Objective:To construct the extracellular region of the human TRAIL cDNA expression vector and express and purify the extracellular region of the TRAIL protein. Methods: The mRNA of TRAIL was extracted from CD3 activated normal human PBMC and used as a template for reverse transcription. After PCR amplification, a 730 bp fragment including extracellular region was obtained and cloned into pGEX-2T.The recombinant vector was named pGEX/TRAILex. The pGEX/TRAILex vector was transformed into E.coli DH5a. After IPTIG induced at lower temperature, the collection of the sonicated extract was purified by using the GST agarose 4B. The purified fusion protein was identified by Western blotting with anti-TRAIL McAb.Results:The pGEX/TRAILex was constructed. After IPTG induced,a high level expression of the extracellular region of the TRAIL protein was obtained, SDS-PAGE analysis showed that the recombinant E. coli could express a 54 kD GST fusion protein which accounted for about 28% of the total cellular protein. The study of solubility of expression protein indicated that GST-Tex was expressed predominantly in the soluble form.The purified production was obtained 2.2 mg/L of culture media and the purity of the GST-Tex was more than 95%. GST/TRATLex protein could be recognized by anti-TRAL McAb in Western blot. Conclusion:The expression of recombinant extracellular domain of the human TRAIL protein may be useful for the study of biological functions of TRAIL and it's biotheraphy in tumor.

Objective To observe the effects of 2 Gy γ-ray irradiation on regulatory T cells (Tregs) and Th17 cells and immune balance of mice.Methods A total of fifty C57BL/6 male mice were randomly divided into two groups,the irradiated group exposed to 2 Gy of whole body γ-ray irradiation,and the control group sham-irradiated.At 1,3,7,14 and 28 d after radiation,changes of peripheral haemogram were detected respectively and Tregs in peripheral blood,thymus and spleen and Th17 cells in spleen were analyzed by flow cytometry.Results Compared with control group,the number of peripheral blood white cells (WBC) and lymphocyte in irradiated group reduced significantly post-irradiation (t =8.89-33.54,P ＜ 0.05),while the cell number of peripheral CD4 + CD25 + Tregs post-irradiation rose but not significantly.Thymic Treg cells increased 1 and 3 d post-irradiation(t =-6.45,-10.59,P ＜0.05),but reduced 28 d post-irradiation (t =5.34,P ＜ 0.05).Splenic Treg cells ascended obviously from 1 to 14 d post-irradiation (t =-6.82-3.89,P ＜ 0.05).After irradiation splenic Th17 cells increased at 1 d,and reached the maximal level at 3 d (t =-2.42,P ＜ 0.05),more obviously than splenic Treg cells.The reduction of Treg/Th17 ratio from 1 to 14 d post-irradiation disturbed Treg/Th17 balance and made it drift to the direction of Th17 (t =4.02-8.04,P ＜ 0.05).Conclusions Treg/Th17 imbalance plays an important role in immune injury induced by irradiation.

Objective To investigate the features of epileptic seizures and eletroencepalogram (EEG) in patients with mitochondrial myopathy encephalopathy,lactic acidosis and stroke-like episodes (MELAS).Methods Forty-four patients with MELAS were diagnosed at the First Hospital of Peking University from November 2007 to August 2013.EEG and head MRI were performed on all patients.The types of epileptic seizure and EEG changes were compared between patients in and outside stroke-like episodes.Results Epileptic seizures occurred in 39 of 44 patients (88.6％) with MELAS,while multitype epileptic seizures were presented in 26 cases (66.7％).In stroke-like episodes,22 patients presented with partial seizures,30 with generalized seizures and 17 with status epilepticus.In nonstroke-like episodes,7 patients presented with partial seizures,14 with generalized seizures and 2 with status epilepticus.The frequency of partial seizures,generalized tonic-clonic seizures,status epilepticus were 47.7％ (21/44),68.2％ (30/44),38.6％ (17/44) in stroke-like episodes and 13.6％ (6/44),27.3％ (12/44),4.5％ (2/44) in nonstroke-like episodes,which have statistical significance (x2 =12.022,14.758,15.103;P =0.001,0.000,0.000,respectively).Abnormal EEGs appeared in all patients.The rates of slow alpha frequency,diffuse δ or θ wave,epileptic discharges were 6.8％ (3/44),43.2％ (19/44),25.0％(11/44) in stroke-like episodes and 31.8％ (14/44),59.1％ (26/44),22.7％ (10/44) in nonstroke-like episodes,respectively.Slow alpha frequency were significantly different between patients in and outside stroke-like episodes (x2 =8.822,P =0.003).Conclusions Epileptic seizures with different types are more common during stroke-like episodes in patients with MELAS.While the rates of epileptic discharges are also common outside the stroke-like episodes.

