Objective To investigate the inhibition effect of silencing Ku80 gene combined with irradiation on growth of esophageal cancer xenografs. Methods shRNA-Ku80 vector was constructed.The expression of Ku80 protein was inhibited by shRNA-Ku80 vector by using Western blotting. 20 BALB/c nude mice were randomly divided into 4 groups, including control group, radiation group, shRNA-Ku80 group and combined group. The growth of esophageal cancer xenografs was observed. The expression of Ku80 was examined in esophageal cancer xenografs by IHC. Results Effective target sequence was selected. The growth of esophageal cancer xenografs was inhibited by shRNA-H2 and radiation, especially in combined group. The inhibition rate of growth in three groups above was 32.0% , 39. 9% and 68. 9% ,respectively. The expression of Ku80 was reduced to 58% by shRNA-H2 in esophageal cancer xenografs ( t = 3.77, P ＜ 0. 05 ). Conclusions Combination of silencing Ku80 and radiation could enhance radiotherapeut effect of esophageal cancer xenografs.

Objective To explore the effect of Buyang Huanwu decoction(BYHWD)on protein kinase B1 (AKT1)and c-Jun amino terminal kinase 1/2(JNK1/2)in rats after focal cerebral ischemia. Methods According to the random number table method,48 Sprague-Dawley(SD)rats were randomly allocated to four groups:normal control group,sham-operated group,model group,traditional BYHWD group(each n=12). The rat model of right focal cerebral ischemia was established by the method of middle cerebral artery occlusion(MCAO). The rats in BYHWD group were ingested with the decoction of BYHWD 14.2 g/kg after 2 hours of the operation(the main ingredients of BYHWD including astragalus mongholicus 120 g,Chinese angelica 6 g,radix paeoniae rubra 4.5 g, rhizoma ligustici wallichii 3 g,safflower 3 g,peach kernel 3 g,earthworm 3 g),once a day for 7 days. Other groups of animals were given the same amount of normal saline orally. After operation,on the 7th day,the animals were killed,and their brains were taken out. The reverse transcription-polymerase chain reaction(RT-PCR)assay was used to detect AKT1 mRNA expression,and immunohistochemical method was applied to measure JNK1/2 protein expression. Results Compared with normal control and sham-operated groups,the level of AKT1 mRNA expression〔absorbance(A)〕was decreased obviously(0.48±0.08 vs. 0.63±0.11,0.61±0.09,both P<0.05),and the number of JNK1/2 positive cells(cell/mm2)was increased significantly(34.13±4.57 vs. 16.15±1.09,16.23±2.05,both P<0.05)in model group;compared with model group,the AKT1 mRNA expression in brain tissue(0.93±0.11)and the number of JNK1/2 positive cells(45.04±5.68)was increased significantly in BYHWD group,the differences being statistically significant(P<0.05 or P<0.01). Conclusion BYHWD can up-regulate expressions of AKT1 mRNA and JNK1/2 positive cells in ischemic brain tissue that is one of the mechanisms in the protection of brain.

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 compare the cost-effectiveness of intervention protocols with or without rehabilitation approaches for burn patients.To explore the pros and cons of the two treatment programs,especially in terms of efficacy.Methods Data on 164 burn patients hospitalized in four hospitals in Hunan Province were collected and compared in terms of the therapeutic interventions employed,the direct medical costs and the effects obtained at the end of the first two months of hospitalization.The patients were divided into an early rehabilitation therapy group and a control group,based on the interventions they received.Results The average total direct medical cost of the early rehabilitation group was significantly lower than that of the control group [((￥)59 380.80 ±196.87) versus ((￥)67 471.62 ±217.23)].The average Vancouver scar scale (VSS) results in the early rehabilitation group were significantly better than those of the controls,but their Zung's Self-rating Anxiety Scale and Barthel index scores were not.The average cost-effectiveness ratio (C/E) of the early rehabilitation group as judged by these scores was lower than that of the control group.Conclusion Early rehabilitation can improve the therapy of burns patients and reduce subsequent expenditures.It is a cost-effective approach to treating burns patients.

