Objective: To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA ( miRNA) ,and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs. Methods: Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini. Results: Raising annealing temperatures 12℃-14℃above the T_m of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels. Conclusion: Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.

Objective:To examine global expression levels of microRNAs(miRNAs) in mouse cerebrum and to provide an important basis for detailed studies of individual miRNAs,their target genes,the miRNA-related regulatory networks in the mammalian central nervous system,and their implications in diseases.Methods:Low molecular weight RNA from cerebrum of five C57BL/6J mice were tailed and reverse transcribed by extended RT-primer.miRNA primers were carefully designed and arrayed on plates according to the Tm of each primer.PCR was carried out at different annealing temperatures using a gradient real-time PCR instrument.The relative expression level of each miRNA was calculated using 5sRNA for normalization.Results:Among the 285 miRNAs detected,260 were positive with varying abundance.Their frequency distribution was approximately a normal distribution.The expression levels of most miRNAs were in accordance with previously published results by microarray.However,the positive rate was higher than that detected by microarray.miRNAs originating from the same hairpin precursors expressed at similar or significantly different levels.Clusters of proximal miRNAs were similar or quite different in abundance.It is suggested that the fate of miRNA after transcription determined their abundance.Conclusion:Using the RNA-tailing and primer-extension PCR array method,we obtained expression profile of miRNA in mouse cerebrum,especially the relative expression data of many low abundant miRNA in mouse cerebrum,which will be of special help for studying the fine-tuning function of low-level miRNAs.

Objective: To further study the effect of apoptin in inducing cancer cell specific apoptosis and the possible applications in cancer therapy. Methods: Apoptin gene was amplified by PCR and inserted into pcDNA3.1(+) with a FLAG tag in front of the multi-cloning-site. Apoptin gene with the FLAG tag was sub-cloned into an adenovirus vector. Several cancer cell lines were transfected with pcDNA3. 1/FLAG/apoptin or infected with apoptin containing recombinant adenoviruses to study the morphologic changes. Ad/apoptin infected cells were also analyzed by flowcytometry after staining with PI. Results: Expressed apoptin was localized in the nucleus of cancer cells. Chromatin condensation occurred 2 or 3 days after Ad/apoptin(+) infection. Cell number in G 2-M phase increased dramatically after Ad/apoptin(+) infection. Conclusion: Apoptin can induce cell cycle G 2-M arrest and chromatin condensation in cancer cells.

Background Choroidal neovascularization (CNV) is the primary pathogenic cause of many fundus diseases.Oxidative stress injury of retinal pigment epithelial (RPE) cells plays important role in angiogenesis of choroid new blood vessels.Oxidative stress injury can active p75NTR receptor, a member of tumor necrosis factors family,resulting in the proliferation of vascular endothelial cells.However, the mechanisms of vascular endothelial cell proliferation remain unclear.Objective This study was conducted to investigate the effect of p75NTR overexpression on CNV and the relative mechanism.Methods The ARPE-19 cell line was used in this study.RPE cells were transfected with p75NTR receptor overexpressed plasmid, and untransfected cells served as the control group.The transfected results were verified by reverse transcription-PCR and Western blot assay.Viability of the cells over time was determined in the p75NTR receptor plasmid transfected group by using BrdU assay.The percentage of apoptotic cells was detected by flow cytometry using Annexin V-FITC/PI fluorescence staining.The percentage of reactive oxygen species (ROS) expression in the cells was detected by using H2 DCFDA fluorescence and flow cytometry.Mitochondrial membrane potential and cytochrome C expression were examined under the confocal microscope.The protein expressions of cleaved caspase-3, Fas and VEGF were determined by Western blot assay.Results The relative expression level of p75 NTR receptor mRNA was (6.11 ±0.77) times higher than that of the control group, and relative expression level of p75NTR receptor protein in the cells in the p75NTR receptor plasmid transfected group was (7.42±0.48) times higher than that in the control group (t=11.49 and 23.17 ,both at P＜0.01).The absorbency values of the p75NTR receptor plasmid transfected group were (93.12±0.56) ％ , (86.30±0.66) ％ , (72.53-±0.86) ％ and (60.77 ±2.81) ％ in 12,24,36 and 48 hours after plasmid transfection, which were significantly lower than 100％ in the control group, and the apoptotic percentages were evidently higher than that in the control group (all at P＜0.05).The relative fluorescence intensity of ROS fluorescence in the p75NTR receptor plasmid transfected group was 2.4 times higher than that in the control group,showing significant difference (t=16.45, P＜0.01).The positive expressing rate of mitomarker (mitochondrial membrane potentials) was 100％ in the control group and (37.30± 2.06)％ in the p75NTR receptor plasmid transfected group, with significant difference between them (t =57.71,P＜0.01).The fluorescence intensity of cytochrome C expression was elevated in the p75NTR receptor plasmid transfected group compared with the control group.Compared with the control group,the expressing levels of cleaved caspase-3 ,Fas and VEGF165 proteins in the cells were significantly raised in the p75NTR receptor plasmid transfected group (all at P＜0.01).Conclusions Overexpression of p75NTR receptors in RPE cells leads to mitochondrial damage and cellular apoptosis and the secretion of VEGF protein, which sequentially promote CNV.P75NTR receptor may be another important regulation pathway in RPE oxygen damage.

