BACKGROUND:Human glial cellline-derived neurotrophic factor (GDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation.
OBJECTIVE:To construct and identify pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector.
METHODS:Human GDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then the GDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP, to generate the bicistronic eukaryotic expression plasmid pIRES 2-GDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by twin PCR. Then VEGF 165 cDNA fragment was cloned into the pIRES 2-GDNF-EGFP, instead of EGFP, to create a double gene co-expressing vector plasmid pIRES 2-GDNF-VEGF 165 containing internal ribosome entry sites. Then pIRES 2-GDNF-VEGF 165 was used to transfect HEK293 cells. RT-PCR and western blot analysis were performed to test the co-expression of double genes.
RESULTS AND CONCLUSION:DNA sequencing analysis demonstrated that the GDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of GDNF gene was 636 bp and the size of VEGF165 gene was 576 bp. Enzyme digestion analysis indicated that, pIRES2-GDNF-VEGF165 bicistronic eukaryotic expression vector inserted GDNF band by Bgl II/Bam HI, inserted IRES-VEGF 165 fragment by Bam HI/Not I, and inserted GDNF-IRES-VEGF165 fragment by Bgl II/Not I. RT-PCR and western blot analysis showed that, after HEK293 cells were transfected with pIRES 2-GDNF-VEGF 165 , double genes were expressed at the mRNA and protein levels. The pIRES 2-GDNF-VEGF 165 bicistronic eukaryotic expression vector is successful y constructed.

[Abstract ] In recent years, induced pluripotent stem cells (iPSCs) technology has played an important role in basic and clini-cal application research of Parkinson′s disease ( PD) and acquired significant progress .The neural progenitor /stem cells or dopamine ( DA) neurons which were obtained through iPSCs technique and direct differentiation technique from somatic cells were used for the study of cell therapy in PD , and good results were achieved .The cell models of DA neurons were established from PD patients carrying LRRK2, PAKK2, PINK or SNCA mutations via iPSCs technology , and the mitochondrial function and morphology , oxidative stress,α-synuclein ( SNCA) accumulation , and other aspects were studied on the pathogenesis of PD .This article briefly reviews the latest pro-gress of iPSCs technology in transplantation for treatment of PD and the establishment of cell model of PD disease , and provides refer-ence for further research .

BACKGROUND:Brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor 165 (VEGF 165 ) are essential genes for celldifferentiation. Virus mediated method has been used numerously in researches, but the security is the most important problem. Eukaryotic expressing vector is a way to solve this question.
OBJECTIVE:To construct and identify pIRES 2-BDNF-VEGF 165 bicistronic eukaryotic expression vector.
METHODS:BDNF genes were obtained from the genomic DNA of human peripheral blood mononuclear cells by PCR. Then, the BDNF cDNA fragment was inserted into the multiple cloning sites of pIRES 2-EGFP to generate the bicistronic eukaryotic expression plasmid pIRES 2-BDNF-EGFP. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by double PCR. Next step was that VEGF 165 cDNA fragment was cloned into the pIRES2BDNF-EGFP instead of EGFP to create a double gene co-expressing vector plasmid pIRES 2-BDNF-VEGF 165 . Then, pIRES 2-BDNF-VEGF 165 was used to transfect HEK293 cells, and RT-PCR and western-blot assay were employed to test the co-expression of double genes.
RESULTS AND CONCLUSION:BDNF and VEGF 165 genes were cloned in this study. The DNA sequencing analysis demonstrated that the BDNF and VEGF 165 were exactly consistent with the sequence recorded in the GenBank. The size of BDNF gene was 744 bp. The VEGF 165 gene was obtained from pIRES 2-VEGF 165-EGFP plasmid by PCR, and the size of VEGF 165 gene was 576 bp. Enzyme digestion analysis indicated that BDNF and VEGF 165 genes were inserted into the expression vector pIRES 2-EGFP correctly and the BDNF and VEGF165 co-expression plasmid was successful y constructed. Then, by transfecting pIRES 2-BDNF-VEGF 165 into HEK293 cells, double genes were expressed at the mRNA and protein level. It provides a novel expression system, which enables further study on the functions of BDNF and VEGF 165 genes.

