Objective:To develop a simple and efficient method for constructing a middle fragment-deleted mutant of MHC class Ⅱ molecule transactivator(CⅡTA)mutant with the 109~(th)to 226~(th) amino acid codons deleted.Methods: Two gene fragments at each end of the deleted CⅡTA gene were obtained by OE-PCR method and were mixed together for 8 PCR cycles without primers to achieve effective overlapping,then 2 primers was added for amplification of the desired fragments.The amplification products were subsequently cloned into eukaryotic vector pIRES for identification.Results: A mutant of CⅡTA with the 109~(th)to 226~(th) amino acids deleted was successfully constructed.Conclusion: This modified OE-PCR technique overcomes some shortcomings of traditional method and is very suitable for constructing mutants with middle fragment deletion,making it worth to be popularized.

BACKGROUND: Oligodendrocyte precursor cells are seed cells for the treatment of white matter injury. The establishment of an efficient and stable in vitro culture method is an important prerequisite for clinical transformation. OBJECTIVE: To investigate the morphological changes of oligodendrocyte precursor cells during passage. METHODS: Four batches of human oligodendrocyte progenitor cells were subcultured from the second generation (P2) to the seventh generation (P7) in vitro. Five pictures of 200-fold field were taken under an optical microscope before each passage. According to the cell morphology, oligodendrocyte precursor cells were divided into three types: bipolar cells (oligodendrocyte precursor cells), multipolar cells (late oligodendrocyte precursor cells) and supra-polar bifurcated cells (immature oligodendrocyte cells). The proportion of oligodendrocyte progenitor cells to the total number of cells was calculated, so as to compare the difference of cell morphology among different generations. RESULTS AND CONCLUSION: In the process of passage from P2 to P7, oligodendrocyte progenitor cells included three types: bipolar cells, multipolar cells and supra-polar bifurcated cells. Among them, bipolar cells and multipolar cells were the main part, and a small number of supra-polar bifurcated cells could be seen in the rest. There were no significant differences in the proportion of bipolar cells, multipolar cells and supra-polar bifurcated cells among P2-P7 (P > 0.05). The cell morphology classification and counting method can be used to preliminarily evaluate that oligodendrocyte progenitor cells have no change in morphology during culture.

Aim To study the inhibition of genistein on proliferation and transcription of c-fos mRNA in human umbilical vascular smooth muscle cells(hUVSMC) induced by monocyte chemotactic protein-1(MCP-1). Methods Growth-arrested hUVSMC were stimulated with MCP-1(10 ?g?L-1) prior to co-treatment with different concentrations of genistein (10,30,90 ?mol?L-1). The response of hUVMSC to these treatments was observed in comparison with that of control group. The proliferation of hUVMSC was evaluated by cell counting. The expression of c-fos mRNA was detected by RT-PCR. Results Low concentration of genistein(10 ?mol?L-1) inhibited the proliferation of hUVSMC and high concentration of genistein(30,90 ?mol?L-1) inhibited the expression of c-fos in hUVSMC induced by MCP-1. Conclusions Genistein could suppress the proliferation of hUVSMC induced by of MCP-1. Its mechanisms may involve the down-regulation of c-fos mRNA expression.

Objective:To construct adenovirus containing murine MHC class II transactivator (CIITA) mutant gene and to observe expression of this gene mediated by adenovirus as well as function of the expression product in vitro.Methods:The type IV CIITAcDNA gene was cloned from peritoneal macrophage of BALB/c mouse by conventional way of molecular biology, and then it was cloned into expression vector pIRES. CIITA mutant gene was constructed by the way of overlap extension by PCR (OE-PCR), and then it was also cloned into expression vector pIRES. By using of pAdEasy-1 system we acquired the deficient recombination adenovirus Ad-CIITAm, containing CIITA mutant gene, which had the ability of infection and the no-loaded control adenovirus Ad-GFP.Then the two adenoviruses strains were processed for a great deal amplification,purification and titer determining. The Ad-CIITAm and Ad-GFP were infected into HeLa cells and Raji cells.Inducible or constitutive expression of HLA-DR molecule and the following changes were observed by the use of flow cytometry.Results:The murine CIITA gene was cloned successfully and the recombinantant adenovirus Ad-CIITAm containing murine CIITA mutant gene was also constructed successfully. It was proved by flow cytometry that expression of HLA-DR molecules on the surface of HeLa cells and Raji cells infected by Ad-CIITAm were all decreased remarkably in comparison with those of control cells infected by Ad-GFP.Conclusion:This experiment proves that expressed murine CIITA mutant mediated by adenovirus could inhibit the expression of MHC II molecules efficiently.

Objective To map out the binding site of P195,which is the major protein on the surface of P.falciparum merozoites,to human erythrocytes,and offer a basis for designing malaria vaccine to blockade invasion of merozoites into human erythrocytes.Methods Eight proteins derived from P195 were expressed in E.coli,and purified by Ni-chelare affinity chromatography.There after,the eight fragments and rabbit serums immunized by which were added into culture medium of P.fatciparum in vitro respectively.Twenty-four hours later,the invasion of merozoite to erythrocyte was observed.Results The antibodies which were induced by three fragments of P195,M6(Amino Acid,AA384～595),M7(AA 595～897)and M11(AA 1397～1563)could inhibit the invasion of P.falciparum merozoite into human erythrocytes.Especially,one fragment of P195,M6,had the ability to inhibit the invasion of P.falciparum merozoite into human erythrocytes.Conclusion M6,a fragment of P195 on the merozoite of P.falciparum may contain a domain thought to be involved in the recognition of human erythrocyte.The domain can be used as a candidate antigen for a malaria vaccine.

