Effects of filament B knockdown on proliferation,migration and apoptosis of mouse MC3T3-E1 cells / 中国组织工程研究

BACKGROUND:Filamin B(FLNB)can crosslink the actin cytoskeleton into a dynamic structure that is essential for the directional movement of cells.It can regulate the proliferation,differentiation and apoptosis of chondrocytes.However,the effect of FLNB on osteoblast proliferation,migration and apoptosis has not been reported. OBJECTIVE:To investigate the effect of FLNB on the proliferation,migration and apoptosis of MC3T3-E1 cells. METHODS:The adenoviral vectors for knockdown of FLNB expression(sh-FLNB1,sh-FLNB2,sh-FLNB3)were constructed and infected with MC3T3-E1 cells.After screened by puromycin drug,the efficiency of FLNB knockdown was detected by western blot and RT-PCR.The MC3T3-E1 cell line with the best efficiency of FLNB knockdown was selected as the stable transient cell line of MC3T3-E1 for subsequent experiments.The cells were divided into blank group,mc3t3 group,sh-NC group(empty vector),and sh-FLNB group(sh-FLNB lentivirus).The blank group was cultured in cell-free α-MEM complete medium;the mc3t3 group was cultured in α-MEM complete medium alone;and the sh-NC and sh-FLNB groups were cultured with α-MEM medium containing 2.5 μg/mL puromycin.After 3 days of culture,cell counting kit-8 assay and cell scratch assay were used to detect the proliferation and migration ability of MC3T3-E1;flow cytometry was used to detect cell apoptosis;and RT-PCR was used to detect the expression of apoptosis-related genes. RESULTS AND CONCLUSION:Western blot and RT-PCR results showed that the efficiency of FLNB knockdown was the best in the sh-FLNB3(P<0.000 1),which was used as a stable cell line for subsequent experiments.Cell counting kit-8 data showed that the proliferative ability of MC3T3 cells was significantly weakened after knockdown of FLNB(P<0.05).Cell scratch assay results showed that the migration ability of MC3T3 cells was significantly decreased after knockdown of FLNB.Flow cytometry and RT-PCR results showed that the apoptotic rate of MC3T3-E1 cells increased after knockdown of FLNB,the expression of pro-apoptotic factor Bax increased significantly,and the expression of anti-apoptotic factor Bcl-2 decreased significantly(P<0.05).To conclude,knockdown of FLNB can reduce the proliferation ability of MC3T3-E1 cells,decrease the migration ability of the cells,and increase cell apoptosis.

Circular RNAs are involved in the pathogenesis of osteoarthritis through intracellular mechanisms / 中国组织工程研究

BACKGROUND:Currently,there is no drug that can completely cure osteoarthritis and its pathogenesis is still unclear.Circular RNAs(circRNAs)are differentially expressed in patients with osteoarthritis and are closely associated with various pathological processes in osteoarthritis.circRNAs play an important role in various physiological and pathological processes,such as chondrocyte homeostasis,extracellular matrix formation,and inflammatory response. OBJECTIVE:To mainly review the effects of circRNAs on pathological factors related to osteoarthritis,as well as the types and expression levels of circRNAs in osteoarthritis. METHODS:Related articles published from 1976 to August 2023 were retrieved from CNKI,WanFang,VIP,PubMed,Medline,Web of Science and Elsevier databases.The keywords were"osteoarthritis,circular RNA,non-coding RNA,synovial tissue,chondrocytes"in Chinese and English,respectively.All the relevant articles were screened,summarized,analyzed,and finally 69 papers were included in the review. RESULTS AND CONCLUSION:circRNAs are non-coding RNAs widely found in eukaryotic cells,with covalently closed continuous loop structure,but with no 5'hat structure and 3'poly A tail,which are involved in multi-gene and multi-target regulatory networks and cannot be degraded by nucleic acid exonucleases(RNase R).circRNAs have a high abundance,high conservativeness and stability,and cell and tissue specificity.circRNAs have biological functions such as acting as molecular sponges for miRNAs,regulating linear RNA transcription and RNA shearing,interacting with RNA-bound proteins,and translating proteins.circRNAs regulate chondrocyte apoptosis and proliferation,degradation of cartilage extracellular matrix,and inflammation and other physiopathologic processes.circRNAs are expected to become biomarkers and potential therapeutic targets for clinical diagnosis and prognosis of osteoarthritis,and may become a new strategy for clinical treatment of osteoarthritis in the future.

