Objective:This study aimed to investigate the effect of differentiation osteogenic bone marrow mesenchymal stem cells (De-BMSCs) transplantation on the promotion of bone formation at the tendon-bone interface after anterior cruciate ligament reconstruction (ACLR), and further explored the molecular mechanism of the enhanced osteogenic effect of De-BMSCs.Methods:BMSCs from femur and tibia of New Zealand White rabbit were subjected to osteogenic induction and then cultured in no osteogenic factor medium; the obtained cell population was termed De-BMSCs. De-BMSCs were induced into osteo-, chondro-and adipo-differentiation in vitro to examine the characteristics of primitive stem cells. ACLR model with a semitendinosus tendon were performed in 48 adult rabbits, three groups were established: control group with alginate gel injectionat the tendon-bone interface, BMSCs group with the injection of alginate gel containing BMSCs, De-BMSCs group with the injection of alginate gel containing De-BMSCs. At 4 and 12 weeks after surgery, rabbits in each group were sacrificed to evaluate tendon-bone healing by histologic staining, micro-CT examination, and biomechanical test. During osteogenic differentiation of De-BMSCs, si-RNA of nuclear factor of activated T cells 2 (NFATc2) si-RNA of nuclear factor of activated T cells 1 (NFATc1) were used to verify the molecular mechanism of enhanced osteogenic effect of De-BMSCs.Results:De-BMSCs exhibited some properties similar to BMSCs including multiple differentiation potential and cell surface marker. At 4 weeks after surgery, the BV/TV value of the De-BMSCs group 0.36±0.01 was significantly higher than that of the control group 0.24±0.03 and BMSCs group 0.30±0.02 (all P<0.05), and the maximum load 40.34±1.19 N and stiffness 20.67±2.14 N/mm were significantly higher than those in the control group 14.88±2.74N, 8.67±2.19 N/mm and the BMSCs group 26.31±1.76 N, 13.81±2.14 N/mm (all P<0.05). At 12 weeks after surgery, the BV/TV value of the De-BMSCs transplantation group 0.47±0.02 was significantly higher than that of the control group 0.30±0.02 and the BMSCs group 0.35±0.03 (all P<0.05), and the maximum load 64.46±6.69 N and stiffness 25.18±3.11 N/mm were significantly higher than those in the control group 41.01±6.12 N, 11.59±2.54 N/mm and the BMSCs group 48.21±4.12 N, 15.89±2.94 N/mm (all P<0.05). During the osteogenic differentiation of De-BMSCs, the expressions of Nanog and NFATc1 were synergistically increased which promoted interaction of NFATc1 and Osterix ( P< 0.05), resulting in the increased expression of osteoblast marker genessuch as COL1A, OCN, OPN (all P< 0.05). Conclusion:De-BMSCs transplantation could promote bone formation at the tendon-bone interface after ACLR,Nanog/NFATc1/Osterix signaling pathway mediated the enhancement of the osteogenic differentiation effect of De-BMSCs.

Objective: Osteoarthritis (OA) is an age-related degenerative disease accompanied by an increasing number of senescent cells and chronic low-grade inflammation. Rhoifolin (ROF) showed considerable inhibition to inflammation, but its role in chondrocyte senescence and OA progress has not been fully characterized. We aimed to evaluate the protective effects of ROF on OA through a series of in vitro and in vivo experiments. Methods: The role of ROF in the expression of senescence-associated secretory phenotype (SASP) factors was investigated using RT-qPCR, western blotting, and ELISA. Chondrocyte senescence was assessed by SA-beta-gal staining. We applied molecular docking to screen candidate proteins regulated by ROF. Meanwhile, SASP factors and cellular senescence were further assessed after the transfection of Nrf2 siRNA. In the anterior cruciate ligament transection (ACLT) rat model, X-ray, hematoxylin-eosin (HE), and Masson's staining were performed to evaluate the therapeutic effects of ROF on OA. Results: We found that ROF inhibited SASP factors expression and senescence phenotype in IL-1 beta-treated chondrocytes. Furthermore, ROF suppressed IL-1 beta-induced activation of the NF-kappa B pathway cascades. Also, molecular docking and knock-down studies demonstrated that ROF might bind to Nrf2 to suppress the NF-kappa B pathway. In vivo, ROF ameliorated the OA process in the ACLT rat model. Conclusions: ROF inhibits SASP factors expression and senescence phenotype in chondrocytes and ameliorates the progression of OA via the Nrf2/NF-kappa B axis, which supports ROF as a potential therapeutic agent for the treatment of OA. (C) 2022 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

