Oligodeoxynucleotide containing unmethylated cytosine phosphate-guanosine motif(CpG ODN) may induce high expression of CD80, CD86, CD83, HLA I and HLAⅡ molecules on dendritic cells(DC) and stimulate DC to produce high level of IL-6, IL-12, TNF-? and IFN-?. CpG ODN is demonstrated in vivo to be a very potent adjuvant for Th1 cells, regulating Th0 cells to develop toward Th1 cells. Its role for DC is characteristics of CpG ODN sequence specificity and species specificity. CpG ODN is, at present, considered as a pathogen associated molecular pattern which binds its specific receptor,Toll-like receptor 9,then functions through TLR/IL-1R signaling pathway. It may represent a new therapeutic drug for broad applications in infectious disease, autoimmune disease, allergy and cancer therapy.

Objective To investigate the distribution and expression of tight junction proteins (including elaudin 1, occludin,ZO-1 and JAM-1) in mucosa of rats with reflux esophagitis (RE), and its underline mechanism in pathogenesis of RE. Methods Two hundred and twenty 8-week-old male Wistar rats were divided into sham operation control group (n=10), acid reflux group (n=70), alkaline reflux group (n=70) and mixed reflux group (n=70). The rats were sacrificed at day 3, 6, 9 and 14 after operation. The successful rate of modeling was assessed by evidence of inflammation in middle and low esophagus. Transmission electron microscopy (TEM) was used to observe the morphological changes of tight junction in esophageal epithelium. The mRNA and protein expressions of tight junction proteins were detected by Western blotting and RT-PCR, respectively. And interleukin (IL)-6 expression was measured by immunohistochemistry. Results At day 14 after the procedure, RE model was established in all executed rats. Successful rate of 100% was achieved. The microscopic observation showed that mucosa was damaged and thickened as the disease progressed. With TEM observation, widened intercellular space was noticed with fewer desmosomes. Elevated expressions of IL-6 and tight junction proteins were found in three model groups compared with control group. Whereas the expression of tight junction proteins in individual cells was gradually decreased with continuing hyperplasia in the basal layer. The mRNA and protein expressions of IL-6 and tight junction proteins were increased gradually as disease progressed. Conclusions The highly expression of tight junction proteins, which involves in the mechanism of RE by playing the role of positive regulation and synergism, may be early molecular event in development of RE. And IL-6 is an inflammatory factor in this process.

AIM: To study the effect of oligonucleotides containing unmethylated CpG motif (CpG ODN) on mouse bone marrow-derived dendritic cells (BMDCs) in anti-HcaF cytotoxicity. METHODS: BMDCs were stimulated by CpG ODN in combination with tumor antigen (TAg). The expression of CD80 on BMDC surface was analyzed by FCAS. Level of IL-12 (p70) in supernatants of BMDC culture was detected by ELISA. The proliferation of T cells was examined by MTT assay. Cytotoxicity of CTL induced by CpG ODN combining with TAg was detected by MTT assay. RESULTS: CpG ODN combining or not combining with TAg up-regulated the expression of CD80 on BMDC surface and stimulated BMDCs to produce a high level of IL-12. CpG ODN-activated BMDC promoted the proliferation of T cells. CTL induced by CpG ODN in combination with TAg appeared strong specific cytotoxicity on Hca-F cells. CONCLUSION: CpG ODN may effectively induce the functional maturation of mouse BMDC in vitro. CpG ODN in combination with TAg can enhance the anti-HcaF cytotoxicity of CTL. [

AIM: To evaluate the effect of staphylococcal enterotoxin B (SEB) or staphylococcal enterotoxin C (SEC) combining with dendritic cells (DC) on T cell functions and in vitro anti-HcaF tumor cytotoxicity of activated T cells. METHODS: S-100 protein expression in DC was detected by immune histochemistry staining. The expressions of I-E~? and CD80 molecules on DC, the expression of CD69 molecule on T cells and the production of IL-2 and TNF-? by T cells were determined with flow cytometry. The proliferation of T cells and its cytotoxicity to HcaF tumor cells were detected by MTT assay. RESULTS: In vitro experiments showed that isolated DC expressed high level of S-100 protein. SEB or SEC-induced DC highly expressed I-E~? and CD80 molecules and that SEB or SEC-induced DC promoted the activation and proliferation of T cells. 100 ?g/L of SEB or SEC was the most effective concentrations to induce T cells to secret IL-2 and TNF-?. The T cells activated by SEB or SEC combined with DC showed significant cytotoxicity to HcaF cells, appearing a stronger role than tumor antigen combined with DC. There was no difference in the role for T lymphocytes between both SEB and SEC. CONCLUSIONS: The results indicate that SEB or SEC combined with DC is an effective way to enhance T cell functions, producing stronger cytotoxicity to HcaF tumor cells than tumor antigen-loaded DC used at present, which offers a forceful evidence for the possibility of superantigen SEB or SEC combining with DC to be applied to clinical tumor immunotherapy.

