Objective To construct humanized monoclonal antibodies against CD 20 and check their affinity to CD 20 antigen and their anti-tumor activity.Methods Based on the computer model , human IgG1 candidates closest to rituximab in crystal structure were selected in the Protein Data Bank ( PDB) .With the selected human IgG 1 candidates as the frame , we modified and transplanted the complementarity determining region ( CDR) of rituximab .First,the target gene fragments were obtained by overlapping PCR.Then, the sequences of the light chains(L) and the heavy chains(H) were inserted in-to the pcDNA3.3 and pOptiVEC vectors.Next, the constructed clones were transfected into 293F cells through transient transfection.After a large-scale cell culture, the mAb was purified by affinity chromatography rProtein A column.The puri-ty and expression level of the humanized antibodies was tested by sodium dodecyl sulfate ( SDS)-polyacrylamide gelelectro-phoresis(PAGE).The affinity of the humanized antibodies to CD20 was assessed with Fortebio assay.Finally, the anti-tumor activity of the constructed antibodies was detected by checking the tumor growth inhibition of the nude mice transplan-ted with tumor .Results Three humanized monoclonal antibodies against CD 20 were expressed and purified successfully . In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 ×103 and 55 ×103 , respectively.The band size of the antibodies matched the expected value.Fortebio assay revealed that the humanized antibodies could bind to CD20 with high affinity (rituximab:6.48 ×10 -9mol/L, L4H7:1.91 ×10 -9mol/L, L5H5:7.35 ×10 -10mol/L,and L5H7:1.91 ×10 -9mol/L).The tumor growth inhibition experiment showed that the anti-tumor activity of L5H7 mAb was better than that of rituximab .Conclusion Three humanized monoclonal antibodies against CD 20 have been successfully construc-ted and expressed.L5H7 mAb possesses high affinity for CD20 and a good ability to kill tumor cells.

Objective:To evaluate the efficacy and safety of Tuina(Chinese therapeutic massage)in the treatment of functional dyspepsia(FD)in children and adults. Methods:Related articles in PubMed,Excerpta Medica Database(EMBASE),Cochrane Library,Web of Science,China Biology Medicine Disc(CBM),Wanfang Academic Journal Full-text Database(Wanfang),China National Knowledge Infrastructure(CNKI),and Chongqing VIP Database(CQVIP)were collected.The retrieval time was from each database's start to March 2022.Two researchers independently screened the literature,extracted the data,and evaluated the risk of bias in the included studies.A meta-analysis was then performed using the RevMan 5.4 software. Results:A total of 19 clinical trials were included,9 of which encompassed studies on adults while 10 were on children with FD,comprising a total of 1961 patients.The findings of the meta-analysis showed that the effective rate of FD in children and adults treated with Tuina was significantly higher than that in the control group[risk ratio(RR)=1.15,95%confidence interval(CI)(1.09,1.21),P<0.001],[RR=1.13,95%CI(1.06,1.21),P<0.001].In addition,the effective rate of FD in children and adults treated with Tuina combined with other treatments was significantly higher than that in the control group[RR=1.14,95%CI(1.07,1.21),P<0.001],[RR=1.12,95%CI(1.02,1.24),P=0.02].In terms of single symptoms,Tuina improved epigastric burning sensation score in adults[standardized mean difference(SMD)=-0.41;95%CI(-0.79,-0.02);Z=2.08;P=0.04]compared with that of the Western medicine group.Compared with children treated with oral Chinese medications(CM)or Chinese patent medicine(CPM),children with FD demonstrated lower scores of epigastric pain[SMD=-0.38,95%CI(-0.56,-0.19);Z=3.96;P<0.001],postprandial fullness[SMD=-0.30,95%CI(-0.50,-0.10);Z=2.88;P=0.004],and early satiety[SMD=-0.26,95%CI(-0.47,-0.06);Z=2.54;P=0.01]after receiving Tuina combined with CM or CPM treatment.No adverse events were reported in the Tuina treatment group,and the follow-up indicated that the symptom scores in the Tuina group improved. Conclusion:Compared with the control group,both Tuina and Tuina combined with other treatments are shown to have better effective rates,lower incidence of adverse events,and better follow-up outcomes.The study results suggest that Tuina may be a clinically viable complementary therapy.However,due to limitations in the number and quality of the included studies,the above conclusions should be verified by further high-quality studies.