目的 发现临床病理生理及治疗学(CPPT)讨论课教学效果好的师资群体及其特征,建立临床医学讨论课师资评价模式.方法 研究对象为2018年参加CPPT神经病学课程的五年制、八年制及5+3年制临床医学生184名,按小班随机分配讨论课老师一名,可为教学医院专科高级或中级职称老师,或学生助教(TA)一名,课程结束后在线填写对授课老师的评价表,结合专科期末考核成绩综合评价师资教学效果.结果 学生对于授课老师的专业能力、对课程的讲授和对学生的帮助三个方面的认可度达到90.2％、86.4％和86.4％;TA带教学生的专科考核平均分显著高于高级职称师资带教成绩,且高分段学生人数的比例高于中级职称老师高分数段学生人数的比例(P<0.05).结论 学生助教参与的CPPT讨论课教学效果好,鼓励多选拔并培养高年级学生当助教参与讨论课的讲授.

Our previous studies discovered that prenatal caffeine exposure (PCE) could induce intrauterine growth retardation (IUGR) and long-bone dysplasia in offspring rats, accompanied by maternal glucocorticoid over-exposure. This study is to explore whether intrauterine high glucocorticoid level can cause endochondral ossification retardation and clarify its molecular mechanism in PCE fetal rats. Pregnant Wistar rats were intragastrically administered 30 and 120 mg/kg day of caffeine during gestational days (GDs) 9–20, then collected fetal serum and femurs at GD20. In vitro , primary chondrocytes were treated with corticosterone (0–1250 nM), caffeine (0–100 μ M), mitogen-inducible gene 6 (Mig-6) siRNA and epidermal growth factor receptor (EGFR) siRNA, respectively, or together. Results showed that the hypertrophic chondrocytes zone (HZ) of PCE fetal femur was widened. Meanwhile, the expression levels of chondrocytes terminal differentiation genes in the HZ were decreased, and the chondrocytes apoptosis rate in the HZ was decreased too. Furthermore, PCE upregulated Mig-6 and suppressed EGFR expression in the HZ. In vitro , a high-concentration corticosterone (1250 nM) upregulated Mig-6 expression, inhibit EGFR/c-Jun N-terminal kinase (JNK) signaling pathway and terminal differentiation genes expression in chondrocytes and reduced cell apoptosis, and these above alterations could be partly reversed step-by-step after Mig-6 and EGFR knockdown. However, caffeine concentration dependently increased chondrocyte apoptosis without significant changes in the expression of terminal differentiation genes. Collectively, PCE caused endochondral ossification retardation in the female fetal rats, and its main mechanism was associated with glucocorticoid (rather than caffeine)-mediated chondrocyte terminal differentiation suppression by the upregulation of Mig-6 and then inhibition of EGFR/JNK pathway-mediated chondrocyte apoptosis.

Objective: This study was undertaken to probe deeply into effects of microRNA-145 (miR-145) expression on chondrocyte apoptosis in osteoarthritis (OA). Methods: A group of 71 OA patients (32 males and 39 females) with age ranging from 49 to 77 years were recruited into this study. Chondrocytes isolated from their OA tissues were separated into four groups: group A (blank control, no transfection), group B (transfection with empty vector pGenesil-1), group C (miR-145 plasmid) and group D (miR-145 inhibitor). Transfection efficiency was observed under fluorescence microscope. Real-time quantitative PCR (RT-qPCR) and western blotting were used to detect expressions of miR-145 and N-cadherin (mRNA) in the chondrocytes of each group. And flow cytometry was utilized to determine cycle and apoptosis of cells in each group. Results: miR-145 expression showed relationship with gender and OA grading (both P < 0.05). miR-145 expression increased in the group C and significantly decreased in the group D compared with that in the group A and group B (all P < 0.05). Interestingly, N-cadherin expression in the group D was increased compared with that in the three groups (all P < 0.05). Moreover, compared with the group A and group B, cell apoptosis was significantly abated and the cell proportion was decreased at the S stage in the group D, but promoted in the group C (all P < 0.05). Also, the group D had lower cell proportion and cell apoptosis rate than the group C (P < 0.05). Conclusion: Inhibition of miR-145 can suppress chondrocyte apoptosis in OA, indicating a new direction for OA treatment.

