Endoscopic esophageal probe dilation followed by intralesional compound betamethasone injection for anastomotic strictures after esophagectomy: a randomized controlled trial / 中华胸心血管外科杂志

Objective:To assess the efficacy, safety of adding intralesional compound betamethasone injection to EEBD to reduce restricture.Methods:77 patients, treated in The first people's hospital of YancHeng from January, 2015 to December, 2018, were randomized to receive EEPD combined with either compound betamethasone injection or placebo injection. A total of 2 ml of compound betamethasone injection or an identical volume of normal saline solution as a placebo was injected per site using a 23-gauge needle immediately after EEPD. Patients and treating physicians were blinded to the treatment. The primary endpoint was the number of dilations required to resolve the stricture、restricture-free survival、time required to resolve the stricture and adverse events.Results:During the 4-years study period, Finally , 74 patients , who were randomized to either the steroid group (37 cases) or placebo group (37 cases), comprised the per-protocol population .The median number of EEPD required to resolve strictures was 2.0( IQR 1.0-3.0) in the steroid group and 3.0 ( IQR 3.0-4.5) in the placebo group ( P<0.001). After 6 months of follow-up, 27.0% of patients who had received steroid injections remained recurrence free compared with 3.5% of those who had received saline injections( P<0.001). The median time of EEPD required to resolve the stricture was 88 days( IQR 0-98 days)in the steroid group and 131 days( IQR 97-157 days)in the placebo group( P<0.001). No adverse events occurred related to the EEPD or steroid injection occurred. Conclusion:Endoscopic esophageal probe dilation combined with compound betamethasone injection shows promising results for the prevention of stricture recurrence in patients of anastomotic strictures.

Endobiliary intraductal radiofrequency ablation to reopen occluded biliary metal stents in malignant biliary obstruction / 中华肝胆外科杂志

Objective To study the safety and feasibility of endobiliary intraductal radiofrequency ablation (RFA) to reopen occluded self-expandable metal stents in patients with malignant biliary obstruction.Methods 11 patients with malignant biliary obstruction and blocked metal stents were prospectively studied.During ERCP, after biliary cannulation, the blocked metal stents underwent RFA using a bipolar radiofrequency probe which was introduced into the stenotic bile duct via a guide wire.This was followed by a balloon to repeatedly remove debris and then endoscopic nasobiliary drainage.The patients were closely observed and followed up.Results RFA was successfully carried out in all the patients and patencies were achieved when compared with pre-RFA.The median post-RFA luminal diameter of the strictures showed significant improvement: 6 (4 ～ 10) mm versus 2 (0 ～ 5) mm, and the mean post-RFA total bilirubin level decreased sharply : (39.4 ± 8.7) μ mol/L versus (130.1 ± 38.2) μmol/L.Following this intervention, 3 patients developed fever, which were controlled with conservative therapy.There was no mortality, haemorrhage, bile duct perforation or bile leak.Of the 11 patients, 3 were dead and 6 were alive at a median follow-up of 187 (75 ～ 304) days.The median stent patency was 135 (75 ～ 203) days and the median survival was 278 (75 ～ 304) days.Four patients had their stents patent at the time of the last follow-up or death.Seven patients had their stents blocked on 113, 124, 154, 203, 96, 135 and 112 days post-procedure.Condusions Endobiliary intraductal RFA is technically feasible and safe to reopen occluded metal stents in malignant biliary obstruction.This efficacy needs to be confirmed by future randomized studies.

