BACKGROUND: Many operations for isolating, purifying and identifying bone marrow mesenchymal stem calls (BMSCs) are complicated and cost much. Also they have great effect on cell activity. Whether whole bone marrow adherent culture can avoid above-mentioned disadvantages remains unclear. At present, many studies huve been done to confirm an effective and low cost method for isolating, purifying and identifying such cells.OBJECTIVE: This study is to in vitro induce and differentiate rat BMSCs by whole bone marrow adherent culture,and to identify the cells.DESIGN: A controlled observational experiment.SETTING: Qingdao University Medical College.MATERIALS: This study was carried out in the Laboratory of Oral Cavity and Laboratory of Molecular Biology (provincial level) Qingdao University Medical College between November 2005 and March 2007. Twenty Wistar rats of either gender, aged 3 to 4 weeks, of SPF grade, weighing 120-150 g, were provided by the Qingdao Laboratory Center. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Fetal bovine serum (FBS, Hangzhou Sijiqing Bioengineering Material Research Institute), alkaline phosphatase (ALP) kit (Nanjing Jiancheng Bioengineering Research Institute), reverse transcription kit (American Promega Corporation) and primer (Shanghai Bioengineering Co.,Ltd.) were used in this study.METHODS: Adult rat BMSCs were isolated and cultured by whole bone marrow adherent culture. They were digested with 2.5 g/L trypse and inoculated at a density of 5 ×107 L-1 in 6-well culture plate. Then, the cells were divided into experimental group and control group. Inducing culture medium was added to experimental group, and the same amount of basic culture medium was added to control group. ① Cell differentiation and calcium tuberculation were observed under the inverted microscope. ② Biological characteristics of induced cells were detected by calcium tubercle Von Kossa and alizarin Bordeaux. ③ALP activity was detected by diazo salt staining. ④Human core binding factor alpha subunit-1 (Cbf α-1), osteocalcin (OCN) and osteoblast-specific Osterix (OSX) mRNA expressions were detected by reverse transcription-polymerase chain reaction (RT-PCR).MAIN OUTCOME MEASURES: ① Induction and differentiation results of cells. ② Biological characteristics of cells induced by rat BMSCs. ③ ALP activity. ④ Cbf α-1, OCN and OSX expressions.RESULTS: ①Inducing culture medium was added in the serial subcultivation. About 9 days later, cell clones were connected to each other. On about 21 to 28 days, some pykno-round mineralized tubercles appeared. Meanwhile,control cells were connected to each other, but they did not form the tubercle. ② In the experimental group, when MSCs were induced for 21 to 28 days, obvious round or oval calcified tubercles were seen by naked eyes. The results of Von Kossa staining exhibited black sediments, and those of alizarin Bordeaux staining exhibited salmon tubercles. Calcium tubercles were not found in the control group. ③The ALP activity after 2 weeks of induction was obviously increased in the experimental group, but was relatively weak in the control group. ④In the experimental group,Cbf α-1, OCN and OSX expressions were significantly increased after induction.CONCLUSION: After being in vitro induced and differentiated by whole bone marrow adherent culture, rat BMSCs exhibited morphological and biological characteristics similar to typical osteoblasts.

Objective:To compare the effect of the new anterior cervical spine memory compression fixation device (GYZ memory alloy plate) and traditional titanium plate on the range of motion (ROM) and stress of the adjacent segment after anterior cervical discectomy and fusion.Methods:An adult male volunteer was recruited for a fee. After excluding cervical malformations, fractures, infections and other diseases, C 3-C 7 thin-layer CT scans were performed. Import the scanned data into the finite element modeling software to establish the finite element model of the physiological group and verify itseffectiveness. After C 5,6 discectomy, the intervertebral fusion device was inserted, and the anterior fixation was assisted by a conventional titanium plate or a new type of fixator. Thus, the finite element model of the traditional titanium plate group and the new fixer group was established. The three models were imported into the finite element analysis software ANSYS 16.0, and a vertical downward axial load of 73.6 N was loaded to simulate the head weight and the torque of 1.0 N·m to simulate the cervical spine flexion, extension, left lateral bending, right lateral bending, left rotation and right rotation.Compare the changes of intervertebral disc ROM and stress in adjacent segments of physiological group, traditional titanium plate group and new type fixator group. Results:The intervertebral disc ROM under six conditions was basically similar to the results of previous studies, and the model was effective. In the adjacent segment C 4,5, the three groups of activities in the flexion, extension, left lateral bending, right lateral bending, left rotation and right rotation conditions were: physiological group 3.9°, 4.2°, 3.7°, 3.7°, 2.2° and 2.2°, traditional titanium plate group 4.6°, 4.7°, 4.3°, 4.4°, 3.3° and 3.1°, and new fixture group 4.4°, 4.3°, 4.0°, 4.2°, 2.8° and 2.7°. The maximum stresses of the intervertebral discs under three different working conditions were: physiological group 1.81, 1.60, 3.99, 2.06, 3.63 and 3.41 MPa, traditional titanium plate group 1.86, 1.67, 4.21, 2.16, 3.82 and 3.63 MPa, and new fixture group 1.84, 1.64, 4.17, 2.14, 3.78 and 3.58 MPa. In the adjacent segment C 6,7, the activities of the three groups in six working conditions were: physiological group 3.1°, 3.2°, 2.5°, 2.5°, 1.2° and 1.3°, traditional titanium plate group 4.2°, 3.7°, 3.4°, 3.0°, 2.1° and 2.2°, and new fixture group 3.5°, 3.3°, 2.5°, 2.7°, 1.8° and 1.9°.The maximum stress of the intervertebral disc under three different working conditions was: physiological group 0.45, 0.66, 1.12, 0.85, 0.84 and 0.82 MPa, traditional titanium plate group 0.62, 0.93, 1.55, 1.24, 1.44 and 1.27 MPa, and new fixture group 0.61, 0.92, 1.54, 1.22, 1.07 and 1.24 MPa. The ROM and disc pressure of adjacent segments in the conventional titanium plate group were higher than those of the new fixator group. Conclusion:Compared with the traditional titanium plate, the new type of anterior cervical memory compression fixator has less effect on the ROM and stress of adjacent segments, which may slow down the process of adjacent segments degeneration to a certain extent.

