In order to construct generally efficient cell immortalization vector, pTP-hTERT, we modified the traditional piggyBac (PB) transposon using artificial synthesis, PCR and enzyme digestion. The modified vector contained the necessary transposon elements, a PB transposase expression cassette, a co-expression selectable element and a human telomerase reverse transcriptase (hTERT) expression cassette. The co-expression selectable element had two markers, enhanced green fluorescent protein (EGFP) gene and puromycin-resistance (Puro) gene, linked by porcine teschovirus-1 2A peptide (P2A). To validate the functionality of vector elements, we transfected pTP-hTERT into HEK293 cell, selected the positive cell clones and then conducted RT-PCR, Western blotting (WB) and Tail-PCR, methylene blue staining and statistic analysis on selected cells. The results of sequencing and cell culture show that the pTP-hTERT was constructed successfully and the positive cell could be selected by puromycin. The WB results, P2A cutting EGFP and Puro fusion protein with high efficiency, reflected the selectable element worked. The sequencing result of Tail-PCR confirmed the vector integrated into the genome through transposition. The results of methylene blue staining and statistic analysis indicated the clone of positive cells triggered by pTP-hTERT significantly increased (P < 0.01) compared with control group. The construction of pTP-hTERT provides an efficient tool for establishing immortalized cell lines and a demonstration for building other eukaryotic plasmids.
Cell Line ; DNA Transposable Elements ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; HEK293 Cells ; Humans ; Plasmids ; Polymerase Chain Reaction ; Telomerase ; genetics ; Transfection

Objective To analyze the clinical features of fungal septicemia in premature infants in a hospital,and provide reference for clinical diagnosis and treatment.Methods Clinical data of 18 premature infants with fungal septicemia in a hospital between January 2011 and December 2013 were analyzed retrospectively.Results Gesta-tional ages of 18 premature infants were 27-36 weeks,birth weights were 1 050 g-3 100 g,8 of whom were very low birth weight infants(VLBWI);all premature infants were treated with broad-spectrum antimicrobial agents,and were given long-term parenteral nutrition before infection,10 with mechanical ventilation,2 with peripherally insert-ed central catheters(PICC).The main clinical manifestations were apnoea,twitch,feeding difficulty,and poor reac-tion;manifestations appeared 3 hours-52 days after birth.13(72.22%)premature infants had abnormal white blood cell count,12 (66.67%)had thrombocytopenia (< 100 × 109/L),18 (100.00%)had elevated C-reactive protein (CRP),the average CRP level was (41 .90 ±26.77)mg/L.The main pathogens were Candida spp .(n = 17, 94.44%),including Candida parapsilosis (n=7),Candida albicans (n =5 ),biovariety of Candida albicans (n =4),and Candida famata (n=1);premature infants were treated with fluconazol and amphotericin B,15 (83.33%) were cured,2 (11 .11 %)improved,and 1 (5.56%)died.Conclusion Fungal septicemia in premature infants are mainly caused by Candida,there is no specific clinical manifestations,clinical symptoms in premature infants with high risk factors should be closely observed,blood routine and CRP should be detected periodically,timely treat-ment with antifungal agents should be given,which are beneficial to achieve good therapeutic effect.

OBJECTIVE:To study improvement effect of modified mahuang chanyi decoction on allergic rhinitis(AR)model guinea pigs and its mechanism. METHODS:32 guinea pigs were randomly divided into normal group (0.9% Sodium chloride in-jection),model group (0.9% Sodium chloride injection),experimental [Modified mahuang chanyi decoction,5.18 g (medicinal materials)/kg] group and control [Fluticasone propionate nasal spray,50 μg/(guinea pig·d)] group. Except for normal group,other groups received ovalbumin 30 mg and calmogastrin 30 mg intraperitoneally to induce AR model. Except for control group,other groups were given relevant medicine intragastrically,twice a day for consecutive 15 d. Nasal symptoms of guinea pigs were ob-served and recorded,and then analyzed and scored. The protein expression of nasal mucosa SHP-1 was detected by immunohisto-chemistry,the serum contents of IL-4 and IFN-γ were determined by enzyme-linked immunosorbent assay. RESULTS:Compared with normal group,model group suffered from AR symptom,and nasal symptoms score was increased;and the protein expression of nasal mucosa SHP-1 and the serum content of IL-4 increased,while the content of IFN-γ decreased(P<0.05). Compared with model group,AR symptom of experimental group and control group were improved;nasal symptoms score,the protein expression of nasal mucosa SHP-1 and the serum content of IL-4 decreased while the content of IFN-γ increased(P<0.05). CONCLUSIONS:Modified mahuang chanyi decoction shows certain improvement effect on AR,and its mechanism is associated the decrease of IL-4 content and the increase of IFN-γcontent in serum.

