To construct a eukaryotic expression plasmid containing the luciferase reporter gene (Fluc) to quickly detect apoptosis. Four amino acids, Asp-Glu-Val-Asp (DEVD), the recognize motif of Caspase-3, were introduced into the middle of the Fluc-C and N fragment. Meanwhile, four amino acids, Asp-Glu-Val-Gly (DEVG), were selected as a negative control. Subsequently, the recombinant gene was cloned into the N and C terminal end of the split intein, and named as pFluc-DEVD and pFluc-DEVG. Then the plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency. Then the apoptosis level was detected by the double luciferase reporter gene and the Western blotting analysis. The results showed that when apoptosis occurred, the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was about 3 times higher than pFluc-DEVG plasmid transfected group. Furthermore, Western blotting detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants. The activation degree of Caspase-3 was closely related to the expression of Fluc, and had a significant statistical difference. These results confirmed that firefly luciferase protein expressed by pFluc-DEVD plasmid can be cleaved by the intracellular Caspase-3 enzyme, and this plasmid can accurately reflect the cell apoptosis level, which provides a useful method for quantitative detection of apoptosis.
Apoptosis ; Genes, Reporter ; Luciferases, Firefly ; Transfection

Objective To report a case of Costello syndrome complicated by curis laxa,and to make a molecular genetic diagnosis.Methods Clinical data were collected from a case of Costello syndrome complicated by cutis laxa.Skin tissues were resected from the patient,and peripheral blood samples were obtained from the patient's parents and 150 unrelated healthy controls.Genomic DNA was extracted from these samples,and all the exons and their flanking sequences of the HRAS gene were analyzed by DNA sequencing.Results The 13-month-old female patient presented with growth retardation,severe malnutrition,coarse facial appearance,severely loose skin over the limbs,and decrease or disappearance of subcutaneous fat.A heterozygous mutation c.34G ＞ T (p.Gly12Cys) was detected in exon 2 of the HRAS gene in the patient,but not in her parents or 150 unrelated healthy controls.Conclusion The c.34G ＞ T (p.Gly12Cys) mutation in exon 2 of the HRAS gene may be responsible for Costello syndrome in the patient.

Objective To investigate the effects of Working Memory Training System on working memory impairment after brain injury. Methods From November, 2013 to March, 2015, 20 patients of brain injury with impairment of working memory were divided into training group (n=10) and control group (n=10). The training group was trained with the Working Memory Training System for four weeks, while the control group did not accept any cognitive rehabilitation. They were tested with digital forwards/backwards, space forwards/backwards, n-back test and Everyday Memory Questionnaire before and after training. Results All of the tests improved more in the training group than in the control group (Z>2.014, P<0.05), except that of digital forwards, as well as the score of Everyday Memory Questionnaire (Z=1.970, P=0.049). Conclusion Application of Working Memory Training System can improve the ability of memory in patients with brain injury, both the working memory and everyday memory.