Cloning Hap gene from non-typeable Haemophilus influenzae and expression of Hap protein in prokaryotic cell / 生物医学工程学杂志

This study was aimed to construct a prokaryotic expressing vector of Hap gene from Nontypeable Haemophilus influenzae, and express and identify the fusion proteins of Hap-His in E. coli. The gene encoding protein Hap was amplified from Nontypeable Haemophilus influenzae ATCC49247 chromosomal DNA by PCR, then it was cloned into prokaryotic expression plasmid pET-32a (+). The recombinant plasmid pET-32a(+)-Hap was transformed into E. coli BL21 and expression was induced by Isopropy-beta-D-thiogalatoside(IPTG). The Hap-His fusion protein expressed so was analyzed by SDS-PAGE and Western-blot. The results showed that the recombinant expressive plasmid pET-32a (+)-Hap was constructed successfully, and the recombinant plasmid expressed Hap-His fusion protein with relative molecule mass 176 000 and mainly existed in inclusion body. This fusion protein could combine with anti-His monoclonal antibody specifically through Western blot analysis. Successful expression of Hap-His fusion protein in prokaryotic cell could lay a basis for further study of immunocompetence of Hap protein and development of NTHi vaccine.

An expression plasmid encoding recombinant immunotoxin IP10-DT390 suppresses the experimental autoimmune encephalomyelitis / 生物医学工程学杂志

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS); it serves as a model for the human multiple sclerosis (MS). In mice, EAE is mediated by T cells specific for various myelin basic proteins which migrate from the periphery to the CNS. In search of a way to prevent the induction and progression of EAE, we observed the effects of recombinant immunotoxin IP10-DT390 on blocking or eliminating the active T cells in the EAE model. In this paper is presented an experimental gene therapy-based model in which the mice were made resistant to EAE induction by plasmid DNA encoding recombinant immunotoxin that was injected into the leg muscles of mice. The new immuno-biological construct could selectively impair autoreactive T-cell homing while the duration of clinical signs is shorter, and the new construct would not affect other components of the immune response. These data demonstrated the effectiveness of the constructs in the treatment of EAE and suggested its usefulness in the treatment of other autoimmune diseases.

Construction of Eukaryotic Expressing Plasmids Encoding HA and HA1 of Influenza A Virus and Their Transient Expression in HEK293 Cells / 华中科技大学学报（医学）（英德文版）

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3. 1 (+) to generate pcDNA3. 1 (+)/HA and pcDNA3.1 (+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1 (+)/HA and pcDNA3.1 (+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3. 1 (+)/HA or pcDNA3. 1 (+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

Suppression of telomerase activity by plasmid-mediated RNA interference / 生物医学工程学杂志

This study was aimed to construct a plasmid expressing siRNA specific for the human telomerase reverse transcriptase (hTERT) gene and to evaluate the ability of small interference RNA(siRNA) for inhibiting telomerase activity in HeLa cells. 64 nucleotides, in which 19 nt were homologous with hTERT gene, were chemically synthesized, annealed and linked into pSUPER to get pSUP-hTE. Then pSUP-hTE was digested with enzyme. We obtained its fragmant concluding promoter and 64nt. So we cloned it into pEGFP-C1 for constructing pEGFP-hTE which contains neo gene and the enhanced green fluorescent protein (EGFP). Recombinant pEGFP-hTE was transfected to HeLa cells. These cells were screened with medium containing G418. When stable colonies appeared, G418-resistant cells were harvested and propagated. At the different cell generations, hTERT mRNA and protein expression, telomerase activity and cell growth activity were analyzed. Compared with control cells, HeLa cells transfected with pEGFP-hTE showed that hTERT mRNA level and hTERT protein expression decreased and telomerase activity reduced by 38%, but the cells growth activity displayed no changes. So pEGFP-hTE could specifically inhibit expression of hTERT and telomerase activity. These results suggested that siRNA targeting hTERT gene might provide a new strategy for cancer biotherapy.

