Effects of adriamycin resistance cell-derived exosomes on the proliferation and migration of osteosarcoma cells through drug resistance transmission / 中华骨科杂志

Objective:To explore the relationship and underlying mechanism between exosomes derived from doxorubicin-resistant osteosarcoma cells and MDR1 and miRNAs. Methods:MG63 and U2OS cell lines were selected to construct doxorubicin-resistant strains, and the 50% inhibitory concentration (half maximal inhibitory concentration, IC 50) of drug-resistant and sensitive strains was detected by MTT, and fluorescence staining was performed at intervals of 15 min between 15 and 120 min to detect the change of fluorescence intensity. RT-PCR and Western Blot were used to detect the expression levels of MDR1 P-gp to verify the drug resistance of osteosarcoma cells. Exosomes were identified by particle size analysis and Western Bolt detection. The endocytosis of PKH26-labeled exosomes from doxorubicin-resistant cells was observed, and the proliferation level and migration of exosomes from doxorubicin-resistant cells co-cultured with osteosarcoma cells were detected by MTT assay and cell scratch assay. The differential expression levels of miRNAs in osteosarcoma-sensitive and drug-resistant cells were verified by sequencing and bioinformatics analysis and RT-PCR assay. Tumor growth, serum exosome identification and mRNA expression level of miR-21-5p in tumor-bearing nude mice between normal osteosarcoma cell group and drug-resistant group, drug-resistant+normal exosome group, drug-resistant+drug-resistant+drug-resistant exosome group were observed. MDR1 expression level in tumor tissue was detected by RT-PCR, Western Blot and immunohistochemistry. Results:The IC 50 of two adriamycin resistant strains were 2.21 vs. 11.81 μg/ml and 0.93 vs. 11.81 μg/ml, respectively, and the fluorescence intensity decreased faster than that of normal strains. The relative mRNA expression levels of MDR1 in two cell lines were normal 1.12±0.16, 1.02±0.11 and drug-resistant 2.15±0.10, 2.127±0.12, respectively. The relative protein expression of P-gp was normal 0.92±0.11, 0.73±0.10 and drug-resistant 0.46±0.03, 0.30±0.04, the differences were statistically significant ( P<0.05). Drug-resistant exosomes can enter osteosarcoma cells through endocytosis and concentrate in the cytoplasm when co-cultured with normal strains. Osteosarcoma cells were co-cultured with drug-resistant exosomes at 2, 4, 6, and 8 μg/ml adriamycin, respectively. Compared with normal group, the proliferation level in drug-resistant group was significantly increased. Compared with the normal cell group 35.95±3.92, 6.72±3.55 and the normal exosome group 51.22±5.55, 19.31±1.93, the drug-resistant cell group 54.20±9.32, 19.24±2.88 and drug-resistant exosome group 76.40±5.41, 30.26±4.87, all had significantly higher cell mobility, the difference was statistically significant ( P<0.05). Exosome sequencing and biogenic analysis of 10 highly upregated miRNAs to validate mRNA expression differences between normal and drug-resistant strains by RT-PCR, showing a significant increase in miR-21-5p expression level of drug-resistant strains (5.89±0.26 vs. 0.99±0.06; 1.05±0.07 vs. 8.80±0.93, P<0.05), the difference was statistically significant ( P<0.05). In MG63 and U2OS, the normal cell group and drug-resistant cell group, and the normal exosome group and drug-resistant exosome group were compared, the tumor volume and the terminal tumor weight of nude mice were increased to varying degrees. MRNA relative expression levels of miR-21-5p in serum exosomes of nude mice after drug intervention were 0.86±0.07 and 0.86±0.05 in normal cell group, respectively. The values were 1.13±0.12, 1.14±0.12 in drug-resistant cell group, 0.71±0.05, 0.75±0.03 in normal exosome group, and 0.90±0.07, 0.93±0.04 in drug-resistant exosome group. Compared with normal and drug-resistant strains, the expression levels of normal and drug-resistant exosome groups were increased, with statistical significance ( P<0.05). Conclusion:The exosomes of drug-resistant cells in osteosarcoma could enhance the proliferation level and migration ability of cells through intercellular transfer of MDR1 and miRNAs. The expression of MDR1 and miR-21-5p in drug-resistant cells and tumor-forming nude mouse serum and tumor tissues were up-regulated which suggested that it might be involved in regulating the drug resistance process of osteosarcoma.

Arthroscopic side-to-side repair of transtendinous rotator cuff tears with remnants preserved / 中华创伤骨科杂志

Objective:To evaluate the clinical efficacy of arthroscopic side-to-side suture with remnants preserved in repair of transtendinous rotator cuff tears.Methods:A retrospective study was conducted to analyze the data of 17 patients who had been treated by arthroscopic side-to-side suture with remnants preserved for transtendinous rotator cuff tear caused by trauma at Sports Medicine Center, The Second Hospital Affiliated to Inner Mongolia Medical University from January 2017 to January 2020. There were 11 males and 6 females with an age of (47.9±8.3) years and a duration from injury to surgery of (50.4±21.3) d. Recorded were range of motion and muscle strength of the shoulder, University of California at Los Angeles (UCLA) shoulder function score, Constant-Murley shoulder function score, visual analogue scale (VAS) pain score, re-tears and complications before operation and at the last follow-up.Results:The 17 patients were followed up for (16.5±3.5) months after operation. Retear of the rotator cuff occurred in 2 patients after operation while MRI showed good healing of the rotator cuff in the other patients with no such postoperative complications as infection or wound dehiscence. At preoperation and the last follow-up, respectively, the range of shoulder flexion was 152.9°±8.5° and 172.4°±5.6°, the abductor muscle strength 3.5 (2.6, 4.1) kg and 6.9 (6.3, 8.3) kg, the external rotator muscle strength (3.8±1.0) kg and (5.9±1.6) kg, the internal rotator muscle strength 3.9 (3.4,4.7) kg and 5.2 (4.5,5.9) kg, the UCLA score (13.2±1.9) points and (30.9±2.4) points, the Constant score (40.1±2.8) points and (86.1±4.6) points, and the VAS score (6.7±0.8) points and (0.9±0.6) points, all showing a significant difference between preoperation and the last follow-up ( P<0.05). Conclusion:In repair of transtendinous rotator cuff tears, arthroscopic side-to-side suture with remnants preserved can lead to significantly improved clinical outcomes in range of motion, muscle strength, functional recovery and pain relief.

MicroRNA-141 is regulated by serum of velvet antler to promote the proliferation of bone marrow mesenchymal stem cells in dexamethasone-induced cell model / 中国组织工程研究

BACKGROUND: The proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) can delay the procession of steroid-induced femoral head necrosis. Besides, microRNA-141 (miR-141) is one of the important regulatory factors to promote cell proliferation. In addition, velvet antler is a traditional Chinese medicine which has significant roles in repairing bone and tissue and improving health. OBJECTIVE: To investigate whether velvet antler serum can regulate the expression of miR-141 to promote the proliferation of BMSCs, and further delay or reverse the progression of steroid-induced femoral head necrosis. METHODS: BMSCs were isolated and cultured from Sprague-Dawley rats. The passage 3 BMSCs were transfected with miR-141 mimic or miR-141 inhibitors, and then real-time PCR and methyl thiazolyl tetrazolium (MTT) assay were performed for detecting miR-141 expression and cell proliferation, respectively. The passage 3 BMSCs were divided into three groups: control group (α-MEM), dexamethasone group (α-MEM+1 μmol/L dexamethasone), and velvet antler serum group (α-MEM+1 μmol/L dexamethasone+15% velvet antler serum). Expression of miR-141 mRNA was detected by real-time PCR at 24 hours after intervention. The proliferation ability of BMSCs was evaluated by MTT assay at 24, 48, and 72 hours after intervention. RESULTS AND CONCLUSION: After transfection with miR-141 mimic, the expression of miR-141 mRNA was upregulated, while the cell proliferation was reduced. After transfection with miR-141 inhibitor, the expression of miR-141 mRNA was downregulated, while the cell proliferation was increased. The expression of miR-141 mRNA was significantly higher in the dexamethasone group than the control group (P < 0.01), while the treatment with velvet antler serum could significantly downregulate the expression of miR-141 mRNA (P < 0.01). The absorbance of BMSCs in the dexamethasone group was significantly lower than that in the control group (P < 0.01), and the absorbance value in the velvet antler serum group was significantly higher than that in the dexamethasone group (P < 0.01). In conclusion, the serum containing velvet antler can downregulate the expression of miR-141 which is upregulated by dexamethasone and then do help to promote the proliferation of BMSCs.

Treatment of acetabular fractures with comminuted posterior wall by pelvic reconstruction plate and T-plate elastic fixation / 中华创伤骨科杂志

Objective:To explore the curative efficacy of pelvic reconstruction plate and T-plate elastic fixation in the treatment of acetabular fractures with comminuted posterior wall.Methods:A retrospective analysis was conducted of the 21 patients who had been treated at Department of Orthopaedics, Affiliated Hospital to the Second Mongolia Medical University for acetabular fractures with comminuted posterior wall from January 2017 to June 2019. They were 15 males and 6 females, aged from 29 to 55 years (average, 41.5 years). According to the Letournel-Judet classification, there were 15 cases of simple posterior wall fracture with dislocation of the femoral head, 4 cases of posterior wall and posterior column fracture, and 2 cases of double-column and posterior wall fracture. The time from injury to surgery averaged 3 days (from 2 to 5 days). The posterior acetabulum was treated by pelvic reconstruction plate combined with T-plate elastic fixation through the posterior acetabular Kocher-Langenbeck approach. Postoperative fracture reduction, fracture union time, function of the affected hip and complications at the last follow-up were evaluated.Results:This group of 21 patients were followed up for 6 to 24 months (average, 15 months). By the Matta imaging scoring, the postoperative reduction of the posterior wall fracture was evaluated as excellent in 18 cases and as good in 3, giving an excellent to good rate of 100%. The fracture union time averaged 10 weeks (from 8 to 12 weeks) for this group. By the improved Merle d'Aubigné & Postel evaluation at the last follow-up, the affected hips scored from 12 to 18 points (average, 16 points), yielding 18 excellent, 2 good and one poor cases, giving an excellent to good rate of 95.2%. There was no major hemorrhage, nerve injury or deep vein thrombosis intraoperatively. During the follow-up, mild ectopic ossification occurred in one case, and subluxation of the femoral head and traumatic arthritis were observed in another, but no patient had other complications like avascular necrosis of the femoral head.Conclusion:In the treatment of acetabular fracture with comminuted posterior wall, pelvic reconstruction plate and T-plate elastic fixation through the posterior acetabular Kocher-Langenbeck approach can lead to fine short-term outcomes.

Cell autophagy is involved in steroid-induced necrosis of the femoral head / 中国组织工程研究

BACKGROUND: Autophagy of osteocytes has been found to be implicated in the pathogenesis of steroid-induced necrosis of the femoral head and closely related to apoptosis.OBJECTIVE: To summarize the research progress of autophagy in the pathogenesis of steroid-induced necrosis of the femoral head by studying the interaction between cell autophagy and cell apoptosis as well as osteocytes.METHODS: A computer-based online retrieval of PubMed, Embase and CNKI databases was performed for relevant literatures published from October 1996 to October 2016 with the keywords of steroid, necrosis of the femoral head, cell apoptosis, cell autophagy, osteocyte in Chinese and English, respectively. The articles concerning steroid-induced necrosis of femoral head and cell autophagy were collected, and the redundant and old researches or Meta analysis were removed.RESULTS AND CONCLUSION: Mammalian target of rapamycin, Beclin-1, microtubule-associated protein 1 light chain 3, bone morphogenetic proteins, fork box protein and transcription gene family and transcription factor 4 are closely related to autophagy. The interaction between autophagy and osteocytes is correlated with steroid dose: the autophagy shows protective factor under the low dose corticosteroids; however, with the increase of the dosage, a large number of apoptotic cells, and the phenomenon of bone loss can been observed. Furthermore, the relationship of cell autophagy with apoptosis and bone mass maintenance is still controversial, which needs to be explored in depth via a series of rational experiments.

Effects of hydrogen on ultraviolet B-induced oxidative damage to skin fibroblasts / 中华皮肤科杂志

Objective To observe the effect of hydrogen on ultraviolet B (UVB)-induced oxidative damage to skin fibroblasts.Methods Primary human skin fibroblasts from foreskin tissues were divided into five groups:normal control group receiving no treatment,hydrogen control group treated with hydrogen-rich saline,UVB group receiving irradiation only,post-treatment group irradiated with UVB followed by hydrogen-rich saline treatment,and pre-treatment group treated with hydrogen-rich saline followed by UVB irradiation.The dose of UVB was 30,60 and 90 mJ/cm2 in the cell proliferation assay and 90 mJ/cm2 in the other experiments.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of fibroblasts,a chemiluminescence method to estimate the activity of superoxide dismutase (SOD) and catalase as well as to determine the level of malondialdehyde in the culture supernatant of fibroblasts,enzyme linked immunosorbent assay (ELISA) to determine the supernatant level of 8-isoprostane-prostaglandin F2α (8-iso-PGF2α),Western blot to detect the expression of heme oxygenase-1 (HO-1) in fibroblasts.One-factor analysis of variance was conducted to assess differences in these parameters among these groups.Results UVB irradiation decreased the proliferative activity (absorbence value at 490 nm) of fibroblasts in a dose-dependent manner.Both the pre-treatment group and post-treatment group showed a statistical increase in proliferative activity of cells compared with the corresponding UVB control groups (all P ＜ 0.05).The activity of SOD and catalase as well as the protein expression of HO-1 were significantly higher (all P ＜ 0.05),whereas the supernatant levels of malondialdehyde and 8-iso-PGF2α were statistically lower (both P ＜ 0.05) in the pre-treatment group and post-treatment group than in the UVB control group.Conclusion Hydrogen may mitigate UVB-induced oxidative damage to skin fibroblasts.

Effect of hydrogen-rich saline on allergic contact dermatitis in mice / 中华皮肤科杂志

Objective To evaluate the therapeutic effect of hydrogen-rich saline on allergic contact dermatitis (ACD) in mice and to explore its underlying mechanisms.Methods Forty mice were equally divided into 4 groups:control group,control treatment group,ACD group and ACD treatment group.ACD was induced by repetitive topical application of dinitrofluorobene (DNFB) to the left ear of mice on day 1,2 and 5.Hydrogen-rich saline was intraperitoneally given to the mice in the ACD treatment group at a dose of 5 ml/kg per day from day 1 to 5.On day 6,the mice were sacrificed,ear tissue was removed from them and subjected to measurement of thickness and weight,detection of tumor necrosis factor (TNF)-α,interleukin (IL)-6,IL-17 and interferon (IFN)-γ expression by enzyme linked immunosorbent assay,as well as numeration of inflammatory cells in lesions after hematoxylin-eosin (HE) staining.Data were processed by SPSS 18.0 software,and statistical analysis was carried out by one-way analysis of variance and least significant difference (LSD) procedure.Results Compared with the control group,the ACD group showed a significant increase in lesion score (7.33 ± 1.53 vs.0,P ＜ 0.05),differences in the thickness ((0.73 ± 0.15) mm vs.(0.13 ± 0.05) mm,P ＜ 0.05) and swelling degree (expressed as tissue weight:(18.67 ± 3.05) mg vs.(3.33 ± 1.52) mg,P ＜ 0.05) between the left and right ear,expressions of TNF-α ((1475.52 ± 233.81) pg/mg vs.(239.01 ± 52.39) pg/mg,P＜ 0.05),IL-6 ((184.65 ± 78.39) pg/mg vs.(42.28 ± 17.64) pg/mg,P＜ 0.05),IL-17 ((628.56 ± 201.44) pg/mg vs.(127.58 ± 50.28) pg/mg,P＜ 0.05) and IFN-γ ((197.72 ± 37.81) pg/mg vs.(24.57 ± 8.31) pg/mg,P ＜ 0.05),and the number of inflammatory cells per square millimetre in the left ear tissue (752.00 ± 166.06 vs.127.33 ± 77.18,P ＜ 0.05).However,hydrogen-rich saline treatment induced a statistical decrease in all of these parameters in the ACD treatment group compared with the ACD group,including lesion score (3.33 ± 0.58,P ＜ 0.05),difference in thickness ((0.46 ± 0.11) mm,P ＜ 0.05) and swelling degree ((11.00 ± 2.64) mg,P ＜ 0.05),expressions of TNF-α ((817.72 ± 101.13) pg/mg,P＜ 0.05),IL-6 ((95.86 ± 36.65) pg/mg,P＜ 0.05),IL-17 ((373.38 ± 126.74) pg/mg,P＜ 0.05),IFN-γ ((63.31± 17.38) pg/mg,P ＜ 0.05) and the number of inflammatory cells per square millimetre (384.00 ± 97.35,P ＜0.05).Conclusion Hydrogen may inhibit the release of inflammatory factors in ACD.