difference was statistically significant(P<0.05).The osteoporosis group+ATF7ip was slightly higher than the osteoporosis group+Control,and the difference was not statistically significant(P>0.05).For the Control group,the osteoporosis group had higher expression of MMP9 In the treatment group,the difference was statistically significant(P<0.05).Conclusions PCR detection results showed that there was a certain correlation between osteoporosis and ATF7ip,and Qianggu Yin had a certain inhibitory effect on its expression.The results of TRAP staining indicated that ATF7ip could promote the formation of osteoclasts,and Qianggu Yin could interfere with the expression activity of ATF7ip and inhibit the formation of osteoclasts.By WB detection,ATF7ip can promote the expression of CSK and MMP9,and there is no obvious correlation with the expression of RANK;compared with the osteoporosis group,Qianggu Yin may inhibit the expression of CSK,RANK and MMP9,so as to achieve the purpose of treating osteoporosis. |
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