Objective To construct anti-IL-4R murine anti-human single-chain variable fragment (scFvs) antibodies through BL21 (DE3) prokaryotic expression system. Methods The anti-IL-4R scFv sequence was optimizated on the basis of previous findings. The optimized scFv sequence was analyzed. The recombinant plasmid pET-32a-scFv was constructed. The recombinant plasmid was detected through enzyme identification, and was turned into BL21 (DE3) prokaryotic expression bacteria to express the pET-32a-scFv recombinant protein in E.coli BL21 (DE3). The purification and renaturation were researched, and SDS-PAGE analysis was studied. The molecular weight of ScFv against IL-4R was analyzed by SDS-PAGE. The expression of the fusion protein was detected by Western-blot assay. Results The length of fusion gene scFv-MLT sequence was 761 bp. The molecular weight of the recombinant expression of proteins of anti-IL-4R single antibody was approximately 45 ku. The recombinant proteins showed high specificity with anti-6 × His-tag antibody. Conclusion This experiment successfully constructs pET-32a-scFv prokaryotic expression system of recombinant protein with high immune reactivity, which provides the basis for further study of anti-IL-4R single chain antibody as drug target.

Objective: To observe the mechanism and characteristics of new bone formation and remodeling process during distraction osteogenesis (DO) in the reconstruction of bone defect in cleft palate (CP). Methods: Sagittal palatal osteoectomy were performed in 12 cats to establish the CP model. The CP defects were reconstructed by intraoral distractors at the rate and rhythm of 0.4 mm?2/day till the transport disc (TD) reached the opposite edge across the defect region. Tetrachloride fluorescent labeling was administered 6 days before euthanasia specimen retrieval at 2, 4, 6, 8, 12 weeks through fixation period. Histological and fluoroscopical study were performed; control groups (2 animals in each group) were set for comparison. Results: The bone defect of CP was successfully reconstructed by DO. Exclusively intramembranous de novo osteogenesis were observed. Soft tissues attaching to TD were elongated simultaneously. No spontaneous repair was observed in control animals. Conclusion: With effective distraction and steady fixation, CP bone and soft tissue defect can be reconstructed by active intramembranous bone formation and remodeling following the applicaion of DO.

OBJECTIVETo recognize the characteristics of desmoplastic small round cell tumor (DSRCT) and improve the standard of diagnosis.

METHODSWe retrospectively reviewed the clinical data on the treatment of 2 patients with DSRCT in terms of their conditions, tissue sources, pathologic characteristics, immunohistochemical methods, clinical manifestation, diagnosis, treatment and prognosis.

RESULTSClinical manifestations were complicated. The 2 patients were mis diagnosed before operation. Their tumors consisted of irregular nests of small and round cells, with nuclear hyperchromatism and scant cytoplasm embedded in a plenty of fibrous connective tissues. The edge of the nest was clear, with different sizes and shapes. Immunohistochemically, the 2 patients were positive for CK or EMA, NSE, des and vim of the epithelium, nerve, muscle and interstitial. They died 9 months after operation.

CONCLUSIONSThe tumor may occur in the abdomen, pelvic cavity and other sites, with different clinical manifestations. Routine examination should be replaced by immunohistochemical test for correct diagnosis of the tumor. Prognosis of most patients is not good.

Adolescent ; Carcinoma, Small Cell ; diagnosis ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Neoplasms, Connective Tissue ; diagnosis ; Retrospective Studies

BACKGROUND:Although several prosthetic methods, such as artificial denture and dental implants, are clinical therapies to tooth loss, they are thought to have safety and usage time issues. With the development of biological and biomaterial sciences, recently, tooth tissue engineering has attracted more and more attention.
OBJECTIVE:To reflect advances and problems of tissue engineering technologies for promotion of tooth regeneration.
METHODS:Using the keywords of“tissue engineering, tooth regeneration”in English and Chinese, PubMed and CNKI databases from 2007 to 2013 were retrieved. A total of 65 literatures addressing tooth regeneration and tissue engineering were col ected, including 25 Chinese articles and 40 English articles. Published early, repetitive, and similar researches were excluded. Final y, 48 articles were included.
RESULTS AND CONCLUSION:The combination of stem cells and suitable scaffolds is widely used in tooth regeneration today, and growth factors or bone marrow which can produce promote tooth regeneration are added as wel , which has achieved partial or whole tooth regeneration. But there are apparent deficiencies in studies which focus on mechanisms behind tooth regeneration.

Objective To investigate the function characteristics of CD4 T cell converted double negative T cell and provide a basis for further insight into the characteristics of mouse converted double negative T cell.Methods The gene expression profile was analyzed by transcriptome sequencing and protein mass spectrometry.The expression of cell active marker CD44,CD69 and OX40 was investigated by flow cytometry and the cytotoxicity of mouse double negative T cell was verified by CFSE staining.Results Mouse CD4 T cell converted double negative T cell expressed cell phenotype that differed from other mature CI4 T cells.Mouse converted double negative T cell expressed high level of active marker of CD44,CD69 and OX40.Cytotoxicity of PrfO DN T was significantly reduced.Conclusions Mouse CD4 T cell converted double negative T cell has distinguishing cell phenotypes,that are not identical to other mature CD4 T cells.Mouse double negative T cell overexpresses cell activation marker and cytotoxic cytokines.The immune suppressive function of mouse double negative T cell is mainly dependent on perforin pathway.

