Objective:To clone human APOE?3 gene for construction of mammalian expression vector pEGFP-N1-APOE?3. Methods:The cDNA encoding APOE?3 was cloned by the methods RT-PCR from total human embryonic brain RNA. The gene was ligated with PMD19-T vector and then was excised from PMD19-T-APOE?3 plasmid and inserted into pEGFP-N1 to construct eukaryotic expression vector. The expression of APOE?3 gene was identified by western-blot. Results:APOE?3 gene sequence and vector were identified correctly by enzyme digestion and sequence analysis. The APOE?3 gene was transfected into HEK293 cells and expressed successfully. Conclusion:The APOE?3 gene is successfully cloned and its eukaryotic expression vector is obtained,which provides basis for the further experimental researches on APOE functions.

Child ; Child, Preschool ; Diagnosis, Differential ; Humans ; Infant ; Medicine, Chinese Traditional ; methods ; standards

Objective To analyze the relevance between the nursing students′ sense of life meaning and death attitudes. Methods 181 nursing students were surveyed with the Purpose In Life Test (PIL) and Death Attitude Profile-Revised (DAP-R), and then did the relevant statistical analysis. Results The nursing undergraduates′overall average of PIL was (91.45 ± 10.54) points, and the neutral acceptance death attitude got the highest scores which were 24.14 ± 5.78, while escape acceptance got the lowest scores which were 11.76 ± 3.61. And there was a significant negative correlation (r =-0.257, P <0.05) between the sense of life meaning and death attitudes. Conclusions There was a significant negative correlation between the sense of life meaning and death attitudes, the more sense of life meaning was, the more positive attitudes toward death would be. So it′s of great importance to provide life-and-death education to improve the nursing students′sense of life meaning and death attitudes.

Objective To investigate whether intervention with two different dosage of atorvastatin may benefit to plasma level of fibrinogen (Fg) in patients with acute cerebral infarction. Methods One hundred and two patients with acute cerebral infarction were randomly assigned into three groups:the control group (33 cases) treated without lipid-lowering drugs, 10 rag (30 cases) and 20 mg (39 cases) atorvastatin group with 3 days of treatment. Before and after the treatment, the plasma levels of Fg and lipids were detected.Results The treatment of 10 mg atorvastatin did not significantly decrease the level of Fg. Compared to the control group, the level of Fg was markedly reduced by 20 mg atorvastatin (21.6% vs 3.2%,P< 0.05). No changes of lipid levels were observed in any group before and after the treatment, whilst there was no relation between the decreasing percentage of Fg and that of TC(r = 0.125 ,P= 0.618), TG(r = 0.147,P = 0.573) or LDL- C (r = -0.279, P = 0.235 ), HDL-C (r = -0.021, P = 0.157). Conclusion Treatment with high dose atorvastatin (20 mg) for 3 days could reduce the level of Fg, and improve the function of vascular endothehum,which may promote the stabilization of atherosclerotic plaque.

Objective To evaluate the levels of pulmonary surfactant protein D (SP-D) and 56-kD human type Ⅰ protein(HTI-56) in bronchoalveolar lavage fluid and serum of children with mycoplasma pneumoniae pneumonia,as well as their potential as biomarkers of mycoplasma pneumoniae pneumonia.Methods A retrospective study was conducted in 57 children with mycoplasma pneumoniae pneumonia from June 2011 to June 2016 in our hospital.In this study,the levels of SP-D and HTI-56 in bronchoalveolar lavage fluid and serum were determined and compared between unilateral lung infection and bilateral lung infection.Results SP-D and HTI-56 levels were significantly higher in bronchoalveolar lavage fluid samples (P ＜ 0.05)compared with bronchoalveolar lavage fluid samples from uninfected lungs (P ＜ 0.05).However,the serum levels of SP-D and HTI-56 in children with unilateral lung infection and bilateral pulmonary infection were not significantly different (P ＞0.05).Conclusion High levels of SP-D and HTI-56 in bronchoalveolar lavage fluid samples from infected lungs may reflect damage to alveolar epithelial cells caused by mycoplasma pneumoniae.

