This study was designed to investigate the impact of ultra-filtration extract mixture from Astragals mongholicus (UEMAM) o radiosensitivity of H22 ascitic tumor in mice to 12C6+ ions radiation. The H22 ascitic tumor model was established in mice by intraperitoneal injection of 0.2 mL H22 ascitic cells. The animals were subsequently divided into 4 groups randomly, treated with normal saline, UEMAM, heavy ion beam radiotherapy and UEMAM plus heavy ion beam radiotherapy, respectively. The body weights, abdomen circumference of the mice were measured and the mouse behavior was monitored every day; survival time was recorded to evaluate life extension effect; flow cytometry technique was used to detect H22 cell apoptosis and cell cycle; protein levels of p53, Bax, Bcl-2 and cleaved Caspase-3 were analyzed by Western blot; the single cell gel electrophoresis was used to detect the level of deoxyribonucleic acid damage (DNA damage). The results suggest that UEMAM significantly increased survival time, and decreased body weights and abdomen circumference over the saline control group. The treatment increased cell apoptosis, cycle arrest and DNA damage compared to the saline control group. UEMAM significantly enhanced the therapeutic effect of heavy ion beam radiation in survival time, and decreased body weights and abdomen circumference in the tumor-baring mice. The combination increased cell apoptosis, cycle arrest and DNA damage compared to the radiotherapy group. The results of Western blot suggest that the treatment significantly enhanced p53-induced apoptotic signals. The experiment discovered that UEMAM could improve radiosensitivity of H22 ascitic tumor through activation of p53-mediated apoptotic signal pathway.
Animals ; Apoptosis ; Astragalus Plant ; chemistry ; Cell Cycle ; Cell Line, Tumor ; DNA Damage ; Ions ; Mice ; Neoplasms, Experimental ; drug therapy ; radiotherapy ; Plant Extracts ; pharmacology ; Radiation Tolerance ; Signal Transduction

Aim To construct and express the eukary-otic expression vector of double-stranded RNA-depend-ent protein kinase (PKR)fusion green fluorescent and analyse its antiviral activity of HBV in vitro.Methods The PKR gene was cloned into an empty expression vector pEGFP-N1 using molecular clone technology. After being confirmed by restriction enzyme digestion and sequencing methods,the recombinant plasmid was named as pEGFP-PKR that was subsequently transfect-ed into HepG2.2.15 cells using LipofectamineTM2000. The expression level of PKR in HepG2.2.15 cells was confirmed by using fluorescent microscopy. Mean-while,HBV DNA and HBsAg/HBeAg were detected by real-time PCR and electrochemiluminescence meth-od,respectively.Results Both restriction enyme di-gestion and sequencing assays showed that the recombi-nant vector pEGFP-PKR was successfully constructed in our study.Fluorescent microscopy observation indi-cated that the fusion protein pEGFP-PKR expressed ef-ficiently in HepG2.2.15 cells.Moreover,compared with the empty vector group,the expression of HBV antigen in supernatants was significantly decreased (P<0.05 ).However,the extracellular HBV DNA ex-pression was not inhibited significantly.Conclusion In vitro,PKR proteion has certain antiviral activity of HBV.

Objective To study the clinical features of acute limited neck motion in children and its differential diagnosis, and to improve doctors' awareness and the early diagnosis of the disease.Methods A total of 420 patients with acute limited neck motion as the chief complaint and atlantoaxial rotary subluxation as the first diagnosis between January 2005 and January 2014 in Nanjing Children's Hospital Affiliated to Nanjing Medical University were enrolled.The clinical manifestations, imaging and consultation data of the 420 patients were retrospectively analyzed.Results There were totally 248 male patients and 172 female patients.The ages ranged from 2 to 14 years old(the mean age was 7.2 years old) ,the course ranged from 2 hours to 2 days.Among 420 patients,353 patients were diagnosed as atlantoaxial rotatory subluxation.Forty-two patients were diagnosed as cervical intervertebral disc calcification.Seven patients were diagnosed as Tourette syndrome.Four patients were diagnosed as intracranial tumors.Three patients were diagnosed as cervical eosinophilic granuloma.Two patients were diagnosed as cervical spinal tuberculosis.Two patients were diagnosed as viral encephalitis.Two patients were diagnosed as benign paroxysmal torticollis.Two patients were diagnosed as Kawasaki disease.One patient was diagnosed as neuroblastoma.One patient was diagnosed as hemophilia A.One patient was diagnosed as drug adverse reaction of metoclopramide.According to Fielding clinical classification,204 patients were type Ⅰ, 122 patients were type Ⅱ,21 patients were type Ⅲ, and 6 patients were type Ⅳ.Conclusions Based on disease distribution of acute limited neck motion in children, atlantoaxial rotatory subluxation seems to be the predominant while non-atlantoaxial rotatory subluxation is not uncommon.Differential diagnoses should be considered to avoid misdiagnosis.For atlantoaxial rotatory subluxation, conservative treatment including continuous mandible occipital belt traction and brace has been proved to be very effective methods.