Objective To explore the role of viral infection in the development of drug eruption in patients with HIV infection, and to evaluate the efficacy of antiviral treatment. Methods This study enrolled 87 HIV-positive patients, including 11 with and 76 without drug eruption, all of whom received highly active antiretroviral therapy(HAART). Clinical data on, baseline CD4+ and CD8+ T cell counts and CD4/CD8 ratio in these subjects were retrospectively analyzed. Results The severity of drug eruption was mild in the 11 HIV-positive patients, with a mean latency period of (14.00 ± 8.10)(range, 8 - 34)days. Of the 11 patients with drug eruption, 7 had liver function impairment, which was not in accordance with the severity of skin lesions. Drug eruption was controlled in all the 11 patients after anti-anaphylactic treatment without withdrawal of antiviral drugs. Compared with 75 HIV-positive patients without drug eruption, the 11 HIV-positive patients with drug eruption showed significantly increased baseline CD4 + T cell counts (493.00 ± 245.68 (range, 42 - 810)/μl vs. 347.81 ± 167.00 (range, 11 - 814)/μl, t = 647.50, P < 0.05), but decreased proportion of patients with baseline CD4+ T cell counts below the lower limit of normal(3/11 vs. 48/75(64.00%), X2 = 3.95, P < 0.05). There were no significant differences between 10 patients with drug eruption and 69 patients without drug eruption in the baseline CD8+ T cell count(1472.30 ± 858.55/μl vs. 1356.59 ± 684.06/μl, P > 0.05), CD4/CD8 ratio(0.40 ± 0.27 vs. 0.29 ± 0.16, P > 0.05), or percentage of patients with a CD4/CD8 ratio below the lower limit of normal (9/10 vs. 68/69 (98.55%), P >0.05). Conclusions The latency period of drug eruption seems to be long in HIV-positive patients receiving HAART, and mild drug eruption can be complicated by liver function impairment in the patients. Relatively high CD4 + counts may be a risk factor for the development and aggravation of drug eruption in HIV-positive patients.

Objective To evaluate the effect of small interfering RNA (siRNA)targeting survivin gene on the expression of survivin and proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431 in vitro.Methods A survivin-specific siRNA was designed and synthesized.Cultured A431 cells were divided into 3 groups to be transfected with 50.0 nmol/L liposome complexes containing survivin-specific siRNA (survivin siRNA group),50.0 nmol/L liposome complexes containing unrelated siRNA (negative control group) and 50.0 nmol/L prepared vesicles (blank control group).Real-time quantitative reverse transcription-PCR (RT-PCR) and Western blot analysis were performed to determine the mRNA and protein expression of survivin in A431 cells,respectively.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate cellular proliferative activity,flow cytometry using annexin-V/propidium iodide (PI) staining to detect cell apoptosis,Transwell assay to estimate migratory and invasive activities of A431 cells,and flow cytometry to detect cell cycle changes.Results At 48 hours after transfection,the mRNA and protein expression of survivin both significantly differed among the survivin siRNA group,negative control group and blank control group (mRNA:0.56 ± 0.15,0.88 ± 0.37,0.90 ± 0.43,F =276.67,P ＜ 0.001;protein:0.59 ± 0.04,0.86 ± 0.05,0.91 ± 0.07,F =243.61,P ＜ 0.001),the survivin siRNA group showed significantly lower mRNA and protein expression of survivin compared with the negative control group and blank control group (all P ＜ 0.05),and there were no significant differences between the negative control group and blank control group (both P ＞ 0.05).Repeated measures analysis of variance showed that the transfection with survivin siRNA could significantly inhibit the proliferation of A431 cells (F =13.19,P =0.004),the proliferation inhibition rate was significantly higher in the survivin siRNA group than in the negative control group and blank control group (both P ＜ 0.05),and no significant difference was observed between the negative control group and blank control group (P ＞ 0.05).At 24 hours after transfection,the apoptosis rate significantly differed among the 3 groups (F =83.97,P =0.002).The survivin siRNA group showed a significantly higher apoptosis rate compared with the negative control group and blank control group (both P ＜ 0.05),and there was no significant difference between the negative control group and blank control group (P ＞ 0.05).At 48 hours after transfection,the survivin siRNA group showed a significantly higher proportion of cells at G2/M phase,but lower number of migratory cells and invasive cells compared with the negative control group and blank control group (all P ＜ 0.05).Conclusion Survivin-specific siRNA can inhibit the expression of survivin gene and the proliferation of A431 cells,promote cell apoptosis,and suppress cell migration and invasion,indicating that survivin may serve as a genetic target for the treatment of cutaneous squamous cell carcinoma.