In recent years, there has been a comprehensive reform of higher medical education in the Medicine School of Wuhan University. According to the need for reform, the teaching of neurology has to be changed from the traditional form to a new form, and be integrated into the clinical pathophysiology and therapeutics (CPPT) courses. Currently neurology in CPPT takes the form of theoretical lectures, case discussions, combined with practical lessons to observe sections under the microscope and clinical practice, for the cultivation of students'!self-learning ability and clinical thinking. In the commissioning process, it exposes some problems in teaching process due to the characteristics of the course in neurology. For example, the knowledge of neuroanatomy is insufficient and review lessons relatively too short, and the teaching effect may be worse due to the fact that teachers have busy clinic work. In addition, students participate in case discussions with less enthusiasm. To solve these problems, we take some measures to promote teaching reform in neurology, such as increasing the review hours of neuroanatomy section in the CPPT neurology, training a group of specialized medical teachers to enrich and stabilize teacher team, adjusting the content and form of discussion class to improve students'!interest and participation, and increasing assistant jobs by the student to assist discussion teaching.

This article briefly introduced the current status of ethics review about the domestic medical research projects involving human,and investigated the common problems in the ethical review of medical research projects involving human,combining with the actual work of the ethics review in the Medical Ethics Committee of Zhongnan Hospital of Wuhan University in recent years.We suggested that management departments and the Ethics Committee should strengthen propaganda and education of the ethical knowledge to researchers,strengthen the supervision of the ethical behavior in the process of research projects,protect the interests of subjects,and ensure the healthy development of the medical research.

Objective To investigate the effect of Src kinase inhibitor 1 on the growth and migration of human chondrosarcoma cell line SW1353 and the molecule mechanism.Methods Human chondrosarcoma SW1353 cells were cultured in vitro and divided into the control group and the experimental group.The effects of Src kinase inhibitor 1 on proliferation and apoptosis of SW1353 cells were assessed by using MTT assay and FITC-Annexin V/PI assay,respectively.The cell migration was measured by using the OrisTM 96-well cell migration assay kit.Western blotting was used to detect the effects of Src kinase inhibitor 1 on Src activation and downstream signaling pathway proteins.Results MTT assay showed that the proliferation of human chondrosarcoma SW1353 cells was significantly inhibited after co-culture with 0.5,1,5 and 10 μmol/L Src kinase inhibitor 1 for 24 h(all P<0.01).Src kinase inhibitor 1 at 1,5 and 10 μmol/L increased the apoptosis of human chondrosarcoma SW1353 cells in a concentration-dependent manner(all P<0.01).The migration of human chondrosarcoma SW1353 cells was also inhibited after co-culture with 1.0 μmol/L Src kinase inhibitor 1 for 24 h.The phosphorylations of Src(Tyr416),ERK1/2,Akt and FAK(Y397) were all attenuated with the treatment of Src kinase inhibitor 1.Conclusion Src kinase inhibitor 1 could inhibit the growth and migration of human chondrosarcoma SW1353 cells in vitro,which was due to the direct inhibitory actions on the activation of Src kinase and Src signaling pathway.Src kinase inhibitor 1 may be effective in the treatment of chondrosarcoma.

Objective: To investigate the effect of recombinant adenovirus-mediated basic fibroblast growth factor (bFGF) gene transfer on the human osteoarthritis (OA) chondrocytes. Methods: Monolayer cultures of human OA chondrocytes were divided into three groups: 1OA control group (chondrocytes without transfection); 2 OA chondrocytes transfected with AdEGFP; 3OA chondrocytes transfected with AdbFGF. Human OA chondrocytes were infected with recombinant adenovirus containing human bFGF gene. The rate of transfection was analyzed by flow cytometry and the diagram of expression was drawn after 48 hours. Six days later, levels of gene expression in culture supernatant were detected. The proliferation of chondrocytes was analyzed by methyl thiazolyl tetrazolium (MTT). The biosynthesis of proteoglycans and type II collagen was observed by toluidine blue staining and immunohistochemistry examination. Results: The expression level of bFGF was high following adenoviral transfection in culture supernatant. The delivery of bFGF promoted the cell proliferation (P<C0.05) and resulted in a significant enhanced biosynthesis of proteoglycans and type II collagen of chondrocytes (P<0.05). Conclusion: Recombinant adenovirus-mediated bFGF gene transfection can greatly promote the cell proliferation and increase matrix synthesis in vitro , which is important to keep the phenotype of chondrocytes.

