Objective To construct a stable synovial cell line MH7A from rheumatoid arthritis(RA)patients using lentiviral vectors that interfere with the expression of tumor necrosis factor receptor associated factor 2(TRAF2),and to study the role of TNF-α-TRAF2 signaling in MH7A abnormal proliferation.Methods Based on the design principles of human TRAF2 gene sequence and shRNA sequence,three pairs of TRAF2 shRNA interference se-quences were designed and synthesized.The primers were annealed by PCR,and a linear vector was obtained by double enzyme digestion PLKO.1-puro.The linearized vector was connected to the annealed primers through Solu-tion I,and the connected products were introduced into receptive cells.The plates were coated,and positive colo-nies were selected for sequencing.Three different recombinant plasmids of PLKO.1-TRAF2-shRNA lentivirus were constructed,and lentivirus packaging plasmids was used to package logarithmic growth phase HEK 293T cells.Vi-rus solution was collected to infect MH7A cells.At the same time,puromycin was used to screen MH7A stable transgenic strains with low TRAF2 expression.CCK-8 method,Western blot,and qPCR were used to detect the proliferation function of MH7A induced by TNF-α and low expression of TRAF2,as well as downstream signal TRAF2,P65 protein expression and mRNA levels.Results PLKO.1-TRAF2-shRNA(1),PLKO.1-TRAF2-shR-NA(2),and PLKO.1-TRAF2-shRNA(3)lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were successfully constructed.The three TRAF2-shRNA lentivirus vector plasmids and control group lentivirus vector plasmids PLKO.1-puro were respectively introduced into the lentivirus packaging plasmid of HEK 293T to obtain virus solution.After infecting MH7A cells with the virus solution,they were treated with puromycin(2.00 μ G/mL)screening and obtaining MH7A stable transgenic plants after 2 days.Through qPCR and Western blot results,it was found that the expression of TRAF2 mRNA and protein in PLKO.1-TRAF2-shRNA(1)MH7A stably transfected cells was significantly reduced compared to the negative control group.The results of CCK-8 and Western blot showed that after knocking down TRAF2 in MH7A,the proliferation of MH7A cells with low TRAF2 expression induced by TNF-α and the phosphorylation level of P65 were significantly reduced.Conclusion A sta-ble transgenic strain of PLKO.1-TRAF2-shRNA(1)MH7A cells was successfully constructed to investigate the role of TNF-α-TRAF2 signal activation in mediating abnormal proliferation of RA synovial cells.

Objective:To analyze characteristics and trends of histopathological diagnosis of adult renal biopsy in Beijing from 2008 to 2020.Methods:A total of 4 652 cases of adult renal biopsy were collected from three hospitals in Beijing between 2008 and 2020. The patients were divided into three age groups: 18-40 years, 40-65 years and≥ 65 years; and also divided into three period: 2008-2011, 2012-2015, and 2016-2020. The pathological characteristics and changes of renal biopsy were analyzed in three age groups at different periods.Results:Among 4 652 cases primary glomerular disease accounted for 81.8%, the membranous nephropathy (MN, 32.4%, 1 509/4 652), IgA nephropathy (IgAN, 29.2%, 1 356/4 652) and minor glomerular abnormalities (MGA, 11.3%, 526/4 652) were the top three pathological types. The overall proportion of MN and diabetic nephropathy (DN) increased from 20.3% and 2.3% in 2008-2011 to 37.3% and 10.1% in 2016-2020) (χ2=99.9 and 96.1, both P<0.01), respectively. For age group 18-40 years, the MN and DN increased from 11.2% and 1.6% in 2008-2011 to 24.7% and 5.5% in 2016-2020 (χ2=32.7 and 20.7, both P<0.01), respectively. For age group 40-65 years the MN and DN increased from 26.6% and 3.2% in 2008-2011 to 41.5% and 13.1% in 2016-2020 (χ2=39.1 and 57.3, both P<0.01), respectively. For age group≥65 years the MN was the most common pathological type in the three periods, fluctuating between 41.3% and 55.0% (χ2=5.2, P=0.08); and DN increased from 0(0/63) in 2008-2011 to 7.5%(22/292) in 2016-2020 (χ2=8.1, P=0.02). Conclusion:The renal biopsy data show that membranous nephropathy and diabetic nephropathy are the most common primary and secondary adult glomerular diseases in Beijing recently.