Objective To investigate the differences in gene expression profiles of peripheral blood from patients with Keshan disease (KD) and the apoptosis mechanism in KD,to obtain diagnostic markers and establish diagnostic centroids plot for KD.Methods RNA was isolated from ten patients with KD diagnosed according to the clinical criteria for KD in China and ten health controls.The expression profiles were evaluated by Agilent 4 ×44K Whole Human Genome density oligonucleotide microarray analysis.The data were extracted by Agilent Feature Extraction Software t test,Pathway studio analysis and prediction analysis for microarray (PAM) were used to identify differently expressed genes,gene pathways,diagnostic markers and establish diagnostic centroids plot.Results Totally 1 570 up-regulated genes and 1 498 down-regulated genes were identified.Thirty-eight enrichment pathways were also identified,and the highest ranked by Pathway studio analysis was related to apoptosis.Six genes involved in apoptosis pathway were up-regulated in KD included ataxia telangiectasia mutated (ATM),cAMP-dependent protein kinase,protein kinase A (PKA),baculoviral IAP repeat-containing 2 (BIRC2),NLR family,apoptosis inhibitory protein (NAIP),BCL2-1ike 11 (Bim),BCL2-related protein A1 (BCL2A1) and down-regulated were 7 which included caspase 8 (CASP8),BCL2 binding component 3 (BBC3),BCL2--associated athanogene (BAG1),BCL2-associated X protein (BAX),BCL2-1ike 1 (BCL2L1),BCL2-related ovarian killer (BOK),and caspase 6 (CASP6).Forty-two diagnostic markers were obtained through PAM analysis.Conclusions Apoptosis related to genes and pathways might play an important role in the pathogenesis of KD.Forty-two markers could be used as molecular markers for the diagnosis of KD,which is important to the diagnosis of KD.

Objective:To analyze the significance of systemic immune inflammatory index (SII) combined with neutrophil lymphocyte ratio (NLR) in the treatment of advanced gastric cancer with PD-1/PD-L1 inhibitors.Methods:The clinical data of 90 patients with stage Ⅳ gastric adenocarcinoma who received immunotherapy from January 2020 to January 2023 were retrospectively analyzed, including 70 males and 20 females, aged from 36 to 80 years, with an average age of (53.76±15.58) years. The clinicopathological features and follow-up data were collected. SPSS 26.0 software was used to conduct statistical analysis. The critical values of NLR, SII, PLR and MLR were calculated, and the overall survival (OS) and progression free survival (PFS) of patients with different levels of markers were analyzed. The independent predictive factors of PFS and OS were determined, and the predictive value of risk factors for PFS and OS in patients with gastric cancer was evaluated.Results:The median follow-up time of all patients was 27.3 months, and the median PFS and OS were 10.0 months and 17.7 months, respectively. The area under the curve (AUC) of NLR and SII for predicting PFS and OS were>0.7, the critical values NLR were 4.75 and 3.85, and SII were 1154.67 and 887.90, respectively. PFS and OS in patients with high NLR, high MLR, high PLR and high SII were lower than those in patients with low levels. ECoG PS≥ 1, high NLR and high SII were independent influencing factors of disease progression or death. The AUC of the combination of NLR, ECoG PS and SII was 0.761, which was higher than that of any single factor. The fewer the number of risk factors, the longer the PFS and OS.Conclusions:NLR and SII are effective predictors of PFS and OS in patients with advanced gastric cancer receiving immunotherapy. Pre treatment detection of NLR and SII can provide reliable guidance for immunotherapy of advanced gastric cancer.

Objective To investigate the effect of extremely low frequency-electromagnetic field (ELF-EMF) on bone mesenchymal stem cells (BMSCs) differentiating into neuron like cells in vitro and research its mechanism.Methods BMSCs were collected from rats by means of whole bone marrow adherent.Flow cytometry was used to assay cell surface marker at passage 3.And then,BMSCs were assigned into four groups:ELF-EMF group,ELF-EMF+U0126 (inhibitor) group,U0126 group and control group;cells were induced by medium (2％ DMSO and 200 μmol/L BHA) for 5 h.In the process of neural induction,ELF-EMF group and ELF-EMF+U0126 group were received 10 Hz,500 GS ELF-EMF stimulation.Besides,ELF-EMF+U0126 group and U0126 group were pretreated with 50 μmol/L extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126.The morphology of BMSCs was observed under inverted microscope.The expression of nestin was detected by immunofluorescent staining and Western blotting to identify and determine the differentiation.Western blotting was applied to detect the preotein level of phosphorylase-ERK1/2 after ELF-EMF exposure.Results BMSCs presented a single long spindle morphology,growing with close whirlpoor-like arrangement;CD90 expression rate was up to 97.9％,while that of CD45 only 4.7％.After induction,each group of cells showed similar shape with neuron-like cells gradually.As compared with the other three groups,ELF-EMF group had significantly higher expression levels of nestin and phosphorylatd ERK1/2 detected by immunofluorescent staining and Westem blotting,respectively (P＜0.05).Meanwhile,the expression levels of nestin among the ELF-EMF+U0126 group,U0126 group and control group were not statistically significant (P＞0.05).Conclusion ELF-EMF could promote neuronal differentiation of mesenchymal stem cells via activation of ERK1/2 signaling pathways.