Objective To investigate the effects of human endostatin(hES)on ovarian cancer cells A2780 growth in vitro and in vivo and the possible mechanism.Methods The coding region for hES was amplified by PCR from human liver tissue and then subcloned into pcDNA3.1 vector.The growth rate of A2780 cells transfected with hES was observed.A2780 cells(1× 10~7/ml)were inoculated subcutaneously into 20 nude mice,and the mice were randomly divided into two groups:the hES-trans-fected group(group A,n=10)and the control group(group B,n=10).One month later,the size of subcutaneous tumor was measured,and the tumor inhibition rate was calculated.The specimens were stained with HE for the histological analysis.Cell apoptosis in ovarian neoplasm transplantation tissues was determined by flow cytometry.The expression of hES and Bcl-2 was detected by Western blot.Results The growth rate of the hES-transfected ovarian cancer cells A2780 was significantly decreased.In group A,growth of tumor in nude mice was significantly slowed as compared with group B(P<0.01),with the tumor inhibition rate being 74% in group A.The tumor necrosis was increased and the basophilia stain of nucleus decreased significantly in group A as compared with group B.The expression of hES was increased significantly,but that of Bcl-2 was decreased significantly in group A.Flow cytometry revealed that hES transfection could significantly incyease the apoptosis rate of tumor cells(P<0.05).Conclusion Transfection of hES could suppress the growth of ovarian cancer cells A2780 in vitro and in vivo,which may be related tO the promotion of apoptosis.

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.

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 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.

This paper was aimed to study effects of different preparation methods on the content of ginsenosides Rg1,Re and Rb1 in Yi-Xin-Shu (YXS) tablets by HPLC-ELSD.HPLC-ELSD was used as the detection method.The separation and content of ginsenosides Rg1,Re and Rb1 were used as indexes.The influences of three different preparation methods (i.e.,defatted alcohol extraction and butanol extraction,alcohol extraction and butanol extraction,alcohol extraction and butanol extraction ammonia solution washing) on the effect of YXS tablets were studied.Then,the same content determination method was used to compare the influence of alkali washing treatment to ginsenosides Rg1,Re and Rb1 among different batches of Panax ginseng.The results showed that a good separation of ginsenosides Rg1,Re and Rb1 component peak of YXS tablets was achieved by three kinds of separation methods.The separation degree was greater than 1.5.Ammonia solution washing had some effect on ginsenosides Rg1,Re and Rb1 content,which made the content of ginsenosides Rg1,Re and Rb1 be 1.5-1.8 times to those without alkali washing.No effect was shown on the content of ginsenosides Rg1,Re and Rb1 during ammonia solution washing.It was concluded that some other ginsenosides can be transferred into ginsenosides Rg1,Re and Rb1 in YXS tablets solution after ammonia solution washing.

Objective To evaluate the therapeutic effect of hepatocyte growth factor(HGF) gene modified placenta-derived mesenchymal stem cells( PMSCs) on limb ischemia in a rabbit model.Methods The placental tissue was digested with enzyme, cultured and passaged.The PMSCs were characterized by surface marker expression.These cells were infected with adenoviral( Ad)-HGF and intramuscular injected for treatment of limb ischemia in a rabbit model.The blood supply of the limb was detected by digital subtraction angiography ( DSA ) and the vessel number was evaluated in histopathological HE staining.Results The results showed that Ad-HGF gene transduction increased the vascular endothelial growth factor ( VEGF ) , basic fibroblast growth factor, bFGF ( bFGF ) and HGF expression in PMSCs. Transplantation of HGF-transduced PMSCs resulted in the increase in vessel density and improvement of blood supply in the rabbit limb ischemia model.Conclusion The therapeutic effect of HGF gene engineered PMSCs on ischemia by enhancing angiogenesis in a rabbit model is evaluated.Transplantation of PMSCs with HGF gene therapy may be a promising strategy for the treatment of ischemia diseases.

Objective To observe the effect of γ-ray irradiation on CD4 + CD25 + regulatory T cells (Tregs),and to investigate the mechanism of immune injury induced by irradiation.Methods The thymus and spleen of C57BL/6 mice were taken and weighted 1-28 d after γ-ray irradiation,and the organ coefficients were calculated.The amount of mouse peripheral WBC measured,CD4 + T cells and Tregs in peripheral and splenic were analyzed by flow cytometry.Results Coefficients of mouse thymus and spleen decreased significantly 1 d post irradiation,and reached to the bottom at 7 d.Coefficients did not recover to control level 28 d after radiation.Peripheral WBC continuously decreased and reached the bottom at 7 d,and did not recover to control level up to 28 d postirradiation.Peripheral CD4 + T lymphocyte temporally reduced at 1 d,while it increased at 7 d,and it approached to control level at 28 d after radiation.Splenic CD4 + T cells slightly reduced at 7 d however,they basically maintained as the same level as control 14 d and 28 d after radiation.Peripheral Tregs ascended at 1 d and reached the peak at 7 d,and reduced at 14 d and 28 d postirradiation,although they still were significantly higher than those of control group.At the same time,splenic Tregs increased significantly and achieved peak value at 1 d,and then gradually decreased and reached the minimum at 28 d after irradiation,which were significantly lower than those of control group( t =2.731,P ＜ 0.05).Conclusions Mouse thymus and spleen were injured severely,and the number of immunocytes decreased after 6 Gy whole body γ-ray irradiation.However,Tregs with immunosuppressive action increased significantly postirradiation,revealing that Tregs were closely correlated with immune function depression and immunomodulation imbalance induced by ionizing radiation.