Objective:To establish Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines using CRISPR/Cas9 technology and 3D retinal organoid culture.Methods:The target site sequence of H9 cell line was verified by polymerase chain reaction (PCR). SgRNAs were designed by CRISPR/Cas9 technique and their activity was detected.The most optimal sgRNA was selected according to the factors such as activity and specificity.After identification of the target vectors by restriction enzyme and sequencing, the target vectors were transferred to the H9 cell line by electroporation.P2A-tdTomato-P2A-iCreERT2 was inserted between Exon4 and 3’-untranslated region of hES-ZLM-001 gene.Knockin positive clones were obtained after drug treatment, enrichment of positive clones.Primers were designed to perform PCR on the target region, and homozygous de-resistant knockin positive cell clones were selected according to the sequencing results and peaks.The 1-A07 cell line was cultured, and then flow cytometry for the proportion of OCT4 positive cells, immunofluorescence for three stem cell molecular markers including SOX2, NANOG, SSEA4, karyotype analysis were carried out to confirm whether the 1-A07 cell line could be used for further experiments.Retinal organoids were obtained by three-dimensional (3D) culture technology and the expression of molecular markers was detected by immunofluorescence at different developmental stages of retinal organoids. Results:The target site sequence of H9 cell line was consistent with that given by Genebank and Ensembl.Sixteen sgRNAs were designed according to the target site sequence of H9 cell line, and finally sgRNA8 and sgRNA12 were selected.The sgRNAs and recombinant plasmids were transfected into the H9 cell line by electroporation, and four homozygous de-resistant knockin positive cell clones were obtained by PCR.Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were successfully obtained.In 1-A07 cell line, the proportion of OCT4 positive cells was about 98.7% by flow cytometry, and the expression of three stem cell markers was positive by immunofluorescence, and the karyotype was normal 46, XX.The results showed that the 1-A07 cell line could be used for further experiments.The Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines were differentiated into tdTomato positive retinal organoids by 3D culture technology.BRN3A positive ganglion cells, CALBINDIN positive horizontal cells and CHAT positive amacrine cells appeared on day 30 of differentiation.RECOVERIN positive photoreceptors arose on day 45 of differentiation.PKCα positive bipolar cells presented on day 90 of differentiation.Ganglion cells were shown in the deep layer of retinal organoids, and horizontal cells, amacrine cells and bipolar cells in the middle layer, and photoreceptors in the top layer.Conclusions:Crx-iCreERT2 fluorescent reporter human embryonic stem cell lines are successfully established and can be differentiated into retinal organoids that express tdTomato red fluorescence through 3D culture technology.Those retinal organoids contain the same types of neurons as normal human retinas, and follow a certain temporal and spatial developmental sequence similar to the developmental rules of normal human retinas.Crx-iCreERT2 fluorescent reporter human embryonic stem cell line is a powerful tool for researching retinal development and diseases and can be applied in treatments for blindness.

Objective To evaluate the correlation between homologous recombination repair protein RAD51 and methotrexate-enhanced radiosensitivity.Methods Western blot and RT-PCR assays were used to detect RAD51 expression in HOS osteosarcoma cells exposed to γ-ray irradiation alone and in combination with methotrexate.Colony formation assay was used to test the survival fraction of HOS cells exposed to γ-rays and methotrexate.Results Methotrexate inhibited both protein and RNA expressions of RAD51,and the combination of radiation and methotrexate enhanced the inhibition of RAD51 expression.Moreover,transfection of cells with RAD51 gene decreased cellular sensitivity to methotrexate and γ-rays.The sensitizer enhancerment ratios after irradiation in combination with methotrexate were 1.51 and 0.99,respectively.Methotrenate was a preferred radiosensitizer to HOS cell.Conclusions RAD51 might be involved in the methotrexate-enhanced radiosensitivity.

