Objective To explore themethod for the objective evaluation of single lim b function after in-jury in forensic m edical practice. Methods The score of activities of daily living (ADL ) w ere graded for a single lim b function after injury from 47 cases.Allcases w ere sim ultaneously evaluated using the differentmethods including Fugl-M eyer m otor function assessm ent (FM A ), w eighting, look-up table (LUT). The correlation w ere com pared betw een ADL and the other threemethods. Results Injured part and the score using the threemethods w ere correlated w ith ADL score (P<0.05). The correlation coeffi-cient (|r| value) show ed highest using LUTmethod, and low est using FM Amethod. Conclusion The loss function of lim b is affected by the injuried parts. Themethods of FM A , w eighting and LUTshow a good accuracy for evaluating the lim b function after injury and the correlation presents higher using LUTmethod.

AIM:To construct a eukaryotic expression plasmid for enhanced green fluorescent protein(EGFP)and ZNF580 fusion protein,and study its subcellular localization in the transfected MGC803 cells.METHODS:The primers were designed according to the cDNA encoding sequence of ZNF580 full-length open reading frame(1-172aa),ZNF580 amino terminus(1-93aa)and ZNF580 carboxyl terminus(94-172aa).The three cDNA segments of PCR were cloned into pGEM-T vector.Then they were subcloned respectively into plasmid pEGFP-C1(enhanced green fluorescent protein).The subcellular localization of the fusion protein in MGC803 cells transfected with the vector was monitored by autofluorescence microscopy.RESULTS:Restricted enzymes analysis and DNA sequencing showed that the sequences of the pEGFP-ZNF580(1-172),pEGFP-ZNF580(1-93)and pEGFP-ZNF580(94-172)transgenic plasmid were correct.The fusion proteins of EGFP-ZNF580(1-172)and pEGFP-ZNF580(94-172)were localized in the nuclei.CONCLUSION:The recombinant eukaryotic expression vector pEGFP-ZNF580 has been successfully constructed.The nuclear localization signal is within amino acid residues 94 and 172 of ZNF580 carboxyl terminus(C2H2 zinc finger domain).

Objective To investigate the effect and safety of alprostadil in the treatment of diabetes complicating chronic kid‐ney disease to provide reference for clinical treatment .Methods 84 cases of diabetes complicating chronic kidney disease in this hospital from September 2013 to January 2015 were selected and divided into the observation group(44 cases) and the control group (40 cases) according to the voluntary principle .The control group used the epalrestat treatment ,while the observation group was combined with using alprostadil on the basis of control group .The effective rate ,serum creatinine ,blood urea nitrogen(BUN) ,uri‐nary albumin excretion rate ,C‐reactive protein(CRP) ,IL‐6 levels and adverse reactions were compared between the two groups .Re‐sults The effective rate of the observation group was 93 .18% ,which was significantly higher than 80 .00% in the control group , the difference was statistically significant (χ2 =4 .251 ,P=0 .005);the CRP and IL‐6 levels after treatment in the observation group were improved ,the difference was statistically significant (P<0 .05);the CRP and IL‐6 levels after treatment in the observation group[(0 .45 ± 0 .05)mg/L ,[(0 .72 ± 0 .11)μg/L] were significantly superior than[(1 .05 ± 0 .14)mg/L ,(1 .25 ± 0 .24)μg/L] in the control group ,the differences were statistically significant(P<0 .05);BUN ,urinary albumin excretion rate ,TNF‐αand endogenous creatinine clearance rate after treatment in the observation group were (6 .41 ± 1 .12)mmol/L ,(41 .12 ± 4 .46)μg/min ,(75 .54 ± 6 .64)ng/L and (92 .94 ± 8 .24)% ,which in the control group were (7 .39 ± 1 .05)mmol/L ,(91 .48 ± 7 .31)mmol/L ,(111 .42 ± 7 .69)ng/L and (81 .55 ± 9 .54)% respectively ,the improvement in the observation group was better than the control group ,the difference was statistically significant (P<0 .05);the occurrence rate of adverse reactions was 18 .18% in the observation group and 17 .50% in the control group ,showing the difference was not statistically significant (P>0 .05) .Conclusion Alprostadil in treating diabetes complicating chronic kidney disease has better effect ,conduces to improve the level of urinary albumin and inflammatory with high safety ,and is worthy of clinical promotion and application .