Semont maneuver was performed in 97 patients with benign paroxysmal positional vertigo involving the posterior semicircular canal.Among 97 patients the Semont maneuver was successful in 69 cases and failed in 28 cases.There were three nystagmus patterns during the third position of the Semont maneuver:orthotropic nystagmus (n=45), no nystagmus (n=42) and reversed nystagmus (n=10);and the effective rates in three groups were 93%, 64% and 0%, respectively (P<0.05).The duration of latency period and nystagmus status in Dix-Hallpike test has no effect on repositioning efficacy ( P>0.05) .

Objective To analyze the pathological diagnosis of patients with clinically diagnosed eczema,to describe the spectrum of skin diseases tending to be misdiagnosed as eczema,and to investigate factors associated with their misdiagnosis.Methods A retrospective study was performed on 400 patients who were clinically diagnosed as eczema and received pathological examination at the dermatology clinic of Peking University Third Hospital from August 2006 to April 2013.Skin biopsy specimens were re-reviewed for these patients,and pathological diagnosis was made in combination with clinical presentations.Results Of the 400 outpatients with clinically diagnosed eczema,110 (27.5％) were finally diagnosed as non-eczema skin diseases pathologically,including 16 cases of psoriasis,13 bullous pemphigoid (BP),11 lichen planus (LP),9 cutaneous amyloidosis,8 mycosis fungoides (MF),14 skin malignancies,and 39 other skin diseases.The highest misdiagnosis rate was observed in people aged 60 to 79 years (33.9％) and lesions at the genital sites (46.2％).Conclusions Many conditions tend to be misdiagnosed as eczema in clinic,including psoriasis,BP,LP,cutaneous amyloidosis,MF and skin malignancies.Misdiagnosis is rather frequent in elderly people and eczematous lesions in genital areas,and pathological examination should be taken actively for uncertain cases.

Objective To construct luciferase reporter plasmid containing synthetic CArG elements and to investigate their radiation-inducible property in tumor cells. Methods Insert chimeric regulation elements consisting of nine tandem-repeat copies of CArG sequence (CCATATAAGG) and CMV IE basal gene promoters into pGL3-Basic vectors to construct luciferase reporter plasmids. Tumor cells(HeLa, A549 and HepG2) were transiently transfected by reporter plasmids using lipofectamine, and transfected cells were irradiated by ?-ray with different doses. After 36 h, we assayed the level of reporter gene expression. Results The CArG elements could successfully induce the expression of a downstream reporter gene following irradiation, with maximal expression seen after 3 Gy irradiation. Conclusion The synthetic CArG elements are responsive to low dose of radiation and are able to enhance down-stream gene expression. It is expected to be the essential potential for future application of radiogenetic cancer therapy.

Objective To analysis the clinical features of valproate (VPA)-induced encephalopathy in elderly people in order to improve our cognition toward it.Methods From March 2003 to March 2011,a total of 10 cases with VPA-induced encephalopathy were retrospectively reviewed and summarized.The data collected included clinical manifestations,biochemistry,EEG and therapeutic effects.Results In the 10 cases,8 were males and 2 females.The age ranged from 65-88 years old (mean age 75.4±10.3 years).7 subjects were on treatment with VPA alone,and the other 3 in combination with other anti-epileptic drugs.The serum VPA level in this study ranged from 62.1-122.7 μg/ml with mean of (92.3 ± 30.1) mg/L (normal range 50-100 mg/L).All subjects presented with confusion and cognitive impairment.The serum ammonia level in this study ranged from 56.7-225.1 μmol/L with mean of (101.4±55.2) μmol/L (normal range 11-32 μmol/L).All cases were with normal liver function.Electroencephalography was characterized by signs of severe encephalopathy with continuous generalized slowing,a predominance of θ and δ activity,occasional bursts of epileptiform discharges and triphasic waves.All cases were improved 3-21 days after VPA withdrawal.Conclusions VPA-induced encephalopathy that manifested in confusion and cognitive impairment is not uncommon in elderly patients and it has a good prognosis and the early withdrawal of VPA lads to improvement in almost all cases.

Objective:To explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.Method:The reformed reconstructed enhanced vector, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer, and hTERT mono-promoter vector, pGL3-basic-EGFP-TK-hTRETp(as controls), were transfected into telomerase(+) nasopharyngeal carcinoma 5-8F cell lines,telomerase(+) human breast cancer MCF-7 cell lines and telomerase(-) normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in maligment tumour cells pre- and post-transfected by enhanced vector . Meanwhile the relationship beteewn TK and telomerase was analyzed.Result:①A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which were more stronger than those of mono-promoter group,pGL3-basic-EGFP-TK-hTRETp,and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. ②Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV.③ After adding GCV, the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line, which was higher than those of mono-promoter, pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP3 and blank controls, but without inhibitory effect in ECV cells transfected by enhanced vector. Conclusion:TK gene expression is regulated by hTERT promoter and CMV enhancer, and then the telomerase activity is reduced and the cancer cells are specifically killed.But it is unclear how the telomerase are down-regulated by TK gene.