Objective To investigate the effect and related mechanism of CD137 stimulation on aortic atherosclerotic plaque calcification in high fat diet fed ApoE-/-mice and on calcification of vascular smooth muscle cells (VSMCs).Methods (1) ApoE-/-mice fed with high fat diet were randomly divided into 3 groups:CD137 activated group (treated by 200 μg CD137 agonist i.p.once per week for 6 weeks,n =5);CD137 inhibited group (anti-CD137 group:200 μg anti-CD137 antibody + 200 μg CD137 agonist,i.p.,once per week for 6 weeks,n =5) and control group (n =5).Von kossa staining was used to observe the calcification of the aortic plaque and VSMCs.Immunohistochemistry was used to observe the expression of BMP-2 and Runx2 which are known mediators of osteogenic differentiation.(2) The mouse aortic VSMCs were obtained by Patch-attaching method.The calcium content was measured by Methylthymol Blue complexone method.The mRNA expressions of bone morphogenetic protein 2 (BMP-2) and Runx2 were measured by real-time fluorescent quantitative PCR (RT-PCR).The protein levels of BMP-2,Runx2 of the VSMCs were determined by Western blot.Results (1) In vivo,the plaque calcified area in ApoE-/-mice was significantly larger in CD137-agonist group than that in control group ((1.75 ± 0.33) × 104 μm2 vs.(0.23 ±0.07) × 104 μm2,P ＜0.01),and this effect was significantly reduced by cotreatment with CD137-antagonist ((0.83 ± 0.30) × 104 μm2 vs.(1.75 ± 0.33) × 104 μm2,P ＜ 0.05).The levels of BMP-2 and Runx2 were all significantly upregulated in CD137-agonist group than in control group (both P ＜ 0.01),again,this effect was blocked by cotreatment with CD137-antagonist (P ＜0.05).(2) Consistent with the in vivo results,VSMCs calcification was also more serious in CD137-agonist group than in control group,which could be significantly attenuated by eotreatment with CD137-antagonist.In VSMCs,calcium content level in CD137-agonist group was higher than in control group ((0.001 3 ± 0.000 2) mmol/mg protein vs.(0.000 7 ±0.000 1) mmol/mg protein,P ＜ 0.01),which could be significantly reduced by co-treatment with CD137-antagonist ((0.000 9 ± 0.000 2) mmol/mg protein vs.(0.001 3 ± 0.000 2) mmoL/mg protein,P ＜0.01).The mRNA and protein levels of BMP-2 and Runx2 were significantly upregulated in CD137-agonist group compared with the control group (P ＜ 0.05),which could be significantly down-regulated by cotreatment with CD-137 antagonist (P ＜ 0.05).Conclusion CD137 activation can promote vascular calcification in high fat diet fed ApoE-/-mice both in vivo and in vitro.

Objective To explore whether CD137-CD137L signaling can promote angiogenesis in atherosclerosis plaque via activating nuclear factor of activated T cells c1 (NFATc1).Methods Apolipoprotein E knock out mice were divided into the following groups:control group (n =5),CD137 activated group(n =5) and CD137 inhibited group (n =5).Immunohistochemistry was performed to detect the expression of CD31 in aortic plaque.Endothelial cells (bEnd.3) were purchased from ATCC and divided into the following groups:control group,IgG isotype control group,CD137 activated group and CD137 inhibited group.Western blot was used to determine total protein and nucleoprotein expression of NFATc1.The expression level of CD137 protein on the surface of endothelial cells was detected by flow cytometry (FCM) and CD137 protein of lysate of endothelial cells was detected by enzyme-linked immunosorbent assay (ELISA).Transwell assay was used to observe the migration ability of endothelial cells.Matrigel tube formation ability of endothelial cells were tested in the following groups:control group,CD137 activated group,silent NFATc1 + CD137 activated group,CD137 inhibited group,and over expressed NFATc1 + CD137 inhibited group.Results (1) In vivo,the expression level of CD31 was significantly higher in the aortic plaque of CD137 activated group than in control group(1 191 ± 187 vs.115 ± 30,P ＜ 0.05),while which was significantly downregulated in CD137 inhibited group (450 ± 92,P ＜ 0.05).(2) The level of nucleoprotein(3.07 ±0.03 vs.1.00 ±0.00,P ＜0.05) and total protein(2.18 ± 0.30 vs.1.00 ±0.00,P ＜0.05) of NFATc1 were significantly higher in CD137 activated group than in IgG isotype control group.The level of nucleoprotein(0.82 ±0.04) and total protein(0.84 ± 0.09) of NFATc1 were significantly lower in CD137 inhibited group than in CD137 activated group(both P ＜ 0.05).(3) FCM results showed that the fluorescence intensity of CD137 on the cell membrane was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells(5 163 ± 329 vs.1 660 ± 162,P ＜ 0.05).(4) ELISA examination showed that the level of CD137 protein was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells ((573.4 ± 23.7) pg/mg vs.(69.5 ± 16.7) pg/mg,P ＜ 0.05).(5) Migration cell number was remarkably higher in CD137 activated group than in IgG isotype control group(1.19 ±0.13 vs.1.00 ±0.00,P ＜0.05) and significantly lower in CD137 inhibited group(0.82 ± 0.06) than in control group (P ＜ 0.05).(6) Values of the formation of the tube length ((5.76 ± 0.18) mm vs.(4.21 ± 0.11) mm,P ＜ 0.05) and branch number (29.38 ± 1.28 vs.21.13 ± 0.96,P ＜0.05) were both significantly higher in CD137 activated group than in the control group.The formation of the tube length ((1.90 ±0.11)mm) and branch number(8.91 ±0.72) were significantly lower in silent NFATc1 + CD137 activated group than in the CD137 activated group (both P ＜ 0.05).The formation of the tube length ((1.28 ± 0.34) mm) and branch number (5.07 ± 0.35) were also significantly decreased in the CD137 inhibited group compared with the CD137 activated group (both P ＜ 0.05).Compared with the CD137 inhibited group,the formation of the tube length ((4.82 ± 0.09)mm) and branch number(24.44 ± 1.05) in the over expressed NFATc1 + CD137 inhibited group was increased (both P ＜ 0.05).Conclusion CD137 can promote the angiogenesis in atherosclerosis plaque by activating NFATc1.

