Analysis of microRNAs and target genes associated with immune response to EVA71 and CVA16 infection in human respiratory epithelial cells / 中华微生物学和免疫学杂志

Objective:To analyze differentially expressed microRNAs (miRNAs) and target genes in human respiratory epithelial cells (16HBE) after enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) infection using high-throughput sequencing.Methods:TargetScan and miRDB databases were used to predict the target genes of miRNAs that were both up-regulated or down-regulated after EV71 and CVA16 infection. The genes that were both up-regulated and down-regulated were screened out. GO and pathway analysis of the target genes were conducted to screen immune-related target genes and their corresponding miRNAs. The target genes and their corresponding miRNAs that were up-regulated or down-regulated in both immune-related GO and pathway were further screened. Some miRNAs and their target genes were selected for qRT-PCR verification.Results:There were 598 target genes of up-regulated miRNAs and 1 311 target genes of down-regulated miRNAs and 62 target genes that might be up-regulated or down-regulated simultaneously were screened out. The number of up-regulated target genes involved in immune-related GO and pathway were 17 and 13, respectively, and the number of corresponding miRNAs were 15 and 17, respectively. There were 58 and 47 down-regulated target genes involved in immune-related GO and pathway, respectively, and the number of corresponding miRNAs were 30 and 42, respectively. Three up-regulated target genes were involved in both immune-related GO and pathway and regulated by four miRNAs. Nine down-regulated target genes were involved in both immune-related GO and pathway and regulated by 13 miRNAs.Conclusions:This study was conducive to elucidate the host-pathogen interaction after EV71 and CVA16 infection, and provided reference for studying the pathogenesis of hand, foot and mouth disease.

Effects of CVA16 infection on m 6A methylation-related protein expression and localization / 中华微生物学和免疫学杂志

Objective:To investigate whether coxsackievirus A 16 (CVA16) infection would affect the expression of N6-methyladenosine (m 6A) methylation-related proteins in human bronchial epithelial cells (16HBE), ICR suckling mice and SCRBA2 humanized mice and influence their subcellular localization. Methods:CVA16 was used to infect 16HBE cells at a multiplicity of infection (MOI) of 0.1 and mice at 10 7 CCID 50/ml. Changes in the expression of methyltransferases, demethylases and methylated reading proteins were analyzed by Western blot. Cellular localization of these proteins was observed using immunofluorescence. Results:The expression of m 6A methylation-related proteins was gradually reduced in CVA16-infected cells with time, but showed no obvious change in ICR suckling mice or SCRBA2 humanized mice. After infection, m 6A methylation-related proteins were redistributed in both the nucleus and cytoplasm and even degraded. Conclusions:CVA16 replication in host cells altered the expression and cellular localization of m 6A methylation-related proteins, which indicated that m 6A modification might be a new potential target for enterovirus therapy.

Expression and purification of rabies virus glycoprotein and analysis of its specific binding capacity to memory B cells / 生物工程学报

We aimed to express and purify three rabies virus glycoproteins with different tags and sizes. After analyzing their binding function, we wish to obtain a rabies virus glycoprotein with higher affinity and ability to specifically bind memory B cells. Experiments were carried out to express full length, as well as the ectodomain RVG by gene engineering method. Combined with the antibody of CD19 and CD27, the candidate protein labeling with fluorescence was used to analyze its binding function. Flow cytometry was used to detect the anti-rabies virus specific memory B cells in PBMCs, and confirm the binding ability between the candidate proteins and anti-rabies virus-specific memory B cells. We successfully constructed three expression vectors pGEX-5X-1-RVG, pET28a-RVG and pET30a-G. Three glycoproteins GST-RVG, His-RVG and His-G were obtained by optimized expression and purification conditions. The antigen specificity of purified GST-RVG, His-RVG and His-G were identified by Western blotting and ELISA. The affinity of these three purified glycoproteins to anti-rabies virus antibody were detected by competitive ELISA. Anti-rabies virus specific memory B cells in positive PBMCs gained from people who had ever been injected with the vaccine can be detected by flow cytometry. Thus, we got a recombinant rabies virus glycoprotein that had high-affinity and could sort antigen specific memory B cells.