Objective To construct cDNA library of the treated Changliver cell by switching mechanism at 5′ end of RNA transcript (SMART) technique and analyze its quality.Methods cDNA of Changliver cell was aquired with reverse transcription polymerase chain reaction (RT-PCR) and long-distance PCR (LD-PCR),then the cDNA library was constructed with SMART cDNA library construction kit.Results Through testing,the high quality cDNA library containing full length cDNA of Changliver cell had been constructed.The titer of the amplified cDNA library was 4.5 × 1010 pfu*ml-1 and the average exogenous inserts of the recombinants was 1.5 kb.Conclusion These results suggest that the Changliver cell cDNA library has a high quality and lays a solid foundation for researching on Changliver cell and screening

Objective To investigate the growth curve,breeding rate,and blood physiological and biochemical parameters in Npc1 gene mutant mice (Npc1-/-) for providing theoretical evidence in research on Niemann-Pick disease type C1 (NPC1) patient.Methods 1) The body mass of Npc1-/-,Npc1+/-,and Npc1+/+ mice (n=120;60♀,60♂) was measured from 0 to 77 days;(2) As Npc1-/-mice were born only by the mating Npc1+/-mice,the breeding rate of Npc1+/-mice was counted here from the 1st to 4th generation;(3) The blood physiological and biochemical parameters were measured on both Npc1-/-and Npc1+/+ mice at 60 days.Results 1) Compared with the wild type controls,the body weight of Npc1-/-mice was progressively increased up to 7 weeks and then decreased,and died around 11 weeks.The body weight of the Npc1+/-and Npc1+/+ mice was increased as time went on.After 4 weeks,the male mice showed a higher weight gain than the females;(2) The generations of Npc1+/-mice had no significant difference in mating-parturition interval,litter size,weaning litter and the number of male and female (P>0.05),but the weaning rate of the 2nd generation was significantly higher than that of the 1st generation (P<0.05);(3) The hematological parameters showed a significant difference only in mean corpuscular hemoglobin (MCH) and mean peroxidase index (MPXI) between the Npc1-/-and Npc1+/+ mice (P<0.05).No significant difference was found in other hematological parameters (P>0.05).Among the biochemical parameters,aspartate aminotransferase (AST),glucose (GLU),lactate dehydrogenase (LDH),potassium (K) and copper (Cu) had a significant difference between the Npc1-/-and Npc1+/+ mice (P<0.05).Conclusions 1) The growth curves of Npc1-/-,Npc1+/-,and Npc1+/+ mice are different due to different genotype and sex;(2) The reproduction rates of Npc1+/-mice have no significant difference among different generations;(3) The blood physiological parameters (MCH,MPXI) and biochemical parameters (UREA,AST,GLU,LDH,K,Cu) are significantly different between Npc1-/-and Npc1+/+ mice.

Objective To investigate the effect of 5-azacytidine(5-AZA) on apoptosis of bone marrow mesenchymal stem cells(BMSCs).Methods BMSCs were isolated from bone marrow of mouse tibia and femur; the expression of MSC specific markers CD44 and CD90 in BMSCs was measured by immunofluorescence staining;BMSCs were cultured in vitro in the medium supplemented with 0,10 and 20 μmol/L 5-AZA for 48 hours.Cell apoptosis was measured with fluorescent labeled inhibitor of caspases (FLICA) apoptosis kit and 4',6-diamidino-2-phenylindole (DAPI) staining ;the expression of apoptosis-related proteins Annexin V and Caspase-3 in the treated BMSCs was detected by Western blot.Results In this study,BMSCs positively expressed MSC specific markers CD44 and CD90.DAPI staining and Caspase-3 staining both showed that 10 and 20 μmol/L 5-AZA markedly increased apoptotic rate of BMSCs;the apoptosis-positive rate in DAPI staining was (21.086 ± 2.601) ％,(34.467 ± 3.724) ％ and (46.512 ± 3.864) ％,the apoptosis-positive rate in Caspase-3 staining was (5.354 ± 0.735)％,(15.462 ± 2.385)％ and (28.190 ± 4.190)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups(all P ＜0.01).Western blot assay showed that Annexin V and Caspase-3 were both markedly upregulated in 5-AZA treated cells;the relative level of Annexin V expression was(26.612 ±2.184)％,(42.873 ±4.313)％ and (50.056 ± 4.457) ％,the relative level of Caspase-3 expression was (19.231 ± 2.683) ％,(38.618 ± 5.385) ％ and(91.235 ± 7.116)％ in the controls,10 and 20 μmol/L 5-AZA groups,and there were significant differences among the control group and 5-AZA treated groups (all P ＜ 0.01).Conclusion The commonly used doses of 5-AZA can induce apoptosis of BMSCs.

