Recombinant PCR applies to fulfill gene recombination by PCR thermal reaction.Over the twenty years,it has branched into three characteristic strategies:splicing by overlapping extension(SOE),jumping polymerase chain reaction(JPCR)and DNA shuffling.Recently,the technique aimed with exploiting natural source of different allele genes is developing up on simplification of experimental procedure,on trap for mutation and variation,and on highthroughput screening with technology of surface display and fluorescent probe.The recombinant PCR is increasesing value in broad range from biological basic research to bioengineering study.

Common warts are close associated with HPVs infection. In this study, we amplified and sequenced the LCR fragment and E2 gene of HPV-2 that infected the patient of extensive common wart with cutaneous horns, and we constructed the recombinant CAT-reporter plasmids pBLCAT-LCR containing HPV-2 prototype or variant LCR and mammalian expression plasmids pcDNA3. 1-E2 containing prototype or variant E2 ORF individually. The promoter activities of HPV-2 variant and the transcriptional repression activities of the mutated E2 protein were evaluated by transient transfection into HeLa cells. The results showed that there were several mutations in LCR and E2 gene of HPV-2 variant. Compared with the prototype, the viral early promoter activity of variant was significantly increased uder the control of LCR. Compared with the wild type E2 protein, the transcriptional repression activities of the mutated E2 protein was abolished partially. We speculate herein that increased promoter activities and decreased repression effect of the mutated E2 protein are linked, at least partially, with the clinical phenotypes of the uncommon huge common wart.
DNA-Binding Proteins ; genetics ; physiology ; Humans ; Mutation ; Oncogene Proteins, Viral ; genetics ; physiology ; Papillomaviridae ; genetics ; Promoter Regions, Genetic ; Repressor Proteins ; physiology ; Warts ; virology

OBJECTIVE: To investigate the effect of oxidative stress on the accumulation of heat shock protein 70 (HSP70) within C2C12 myogenic cells. METHODS: Heat shock response (42 degrees C for 1 h and recovery for 12 h at 37 degrees C) was used to induce the expression of heat shock protein 70. We constructed a recombinant plasmid of HSP70 with enhanced green fluorescent protein (EGFP). After being transfected transiently into C2C12 cells, immunoblotting was used to detect the expression of HSP70 induced by heat shock response and transfection. Immunocytochemistry, fluorescent microscopy and immunoblotting were used to detect the translocation of HSP70. RESULTS: Immunoblotting showed that the overexpression of HSP70 was induced by heat shock response and transient transfenction. HSP70 localized within the cytoplasm of the normal cells, but HSP70 translocated from the cytoplasm to the nucleus and the nucleolus at 1 h after the treatment of oxidative stress (0.5 mmol/L H2O2) by using immunocytochemistry, fluorescent microscopy and immunoblotting for cellular partial proteins. CONCLUSION Oxidative stress may induce the accumulation of heat shock protein 70 within the nucleolus.
Cell Nucleolus ; metabolism ; Cells, Cultured ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Myoblasts ; cytology ; metabolism ; Myocytes, Cardiac ; cytology ; metabolism ; Oxidative Stress ; physiology

Aviation ; Humans ; Physical Fitness ; Resistance Training ; Schools ; Students

AIMTo investigate the preventive effects of the cerebro cellular growth peptide (CCGP) on gentamycin-induced inner ear damage in guinea pigs, and to clarify its mechanism.

METHODSThe hypoacusis severity and enzymatic activity in the cochlear hair cells were examined by brainstem auditory evoked potential (BAEP) and histochemistry, respectively. The damaged hair cells was counted in three groups.

RESULTSCCGP reduced the elevated BAEP reaction thresholds. It protected activities of mitochondrial succinate dehydrogenase and lysosome acid phosphatase in the cochlear hair cells. The number of damaged hair cells in the CCGP group was less than that in the gentamycin (GM) group.