Objective: To explore the interaction effects and possible sex differences in childhood emotional overeating and polygenic influences on adolescent pubertal timing and tempo. Methods: In March 2016 (T0), all participants were recruited from grades 1 to 3 from two primary school of Bengbu, Anhui Province using cluster sampling, and follow up surveys were conducted once per year (T1, T2, T3). Emotional overeating was assessed at T1 and pubertal development was assessed annually (breast Tanner stage in girls and testicular volume in boys). The nonlinear growth model was used to estimate pubertal timing and tempo. Polygenic risk scores were calculated based on 17 SNPs for early pubertal timing. Hierarchical linear regression was performed to examine the interaction effects between childhood emotional overeating and polygenic risk scores on pubertal timing and tempo. Results: The complete data of 896 children were analyzed, including 373 boys (41.60%) and 523 girls (58.40%). A total of 203 (22.7%) children reported emotional overeating behavior at T1. After adjusting for several variables including early life adversity, delivery mode, and birthweight, only emotional overeating was associated with accelerated pubertal tempo among girls with a high genetic risk (B=0.19, 95%CI=0.07~0.32， P<0.01), although there was no association with pubertal timing (B=0.14, 95%CI=-0.12~0.41，P=0.28). In girls with a low genetic risk and boys, no evidence was found to support interaction effects between childhood emotional overeating and polygenic influences on pubertal timing and tempo (P＞0.05). Conclusion Emotional overeating was associated with a faster pubertal tempo in girls who had a high genetic risk of early pubertal development.

Objective: To evaluate the development trajectory of sugar-sweetened beverage (SSB) intake in childhood, and to explore the influence of different SSB intake patterns on childhood obesity. Methods: In 2016, a follow-up cohort study was carried out in two primary schools in Bengbu, Anhui Province. Three annual follow-ups were conducted in 1 263 children at baseline, and 997 children were included in the final analysis. Parental and student questionnaires were used to obtain basic information related to the children s consumption of SSBs. A group-based trajectory model (GBTM) was applied to classify the development trajectory of SSB intake patterns in childhood. Multiple linear regression analyses were performed to assess the correlation between different SSB intake patterns and childhood obesity. Results: GBTM identified four childhood SSB intake patterns, namely, the "persistently-low group (n=822), “decreasing-after-increasing” group (n=20), “gradually-decreasing” group (n=106), and “increasing” group (n=49). In the decreasing-after-increasing group and the gradually-decreasing group, baseline BMI levels and BMI levels obtained at the three follow-ups were significantly higher than those observed in the persistently-low group (F=6.26, 5.90, 5.99, 5.87, P<0.01). There were sex differences in the association between SSB intake patterns and the children s BMI levels. Among girls, after adjusting for confounding factors, the gradually decreasing group increased by 1.20 kg/m 2(B=1.20，95%CI=0.25-2.15, P=0.01) when compared with the persistently low group at the third follow-up. Among boys, no statistically significant association was found between SSB intake patterns and BMI levels (P>0.05). Conclusion Sex differences were observed with respect to the association between SSB intake patterns and obesity in children. Girls with a higher SSB intake had a significantly increased risk of obesity. Further studies are needed to explore the physiological mechanisms underlying sex differences, to provide the theoretical basis for developing intervention programs to prevent childhood obesity.