Objective:To investigate the role of mitofusion 2 (Mfn2) in high glucose (HG)-induced endoplasmic reticulum stress (ERS) and apoptosis of podocytes.Methods:(1) Streptozocin was used to induce a diabetes mellitus (DM) rat model. Renal histopathological changes in rats were observed by HE staining. Expression of Mfn2 and CCAAT/enhancer-binding protein homologous protein (CHOP) in glomeruli was observed by immunohistochemistry. Protein levels of Mfn2, protein kinase RNA-like ER kinase (PERK), phospho(p)-PERK, and CHOP in glomeruli were analyzed by Western blotting. (2) Conditionally immortalized human podocytes (HPC) cultured in vitro were divided into control, mannitol (MA) and HG groups. Expression of Mfn2 was observed by immunofluorescence. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. (3) HPC were divided into control, HG, HG+Mfn2-Myc plasmid-transfected and HG+control plasmid-transfected groups. Protein levels of Mfn2, p-PERK, PERK and CHOP in HPC were analyzed by Western blotting. Expression of CHOP was observed by immunofluorescence. Mitochondrial membrane potential in each group was observed by mitochondrial membrane potential assay kit with JC-1. Podocyte apoptosis in each group was evaluated by flow cytometry with AnnexinⅤ-PE/7AAD double staining method. Results:(1) Compared with the control group, the glomerular mesangial matrix of the DM group rats was significantly proliferated, and the expression of Mfn2 was down-regulated with the expression of ERS-related proteins p-PERK/PERK and CHOP up-regulated (all P<0.05). (2) Compared with the control group, Mfn2 was down-regulated and p-PERK/PERK and CHOP were up-regulated in HPC of HG group (all P<0.05). Apoptosis of HPC was also increased in HG group. There was no significant difference in the above indicators between the control group and the mannitol group (all P>0.05). (3) Compared with the HG group, mitochondrial membrane potential of HPC was alleviated and apoptosis of HPC was decreased in HG+Mfn2-Myc plasmid-transfected group ( P<0.05). P-PERK/PERK and CHOP were down-regulated in HG+Mfn2-Myc plasmid-transfected group (both P<0.05). There was no significant difference in the above indicators between the HG group and the HG+control plasmid-transfected group (all P>0.05). Conclusions:Mfn2 down-regulation in HG-stimulated podocytes may induce ERS to increase apoptosis of podocytes. Up-regulation of Mfn2 can alleviate the HG-induced ERS and apoptosis in podocytes.

Objective:To construct a set of weighted trigger index system for dynamic adjustment of medical service price,so as to provide references and basis for price adjustment of complex medical service projects in Tangshan.Methods:The Delphi expert consultation method was used to demonstrate and determine the index system,and the difference comparison method and product method were used to determine the weight coefficient for each index.Results:The trigger index system of dynamic adjustment of medical service price including 4 first-level indicators,10 second-level indicators and 29 third-level indicators,and the weight coefficient of each indicator was given.Conclusion:The trigger index system of dynamic adjustment of medical service price in Tangshan has high scientificity and reliability,which is helpful for Tangshan to establish a sensitive and moderate price dynamic adjustment mechanism.Considering the affordability of all parties in the price adjustment of medical services in Tangshan,it is conducive to strengthening the total price control and structural adjustment of medical services in Tangshan.

Objective:This study aims to analyze the threshold changes in distortion product otoacoustic emissions（DPOAE） and auditory brainstem response（ABR） in adult Otof-/- mice before and after gene therapy, evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment. Methods:At the age of 4 weeks, adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene, delivered via dual AAV vectors through the round window membrane（RWM）. Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment. Results:AAV-PHP. eB demonstrates high transduction efficiency in inner ear hair cells. The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice. The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz. Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment. Conclusion:Gene therapy significantly restored hearing in adult Otof-/- mice, though the surgical delivery may cause transient hearing damage. Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.
Mice ; Animals ; Otoacoustic Emissions, Spontaneous/physiology* ; Hearing/physiology* ; Ear, Inner ; Hearing Loss/therapy* ; Genetic Therapy ; Auditory Threshold/physiology* ; Evoked Potentials, Auditory, Brain Stem/physiology* ; Membrane Proteins

Objective To study the mechanical properties and biological characteristics of 3D-printed porous β-tricalcium phosphate ［β-Ca3(PO4)2, β-TCP］ scaffolds, so as to provide guidance for the design of composite scaffolds in animal experimentation. Methods Poly 1,8-octanediol citrate (POC), a kind of novel biodegradable materials, was used as the adhesive. The 3D-printed porous β-TCP scaffolds were fabricated by fused deposition modeling (FDM) technology, and Gly-Arg-Gly-Asp-Ser (GRGDS), a kind of polypeptides, was added into the scaffolds to improve the adhesive property of cells. The optical microscope and scanning electron microscope (SEM) were used to observe the micro-pore architectures of those scaffolds. The material testing machine was used to conduct compressive test on the scaffolds, and the water contact angles of the scaffolds were measured. The cell adhesion rate and proliferation rate of the scaffolds were also tested by in vitro cell experiment. The model of SD rat skull defects was repaired by the scaffolds, and the osteogenic ability in vivo was further studied. Results The GRGDS, remaining active, was evenly distributed in the composite scaffolds. The micro-pore architectures of the polypeptide modified scaffolds changed, with improvement in cell adhesion rate, while the compressive modulus, water contact angle and osteogenic ability in vivo of the scaffolds were not obviously affected. Conclusions The cell adhesion capacity of β-TCP composite scaffolds modified by polypeptide improved significantly, while the mechanical properties and hydrophilicity, osteogenic ability in vivo of the scaffolds were not affected very much. These research results provide new ideas for reconstruction of scaffolds for repairing bone defects in clinic, and a laboratory basis for further clinical application of this scaffold.