Objective To explore the clinical diagnosis and treatment of cyclic vomiting syndrome in children.Methods Thirty children proved with cyclic vomiting syndrome admitted from January,1998 to January,2003 were analyzed retrospectively.Results Cyclic vomiting syndrome was most likely to occur in 3-12 years old.The male to female ratio was 3∶2.The clinical manifestations were recurrent vomiting.Twenty-one cases had inducements,while 9 cases had not inducements.It was safe and efficient that curing cyclic vomiting syndrome with cyprohetadine and amitriptyline.Conclusions If these children with cyclic vomiting syndrome are inefficient to treatment,excluding metabolizable diseases,gastrointestinal,neurological diseases,they may be diagnosed cyclic vomiting syndrome,and cured with cyprohetadine and amitriptyline.

Colchicine ; Forensic Medicine

Objective To investigate the diagnosis and the microsurgical treatment of intramedullary hemangioblastoma in cervical spinal cord.Methods The signs of MRI,and the results of operations were analysed in 26 patients with the tumors.Rusults The tumors can be classified into two types:Solid type (14 cases)and cystic type(12 eases).All the tumors underwent total removal and were all hemangioblastoma confirmed by histopathologic examinations.Postoperatively,neurological status were improved in 17 patients, remained in 7 cases and worse in 2 cases.Conclusion For intrameduUary hemangioblastoma of cervical spinal cord MRI is of significant importance in the diagnosis of localization and the nature of the tumors which is conductive to selecting appropriate operative methods.There is high risk in operating at cervical section,but microsurgical total resection is the optimal method to stop the development of the clinical presentation.Opera- tive methods varied with the different typer of the tumor.It is the most important principal that dissection is performed along the correct interface and the tumor should be removed en bloc after it is devascularized.

Background With the changes of diet and living style,the diabetes has become the major diseases affecting human health.Diabetic cataract is a common complication of diabetes. Objective The present study was to investigate the difference of lens proteomics between diabetic cataract and age related cataract using two dimensional electrophoresis (2-DE) and mass spectrometry in order to postpone happening of diabetic cataract and offer the effective approach to the prevention and therapy of diabetic cataract. Methods The lenses were obtained from 8 diabetic patients and 12 age-related cataract patients during the surgery to extract the protein by lysis and centrifugation.The lens proteins were separated using immobilized pH gradients 2-DE.Image analysis was carried out using PDQuest Advanced-8.0.1 software package.Significant difference of the crystallines was identified by matrixassisted laser adsorption/ionization time of-flight-mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprint combined with protein database. Results The maps of 2-DE showed that lens proteins of diabetic cataract and age related cataract were in the section of pH 5-9 with the relative molecular weight 14000-97000;while relative molecular weight of more abundant crystalline was localized at 20000-31000.About 3 differential protein spots were detected by image analysis software.Two crystallines,αB and βB1 crystallin,were identified using MALDI-TOF-MS.Conclusions Proteomic analysis of lens can be accomplished and the proteins can be well separated,moreover,differential proteins can be analyzed using 2-DE and mass spectrometry between diabetic cataract and age related cataract.These results indicate that αB and βB1 crystallin proteins accelerate the development of diabetic cataract.This technique offers a new avenue for clarity of lens proteins of diabetic cataract other than age related cataract.

0.9). Conclusions CT perfusion imaging could quantify the hepatic blood flow.The accuracy is similar to electromagnetic flowmeter,and it could effectively reflect physiological or pathological hemodynamies of liver.CT perfusion is noninvasive,high reproducible and convenient,which could be widely used in clinical practice.

Objective To compare the efficacy and safety of intravenous thrombolysis on cardiogenic cerebral infarction and noncardiac infarction by recombinant tissue plasminogen activator (rt-PA). Methods Comparations of NIHSS, mRS and adverse events before and after treatment were made between the cardiogenic group and the noncardiac group. Results No significant differences in the NIHSS and mRS were found between the two groups. The incidence of brain hernia and dermatorrhagia in the cardiogenic group was higher than that in the noncardiac group. Conclusion Rt-PA therapy in cardiogenic cerebral infarction was effective and safe in spite of higher incidence of hemorrhage and brain hernia.

Objective:To explore therapeutic effect and safety of endovascular stenting on transient ischemic attack (TIA) caused by atherosclerotic vascular stenosis .Methods:A total of 100 patients with TIA caused by vascular ste-nosis in our hospital from Jan 2011 to Feb 2013 were enrolled ,and equally divided into combined treatment group (received endovascular stenting combined medication ) and routine treatment group (received medication treat-ment) .After 12-month treatment ,recurrence rate of TIA ,incidence rate of stroke and vascular stenosis rate before and after treatment were compared between two groups .Results:Compared with before treatment ,there was no significant change in all above-stated indexes after treatment in routine treatment group;were significant reduction in vascular restenosis rate [ (73.31 ± 12.76)% vs .(25.01 ± 5.73)% ] in combined treatment group ,and it signifi-cantly reduced than that of routine treatment group (74.33 ± 12.96)% ,P<0.01 both ;during the 12-month follow-up ,compared with routine treatment group , there were significant reductions in percentages of recurrent TIA (16.0% vs .2.0% ) and cerebral stroke (12.0% vs .0) in combined treatment group ,P<0.05 both Conclusion:En-dovascular stenting can significantly improve clinical therapeutic effect and prognosis in patients with atherosclerotic vascular stenosis ,and is worth clinical extension in some condition .