Objective To explore the regulation mechanism for miR - 125a - 5P in epidermal growth factor receptor(EGFR)signaling pathway in medulloblastoma. Methods The potential targets of miR - 125a - 5P in the EGFR signaling pathway were predicted by TargetScan and Sanger software,there were 3 groups:control group,non -sense group and miR - 125a - 5P group. Their relationship,between miR - 125a - 5P and cyclin - dependent kinase in-hibitor 2B( CDKN2B),E2F transcription factor 3( E2F3),mitogen - activated protein kinase 14( MAPK14)and growth factor receptor - bound protein 10(GRB10),were tested by luciferase experiments. After miR - 125a - 5P oligo-nucleotide was transfected to D341 cells,miR - 125a - 5P level was detected by reverse transcription polymerase chain reaction. Then the thiazolyl blue tetrazolium bromide assay was used to draw the cell growth curves,and Transwell assay was used to detect cell migration ability. The expression levels of GRB10,EGFR,phosphatidylinositol 3 - kinase(PI3K) and Ras were tested by Western blot method. Results The results of luciferase experimental results showed that GRB10 was the only target gene of miR - 125a - 5P. After miR - 125a - 5P being transfected,the D341 cell prolifera-tion obviously declined markedly. Compared with control group[(38. 16 ± 7. 47)% ]and the non - sense group [(36. 79 ± 8. 94)% ],cell migration rate in the miR - 125a - 5P group was lowest[(13. 59 ± 4. 41)% ],and there was a significant difference among 3 groups(χ2 = 11. 495,P < 0. 05);in the miR - 125a - 5P group,the expression level of EGFR increased 1. 67 times,GRB10,PI3K and Ras levels were reduced to 23% ,61% and 42% . Conclusion miR - 125a - 5P can inhibit tumor growth by silenced GRB10 expression targeting EGFR downstream signaling pathways in medulloblastoma.

This paper provides a brief overview of the current research activities which focused on the bio-application of gold magnetic nanocomposite particles. By combining the magnetic characteristics of the iron oxide core with the unique features of nano-gold particles such as targeting by surface modification and optical properties, such composite nanoparticles have a wide range of applications in cancer hyperthermia, CT and MRI imaging, bio-separation, bio sensors, gene diagnosis, drug targeting and many other biomedical fields.
Diagnostic Imaging ; Drug Delivery Systems ; Ferric Compounds ; chemistry ; Gold ; chemistry ; Humans ; Magnetics ; Nanocomposites ; chemistry ; Nanoparticles ; chemistry ; Neoplasms