Objective: To explore the relationship between dietary patterns and central precocious puberty in children, and to provide a scientific basis for dietary prevention of precocious puberty. Methods: A case-control study was conducted, among 35 newly diagnosed central precocious puberty girls from May to December 2019 as the case group, and 70 healthy girls with normal development as the control group. Physical development examination, parent questionnaire survey and child interview were carried out. Dietary information was assessed using a simplified food frequency questionnaire(FFQ). Principal component analysis was used to identify children s dietary patterns, and multiple Logistic regression was used to assess the association between dietary patterns and precocious puberty. Results: Three different dietary patterns have been established, namely "snack and processed food type", "animal protein type" and "nutritional tonic type" dietary patterns, respectively. After adjusting for covariates such as age and BMI, Logistic regression analysis showed that the "snack and processed food type" dietary pattern was positively correlated with precocious puberty(OR=10.81, 95%CI=2.59-45.15, P<0.01). There was a negative correlation between "animal protein type" and precocious puberty(OR=0.24, 95%CI=0.06-0.91, P=0.04), while the association between "nutritive tonic" and precocious puberty was not statistically significant(OR=0.28, 95%CI=0.07-1.05, P=0.06). Conclusion Children s dietary patterns were related to precocious puberty." Snack and processed food "dietary pattern with a high intake of fried foods, puffed foods, foods containing preservatives or pigments, western fast foods, chocolate and products, was closely related to central precocious puberty.

Objective:To investigate the role of neurite outgrowth inhibitor (Nogo)-oligodendrocyte myelin glycoprotein (Omgp)/Ras homologous (Rho)-Rho-associated coiled-coil forming protein kinase (Rock) signaling pathway in acute brain injury of carbon monoxide (CO) poisoning rats and treatment feasibility with Rho kinase inhibitor hydrochloride fasudil.Methods:According to random number table method, 135 healthy male SD rats were divided into three groups: a normal control group, a CO poisoning group and a fasudil treatment group ( n=45). Rat models of acute severe CO poisoning were established in the CO poisoning group and fasudil treatment group by inhalation method in a hyperbaric oxygen chamber. All rats received hyperbaric oxygen therapy for two weeks. Rats in the farsudil treatment group were intraperitoneally injected with hydrochloride farsudil for intervention (15 mg/[kg·d], once a d for 2 weeks), while those in the CO poisoning and normal control groups received the same volume of normal saline. The ultrastructures of rat brain tissues were observed by transmission electron microscopy one week after modeling. Staining intensities of Nogo- and OMgp-positive cells were detected by immunohistochemistry, and those of Rock-positive cells were analyzed by immunofluorescence one d, one week, one month and two months after modeling. The protein expressions of Nogo, OMgp and Rock in brain tissues were detected by Western blotting one d, one week, one month and two months after modeling. Results:In the CO poisoning group, the ultrastructures of brain tissues and blood-brain barrier were damaged obviously, and the changes in nucleus, mitochondria and synaptic structure were obvious; while fasudil treatment could effectively maintain the integrity of ultrastructures and functions of brain tissues, and reduce brain edema. One d, one week, one month and two months after modeling, the staining intensities of Nogo, OMgp and Rock positive cells and protein expression levels of Nogo, OMgp and Rock in the CO poisoning group were significantly higher than those in the normal control group at the same time point ( P<0.05); the staining intensities of Nogo, OMgp and Rock positive cells and protein expression levels of Nogo, OMgp and Rock in the fasudil treatment group were significantly lower than those in the CO poisoning group at the same time point ( P<0.05). Conclusion:The activation of Nogo-OMgp/Rho-Rock signaling pathway related molecules (Nogo, OMgp and Rock) is closely related to acute brain injury caused by CO poisoning; hydrochloride fasudil can effectively down-regulate the protein expressions of Nogo, OMgp and Rock, therefore obviously alleviate brain injury after CO poisoning.

BACKGROUND: Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). METHODS: Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. RESULTS: Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7–33 nm in width and 130–600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. CONCLUSION: This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.
Actin Cytoskeleton ; Blotting, Western ; Bone Regeneration ; Cell Count ; Cell Culture Techniques ; Circular Dichroism ; Endopeptidase K ; Fluorescent Antibody Technique ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Osteogenesis ; Sincalide ; Spectrum Analysis