Objective To investigate the methods and results of hand reconstruction by transplanting the fostered residual finger and the second toe,which share the same blood supply system.Methods From January,2014 to January,2016,3 cases with destructive hand injuries in our hospital;and debridement was performed in one session during the early stage of trauma,with skin grafting to cover the wrist stump and foster the residual finger to a branch of the dorsalis pedis artery.The first dorsal metatarsal artery around the toe web was identified and then gradually isolated proximally with dissection of the branches of the dorsalis pedis artery supplying the fostered finger until the convergence of the two blood vessels.Then,reconstructed hand by residual finger and combined second toe transplantation.Results After a follow-up period of 3 months to 2 years,the reconstructed hand had a good appearance and was able to complete basic mundane actions.The function of the limb was restored to a large extent andthe patient was satisfied with the reconstructed hand.Conclusion Transfer of a fostered residual finger and combined second toe which have the same blood supply system for hand reconstruction as a new method need to anastomose only one set of arteries,and it is feasible with the good appearance and function of the reconstructed hand.

Objective To establish an eukaryotic vector of monkey B virus glycoprotein D gene and analyze the expression of gD gene in human embryonic kidney 293T cells.Method First, the protein of monkey B virus glycoprotein D was obtained by gene synthesis.The gene fragments were digested with Pst I and Not I, and ligated to pEGPF-N3. Then, the recombinant plasmid pEGPF-N3-GD was transfected into 293T cells.The expression of gD protein in the cells was detected by Western blot, and the expression localization was investigated using laser scanning confocal microscopy. Results The recombinant plasmid pEGPF-N3 carrying gD gene was successfully constructed, and normally expressed in the 293T cells.Conclusions Glycoprotein D of monkey B virus is expressed successfully in the 293T cells and the protein is located on the cell surface.It may be useful for the preparation of specific recombinant antigen to the glycoprotein D of monkey B virus on cell surface, and can be also used for preparation of antigen slide for detection of monkey B virus.

Objective:To investigate the difference of accuracy between magnetic induction Freehand-3D ultrasound and two-dimensional ultrasound in measuring the volume of thyroid model.Methods:Forty thyroid models were established using porcine liver, and the Archimedes procedure was set as gold standard in the measurement of the volume of each model. The accuracy of measurement of the porcine thyroid model volume between two-dimensional ultrasound and magnetic induction Freehand-3D ultrasound were compared.Results:There were no significant differences in the accuracy of measurements of thyroid model volume among two-dimensional ultrasound, magnetic induction Freehand-3D ultrasound and Archimedes procedure (all P>0.05). Compared with the Archimedes procedure, magnetic induction Freehand-3D ultrasonic method showed higher correlation coefficient of the measurement of thyroid model volume ( r=0.998). Bland-Altman analysis showed the lower measure error with a relative error of 3.42% and range of -9.57% to 12.07%. And the limits of agreement were (-1.253, 0.999) in the magnetic induction Freehand-3D ultrasonic measurement. Conclusions:Compared with two-dimensional ultrasound, the magnetic induction Freehand-3D ultrasound show higher accuracy in the measurement of the volume of the thyroid model.

Objective To observe if fluoxetine has a potency to inhibit the progression of Lewis lung cancer by combining the fluoxetine and cisplatin to treat the mice bearing Lewis lung cancer with depression .Methods We devel-oped a mouse model of Lewis lung cancer with depression which was intervened with cisplatin and fluoxetine , and the indi-cators related to cancer and depression were tested .Social interaction test was used to measure the behavioral changes of the depressed model mice .The serum cortisol and IL-6, BDNF mRNA in the hippocampus , and NF-κB and VEGF in the tumor tissue were selected for investigation and comparison .Results The mice which were induced by social defeat exhib-ited social avoidance behavior in the social interaction test .The cortisol and IL-6 level in both combination groups was de-creased compared with that in the model group (P<0.05), and the cortisol and BDNF mRNA in the combination group in-creased signifuicantly ( P <0.05 ) .There were no statistically significant differences in the tumor weight , NF-κB and VEGF in tumor tissues between the single and combination groups (P>0.05).Conclusions Fluoxetine has antidepres-sant effect by decreasing the high level of serum cortisol and promoting the BDNF mRNA expression in the hippocampus . However , fluoxetine is not found to have the potency to inhibit the expression of NF -κB related with the progression of tumor.