Construction of eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus and their transient expression in HEK293 cells / 华中科技大学学报（医学）（英德文版）

In order to explore the feasibility and protective efficiency of influenza DNA vaccine, we constructed eukaryotic expressing plasmids encoding HA and HA1 of influenza A virus (A/PR/8/34) and studied their expression in HEK293 cells. HA and HA1 genes were amplified by RT-PCR and cloned into pcDNA3.1(+) to generate pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1, respectively. After verification of the cloning fidelity by restriction endonuclease digestion, PCR, and sequencing, pcDNA3.1(+)/HA and pcDNA3.1(+)/HA1 were transfected into HEK293 cells using PolyFect Transfection Reagent. Immunofluorescence assay was used to detect the transient expressing cells. Fluorescence microscopy revealed strong expression of target gene in HEK293 cells transiently transfected with either pcDNA3.1(+)/HA or pcDNA3.1(+)/HA1. Therefore, the results confirm the successful construction of eukaryotic expressing plasmids capable of driving the eukaryotic expression of influenza virus antigen HA and HA1, which is likely to provide a basis for both further investigation of the mechanism of influenza viral infection and the development of influenza DNA vaccine.

Genetic Construction and Characterization of Murine Interleukin-18 Immunotoxin with a Truncated Pseudomonas Exotoxin / 航天医学与医学工程

Objective To construct a new recombinant immunotoxin expression vector fused with a murine interleukin18(IL18) gene and a truncated pseudomonas exotoxin (PE38) gene, and examine the expression of IL-18-PE38 fusion protein in Escherichia coli (E. coli). Method Murine IL-18 (mIL-18) cDNA was cloned from murine liver tissue through reverse transcription-polymerase chain reaction (RT-PCR). The mIL-18 cDNA was ligased with a PE38 gene carried by PRKL expression vector through T4 DNA ligase and constructed into fusion protein expression plasmid PRKL-IL18-PE38. The recombinant vector was identified by restriction endonucleases digestion, PCR and DNA sequencing. After transformed into E.coli BL21 and induced by IPTG, the expressed product was obtained and the molecular weight and specificity were determined by SDS-PAGE and Western-blotting. Result The new recombinant immunotoxin expression vector was constructed successfully. DNA sequencing revealed that the mIL-18 and PE38 gene were consistent with NCBI Gene Bank. The IL-18-PE38 fusion protein was expressed in E.coli BL21, and Western-blotting analysis indicated that the molecular weight of the expression product is about 56 kDa, and could react with the specific antibody against mIL-18. Conclusion IL-18-PE38 recombinant immunotoxin expression vector will provide the basis for study on the targeted cytotoxic activity to Th1 cells and may have some potential value in the treatment of Th1 cell-mediated autoimmune diseases.

Apoptosis in Raji cell line induced by influenza A virus / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To study the apoptotic effects of influenza A virus on the Raji cell line.</p><p><b>METHODS</b>Cultured Raji cells were infected with influenza A virus at a multiplicity of infection (m.o.i) of 20 and the effects of apoptosis were detected at different time points post infection using the following methods: electron microscope, DNA agarose gel electrophoresis, PI stained flow cytometry (FCM) and Annexin-V FITC/PI stained FCM.</p><p><b>RESULTS</b>Raji cells infected with influenza A virus showed changes of morphology apoptosis, DNA agarose electrophoresis also demonstrated a ladder-like pattern of DNA fragments in a time-dependent manner. PI stained FCM showed "apoptosis peak" and FITC/PI stained FCM showed apoptotic cells. Quantitative analysis indicated that the percentage of apoptotic Raji cells increased after infection, and cycloheximide (CHX), an eukaryotic transcription inhibitor, could effectively inhibit the apoptotic effects of influenza A virus in vitro.</p><p><b>CONCLUSIONS</b>Influenza A virus can induce apoptosis in Raji cell line suggesting that it may lead to a potential method for tumor therapy.</p>

Tanshinol induces proliferation,apoptosis and NF-?B activation in cultured rat hepatic stellate cells after IL-1? inducement / 第三军医大学学报