Objective:To explore the relationship between development of social skills and executive function and theory of mind in children and adolescents with autism.Methods:Forty-six children and adolescents with autism aged 6 to 17 years were recruited.The diagnosis was made according to the Diagnostic and Statistical Manual of Mental Disorder,Fourth Edition(DSM-Ⅳ),and severe mental disorders were excluded by the screening of the Schedule for Affective Disorders and Schizophrenia for School-Age children (K-SADS).The Rey Complex Figure Test (RCFT),Trail-Making Test,Verbal Fluency Test,Stroop Color-word Task,the First-Order Belief Test and the Second-Order Belief Test were used to assess executive function and theory of mind,and Social Response Scale (SRS) was used to assess social skills.The correlation between results of Executive Function and SRS was analyzed.The SRS scores between groups passing and without passing of First-Order Belief Test and Second-Order Belief were compared.Results:In the aspect of Executive Function,RCFT scores were positively correlated with autistic behavior factor score of SRS (r =0.31-0.41,Ps ＜ 0.05),meanwhile,immediate recall structure scores of RCFT were positively correlated with social motivation factor scores of SRS (r =0.30,P ＜ 0.05),delayed recall structure scores of RCFT were positively correlated with SRS total scores(r =0.34,P ＜ 0.05).In Stroop Colorword Task,errors of the first task were positively correlated with social cognitive factor scores of SRS (r =0.32,P ＜ 0.05).There were no correlation between other Executive Function results and SRS scores (r =-0.21-0.24,Ps ≥0.05).In the aspect of Theory of Mind,there was no significant difference in SRS scores between groups passing or without passing (t =-0.68-1.73,Ps ≥ 0.05).Conclusion:The development of social skill in children and adolescents with autism may have relationship withexecutive function,but have no relationship with the theory of mind.

ObjectiveTo explore the Apolipoprotein-E （ApoE） gene polymorphism in patients with Alzheimer's disease （AD）, vascular dementia (VD) or mild cognitive impairment (MCI). MethodsPeripheral blood was taken from patient with AD, VD or MCI to determine the ApoE genotypes. ResultsThe most of the patients were ε3/ε3 genotype, while the ε2/ε2 and ε4/ε4 could not be detected. ε3/ε4 genotype （P=0.001） and ApoE ε4 allele （P=0.013） was more frequent in AD than in MCI. ApoE ε4 was more frequent in VD than in MCI （P=0.044）. ConclusionApoE ε4 allele is a risk factor in AD, and may be associated with VD and MCI.

30%) might be a risk factor in HAPE susceptibility.

Objective To construct human cytomegalovirus(HCMV)Han?ΔUS12?BAC strain and to study the role of US12 in HCMV replica?tion in human embryonic lung fibroblasts(HELF). Methods Kanamycin?resistant gene was amplified with primers containing homology arms se?quence flanking US12 and then electroporated into E.coli DY380?Han?wt?BAC competent cells. Successfully recombinant Han?ΔUS12?BAC clones were identified by PCR,sequenced for confirmation Han?ΔUS12?BAC plasmids were electroporated into HELF to produce infectious viri?ons. Han?ΔUS12?BAC and Han?wt?BAC were inoculated onto HELF at the multiplicity of infection of 0.1 pfu/cell. The viral titer in the supernatant was measured by TCID50 assay and growth kinetics of the viruses in HELF was studied. Results Han?ΔUS12?BAC clone was successfully con?structed. Han?ΔUS12?BAC was reconstructed in HELF to generate infectious virions. Growth kinetics assay indicated that Han?ΔUS12?BAC and Han?wt?BAC showed no differences in their growth and dissemination in HELF. Conclusion US12 in HCMV clinical strain Han is dispensable for HCMV growth and dissemination in HELF.

Objective: To investigate the distribution of human papillomavirus (HPV) infection characteristics and genotypes in Shenyang area of Liaoning province. Methods: HPV genes were detected in cervical exfoliated cells from 55, 548 patients by amplification and diversion hybridization. Results: A total of 9, 566 patients were positive for HPV infection with a positive rate of 17.22%. Additionally, the positive rate of high risk HPV infection was 14.57% and the positive rate of single genotype HPV infection was 13.63%. Totally, 12, 360 HPV viruses were detected. Among them, 10, 879 HPV viruses were classified into high risk genotypes (10, 879 out of 12, 360, 88.02%). The genotypes in women with ages less than 20 were 16/11/6/51/58/52 genotypes; the susceptible HPV genotypes in other women were 16/58/52/53/39/51/81 genotypes. Conclusions HPV infections in Shenyang are mainly infections with high risk viruses and single infection. The infection rate and genotype distribution of HPV are different in different age groups. More suitable HPV vaccine prophylaxis can be taken according to the epidemic characteristics of HPV in this area.

Objective To evaluate the applicability of Han strain bacterial artificial chromosome (Han-BAC) in small fragment mutation of the human cytomegalovirus (HCMV) genome. Methods A 31-bp long fragment of LUNA between UL80 and UL82 in the HCMV was chosen as the mutation target. Kanamycin resistance gene sequence flanking the homologous arms of the target neighbor sequence was used to replace the target sequence. Electronic transformation was used to rescue the mutated virus. Reverse transcription PCR and cDNA clone sequencing were used to identify the mRNA expression and the 3' terminal structures of LUNA,UL80,and UL82 transcripts. Results The 31 bp fragment was replaced precisely by the kanamycin resistance gene sequence. The efficiency of mutation was more than 3％. LUNA-mutated virus was rescued successfully. Transcriptions and 3' terminal structures of the UL80 and UL82 transcripts of the mutant virus were the same as those of its original virus. Conclusion The sequence at the transcription start site of LUNA was replaced successfully. The HCMV Han-BAC supports fragment mutation as small as 31 bp with a relatively high efficiency.