Objective To discuss the change of androgen level in postmenopausal women with coronal atherosclerosis heart dis-ease,and to discuss its clinical significance.Methods According to the results of coronary angiography,218 cases of possible coro-nal atherosclerosis heart disease patients were divided into the observational group with 93 cases of coronal atherosclerosis heart disease patients,and the control group with non-coronal atherosclerosis heart disease patients.The levels of TC,TG,LDL-C,HDL-C,testostemne and andmstenedione in blood were investigated in two groups.Results The level of blood glucose was not statisti-cally different between the two groups(P >0.05),however,the levels of systolic and diastolic blood pressure,TC,TG,LDL-C and HDL-C were statistically different between the two groups (P < 0.05 ).Conclusion The level of androgen in postmenopausal women with coronal atherosclerosis heart disease are higher than postmenopausal women without coronal atherosclerosis heart dis-ease.Androgen detection is useful for the monitoring and diagnosis for postmenopausal women with coronal atherosclerosis heart disease.

Objective To construct and identify recombinant vaccine Bifwlobacterium bifidum(Bb)pGEX-Sj14-3-3 of Schistosoma japonicum(Sj). Methods Total RNA was extracted from adult Sj, antigen encoding gene Sj14-3-3 was amplified by RT-PCR and cloned into Escherichia coli (E. coli)-Bb shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Sj14-3-3. The recombinant plasmid was transformed into E. coli BL21 (DE3).The plasmid was extracted and identified by using BamH I and EcoR I. Then pGEX-Sjl4-3-3 was electroporated into Bb to construct recombinant Bb (pGEX-Sj14-3-3) vaccine. The extracted plasmid of the recombinant Bb (pGEX-Sj14-3-3) vaccine was identified by PCR, and the size of the products was compared with Sj14-3-3 gene of adult worms.Results Sj14-3-3 of 399 bp in length was amplified by RT-PCR. The products were digested by BamH I and EcoR I , and the fragments length of plasmid pGEX-Sj14-3-3 vector was 4947 bp, and of Sj 14-3-3 gene was 399 bp.The product of 399 bp Sj14-3-3 gene was also amplified by PCR from template of the extracted plasmid of the recombinant Bb(pGEX-Sj14-3-3 ) vaccine. The size of the product obtained was just the same as expected.Conclusion The recombinant Bb(pGEX-Sj14-3-3) vaccine of Sj is successfully constructed.

Objective To construct and identify the transgenic plant vector recombinant pBI-Eg95 plasmid of Echinococcus granulosus. Methods Total RNA was extracted from hydatid cyst protoscoleces of Echinococcus granulosus after sonication. A couple of specific primers were designed on the basis of known sequences of Eg95 gene. The desired gene was amplified by PCR technique from the cDNA, and then was cloned into the plant expression vector pBI121 to construct the recombinant pBI-Eg95 plasmid. The recombinant plasmid was electroporated into Agrobocterium tumefaciens (At) LBA4404 strain. The positive recombinant clones were confirmed by restriction endonuclease digestion and characterized by PCR. Results For RT-PCR, a specific band around 471 bp was amplified. The result of DNA sequencing of Eg95 showed the identity with the published sequence. The same band was obtained by restriction endonuclease digestion and PER from the plasmids of positive recombinant At(rAt). Conclusions The recombinant pBI-Eg95 plasmid was successfully constructed, and it provides the basis to further research of the transgenic plant vaccine of Echinococcus granulosus.