AIM:To study the effects of noninvasive delayed limb ischemia preconditioning ( NDLIP) on ani-mal cardiac function , myocardial morphology and myocardial apoptosis after myocardial infarction ( MI ) .METHODS:Healthy SD male rats [n=45, weighing (250 ±10) g] were randomly divided into 3 groups:MI group:the animal model of MI was established by surgical ligation of left anterior descending artery ( LAD) after 2 weeks;NDLIP group:after the success of the MI animal model , NDLIP was carried out every other day until the 4th, 6th and 8th weeks;sham group:as the negative control group , the animals were taken heart LAD threading but no ligation .All rats were fed conventionally .At the end of the 4th, 6th and 8th weeks, all rats were made ventricular intubation , and then the hemodynamic parameters were recorded .The blood samples were withdrawn from the abdominal aorta and the serum was separated via centrifugation . The serum contents of Bcl-2 and Bax were measured by ELISA .Left ventricular anterior wall was homogenized .The mito-chondrial respiratory chain complexes Ⅰ,Ⅱ,Ⅲand Ⅳin the myocardial tissues were detected by ELISA .RESULTS:At the end of the 4th, 6th and 8th weeks, compared with MI group, left ventricular systolic pressure in NDLIP group was significantly increased , while left ventricular end-diastolic pressure in NDLIP group was significantly decreased ( both P<0.05).Mitochondrial respiratory chain complexesⅠ, Ⅱ, Ⅲ and Ⅳ in NDLIP group were significantly increased (P<0.05).The serum level of Bcl-2 in NDLIP group was significantly increased and Bax level was reduced remarkably (both P<0.01) .CONCLUSION:NDLIP improves the hemodynamic indexes , promotes the mitochondrial respiratory function and inhibits cell apoptosis , thus improving the prognosis of MI .

Aim To study the preventative effects of noninvasive delayed limb ischemic preconditioning ( NDLIP) on sudden cardiac death in rats with myocar-dial infarction. Methods Thirty healthy SD male rats weighting ( 250 ± 10 ) g were randomly divided into 3 groups:① myocardial infarction ( MI ) group: animal model of MI was established by making surgical ligation of animal LAD. ② MI plus NDLIP group: after the success of the animal model of MI, NDLIP was carried out every other day until 4th week. ③Sham group:as the negative control group, animals were taken heart LAD threading but no ligation. All rats were fed con-ventionally. At the end of 4 weeks, three groups of rats were administered with metaraminol ( 0. 2 mg · min-1 ) . ECG, drug cumulant of sudden death and death onset time were recorded. After sudden death, blood samples were withdrawn from abdominal aorta and serum was separated via centrifugation. ELISA method was used to measure serum caspase-3 , HSP70 and SOD concentration. Results While metaraminol led animal cardiac sudden death, the rats heart rate ( HR) kept declining with the increase of dosage of metaraminol during the administration period. Rat HR of MI+NDLIP group [ ( 479 ± 8 ) vs ( 416 ± 19 ) beat ·min-1 , ( 446 ± 32 ) vs ( 370 ± 20 ) beat · min-1 , (376 ± 53) vs (305 ± 29) beat·min-1, (307 ± 63) vs (244 ± 33) beat·min-1, (283 ± 45) vs (121 ± 35 ) beat · min-1 , P <0. 01 ] was markedly higher than that of MI group at 0 , 5 , 10 , 30 , 50 min before death. Compared with MI group, drugs cumulant to sudden death and death onset time of MI + NDLIP group [ ( 14. 58 ± 3. 03 ) vs ( 10. 76 ± 2. 73 ) mg, (72. 9 ± 15. 2 ) vs ( 53. 8 ± 13. 6 ) min, P <0. 01 ] were significantly increased. Compared with MI group, serum caspase-3 content of MI+NDLIP group was sig-nificantly reduced [ ( 2. 01 ± 0. 52 ) vs ( 2. 34 ± 0. 38 )μg·L-1 , P<0. 01 ]; HSP70 levels were remarkably increased [ ( 3. 01 ± 0. 58 ) vs ( 2. 70 ± 0. 43 ) μg · L-1 , P <0. 05 ]; SOD levels were significantly im-proved [(1. 99 ± 0. 65) vs (1. 70 ± 0. 58) mg·L-1, P<0. 01 ] . Conclusion NDLIP can prevent sudden cardiac death after myocardial infarction in rats, which may be mediated by reducing the myocardial cell apop-tosis, increasing protective protein expression and en-hancing antioxidant capacity.

OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection