OBJECTIVE: To investigate the effect of estradiol (E2)/estrogen receptor 1 (ESR1) on the proliferation of human chondrocytes and explore the molecular mechanism. METHODS: The Ad-Easy adenovirus packaging system was used to construct and package the ESR1-overexpressing adenovirus Ad-ESR1. Western blotting and qPCR were used to detect the expression of ESR1 protein and mRNA in human chondrocyte C28I2 cells. In the cells treated with different adenoviruses, the effects of E2 were tested on the expressions of proteins related with cell autophagy and apoptosis and the phosphorylation of ERK signaling pathway using Western blotting. Immunofluorescence assay was used to observe the intracellular autophagic flow, flow cytometry was performed to analyze the cell apoptosis rate and the cell cycle changes, and qPCR was used to detect the expressions of PCNA, cyclin B1 and cyclin D1 mRNAs. The inhibitory effect of the specific inhibitor of ERK on the expressions of autophagy- and apoptosis-related genes at both the protein and mRNA levels were detected using Western blotting and qPCR. RESULTS: Transfection with the recombinant adenovirus overexpressing ESR1 and E2 treatment of C28I2 cells significantly enhanced the expressions of autophagy-related proteins LC3, ATG7, promoted the colocalization of LC3 and LAMP1 in the cytoplasm, increased the expressions of the proliferation-related marker genes PCNA, cyclin B1 and cyclin D1, and supressed the expressions of cleaved caspase-3, caspase-12 and pERK. RNA interference of ESR1 obviously lowered the expression levels of autophagy-related proteins in C28I2 cells, causing also suppression of the autophagic flow, increments of the expressions of apoptosis-related proteins and pERK, and down-regulated the expressions of the proliferation marker genes. Blocking ERK activation with the ERK inhibitor obviously inhibited the effects of E2/ESR1 on autophagy, proliferationrelated gene expressions and cell apoptosis. CONCLUSIONS The targeted binding of E2 with ESR1 promotes the proliferation of human chondrocytes possibly by inhibiting the activation of ERK signaling pathway to promote cell autophagy and induce cell apoptosis.
Adenoviridae ; metabolism ; Apoptosis ; Autophagy ; Autophagy-Related Protein 7 ; metabolism ; Cell Line ; Cell Proliferation ; Chondrocytes ; cytology ; metabolism ; Estradiol ; metabolism ; Estrogen Receptor alpha ; metabolism ; Humans ; Lysosome-Associated Membrane Glycoproteins ; metabolism ; MAP Kinase Signaling System ; Microtubule-Associated Proteins ; metabolism ; Transfection

Objective To compare the effects of different anesthesia techniques on early prognosis in patients undergoing hip joint replacement. Methods The demographic, preoperative and postoperative data of 478 patients, aged 18-95 yr, of American Society of Anesthesiologists physical statusⅠ-Ⅳ, who underwent elective unilateral hip joint replacement in Tongji Hospital from May 2014 to December 2016, were retrospectively analyzed. Patients were divided into general anesthesia group (group GA, n=197), peripheral nerve block group ( group PNB, n=147) and peripheral nerve block combined with general an-esthesia group ( group PNB+GA, n=134) . The amount of crystalloid solution and colloid solution infused, consumption of sufentanil and requirement for vasoactive agents were recorded during operation. The dura-tion of anesthetic recovery room stay, length of hospital stay before and after operation and total length of hospital stay were recorded. The development of complications within 48 h after operation, therapy after ad-mission to intensive care unit and in-hospital fatality were also recorded. Results Compared with group GA, the intraoperative consumption of sufentanil was significantly decreased in group PNB+GA, and the a-mount of crystalloid solution infused, urine output, consumption of sufentanil, requirement for vasoactive agents and incidence of postoperative hypoxemia, pulmonary infection and acute cerebral infarction were significantly decreased in group PNB+GA ( P<0. 05) . Compared with group PNB+GA, the consumption of sufentanil, requirement for vasoactive agents and incidence of postoperative hypoxemia, pulmonary infec-tion and acute cerebral infarction were significantly decreased in group PNB (P<0. 05). Conclusion Compared with general anesthesia or with peripheral nerve block-general anesthesia, peripheral nerve block is more helpful in improving early prognosis in patients undergoing hip joint replacement.

Objective Indoxyl sulfate (IS) is associated with endothelial damage, NF-κB activation and induces the development of atherosclerosis. The purpose of this study was to investigate the relationship between serum IS levels and the severity of coronary artery stenosis and the relationship among IS and various cardiovascular risk factors. Methods Serum IS concentrations were measured using ultra performance liquid chromatography in 191 consecutive patients presenting with stable angina. The associations between serum IS levels and angio-graphic indexes of the number of diseased vessels, modified Gensini scores and calcium scores were determined. Results Patients with significant coronary artery stenosis were found to have higher serum IS levels than those with normal coronary arteries. Multivariate analysis showed that serum IS levels were found to be independently associated with the presence and severity of coronary artery disease (CAD). Furthermore, statistically significant correlation was observed between the serum IS levels and age, Agatston calcium score, volume calcium score, modifed Gensini score, coronary lesions, coronary disease and Framingham-10 year risk score. Conclusions Se-rum IS levels are significantly higher in the presence of CAD and correlate with the severity of the disease and coro-nary atherosclerosis scores,which suggests that increased serum IS may be involved in the pathogenesis of coronary atherosclerosis.

Objective To investigate the effect of sterol regulating element binding protein (SREBP1c) and its ac-tive form (SREBP1cm) on human protein kinase R-like endoplasmic reticulum kinase (PERK).Methods Re-porter victors of PERK promoter and its truncations were constructed with pGL 3-basic and co-transfected with internal reference pRL-SV40 into cell and luciferase activity was detected .pcDNA3.1 ( -)-SREBP1c or pcDNA3.1 ( -)-SREBP1cm was co-transfected with PERK promoter transcriptional activity core regions and the detection of dual -lu-ciferase reporter gene was used to analyze the regulation of SREBP 1c/1cm on PERK promoter transcriptional activity . The expression level of PERK mRNA and protein were detected by RT-PCR and Western blot .Results PERK pro-moter and truncations were successfully constructed into pGL 3-basic, and PERK promoter core area of transcription-al activity had determined;Dual-luciferase report gene showed that SREBP 1c inhibited PERK promoter transcrip-tional activity and SREBP1cm promoted PERK promoter transcriptional activity .RT-PCR and Western blot showed that SREBP1c decreased PERK mRNA and protein expression , but SREBP1cm increased PERK mRNA and protein expression, which was consistent with the detection of dual-luciferase report gene .Conclusions SREBP1c and SREBP1cm have a opposite regulation effect on PERK promoter transcriptional activity and its expression .

BACKGROUND:Herba epimedi, a traditional Chinese medicine, has a long time in dealing with various orthopedic disorders. Icarinwithmany biological activites is one of the most important compositions of Herba epimedi. OBJECTIVE:Toinvestigate the effects of icarin on osteogenic differentiation of mesenchymal stem cels and the underlying mechanisms. METHODS:Bone marrow mesenchymal stem cels were treated using icarin with or without osteogenic mediumin vitro. Osteogenic differentiation markers, including runt-related transcription factor 2, osteocalcin and osterix, were detected by real time-qPCR. Alizarin red staining was used to measure calcium nodes generated by osteoblasts induced frombonemarrow mesenchymal stem cels. The proximal tibia bone structure of rats fed with icarin (2 mgperday) for 5 weeks was detected and analyzed by MicroCT. RESULTS AND CONCLUSION:Icarin was able to promote the expression of genes related to osteogenic differentiation in the absence or presence of osteogenic induction. Icarin could obviously increase the quantity of calcium nodes whenmesenchymal stem celswere cultured in the osteogenic medium. The animal experiment showed that icarin improved formation of trabecular bone.

Objective To investigate the effect of calcitonin gene-related peptide ( CGRP ) in inducing os-teogenic differentiation of rat precartilaginous stem cells in vitro and the underlying mechanisms. Methods Rat pre-cartilaginous stem cells ( PSCs) were cultured in complete osteogenesis medium containing DMEM/F-12 medium and different concentrations (0, 10-8,10-9,10-10mol/L) of CGRP, the morphology changes of PSCs were observed. The proliferation of PSCs was examined at different time points by CCK-8. All the PSCs were then randomly assigned to an experimental group and a control group. The PSCs in the experimental group were cultured in complete osteogenesis medium with 10-10 mol/L CGRP , while the control group cultured merely in complete osteogenesis medium was re-ceived no special intervention. Both groups were stained by Alizarin Red and the expression of alkaline phosphatase (ALP) was detected. The osteogenic genes (RUNX2,OPN and BGP) were measured by use of RT-PCR. The activa-tion of Wnt/β-catenin signaling pathway was tested by using Western blotting to evaluate the effect of CGRP . Results Compared to the control group ( the concentration of CGRP was 0 mol/L) , the concentration of ALP was significantly higher in the experimental group, calcium deposition was significantly more obvious, and the expression of the osteogenic genes such as RUNX2,OPN and BGP as well as theβ-catenin protein expression were up-regulated significantly. However, CGRP had no effect on cell proliferation. Conclusion CGRP activated Wnt/β-catenin sig-nal pathway and induced osteogenic differentiation of precartilaginous stem cells.

The present paper is aimedto investigate the effect of basic fibroblast growth factor (bFGF) on proliferation, migration and differentiation of endogenous neural stem cell in rat cerebral cortex with global brain ischemia-reperfusion. A global brain ischemia-reperfusion model was established. Immunohistochemistry was used to observe the pathological changes and the expression of BrdU and Nestin in cerebral cortex. RT-PCR was used to measure the NSE mRNA in brain tissue. The results of measurements indicated that in sham operation group, there was no positive cell in cerebral cortex, and the content of NSE mRNA did not change. In the operation group, the expression of BrdU and Nestin increased significantly at the end of the 3rd day, and peaked on the 7th day. NSE mRNA expression did not significantly increase. In bFGF group, compared with sham operation group and model group, the number of BrdU-positive and Nestin-positive cells increased significantly at each time point (P<0. 05), and peaked at the end of the 11th day, and the content of NSE mRNA increased significantly (P<0. 05). This research demonstrated that the proliferation of endogenous neural stem cells in situ could be induced by global cerebral ischemia and reperfu- sion, and could be promoted and extended by bFGF. In additiion, bFGF might promote endogenous neural stem cells differentiated into neurons.
Animals ; Brain Ischemia ; pathology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cerebral Cortex ; cytology ; metabolism ; pathology ; Fibroblast Growth Factor 2 ; pharmacology ; Nestin ; metabolism ; Neural Stem Cells ; drug effects ; Rats ; Reperfusion Injury

Objective To observe the effects of different concentrations of glucose on the proliferation and differentiation of primary osteoblasts.Methods The identification of mouse primary osteoblasts was performed by alkaline phosphatase (ALP)staining and Von Kossa staining.Treating osteoblasts with different dose of glucose (5.5,15.5,25.5 mmol/L),the osteoblasts proliferation,ALP staining,and Runx2,OB markers ALP and OCN mRNA expression were observed.Real-time PCR was used for the determination of Runx2,OB markers ALP and OCN mRNA expression.Results With the increasing glucose concentrations,the osteoblasts cell proliferation was decreased.Compared with 5.5 mmol/L normal glucose,the ALP staining in 15.5 mmol/L group and 25.5 mmol/L group were decreased.The expressions were decreased by (36.7±6.2)％ and (38.3±2.2)％ in Runx2 mRNA,(26.7±7.2)％ and (40.4±4.3)％ in OCN mRNA respectively.ALP in 15.5 mmol/L group was reduced by (33.3±10.2)％,but increased by(50.8±10.4) ％ in 15.5 mmol/L group.Conclusions High glucose may decrease osteoblasts proliferation and activity,which may be one of the key pathogenesis factors of diabetic osteoporosis.

BACKGROUND:Current research has shown that many cellgrowth factors can promote the proliferation and differentiation of epiphysis stem cells, and this regulation is closely related to Sox-9.
OBJECTIVE:To investigate the effects of smal interfering RNA-induced specific silence of Sox-9 gene on the proliferation and apoptosis of epiphysis stem cells.
METHODS:Epiphysis stem cells were isolated from the ring of La Croix with enzyme digestion and purified with magnetic activated cellsorting, identified by immunocytochemistry assay. The cells in good conditions were selected to be transfected by Sox-9 silenced by smal interfering RNA with fluorescent marker, and then were observed under the fluorescent microscope to check the transfection efficiency. Next step, epiphysis stem cells were divided into 2 groups:group one was cultured normal y as control group;group two was transfected by Sox-9 silenced by smal interfering RNA through LipofectamineTM 2000 as experimental group.
RESULTS AND CONCLUSION:The primary epiphysis stem cells were separated and purified, which were of stable growth and tightly attachment. The results of immunohistochemistry and immunofluorescence showed the epiphysis stem cells expressed cel-specific markers, fibroblast growth factor receptor 3. After transfection, reverse transcription-PCR results showed that Sox-9 gene expression in epiphysis stem cellwas inhibited specifical y;3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide results showed that cellproliferation in experimental group decreased significantly compared with the control group (P<0.05), and the cellapoptosis detected by flow cytometry showed that the experimental group increased as compared with the control group (P<0.05). Sox-9 gene plays an important role in regulating the proliferation and apoptosis of rat epiphysis stem cells.

Objective To study the impacts of dynamic compressive stress on the mRNA expression of osteopontin ( OPN ),runt related gene 2 ( Runx2 ),osteocalcin ( OC ),osterix,alkaline phosphatase (ALP) and bone morphogenetic protein 2 (BMP-2) in the osteoblasts of Sprague-Dawley (SD) rats. Methods Osteoblasts extracted from skull periosteum tissue of neonatal SD rats were digested using trypsin and collagenase （Ⅰ）,then were subcultured and amplified in vitro.ALP staining and alizarin red staining were performed to identify the purified cells.The cells were treated with compressive stress at 20,50 or 100 mmHg for 24 h.The expression levels of OPN,Runx-2,OC,osterix,ALP and BMP-2 were measured and quantitatively analysed using a real-time quantitative polymerase chain reaction. Results Under 20 mmHg of dynamic compressive stress the expression levels of OPN,Runx2,OC,osterix,ALP and BMP-2 all were elevated compared with the control group.The peak expression oecured under 50 mmHg pressure. The expression levels did not change significantly compared with the control group under 100 mmHg pressure. Conclusions Moderate dynamic compressive stress can promote the expression of OPN,Runx-2,OC,osterix,ALP and BMP-2 mRNA in osteoblasts,which might be an important mechanism for promoting the union of fractures.