AIM:To investigate the renal function and pathological changes in Npc1 mutant ( Npc1-/-) mice. METHODS:Different genotypes of Niemann-Pick disease type C1 (Npc1) mice were identified by PCR.Subsequently, the renal function of Npc1-/-and Npc1 +/+mice at postnatal day 60 ( P60) was evaluated by measuring the activity and con-tent of important indicators in the serum including ALT , AST, LDH, urea, UA and Cr.Furthermore,β-galactosidase stai-ning and Masson staining were performed to examine the aging and fibrosis of the renal tissues , respectively .RESULTS:Compared with the Npc1 +/+mice, the body weight and kidney weight had a significant reduction ( P<0.01) in the Npc1-/-mice.The results of hepatic and renal functions showed that the activities of ALT , AST and LDH, and contents of urea, UA and Cr had marked increases (P<0.05) in the Npc1-/-mice.Moreover, the results of senescence-associatedβ-galacto-sidase staining in the renal tissues demonstrated accelerated aging in the Npc1-/-mice (P<0.01), and these results were confirmed by Masson staining, which clearly showed the formation of collagen fibers (P<0.01).CONCLUSION:Muta-tion of the Npc1 gene results in abnormal lipid metabolism , which accelerates kidney senescence by promoting fibrosis in the renal tissue and subsequently causes reduction in renal function .

Objective To develop a method of studying fiber projecting in the spinal cord duiring chicken embryo development.Methods At embryonic incubation 3 day (E3), pCAGGS-green fluorescent protein (GFP) plasmid was injected into the spinal cord using in vivo electroporation.Three days after transfection (E6), GFP-positive embryos were collected under a stereo fluorescence microscope .Subsequently , the spinal cord was separated from the embryos and cut from the roof plate as an open book .After fixed with 4%paraformaldehyde ( PFA) for one hour , the opened spinal cords were used for immunohistochemistry with N-cadherin antibody and with DAPI for nuclei .Finally, the nerve fiber projecting was photographed and analyzed under a fluorescence microscope . Results Based on the opened spinal cord and immunostaining in the cryosection , we observed that the nerve fibers projected across the midline of the floor plate and reached to the sulcus terminalis along the white matter of the contra side .The immunoreaction against N-cadherin indicated that overexpression of GFP has no significant effect on chicken embryonic development .Conclusion A new method to study fiber projecting in the developing chicken spinal cord is established successfully in this study .

Objective:To explore the role of N-cadherin and β-catenin in the formation of spinal commissural axon projection during chicken embryonic development.Methods:Fertilized eggs were cultured for three days(stage22),N-cadherin orβ-catenin inter-ference plasmid was injected into the neural tube and in vivo electroporation was performed.Three days after the electroporation, embryos were collected,fixed with 4%PFA,embeded with OCT,and cut into frozen sections.Four groups ( knockdown of N-cadherin orβ-catenin or both of them,and control) were included in this study.Immunohistochemistry method was used to analyze the protein expression result of N-cadherin or β-catenin.The changes of spinal commissural projections were observed with GFP fluorescence.Results:During chicken embryonic development,knockdown of N-cadherin inhibited the expression of β-catenin in the spinal cord.The commissural nerve fibers projecting to the contralateral side of the spinal cord was impaired after knockdown of N-cadherin or β-catenin;this phenotype was similar after knocking down both of them.Conclusion: N-cadherin is implicated in the formation of spinal commissural projection in the developing spinal cord,possibly viaβ-catenin.

Brain-computerinterface(BCI) is a kind of direct channel for information communication and control established between the human brain and computer or other electronic equipment.BCI is a novel information communication system which does not depend on the conventional brain information pathways.The asynchronous brain-computer interface technology is based on alpha wave control,and can automatically switch system mode between working and idle and select the larger EEG signal associated with motion imagination.In this paper,the basic knowledge of BCI and alpha wave-based asynchronous BCI technology were introduced.The key technology and application prospect of the novel alpha wave-based asynchronous BCI technology were summarized,and the status and existing problems were analyzed.