Objective The incidence of cardiovascular and cerebrovascular diseases in postmenopausal women with type 2 diabetes is grim.The study was designed to explore the effect of ω-3 polyunsaturated fatty acids (PUFA) on endothelial function in postmenopausal women with type 2 diabetes. Methods 50 patients admitted to Dingli Medical College of Wenzhou Medical Univer-sity from March 2014 to October 2014 were divided into group A and Group B by random number table .Cross-design of two stages ( I, II) was applied in the investigation .At stage I(3 months ahead of the experiment ), Group A took oral ω-3 PUFA while Group B took placebo .At stage II ( 3 months after the experiment ) , Group B was given oral ω-3 PUFA, while Group A was given placebo .T1 and T3 time was the beginning of the stage I and stage II experiment , while T2 and T4 time was the end of stage I and stage II experiment .At the beginning and end of each stage , detection was made on LDL-C, TG, IL-6, plasminogen activator inhibitor-1 (PAI-1) and endothelium-dependent flow-mediated vasodilation (FMD). Results After the intervention on Group A at stage I , FDM at T2 time was significantly increased compared with that at T 1 time([7.23 ±3.28]% vs [3.62 ±2.13]%, P<0.05), while all the other indexes at T2 time decreased significantly in comparison with T1 time: LDL-C ([2.85 ±0.47]mmol/L vs [3.36 ±0.57] mmol/L), TG([2.41 ±1.06]mmol/L vs [2.96 ±1.12] mmol/L), IL-6([2.83 ± 0.30]ng/L vs [3.42 ±0.32]ng/L), PAI-1 ([7.23 ±3.28]ng/L vs [3.62 ±2.13]ng/L) (P<0.05).After receiving ω-3 PUFA intervention on Group B at stage II , FDM at T4 time was significantly increased compared with that at T 3 time([6.88 ±2.06]% vs [3.60 ±2.18]%, P<0.05), while all the other indexes at T4 time decreased significantly in comparison with T3 time: LDL-C ([3.26 ±0.77]mmol/L vs [3.63 ±0.73] mmol/L), TG([2.28 ±0.94]mmol/L vs [2.77 ±1.25] mmol/L), IL-6([2.91 ± 0.48]ng/L vs [3.30 ±0.52]ng/L), PAI-1 ([45.7 ±24.4]ng/L vs [56.3 ±24.4]ng/L) (P<0.05).Two-period crossover design analysis of variance showed that there was significant difference effect on LDL -C(F=2.754, P=0.019), TG(F=3.115, P=0.011), IL-6(F=1.825, P=0.032), PAI-1(F=2.324, P=0.023) and FMD(F=3.784, P=0.006)between ω-3 PUFA and placebo . Conclusion ω-3 PUFA can improve endothelial function in postmenopausal women with type 2 diabetes , which is of great significance for the primary prevention of cardiovascular disease .

Objective To construct a prostate cancer specific oncolytic adenovirus armed with a fusion protein gene , PSA-IZ-CD40L, and to evaluate its oncolytic efficiency and immune activation ability in vitro.Methods Prostate Specific Antigen (PSA) gene, CD40L-N and CD40L-C genes were obtained from cDNA of LNCaP cells and Jurkat cells using poly-merase chain reaction (PCR) or nested-PCR, respectively.PSA,Linker,CD40L-N and CD40L-C were linked sequentially to generate fusion protein gene PSA-IZ-CD40L (PL) by overlapping PCR.Then, prostate specific oncolytic adenovirus PL-carrying gene, Ad-PL-PPT-E1A,was constructed using the oncolytic adenovirus system , which was based on Adeasy sys-tem.PC3M cells were infected by Ad-PL-PPT-E1A at serial multiplicity of infection (MOI), and the apoptosis was detec-ted by flow cytometry at several time points post-infection.For immune activation detection , PC3M cells were infected with Ad-PL-PPT-E1A at a MOI of 50, and the cell lysate was collected at 48 h post-infection.Peripheral blood mononuclear cells derived (PBMCs) from healthy donors were stimulated by the lysate from PC 3M cells or Ad-PL-PPT-E1A infected PC3M cells before proliferation was assayed using cell counting kit-8 (CCK8).Results Fusion protein gene, PSA-IZ-CD40L, was successfully constructed and cloned into the prostate cancer specific adenovirus to generate Ad -PL-PPT-PL. The expression of E1A and PL protein could be detected by reverse transcription PCR and Western-blotting.Cytopathic effect was observed in PC3M cells infected with Ad-PL-PPT-E1A.Furthermore, the apoptosis rate reached 70.67% ± 2.98%at 48 h post-infection with 200 MOI Ad-PL-PPT-E1A.Compared with the lysate of PC3M cells, that from Ad-PL-PPT-E1A infected cells could promote the proliferation of PBMCs .Conclusion We have constructed a prostate cancer spe-cific oncolytic adenovirus armed can fusion protein gene PL , Ad-PL-PPT-E1A, which could kill PC3M cells effectively and enhance the proliferation of PBMCs in vitro.