Objective To systematically evaluate the efficacy and safety of different non-steroidal anti-inflammatory drugs (NSAIDs) in middle-aged and old Chinese patients with osteoarthritis(OA). Methods A systematic literature search was conducted through PubMed, Cochrane Library, CNKI, Wan Fang Data and VIP databases to collect randomized controlled trials with non-steroidal anti-inflammatory drugs in middle-aged to old Chinese OA patients. The search time was from the establishment of the database to November 17, 2020. Two researchers independently carried out literature screening, data extraction and literature quality evaluation. Bayesian network meta-analysis was conducted with R3.6.0 software. Results 28 RCTs were included with 2531 patients. Based on the last follow-up pain visual analogue scale (VAS) score, the ranking chart showed that Etoricoxib had the highest probability of having the lowest pain VAS score (88.55%). In terms of total effective rate, the ranking chart showed that the probability of Etoricoxib as first choice was the highest (92.49%). As far as safety, diclofenac sodium patch had the lowest adverse effects rate (59.10%). Conclusion The results of this study indicated that Etoricoxib was the most effective treatment for middle-aged and old Chinese OA patients. It can significantly reduce the OA pain. Diclofenac sodium patch had the least adverse effects.

ObjectiveTo investigate the effect and mechanism of Yishen Tongluo prescription in protecting mice from oxidative stress injury in diabetic kidney disease （DKD） via the nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1）/NAD（P）H quinone oxidoreductase 1 （NQO1） signaling pathway. MethodsSpecific pathogen-free （SPF） male C57BL/6 mice were assigned into a control group （n=10） and a modeling group （n=50）. The DKD model was established by intraperitoneal injection of streptozotocin. The mice in the modeling group were randomized into a model group， a semaglutide （40 μg·kg^-1） group， and high-， medium-， and low-dose （18.2， 9.1， 4.55 g·kg^-1， respectively） Yishen Tongluo prescription groups， with 10 mice in each group. The treatment lasted for 12 weeks. Blood glucose and 24-h urine protein levels were measured， and the kidney index （KI） was calculated. Serum levels of creatinine （SCr）， blood urea nitrogen （BUN）， alanine aminotransferase （ALT）， and aspartate aminotransferase （AST） were assessed. The pathological changes in the renal tissue were evaluated by hematoxylin-eosin， periodic acid-Schiff， periodic acid-silver methenamine， and Masson’s trichrome staining. Enzyme-linked immunosorbent assay kits were used to measure the levels of β2-microglobulin （β2-MG）， neutrophil gelatinase-associated lipocalin （NGAL）， kidney injury molecule-1 （KIM-1）， liver fatty acid-binding protein （L-FABP）， nitric oxide synthase （NOS）， glutathione （GSH）， total antioxidant capacity （T-AOC）， and 8-hydroxy-2'-deoxyguanosine （8-OHdG）. Immunohistochemical staining was performed to examine the expression of Kelch-like ECH-associated protein 1 （Keap1） and malondialdehyde （MDA）. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR） and Western blot were employed to determine the mRNA and protein levels， respectively， of factors in the Nrf2/HO-1/NQO1 signaling pathway. ResultsCompared with the control group， the DKD model group showed rises in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with glomerular hypertrophy， renal tubular dilation， thickened basement membrane， mesangial expansion， and collagen deposition. Additionally， the model group showed elevated levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， lowered levels of GSH and T-AOC， up-regulated expression of MDA and Keap1， and down-regulated expression of Nrf2， HO-1， NQO1， and glutamate-cysteine ligase catalytic subunit （GCLC） （P<0.05）. Compared with the model group， the semaglutide group and the medium- and high-dose Yishen Tongluo prescription groups showed reductions in blood glucose， 24-h urine protein， KI， SCr， BUN， and ALT levels， along with alleviated pathological injuries in the renal tissue. In addition， the three groups showed lowered levels of β2-MG， NGAL， KIM-1， L-FABP， NOS， and 8-OHdG， elevated levels of GSH and T-AOC， down-regulated expression of MDA and Keap1， and up-regulated expression of Nrf2， HO-1， NQO1， and GCLC （P<0.05）. ConclusionYishen Tongluo prescription exerts renoprotective effects in the mouse model of DKD by modulating the Nrf2/HO-1/NQO1 signaling pathway， mitigating oxidative stress， and reducing renal tubular injuries.