CONCLUSIONCCGP can reduce GM ototoxicity. The mechanism may be associated with the protective activity of mitochondrial enzyme, the maintenance of lysosome intactness, energy metabolism of the cochlear hair cells, and reduction of autolysis of hair cells induced by hydrolase over flowing from lysosome.

Animals ; Evoked Potentials, Auditory, Brain Stem ; Female ; Gentamicins ; toxicity ; Guinea Pigs ; Hair Cells, Auditory ; drug effects ; physiology ; Male ; Nerve Growth Factor ; pharmacology

OBJECTIVE: To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes. METHODS: Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model. RESULTS: Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS. CONCLUSION Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.
Cell Line ; Cell Membrane ; metabolism ; Humans ; Interleukin-1beta ; biosynthesis ; metabolism ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism ; Phosphoproteins ; metabolism ; physiology ; RNA-Binding Proteins ; metabolism ; physiology ; Tumor Necrosis Factor-alpha ; biosynthesis ; metabolism

OBJECTIVE: To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells. METHODS: After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays. RESULTS: After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis. CONCLUSION The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.
Animals ; Apoptosis ; physiology ; Cell Proliferation ; Cells, Cultured ; Down-Regulation ; Mice ; Myoblasts ; cytology ; Myocytes, Cardiac ; cytology ; Oligonucleotides, Antisense ; Phosphoproteins ; biosynthesis ; genetics ; RNA-Binding Proteins ; biosynthesis ; genetics ; Transfection

Objective:To compare the clinical effect of anchor fixation of medial collateral ligament（MCL）and non-repaired treatment combined with arthroscopic anterior and posterior cruciate ligament reconstruction in the treatment of type KD-IIIM（Schenck classification）dislocation of knee joint.Methods:A retrospective case-control study was conducted to analyze the clinical data of 41 patients with type KD-IIIM dislocation of the knee joint admitted to Xi'an Honghui Hospital of Xi'an Jiaotong University from September 2015 to September 2017. There were 26 males and 15 females，with the age range of 15-62 years［（37.7±13.9）years］. A total of 21 patients were treated by arthroscopic anterior and posterior cruciate ligament reconstruction and anchor fixation of MCL（repaired group）and 20 patients by arthroscopic anterior and posterior cruciate ligament reconstruction without MCL repair（non-repaired group）. The perioperative complications were observed. Comparisons between the two groups were made before operation，4 months after operation and at the last follow-up in terms of Lysholm score，International Knee Documentation Committee（IKDC）score，Visual Analogue Scale（VAS）and range of motion of joint flextion and extension. The medial opening distance of stress X-ray of lower limbs was also observed at the last follow-up.Results:All patients were followed up for 48-66 months［（54.4±5.1）months］. No perioperative complications were noted. The Lysholm score，IKDC score，VAS and range of motion of joint flextion and extension showed no statistical differences between the two groups before operation（ P>0.05），and all were improved from the preoperative level at 4 months after operation and at the last follow-up（ P<0.05）. The Lysholm score in repaired group were（69.7±5.1）points and（83.8±4.9）points at 4 months after operation and at the last follow-up，significantly higher than those in non-repaired group［（61.0±5.5）points and（74.6±6.0）points］（ P<0.05）. The IKDC score in repaired group was（71.8±4.0）points at 4 months after operation，significantly higher than that in non-repaired group［（71.1±3.9）points］（ P<0.05）. The IKDC score in repaired group was（82.3±5.1）points at the last follow-up，similar with that in non-repaired group［（83.2±4.0）points］（ P>0.05）. The VAS in repaired group was（2.5±0.6）points at 4 months after operation，significantly decreased in comparison with（3.2±1.2）points in non-repaired group（ P<0.05）. The VAS in repaired group was（2.0±1.4）points when compared to（2.2±1.3）points in non-repaired group at the last follow-up（ P>0.05）. The range of motion of joint flextion and extension in repaired group was（107.6±6.9）° and（125.9±7.8）° at 4 months after operation and at the last follow-up，significantly increased in comparison with（89.6±4.0）° and（120.9±5.2）° in non-repaired group（ P<0.05）. The medial opening distance in repaired group was（2.3±0.2）mm at the last follow-up，significantly decreased when compared to（3.1±0.2）mm in non-repaired group（ P<0.05）. Conclusion:For type KD-IIIM knee dislocation，arthroscopic anchor fixation of MCL or without MCL repair combined with anterior and posterior cruciate ligament reconstruction are both effective in satisfactory functional recovery，but MCL repair contributes to earlier function recovery，faster pain relief and more reliable joint stability.