Objective To study the curative effect of XRCC2 gene silencing mediated by shRNA combined with radiation on human colonic trans?planted carcinoma in nude mice. Methods Colonic carcinoma T84 cells were transfered into BALB/c nude mice to establish a tumor xenograft mod?el in vivo. Mice were divided into three groups:control,shRNA?SC and shRNA?XRCC2 and exposed to X?ray radiation. The change of volume and weight of the xenografts were examined after receiving radiotherapy and the pathological analysis of tumor tissues were conducted. Results Tumor xenografts transfected with shRNA?XRCC2 in nude mice grew slowly. The xenograft volume in the shRNA?XRCC2 group was decreased significant?ly from day 12 to day 28 after radiotherapy compared with the control group(P<0.01). The xenograft weight in the shRNA?XRCC2 group was small?er than in the control group,with statistically significant difference(t=18.843,P<0.01). The inhibited rate of xenografts in the shRNA?XRCC2 group(56.25%),was markedly higher than that in the shRNA?SC group(4.69%). Pathological analysis of colonic transplanted carcinoma showed that nuclear atypia was not obvious,karyokinesis was decreased and small areas of necrosis were present in tumor xenografts treated with shRNA?XRCC2 transfection. Conclusion XRCC2 gene silencing combined with radiation has significant inhibition effect on colonic transplanted carcino?ma in nude mice.

objective To assess the DNA damage of radiation workers in different grade hospitals,and to explore the correlation between the types of work or work time and the levels of DNA damage.Methods DNA single strand break were detected by using alkaline single cell gel electrophoresis(SCGE),and the comet was analyzed with CASP(Comet Assay Software Project).TDNA,TL,TM and OTM were calculated.Results The parameters of SCGE in the radiation greup were higher than those of control group(F=3.93,P<0.01).The significant difference was found not only among the different types of work or difierent work time,but also among the different grade hospitals(F=1.83,1.9 1,P<0.05).Conclusions Various levels of DNA damage could be detected in the radiation workers of the two hospitals.DNA damage of radiation workers is less serious in the higher-grade hospital than the lower grade one.Different types of work or work time might affect the DNA damage level.

Objective To study the co-effects of methylene blue(MB) and exposure with the cold light source on cell DNA damage, and to explore the mechanism involved. Methods The alkaline single cell gel electrophoresis was used to determine cell DNA damage. Apoptosis of the cells was determined by flow cytometry analysis using Annexin V-FITC/PI staining. DCFH-DA probe was used to determine endocellular Reactive Oxygen Species (ROS). Results The levels of DNA damage in the SGC7901 adenocarcinoma cells treated with methylene blue in the light were significantly increased compared to that of control ( F = 8.39, P＜0.05 ). The DNA damage levels were related to the length of time of light exposure, and the damage was recovered to a certain level after light withdrew. Cell apoptosis ( x2=7.71,P ＜0.05)and endocellular ROS level (F = 34.11, P＜0.01＝ increased significantly in the exposure group. Conclusions Methylene blue chromoendoscopy can induce DNA damage and cell apoptosis, and the mechanism may be associated with ROS produced by the photochemical reaction.

Objective To evaluate the effect of hydrogen on mitochondrial fusion during myocardial ischemia-reperfusion (I/R) in aged rats.Methods One hundred and fifty pathogen-free healthy male Sprague-Dawley rats,aged 18 months old,weighing 400-500 g,were divided into 5 groups (n=30 each) using a random number table:control group (group C),sham operation group (group S),group I/R,normal saline group (group NS) and hydrogen-rich saline group (group H).Group C received no treatment.The anterior descending branch was only exposed but not ligated in group S.Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 rmin followed by reperfusion in I/R,NS and H groups.Hydrogen-rich saline 1 ml/100 g was injected intraperitoneally at 5 min before reperfusion in group H,while normal saline 1 ml/100 g was injected intraperitoneally at 5 min before reperfusion in group NS.The rats were sacrificed at 12 and 24 h of reperfusion,and hearts were removed for examination of the pathological changes and for determination of apoptosis in cardiomyocytes (by TUNEL) and expression of Mfn1 and Mfn2 protein and mRNA in myocardial tissues (by Western blot or real-time polymerase chain reaction).The apoptosis index was calculated.Results Compared with C and S groups,the apoptosis index of cardiomyocytes was significantly increased and the expression of Mfn1 and Mfn2 protein and mRNA in myocardial tissues was down-regulated at 12 and 24 h of reperfusion in I/R,NS and H groups (P＜0.05).Compared with NS and I/R groups,the apoptosis index of cardiomyocytes was significantly decreased and the expression of Mfn1 and Mfn2 protein and mRNA in myocardial tissues was up-regulated at 12 and 24 h of reperfusion in group H (P＜0.05).The pathological changes of myocardial tissues were significantly attenuated in group H when compared with group I/R.Conclusion The mechanism by which hydrogen attenuates myocardial I/R injury is related to promoting mitochondrial fusion and inhibiting apoptosis in cardiomyocytes of aged rats.