Objective To study the effect of heavy ion radiation on proliferation and apoptosis of human peripheral blood derived T lymphocytes and the mechanism .Methods T lymphocytes were isolated from heparinized whole blood samples by density gradient centrifugation using Ficoll before being irradiated with heavy ion beams 12 C.The accumulated absorbed dose (dose-rate values=0.5 Gy/min, and meanLET=29 keV/μm).12 h and 24 h post-infection, total RNA of T lymphocytes was isolated , and the apoptosis related gene expression , including Bcl-2, Bax, Caspase3, Caspase8 and Caspase9, was detected by RT-RT-PCR.24 h and 48 h after irradiation, the proliferation was analyzed by CCK 8 kit.The cell apoptosis was detected by flow cytometry after being labeled with AnnexinV-PE/7-AAD or AnnexinV-FITC/PE.The expression of Bcl-2, Bax and Caspase3 was also assayed by RT-PCR.Results Data showed that heavy ion radiation could inhibit the proliferation of T lymphocytes obviously , and the inhibition ratio in cells that received 2 Gy dose was much high-er than in cells that received 1 Gy dose.Furthermore, heavy ion radiation promoted the apoptosis of T lymphocytes signifi-cantly.The results of RT-PCR showed that the mRNA expression of Bcl-2 was down-regulated in heavy ion radiation T lym-phocytes while the expression of Bax and Caspase 3 was up-regulated.Conclusion Heavy ion radiation can inhibit the pro-liferation and promote the apoptosis of human peripheral blood derived T lymphocytes .

Objective To study the role of endothelial cells on the inflammatory cytokine release in septic shock through the septic shock serum stimulating human primary endothelial cells (HPAEC) and peripheral blood mononuclear cells(PBMC).Methods PBMC isolated from healthy people by density gradient centrifugation.HPAEC cell surface markers CD144 and von Willebrand factor(vWF) molecule expression by RT-PCR and Western blot.Serum levels of IL-6,TNF-α,MCP-1 from septic shock patients and healthy human detected by ELISA.HPAEC and PBMC were stimulated with the isolated serums and LPS,respectively.ELISA was used to detect the supernatant IL-6,TNF-α,MCP-1 levels.HPAEC membrane molecules ICAM-1 expression was detected by flow cytometry with serum shock and LPS stimulation.Supernatant levels of IL-6,TNF-α,MCP-1 of HPAEC with S1P1 receptor agonist CYM-5442 pretreatment was detected by ELISA after shock serum stimulation.Results Endothelial cell markers CD144 and vWF molecules could be detected in the HPAEC.Levels of inflammatory cytokines IL-6,TNF-α,MCP-1 in patients with septic shock serum were significantly higher than healthy people (P＜0.01).PBMC and HPAEC with LPS or shock serum treatment respectively,compared with normal group,levels of inflammatory cytokines in the culture supernatant were significantly higher(P＜0.01).For PBMC,the level of inflammatory cytokines between shock group and LPS group were not significantly different (P＞0.05).But for HPAEC,levels of inflammatory cytokines in the supernatant of the shock group compared to the LPS group was significantly higher (P＜0.01).Similarly,when two cells after LPS stimulation,IL-6,TNF-α levels of HPAEC's supernatant were significantly lower than PBMC' s (P＜0.01),MCP-1 levels was no difference (P＞ 0.05).But when the stimulation of shock serum,HPAEC of IL-6,TNF-α levels and PBMC no significant difference (P ＞0.05).MCP-1 was significantly increased (P＜0.01).Shock patients serum stimulation S1P1 receptorspecific agonist CYM-5442 pretreatment of HPAEC with pretreatment of S1P1 receptor specific agonist CYM-5442,the culture supernatant of inflammatory cytokines IL-6,TNF-α,MCP-1 levels were significantly lower (P＜0.01).Conclusion Endothelial cells may play a central role on the release of inflammatory cytokine during septic shock.