Objective To study the effects of internal exposure of 18F-FDG (18F-2-deoxy-2-fiuoroD-glucose) on the protein expressions of P53,XIAP (X-linked inhibitors of apoptosis protein) and GADD (growth arrest and DNA damage)45 in Lewis lung carcinoma,and to explore the possibility of applying 18F-FDG as a radiotherapy drug in vivo.Methods Lewis lung cancer transplanted tumor models were established via subcutaneous injection of 0.2 ml of 2 × 107/ml Lewis lung carcinoma cells at left hind limbs of 48 C57BL/6 mice that were randomly divided into high dose group,low dose group and control group with 16 mice each.After 7 d of cancer cell inoculation,18.5 × 107 Bq and 9.25 × 107 Bq of 18F-FDG in 0.2 ml saline or equal volume of physiological saline was intraperitoneally injected into three group mice,respectively.22 d post inoculation,the protein expressions of P53,XIAP and GADD45 were immuohistochemically detected by using SP approach.Results There was significant difference among the protein expressions in each group (x2 =8.30,16.02,7.68,P ＜0.05).The mean integral optic density of protein expression increased from 0.089 ± 0.033 in control group to 0.315 ± 0.028 in high dose group for P53,and from 0.126 ± 0.023 to 0.383 ±-0.035 for GADD45,but it decreased from 0.422 ± 0.034 to 0.149 ± 0.055 for XIAP.There was significantly difference of these protein expressions in each group (P53:F=5.26,P＜0.05;XIAP:F=4.29,P ＜0.05;GADD45:F=5.98,P ＜0.05).Conclusions 18F-FDG can up-regulate the expressions of P53 and GADD45 proteins and down-regulate the expression of XIAP protein in tumor cells,and it inhibits tumor growth by promoting cell apoptosis in the Lewis lung carcinoma tissue with a P53-dependent manner.

Objective To investigate the outcome of postoperative hypopituitarism and hormone replacement in patients with pituitary adenoma,and to analyze the potential factors related to postoperative hypopituitarism.Methods A total of 215 postoperative patients with pituitary adenoma were analyzed.Pituitary functions( including gonadal,thyroid,and adrenal axes ) were asessed by strict criteria.Data of surgery history and hormone replacement situation were collected for statistical analysis.Results The prevalence of hypopituitarism was 54.0％,including 36.7％ hypogonadism,32.6％ hypothyroidism,and 28.4％ hypoadrenalism.Replacements of gonadal steroid,glucocorticoid,and thyroxine were carried out in 25.6％,84.3％,and 80.6％ of the cases,respectively.Univariate analysis showed that male sex and large tumor were related to hypopituitarism. Conclusion After pituitary adenomectomy,approximately half of the patients present anterior pituitary dysfunction,while quite a number of them have not been treated appropriately.

Objective To explore the effect of thyroxine (TH) on the migration of hippocampal neurons in newborn rat exposed to tritiated water (HTO).Methods The hippocampal neurons from neonatal rats were primarily cultured,7 days later,randomly divided into control group,HTO group,TH group and HTO + TH group(3.7 × 105 Bq/ml HTO and 0.3 μg/ml TH were simultaneously added).After 24 h,the distance of neuronal migration was measured with Leica AF 6000,the expressions of BDNF and Reelin mRNA in neurons were analyzed with reverse transcription polymerase chain reaction (RT-PCR),the expression of β-tubulin protein in neurons was assayed with Western blot and immunocytochemical staining.Results Compared with control group,the expression of Reelin mRNA,BDNF mRNA and β-tubulin in HTO group were significantly reduced(t =5.80,5.48,5.47,P ＜ 0.01),but those in HTO + TH group and TH group were obviously increased (t =7.75,12.06,13.65,P ＜ 0.01 ;t =4.34,5.47,5.65,P ＜0.01)and higher than that in HTO group (t =2.92,10.32,8.76,P ＜ 0.01 ;t =18.07,20.55,40.13,P ＜0.01).Accordingly,the neuronal migration distance in HTO group was much shorter than that in control (t =8.62,P ＜ 0.01),and in HTO + TH group and TH group was far longer than that in control(t =7.64,4.93,P＜0.01).Moreover,the neuronal migration distance in HTO + TH group was notably elongated in comparison with that in HTO group(t =11.32,12.31,P ＜ 0.01).Conclusions Thyroxine may promote the migration of hippocampal neurons in newborn rat exposed to HTO.