Beverages consumption among grade four and grade five primary school students in six cities of China / 中国学校卫生

Objective@#To understand the status on beverages consumption among grade 4 and grade 5 primary school students in six cities of China, and to provide evidence for nutrition education and intervention strategies.@*Methods@#A multi-stage stratified cluster random sampling method was used to select 12 197 grade 4 and grade 5 primary school students from 72 primary schools in Beijing, Guangzhou, Nanjing, Chongqing, Jinan and Harbin. All the participants were investigated with a self-administered questionnaire survey of dietary behaviors.@*Results@#The proportion of students who consumed beverages at home, school and elsewhere was 92.5%, 51.4% and 70.6% respectively. The most popular beverages at home were milk, fruit & vegetable drinks, tea drinks (69.4%, 46.6%, 39.6%); the most popular beverages at school were milk, fruit & vegetable drinks, tea drinks (30.5%, 13.0%, 12.7%) while the most popular beverages in other places were milk, tea drinks, fruit & vegetable drinks(37.4%, 29.6%, 28.1%). The top five reasons for choosing beverages were taste delicious, healthy & nutritious, clean, choice of peers and family members(72.3%, 50.8%, 38.4%, 21.9%, 21.6%, respectively).@*Conclusion@#Consumption of drinking beverages is popular among students, most of which are unhealthy. Therefore, nutrition education for students and parents should be encouraged aiming to help students choose healthy drinks and eating behaviors.

Establishment and application of co-transfection screening method for phytoestrogen active constituents / 中国中药杂志

<p><b>OBJECTIVE</b>To establish a highly sensitive screening method for phytoestrogen active constituents and to primarily screen the phytoestrogenic active constituents from the chickpea extractions by the method.</p><p><b>METHOD</b>Human ERalpha cDNA was cloned using MCF-7 total RNA as the template by RT-PCR and then was constructed into a pcDNA3 and named as pERalpha. The cell line MCF-7 was co-transfected with pERalpha and the reporter plasmid pERE-Luc which carrying the estrogen response element (ERE) plus the luciferase reporter gene. The luciferase activity was then assayed. The model was optimized by changing the ratio of two plasmids. The feasibility of the optimized model was further proved by the several known phytoestrogen compounds including fermononetin, biochanin A and genistein, et al. As an application of the model, the phytoestrogen activity of the extracts of the chickpea was assayed.</p><p><b>RESULT</b>The recombinant plasmid (pERalpha) can enhance luciferase activities of pERE-Luc transfected MCF-7 cells. The highest transfection efficiency and luciferase activity were found at the ratio of 10:1 (pERE-Luc: pERalpha), the luciferase activity was improved five times as high as the unique pERE-Luc transfection. The co-transfection screening model also indicated that fermononetin, biochanin A and genistein could induce ERE-driven luciferase activity and ICI 182,780 suppressed the induced transcription. As the application of the model, the results showed that the ethanol (70%) total extraction, the ethyl acetate extraction and the ligarine extraction of the chickpea can induce ERE-driven luciferase activity. Concurrent treatment with ICI 182,780 abolished the induced luciferase activity.</p><p><b>CONCLUSION</b>A phytoestrogen active constituent screening mode have been established based on co-transfection method. It is sensitive to assay the phytoestrogen active constituents and can be applied to screen the active component of phytoestrogens.</p>

cDNA Cloning and Sequence Analysis of Human Bactericidal/Permeability-increasing Protein / 微生物学通报

Human bactericidal/permeability-increasing protein(hBPI)cDNA was amplified by reverse transcription(RT)and touchdown PCR(TD-PCR)from blood stem cells collected from healthy human of Uygur nationality in Xinjiang Uygur Autonomous Region of China, and then was subcloned into pEGFP-N1 vector,hBPI cDNA sequence consists of 1,464bp.Comparison with other 4 hBPI cDNA sequences registered in GenBank identified 99% homology in DNA sequence.However,there were two base substitutions(nucleotide 576G→C,nu- clotide 676A→G),one of which resulted in an amino acid(residue 185 Lys→Glu).