Objective To investigate the effects of recombinant vaccine Bifidobacterium bifidum (Bb) pGEX-Sj14-3-3 on splenocyte apoptosis in BALB/c mice.Methods Ninety-six BALB/c mice were randomly divided into two groups according to their body mass:per os group (PO) and intranasal immunization group (IN),with 48 mice in each group.All mice were orally and intranasally immunized with recombinant vaccine Bb(pGEX-Sj14-3-3).Four mice in each group were sacrificed on weeks 0,2,4,6,8,10,12,14,16,18,20 and 22,respectively,after immunization,and splenocytes were separated and cultured with or without ConA stimulation.The apoptotic rates of splenocytes were detected by flow cytometry.Results It showed that apoptotic level of splenocytes in both groups remarkably increased after 2-4 weeks without ConA stimulation (PO:0.069 ± 0.005,0.076 ± 0.010; IN:0.037 ± 0.002,0.075 ± 0.002),and the value reached the peak on the 4th week,and the differences were statistically significant compared with that of week 0(all P ＜ 0.05).Apoptotic level of splenocytes in both groups with ConA stimulation increased after 2-6 weeks(PO:0.089± 0.006,0.098 ± 0.010,0.060±0.007; IN:0.054 ± 0.001,0.093 ± 0.003,0.058 ± 0.012),and the value also reached the peak after 4 week,respectively.The differences were statistically significant compared with that of week 0 (all P ＜ 0.05).Apoptotic level of splenocytes in each group with ConA stimulation was significantly higher than that without ConA stimulation.Conclusion It is suspected that the recombinant vaccine Bb(pGEX-Sj14-3-3) may inhibit apoptosis of splenocytes in mice immunized orally or intranasally.

ObjectiveTo study the dynamic changes of IgG,IgG subclasses,IgE and IgA in sera of BALB/c mice immunized with recombinant Bifidobacterium bifidum (Bb) pGEX-Sj14-3-3 vaccine of Schistosoma japonicum.MethodsNinty six BALB/c mice were randomly divided into two groups:oral immunization group and intranasal immunization group,48 mice in each group.Mice were orally and intranasally immunized with recombinant Bb(pGEX-Sj14-3-3) vaccine,respectively.Four mice from each group were sacrificed,respectively,on weeks 0,2,4,6,8,10,12,14,16,18,20 and 22 after immunization and their sera from the eyeballs were collected.The levels of IgG,IgG subclasses,IgE and IgA were assayed with routine Enzyme-linked immunosorbent assay(ELISA).ResultsThe titers of IgG,IgG1,IgG2a,IgG2b,IgG3,IgE and IgA in both groups increased during the 2 - 22th,2 - 14th,2 - 22th,2 - 22th,2 - 20th,2 - 22th,2 - 22th weeks,respectively.The values reached the highest level on weeks 8,6,6,4,8,10 and 6,respectively,in the oral group,and the values were (0.065 ±0.001,0.021 ± 0.002,0.011 ± 0.001,0.015 ± 0.003,0.014 ± 0.002,0.011 ± 0.001,0.013 ± 0.002),respectively,as compared with the values on week 0(0.032 ± 0.001,0.015 ± 0.002,0.005 ± 0.002,0.005 ± 0.001,0.006 ± 0.001,0.006 ± 0.001,0.005 ± 0.001 ),the differences were statistically significant(P ＜ 0.05 or ＜ 0.01 ) except that of IgG1 and IgG2b.In the intranasal group these values reached the highest levels on weeks 4,6,4,4,8,10 and 8,respectively,and the values were (0.064± 0.003,0.022 ± 0.002,0.012 ± 0.003,0.019 ± 0.001,0.013 ± 0.001,0.015 ± 0.001,0.014 ± 0.003),respectively,as compared with the value on week 0,the differences were statistically significant(P ＜ 0.05 or ＜ 0.01 ) except that of IgG1 and IgA.ConclusionsTypes Th1 and Th2 mixed type immune responses can be induced in mice by immunization with the recombinant Bb(pGEX-Sj14-3-3) vaccine by early period of immunization (2th - 10th week).