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 screen and isolate the radioresistance related genes of IRM-2 mice.Methods cDNA library of IRM-2 mice was constructed by SMART technique.Total RNA was isolated from spleens of IRM-2 male mice.The first-strand cDNA was synthesized by using PowerScript reverse transeriptase,and double-strand cDNA was synthesized and amplified by long PCR.The PCR products were purified,digested with restriction enzyme Sfi I.The ds-cDNA fragment lessthan 500 bp was fractionated and ligated to the Sfi I-digested pDNR-LIB vector.The ligation mixture was transformed into E.coil DH5α by electroporution transformation to generate the unamplified cDNA library.The quality of cDNA library was identified by PCR technique.130 clones from cDNA library were sequenced and compared with GenBank database.Results The cDNA library contained 2.25 x 106 independent clones with an average insert size of 1.2 kb.The ratio of recombination and full-length was 95% and 55%,respectively.21 pieces of EST sequences from cDNA library were not the same as the known mice genes and registered into GenBank EST database,with registered number DW474856-DW474876.Conclusions cDNA library of IRM-2 mice has been constructed successfully.21 pieces of EST implies that radioresistance correlative genes may be in IRM-2 mice,which will lay a foundation for isolating and identifying radioresistance related genes in further study.

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 study the combined effect of interleukin-21 gene transfer and ionizing radiation on the growth of cervical carcinoma HeLa cells.Methods Previously constructed Ad-IL-21 gene was amplified by infecting 293A cells and the titer was measured by TCID50 method. HeLa cells were transfected with Ad-1L-21 and then irradiated with 6 Gy 137Cs γ-rays.The cells were divided into 5 groups,including blank control,Ad-LaeZ group,Ad-IL-21 group,radiation group and Ad-IL-21 combined with radiation group (combination group).The cell growth,cell cycle,apoptosis,and the expressions of IL-21 gene and protein in HeLa cells were detected.Results Ad-IL-21 was successfully amplified and the titer of Ad-11.-21 was 9 × 1010 pfu/ml.Compared with Ad-IL-21 group and radiation group,the cell growth of combination group was significantly inhibited at 96 h after transfection ( F =85.26,72.98,P ＜ 0.05 ).The cells in combination group were arrested in G1 phase and decreased at S phase( F =36.69,34.83,P ＜ 0.05),while the cellular apoptosis increased markedly ( F =28.23,25.57,P ＜ O.05 ). The gene expression of 1L-21 in the combination group was 1.54- and 2.43-fold of Ad-IL-21 group and blank control group,respectively (F=22.31,36.65, P ＜ 0.05 ), while the protein expression of IL-21 in the combination group was 1.62-fold and 2.31-fold of Ad-IL-21 group and blank control group,respectively ( F =27.36,35.86,P ＜ 0.05 ).Conclusions Ad-IL-21 gene transfection combined with radiation has synergic effect on the inhibition of cervical carcinoma cell growth.

Objective To study the influence of irradiation on the osteoblast function by the gene expression changes of RANKL and OPG.Methods Bone marrow stromal cells were induced to develop into early and mature osteoblasts in vitro.The characterization of osteoblasts was indentified by ALP staining.The RANKL and OPG mRNA levels in early and mature osteoblasts, which exposed to 0 -4 Gy radiation were determined by RT-PCR.Results Bone marrow stromal cells had been induced to early and mature osteoblasts by osteoblast differentiation medium in vitro.In early stage of osteoblast, RANKL mRNA expression levels treated with 1Gy irradiation was 2.83-fold higher than those other irradiation dosage groups.The RANKL mRNA expression levels of each group in early stage of osteoblasts were significantly higher than those in the mature counterpart ( t = 8.34 - 103.57, P ＜ 0.05 ).The ratio of RANKL/OPG mRNA was obviously greater in early osteoblast compared with the mature cells ( t = 2.84 - 20.99, P ＜0.05 ), and it was the highest in 1Gy irradiation treated early osteoblast.Conclusions Radiation exposure of the early osteoblasts promotes osteoclasts function and results in the bone loss.