Objective To understand the infection situation of human T lymphocyte virus(HTLV) among blood donors in Zhongshan area.Methods Blood samples from 40 874 blood donors in Zhongshan from March to December 2016 were screened for HTLV antibody by using,enzyme linked immunosorbent assay(ELISA).The positive samples were reexamined two times,and specimens with positive results of reexamination were detected by using immunohistochemical method(CLIA).Then the positive samples were confirmed by Western blot(WB),and confirmed positive samples were judged as infection.Results Of all 40 874 cases of voluntary blood donors,21 cases were positive with HTLV antibody detected by ELISA,the positive rate of ELISA was 0.05%.Five cases were positive detected by CLIA method.One case was confirmed by WB,and the infection rate was 0.002 4%.Conclusion In order to ensure the safety of blood transfusion and reduce blood transfusion infection of HTLV,it might be necessary to perform HTLV screening in first-time blood donors in Zhongshan area.

Objective To assess the value of magnetic resonance diffusion weighted imaging(DWI) and apparent diffusion coefficients(ADC) values of hepatic alveolar echinococcosis(HAE) in children.Methods 20 cases of children(≤14 years) with HAE were collected in this restrospective study.PNM staging was determined, the HAE peripheral area of DWI lesions with different P stages was observed, and the ADC value of the peripheral area was measured.The comparison of alveococcus lesions in different stages of DWI with continuous edge degree and ADC value difference was done to evaluate the growth activity.Results There were 5 cases of P1 lesions, 7 cases of P2 lesions, 2 cases of P3 lesions and 6 cases of P4 lesions.DWI features of peripheral area were as follows: High signal ring band between HAE lesion edge and adjacent normal hepatic parenchyma was observed.P1 lesions showed almost complete obviously high signal peripheral area, indicating the most active proliferation, P2 and P3 lesions of peripheral area were continuous and with high signal, and still had obvious growth activity.P4 lesions of peripheral area were not continuous, while the signal decreased, indicating the activity also decreased.The highest ADC value was detected in P1 lesions group of and the ADC value of P2 lesions group were lower than P1, and the ADC value of P4 lesions group were the lowest.P3 lesions samples were too small and thus no statistical analysis was done.Differences of ADC value between the three groups were statistically significant (P<0.05).Conclusion DWI image features could be used to assesse the growth activity of HAE in children with different stages to a certain extent.ADC values measurement provides important reference value for evaluating the growth activity at various stages of the lesions.

Objective To investigate the roles of SENP1 in regulation of biological characteristics of NK cells.Methods Lentivirus-mediated-Senp1-small-hairpinRNA (shRNA) transduction was applied to NK92 cells.The expression of SENP1 in NK92 cells was determined by real-time PCR and Western blot.The proliferation of NK92 cells was detected by CCK-8 assay.The apoptosis of NK92 cells was determined by Annexin Ⅴ and PI labeling.The cytotoxicity of NK92 cells against K562 cells was evaluated by luciferase reporter assay.Results Treatment of NK92 cells with IL-21 resulted in SENP1 upregulation.Lentivirus mediated SENP1 knockdown reduced proliferation and increased apoptosis in NK-92 cells,but SENP1 inhibition had slight impact on the cytotoxic ability of NK92 cells to kill K562 cells.Conclusion SENP1 mediates the regulatory effect of IL-21 on the proliferation and survival of NK92 cells.