Objective To investigate the effects of tanshinol on the proliferation,apoptosis and NF-?B activation in rat hepatic stellate cells(HSCs) after IL-1? inducement,and to elucidate the anti-fibrotic molecular mechanisms of tanshinol.Methods The rat HSCs was isolated with collagenase in situ liver recirculation perfusion and cultured in vitro.The cells were divided into 5 groups: normal control,IL-1? treatment group(10 ng/ml),and tanshinol group 1,2 and 3.The later 3 groups were pretreated with tanshinol at the concentrations of 0.062 5,0.125 and 0.25 mmol/L respectively followed by 10 ng/ml IL-1? treatment 1 h later.MTT colorimetric assay was used to detect the proliferation of HSCs.AO/EB immunoflurorescence microscopy and combination Annexin-V-FITC/PI double-labelimmunofluorescence with flow cytometer were employed to examine the apoptosis of HSCs.Synthesis and secretion of collagen Ⅲ were detected by the quantitative immunocytochemical assay and ELISA respectively.The amounts of cytoplasm p-I?B? and NF-?B p65,and nuclear NF-?B p65 in HSCs were determined by Western blotting.Immunocytochemical staining and Western blotting were used to observe nuclear translocation of NF-?B p65.Results IL-1? increased the proliferation of HSCs(P

The changes of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha(6-keto-PGF1 alpha) in the lungs of rats drowned in hypothermic-sea-water / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To observe changes of thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha(6-Keto-PGF1 alpha) and TXB2/6-Keto-PGF1 alpha (T/P) in lungs of rats drowned in hypothermic sea water and to assess their influence on the blood-gas.</p><p><b>METHODS</b>Rats of different groups were drowned nearly to death in hypothermic sea water and then taken out of the water rapidly, observed at room temperature, after that the following steps were taken in 5, 15, 30, 60, 240 min and 360 min groups, that were 1 ml arterial blood taken from left heart for blood-gas analysis including pH, PaO2 and PaCO2, rectal temperature observed; at last, the ratio of left dry lungs with left wet lungs was assessed, TXB2 and 6-Keto-PGF1 alpha in right lungs were examined in all above groups and dead group(14 rats dead, only 4 examined).</p><p><b>RESULTS</b>The rectal temperature[(20.13 +/- 0.48) degree C], pH(6.68 +/- 0.03), PaO2[(45.00 +/- 6.30) mm Hg)], TXB2[(97.46 +/- 17.46) ng/L] and 6-Keto-PGF1 alpha[(25.59 +/- 8.12) ng/L] dropped to the lowest point in the 5 minutes group(P < 0.01), while PaCO2[(89.18 +/- 5.10) mm Hg] reached the highest point(P < 0.01), all above items from 5 minutes group then showed a recovering tendency, but only the pH in 240 minutes and 360 minutes groups as well as TXB2 in 360 minutes group and dead group reached near the level of normal control groups (P > 0.05); T/P had a rising tendency and reached the highest point in the 360 minutes group.</p><p><b>CONCLUSIONS</b>The production and secretion of TXB2 and 6-Keto-PGF1 alpha were influenced by hypothermia, hypoxemia and acidosis, the imbalance of T/P could be one of factors influencing the improvement of blood gas index.</p>

The Therapentic Effect of Influenza A Virus on Murine S180 Ascites Tumor / 中国肿瘤生物治疗杂志

Objective: To investigate the immunological mechanism of influenza A virus for murine S180 ascites sarcoma. Methods: After inoculation with S180 sarcoma cells, mice were i. p. injected with influenza A virus or vehicle 15 days. The average living time and survival rate of the mice were examined. The levels of IL-2, IL-6 and TNF-? were detected. The sarcoma cell's apoptosis was detected by DNA ladder, flow cytometry (FMC) , fluorescent microscope and e-lectron microscope (EM). Results: The average living time and survival rate of the mice injected with Influenza A virus were significantly longer or higher than that of the controls. The levels of IL-2, IL-6 and TNF-? also had the same differences. The apoptosis cells were detected by EM and fluorescent microscope. Sub-diploid peaks were observed by FCM a-nalysis and DNA ladder was seen after electrophoresis in the ascites cells. Conclusion: Our results demonstrated that the feasibility and potential of delivery of influenza A virus as a general means for the treatment of S180 ascites sarcoma.