OBJECTIVETo construct anti-CD20scFv/CD80/CD28/zeta recombinant gene modified T cells, test its effectiveness of eradicating CD20+ lymphoma cells and provide a probably new approach to tumor adoptive immunotherapy.

METHODSCD28-zeta cDNA were amplified from vector pBULLET and inserted into pLNCX vector that contained anti-CD20scFv/CD80 gene. The recombinant vectors were transduced into PA317 cells and high titer retroviruses were obtained to infect human peripheral blood T lymphocytes. Resistant T cells were obtained by G418 selection at one week. Then transduced T lymphocytes and lymphoma cell lines Daudi Raji were cocultured. The cytotoxicity and cytokine production of transduced T cells were determined by non-radio-activation cytotoxicity assay and ELISA respectively.

RESULTSThe recombinant eukaryotic vector was constructed successfully as proved by enzyme digestion analysis and sequencing. These T cells were able to lyse CD20+ target cells and secrete high levels of IL-2 and IFN-gamma in vitro.

CONCLUSIONRecombinant gene modified T cells can be constructed successfully. It can specially kill CD20 positive lymphoma cells in vitro.

Antigens, CD20 ; genetics ; immunology ; B7-1 Antigen ; genetics ; immunology ; CD28 Antigens ; genetics ; immunology ; Cell Line ; Cytotoxicity, Immunologic ; Genetic Vectors ; Humans ; Immunotherapy, Adoptive ; Plasmids ; genetics ; T-Lymphocytes ; immunology ; metabolism ; Transfection

BACKGROUNDThe present study was aimed to investigate the usage of aspirin for the secondary prevention of ischemic stroke, evaluate the correlated factors, and analyze the reasons for not taking and irregularly taking aspirin.

METHODSThe patients in this group were all stroke survivors who have formerly been diagnosed with a cerebral infarction or transient ischemic attack (TIA) in our hospital. We investigated their use of aspirin over a three-year period following their hospitalization. According to the patients' aspirin usage, they were divided into treatment and non-treatment groups. In addition, the reasons for not taking or irregularly taking aspirin were analyzed in the two groups.

RESULTSA total of 1240 patients were studied, including 367 (29.60%) in the treatment group and 873 (70.40%) cases in the non-treatment group. In addition, 201 (16.20%) cases in the treatment group had been regularly taking aspirin (50 - 325 mg of aspirin daily) for 1 to 3 years or longer. The results demonstrated that the main reasons for not taking aspirin in this study were related to patients' concerns regarding the side effects of taking aspirin (46.45%), as well as the doctors' inadequacy in informing their patients to take aspirin (38.71%). The major reasons for patients to irregularly take aspirin were that the doctors did not notify the length of aspirin usage to their patients (41.57%), and that doctors did not prescribe aspirin upon the patients' follow-up visit (26.51%).

CONCLUSIONThe most effective way to increase patient's compliance for aspirin consumption is to promote the guidelines for stroke treatment and to relay these advances in stroke therapy to the patient.

Aged ; Aspirin ; therapeutic use ; Female ; Humans ; Male ; Middle Aged ; Patient Compliance ; Retrospective Studies ; Secondary Prevention ; methods ; Stroke ; prevention & control ; psychology