Objective To evaluate the correlation between positive end-expiratory pressure (PEEP) level and postoperative pulmonary complications (PPCs) in patients undergoing thoracoscopic lung surgery. Methods The clinical data of patients who underwent elective thoracoscopic lung surgery at West China Hospital of Sichuan University from January 2022 to June 2023 were retrospectively analyzed. Patients were divided into 2 groups according to intraoperative PEEP levels: a PEEP 5 cm H2O group and a PEEP 10 cm H2O group. The incidence of PPCs in the two groups after matching was compared using a nearest neighbor matching method with a ratio of 1∶1, setting the clamp value as 0.02. Results A total of 538 patients were screened, and after propensity score-matching, a total of 229 pairs (458 patients) were matched, with an average age of 53.9 years and 69.4% (318/458) females. A total of 118 (25.8%) patients had PPCs during hospitalization after surgery, including 60 (26.2%) patients in the PEEP 5 cm H2O group and 58 (25.3%) patients in the PEEP 10 cm H2O group, with no statistically significant difference between the two groups [OR=0.997, 95%CI (0.495, 1.926), P=0.915]. Multivariate logistic regression analysis showed that PEEP was not an independent risk factor for PPCs [OR=0.920, 95%CI (0.587, 1.441), P=0.715]. Conclusion For patients undergoing thoracoscopic lung surgery, intraoperative PEEP (5 cm H2O or 10 cm H2O) is not associated with the risk of PPCs during hospitalization after surgery, which needs to be further verified by prospective, large-sample randomized controlled studies.

OBJECTIVE To establish a method to detect the blood concentration of ibrutinib and apply it to the clinic. METHODS Using zanubrutinib as internal standard， the concentration of ibrutinib was detected by high performance liquid chromatography （HPLC） after plasma samples were processed by solid-phase extraction. The separation was performed on an Agilent 5 TC-C18（2） column with acetonitrile-0.5% potassium dihydrogen phosphate solution （43∶57， V/V） as the mobile phase at a flow rate of 1 mL/min， a detection wavelength of 260 nm， a column temperature of 40 ℃ ， a sample size of 20 μL， and a run time of 25 min. The concentration of ibrutinib was measured in the plasma of 9 patients with non-Hodgkin’s lymphoma 2 h after drug administration on the 30th day by the above method. RESULTS The linear range of the assayed mass concentration of ibrutinib was 10-500 ng/mL （R 2＝0.998 9）， the lower limit of quantification was 10 ng/mL， and the RSDs of the intra-batch and inter-batch precision tests were not higher than 12.77%. The recoveries of the extraction were 74.80% and 97.70%， with both RSDs＜2.90%， and the RSDs of the stability tests were not higher than 7.10%. The peak plasma concentrations of 9 patients were 15.341-279.628 ng/mL. CONCLUSIONS The established HPLC method is simple and rapid， and can be used for the determination of ibrutinib concentration in plasma samples.

Objective To investigate the effects and related mechanisms of mitochondrial-derived peptide MOTS-c on glycochenodeoxycholic acid (GCDCA)-induced injury in human hepatocytes (THLE-3 cells). Methods THLE-3 cells were cultured in vitro and treated with different concentrations of GCDCA and MOTS-c. The optimal concentrations of GCDCA and MOTS-c were determined by cell counting kit (CCK)-8 method. Subsequently, THLE-3 cells were treated or pre-treated with GCDCA (200 µmol/L), MOTS-c (15, 30, 60 µmol/L), the multidrug resistance protein 2 (MRP2) inhibitor Probenecid (500 µmol/L), and the nuclear factor erythroid 2-related factor 2 (Nrf2) inhibitor ML385 (10 µmol/L). Cell proliferation was assessed by CCK-8 method. Lactate dehydrogenase (LDH) levels in the culture medium were measured by biochemical method. Cell apoptosis rates were determined by flow cytometry. MRP2 messenger RNA (mRNA) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). MRP2 and Nrf2 protein expression levels were analyzed by Western blotting. Results As the concentration of GCDCA increased, the proliferation activity of THLE-3 cells gradually decreased, while LDH activity in the culture medium and apoptosis levels increased, and the expression levels of MRP2 in the cells decreased (all P<0.05). Treatment with 30 and 60 µmol/L MOTS-c significantly enhanced the proliferation activity of THLE-3 cells exposed to GCDCA, upregulated the expression of MRP2 and Nrf2, and reduced LDH activity and apoptosis levels (all P<0.05). Co-treatment with Probenecid partially reversed the protective effects of MOTS-c on GCDCA-induced THLE-3 cells injury, while co-treatment with ML385 partially inhibited the induction of MRP2 expression by MOTS-c in THLE-3 cells exposed to GCDCA. Conclusions MOTS-c may alleviate GCDCA-induced injury in human hepatocytes (THLE-3 cells), and its mechanism may be related to the upregulation of MRP2 expression mediated by Nrf2.