The two-dimensional (2D) cell culture model is currently used to study cellular processes and drug screening for human diseases. However, the growth of cells is affected by many factors. For conventional 2D cell culture, many of the difficulties are encountered in accurately replicating the cell function in three-dimensional (3D) tissues. Compared with 2D cell culture, much attention is paid to the cell-to-cell and cell-matrix interactions for 3D cell culture systems, which can more closely mimic the growth environment for cultured cells. Therefore, the 3D cell culture system was more suitable for a variety of applications such as drug screening and cell proliferation. In this work, we prepared microarray-structured polymer films with different geometric structures by nanoimprint lithography and used the films as cell culture platforms for the culture of 293T cells. Through the adjustment of the surface morphology and water contact angle of the prepared films, the regulation of the morphological changes of cell growth was successfully realized. Experimental results demonstrated that the hydrophilic films with 10 μm-pillar microstructure are applicable to 3D cell culture, whereas the hydrophobic films with 3 μm-pillar microstructure are only suitable for 3D culture of cells with a smaller size and stiff cuticular layer. In addition, cells tended to the formation of spheroids on the hydrophobic films, while cells usually adhered to the surface and grew on the hydrophilic films. This work represents further technological progress in the development of 3D cell culture, thereby facilitating future studies of physiologically relevant processes.
Cell Culture Techniques ; Cell Proliferation ; HEK293 Cells ; Humans ; Polymers

Currently,there is no study on the unified coding of the spinal nerve and its main branches.The positions of spinal nerve were encoded according to the basic anatomical principles from top to bottom,from inside to outside and from front to back,with reference to AO bone classification and somatic artery coding and injury classification system.The segmental coding of spinal nerves was decided by its branches and running characteristics.The spinal nerve injuries were encoded by a combination of numbers and letters.The first number represented the region,the second represented the injured nerve,and the third represented the specific segment of injured nerve.The injuries of spinal nerve were divided into 5 categories according to severity.The first letter indicated the category of injury,and the second indicated the orientation of the injured nerve.The functional score scale of the innervation area was prepared based on the evaluation of motor function,sensory function and the results of neurophysiological examination.This scale was used to classify the neurological injuries,guide the clinical treatment and evaluate the prognostic outcomes.This coding and classification system can clearly and comprehensively describe the location and type of spinal nerve injuries,and it is convenient for the diagnosis of nervous system damage.It also has important reference value for treatment and prognosis evaluation.

Objective To evaluate the treatment of lateral tibial plateau fractures involving the posterolateral condyle with double plate fixation via the combined anterolateral and posterolateral approaches.Methods From January 2013 to August 2015,12 patients with lateral tibial plateau fracture involving the posterolateral condyle were treated by double plate fixation via the combined anterolateral and posterolateral approaches.They were 8 males and 4 females,with an average age of 36.9 years (range,from 23 to 58 years).According to the Schatzker classification,we had 8 ones of type Ⅱ,3 ones of type Ⅴ and one of type Ⅵ.Firstly,the posterolateral condylar fractures were stabilized temporarily by the Kirschner wire after reduction through the posterolateral approach.Secondly,the lateral tibial plateau fractures were dealt with via the anterolateral approach,and fixated with a proximal tibial locking plate.Finally,a T-shaped plate for the distal radius was used to support the posterolateral condyle.The clinical and radiographic results were evaluated using the Hospital for Special Surgery (HSS) knee score and the Rasmussen Score,respectively.Results The average operative time was 143.3 min (range,from 110 to 210 min);the average intraoperative blood loss was 190.0 mL (range,from 100 to 300 mL).The follow-ups for the 12 cases ranged from 12 to 24 months (average,16.6 months).All the cases obtained clinical healing after 12 to 24 weeks (average,16 weeks).At the last follow-up,the HSS knee-scores ranged from 65 to 98 points (average,84.6 points),giving 7 excellent cases,3 good ones and 2 fair ones.The Rasmussen radiological scores ranged from 13 to 18 points (average,17.1 points),giving 8 excellent and 4 good cases.Conclusions The combined anterolateral and posterolateral approaches can offer excellent exposure for articular reduction and fixation with double plates,achieving satisfactory radiological and functional results in lateral tibial plateau fractures involving the posterolateral condyle.First reduction of the posterior lateral condylar fractures,followed by reduction and fixation of the anterolateral plateau fractures and finally fixation of the lateral condylar fractures,can lead to effectively improved knee function for the patients.