Objective To observe the therapeutic effect of recombinant basic fibroblast growth factor (bFGF), interleukin-1 receptor antagonist protein (IL-Ra) and insulin-like growth factor (IGF)-1gene transfer in the rabbit model of osteoarthritis (OA). Methods OA was induced in NZW rabbits by anterior cruciate ligament transaction in the knees. Adenoviral vectors encoding human bFGF, IL-1Ra and IGF-1 were injected intraarticularly into the knee joints after dividing into different groups. After 3 weeks,rabbits were sacrificed and the expression of interest genes and glycosaminoglycan (GAG) content were examined in synovial fluids. The Mankin score and type Ⅱ collagen immunohistochemistry examination for cartilage were done. Results The transfected genes were expressed in synovial fluids after gene delivery.The matrix of cartilage was degraded in OA control group. The Mankin score and GAG content were (8. 60± 1.14) and (69. 96 ±8. 32) mg/L in OA control group, respectively. The individual bFGF gene transfer reduced the Mankin score (P ＜0. 05) and the value was (6. 00 ±0. 71 ). The combined use of bFGF with IL-1Ra and IGF-1 gene delivery further reduced the Mankin score (P ＜0.05 ) and the value was (3. 80 ±0. 84). The bFGF had no significant effect on reducing the GAG release from matrix. When two or three genes were used together, there was greater inhibition on the degradation of matrix and reducing the GAG release into synovial fluids. Conclusion Individual bFGF gene transfer can play a role in treating osteoarthritis. Local intraarticular administration of adenoviral vectors encoding bFGF, in combination with IL-1Ra and IGF-1, significantly suppressed OA progression. These results suggest that gene combination may be a potential treatment for OA.

骨关节炎(osteoalthritis,OA)是中老年人最常见的关节疾病,病理上表现为软骨细胞和软骨基质的退行性改变,最终导致关节软骨结构破坏和功能障碍.

Objective: To study the separation,digestion and cultivation of human osteoarthritis chondrocytes,to compare biological characteristics with human normal chondrocytes and to evaluate their biological activity.Methods: The articular cartilage was digested one by one with the 0.05% and 0.2% collagenaseⅡ to separate chondrocytes in the culture medium including fetal bovine serum.The viability rate of the primary chondrocytes was detected.During the cultivation in vitro,the changes of the chondrocytes shape,growth and proliferation were observed;the changes of the collagen type Ⅱ and aggrecan were investigated and detected.Cell cycle,DNA ploidy and apoptosis of specimens in the presence or absence of IL-1β were evaluated by flow cytometry.Results: ① After the articular cartilage was completely dissolved by the enzymatic two-step digestion,the chondrocytes suspension were harvested.The viability rate of primary osteoarthritis chondrocytes was 82% on average,which was less than 95% of normal(P0.01).② The primary and passage osteoarthritis chondrocytes proliferated more slowly than the primary normal ones(P0.01).Their immunohistochemical staining of collagen type Ⅱ and heterochromia to toluidine blue O was both weaker than the normal.The fourth generation osteoarthritis chondrocytes had lost their special biological activity.③ The apoptotic rate of osteoarthritis chondrocytes was 6.9%,but that of normal was only 0.5%.After induced by IL-1β,the apoptotic rate of osteoarthritis chondrocytes increased to 27.4%,but that of normal increased to 12.7% only.Conclusion: The method of the enzymatic two-step digestion has advantages in the high cellular viability rate,low contamination and simple procedures.The osteoarthritis chondrocytes separated and cultured have biological characteristics of human osteoarthritis degenerated cartilage and can be regarded as the best experiment object for the research of osteoarthritis at the cell level.