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Objective To investigate the osteogenic differentiation potency of ligament cells in thoracic ossification of the ligamentum flavum (TOLF) and analyze further by using transcriptome high-throughput sequencing.Methods Clinically,the patients with non-TOLF and TOLF (n=10 in each group) who underwent surgery in our hospital from October 2015 to April 2016 were included in this study.The primary ligament cells that derived from the two groups were separately cultured and induced osteogenesis with 15％ strength of cyclic mechanical stress for 12h and 24h using a device called Flexcell FX-4000.The ALP activity was determined to evaluate the osteogenesis using quantitative analysis and ALP staining assay.Real-time PCR and westernblotting were used to detect the mRNA and protein expression of osteogenic-related genes including ALP,BMP-2 and Osteocalcin.Then,three patients in each group were included in the study of transcriptome high-throughput sequencing and bioinformatics analysis using Illumina HiSeqTM 2500 sequencing platform to compare further.Results The morphology of the cells that derived from two groups was basically similar,all presented an elongate spindle-shape.To evaluate the ostogenesis,ALP activity assays including quantitative and staining assays were performed.Under microscope,the ALP staining in the TOLF group was higher than non-TOLF group and increased with the longer duration of stress induction.The result of semi-quantitative analysis showed the stained area and positive cells in TOLF group were more than non-TOLF group significantly at 0 h,and were increased with the induction.The results of quantitative analysis showed ALP activity in the TOLF group was significantly higher than non-TOLF group and were increased with the induction significantly all the time.But no significant change in ALP staining or quantitative analysis was found in non-TOLF.The results of real-time PCR indicated that the expression of ostegenic markers above in the TOLF group was more than non-TOLF group significantly except the expression of OCN at 0 h.The expression of the three ostegenic markers in TOLF group was increased with the stress induction for 12 h and 24 h significantly except the expression of BMP-2 and OCN at 12 h.The results of western-blotting indicated that the expression of the three ostegenic markers above in the TOLF group was more than non-TOLF group significantly except the expression of ALP at 0 h.The expression of the three ostegenic markers in TOLF group was increased with the stress induction for 12 h and 24 h,but only the expression of ALP at 24 h was significant.And no significant change in the expression of mRNA and protein was found in non-TOLF group.In the transcriptome analysis,671 genes of TOLF group were up-regulated and 314 genes were found to be down-regulated compared to the control group.In addition,22 significant GO terms associated with upregulated genes were found to be closely related to ossification.Conclusion TOLF ligament cells have high osteogenic differentiation potency,which could express obvious osteogenesis-related gene spectrum,and differentially expressed genes including L1RL1 、PTHLH、DKK1 、BMP6、SPP1 and FGF1 may be related with the osteogenic potency of ligament cells in thoracic ossification.
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Objective To compare the effect of breast conserving surgery and modified radical mastectomy on the clinical efficacy of patients with early breast cancer.Methods CNKI,Chinese Journal Full-text Database (CJFD),China Biology Medicine Disc (CBMdisc),China Dissertation Database (Chinese Dissertation Database,CDDB) google academic and other databases were comprehensive searched.And then the search time was limited to between January 1,2015 and September 1,2017.Key words can be locked for breast conserving surgery,improved surgery for breast surgery,case-control studies,etc.,and then meet the conditions of the literature into the study,for a retrospective analysis.The authors reviewed the literature independently,extracted data and evaluated the risk of bias,and used Review Manager 5.3 software for systematic analysis.Results A total of 1 093 patients with early breast cancer were enrolled in the study.The Meta-analysis showed:there was a significant difference in operation time between the two groups(MD =-30.71,95％ CI:-31.96--29.46,P ＜ 0.01);there was a significant difference in intraoperative blood loss between the two groups(MD =-53.30,95％ CI:-55.38--51.22,P ＜ 0.01);there was a significant difference in postoperative hospital stay (MD =-5.66,95％CI:-7.17--5.17,P ＜0.01) and the incidence of complications (OR =0.30,95％ CI:0.19-0.47,P ＜ 0.01)compared with modified radical mastectomy in early breast cancer patients.There was no significant difference between the two groups in the postoperative recurrence and metastasis (OR =0.78,95％ CI:0.54-1.13,P =0.19).Conclusions In the choice of surgical methods,breast-conserving therapy is better than modified radical surgery,and postoperative recurrence and metastasis rate has no significant difference.
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Objective To investigate the effects of different magnitudes of mechanical stress on human interverte-bral disc degeneration. Methods The human intervertebral disc cells were subjected to different magnitudes of mechanical stress (0, 6%, 12%, or 18%elongation) for 24 h using a Flexercell Strain Unit. The mRNA expressions of anabolic genes (col-lagen-1A1, collagen-2A1, aggrecan and versican) and catabolic genes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5) were examined by real-time PCR and Western blot methods. Results The expression levels of collagen-1A1 and collagen-2A1 were increased at 12%of mechanical stress, and collagen-2A1 was decreased at 18%of mechanical stress compared with those of control. The mRNA expressions of catabolic genes, MMP-13 and ADAMTS-5, were increased at 12%and 18%of mechanical stress than those of control. The mechanical stretch induced a magnitude-dependent increase in ADAMTS-4 synthesis, which was finely tuned by stretching-triggered activation of distinct mitogen-activated protein kinase cascades. Specifically, an ERK1/2 specific inhibitor, U0126, significantly inhibited the stretching-induced ADAMTS-4 expression, whereas the inhibitors of p38 and JNK, SP6000125 and SB203580, showed only slightly effect on the stretching-induced ADAMTS-4 expression. Conclusion The different magnitudes of mechanical stretch exhibited different effects on the bio-logical behavior of intervertebral disc cells, which profoundly affects the intervertebral disc degeneration.