A study on the morphological characters of immortalized mandibular condylar chondrocyte / 华西口腔医学杂志

<p><b>OBJECTIVE</b>The purpose of this study was to compare the morphological character between immortalized mandibular condylar chondrocyte (IMCC) and primarily cultured mandibular condylar chondrocyte (MCC).</p><p><b>METHODS</b>The phase contrast microscope, photomicroscope and transmission electron microscope were used to observe the morphological character of IMCC and MCC. The highresolution pathological image and word report system-1000 (HPIAS-1000) was used to compare the size of IMCC and MCC.</p><p><b>RESULTS</b>The phase contrast micrography showed that MCCs in primary culture underwent distinct morphological changes with respect to shape, size, and density of the cells. The majority of MCCs were in polygonal shape earlier in culture, while more fusi-form and spindle-shaped cells were found after 4-5 passages. While IMCCs were polygonal-shaped, similar to MCCs. Subculture, freezing and recovering had no effect on cellular shape of IMCC. Transmission electron microscopy indicated that MCC had chondrocyte-like phenotype, while IMCC looked like prechondroblast or immature chondrocyte. Some of IMCCs had irregular nucleus, and the proportion of nucleus/cytoplasm changed. By analysis of HPIAS-1000, the diameter and area of IMCC were obvious smaller than those of MCC (P < 0.01).</p><p><b>CONCLUSION</b>IMCC retain the main morphological character of MCC, and also keep a stable phenotype, which belong to immature chondrocytes, similar to cells in the proliferative zone.</p>

Human telomerase reverse transcriptase(hTRT) mRNA expression in squamous cell carcinoma and the relationship between hTRT and PCNA expression / 实用口腔医学杂志

砄bjective: To investigate Human telomerase reverse transcriptase(hTRT) mRNA expression in oral squamous cell carcinoma(OSCC) and the relationship between hTRT activity and proliferation cell nuclear antigen(PCNA) expression.Methods: The expression of hTRT mRNA and PCNA was detected by in situ mRNA hybridization and immuno histochemical technique in 52 cases of OSCC and 22 cases of OSCC adjacent tissues including histologically normal mucosas and dysplastic lesions. Results: hTRT mRNA was observed in 82.7% cases of OSCC, 41.7% of dysplasia and 20% of normal mucosa( P

The phenotype characteristic of mandibular condylar chondrocytes in vitro / 实用口腔医学杂志

Objective: To study the phenotype characteristic of mandibular condylar chondrocytes (MCCs) in vitro. Methods: MCCs of two-week-old New Zealand rabbits were isolated with enzyme digestion and cultured in DMEM. Phase contrast microscopy, image analysis system, MTT assay and immunocytochemistry method were used to compare MCCs of different passages in cellular shape and size, growth curve and expression of type I and type II collagen. Results: The majority of MCCs in earlier passages (1～3 passage) were polygonal, while more fusiform and spindle-shaped cells were found after 5～6 passages. MCCs in earlier passages were smaller than those in sixth passages. The proliferation rate of MCCs decreased with passaging. There were more type II collagen positive cells in 1 ～ 3 passages, while more type I collagen positive cells in 7th passage. Conclusion: The changing characters of MCCs in vitro such as cellular shape and size and expression of type I and II collagen are similar to those of MCCs in vivo from proliferating zone to hypertrophic zone. MCCs in later passages may be with dedifferentiation.

The significance of PCD and mRNA transcription of p 53 gene during development of normal palate and cleft palate in C57BL/6N mouse / 实用口腔医学杂志

砄bjectives:To observe the difference of programmed cell death (PCD) and the expression of related gene p 53 in the development of normal palate and in the formation of cleft palate.Methods:The model of the development of cleft palat was estabished with retinoid acid (80 mg?kg 1 for each pregnant mouse). The palate samples were obtained at GD13 14 (at 13rd day 14 hours of prenancy),13 22 ,14 8,14 14 ,14 33 ,15 8,15 22 and 16 8 respectively.PCD was detected with TUNEL staining,while the mRNA transcription of p 53 was observed by in situ hybridization.Rssults:In early development of palate process,the positive index of PCD in the cleft palate samples was significantly higher than that in the normal ( P 0.05) between the two groups.Conclusion:The proliferation of mesenchymal cells of the early developmental palate process may be inhibited due to the abnomal PCD in the formation of cleft palate.The mechanism of PCD in this study may be a p 53 independed pathway.