Influence of indoor decoration volatile pollutants in reproductive function and sperm apoptosis of male mice / 吉林大学学报(医学版)

Objective:To simulate the pollution environment of indoor decoration volatile pollutants,and to explore the influence of three main indoor decoration volatile pollutants formaldehyde,xylene and ammonia in the male mouse reproductive function.Methods:A total of 20 male ICR mice were randomly divided into experimental group(exposed to 60 mg·m-3formaldehyde,50 mg·m-3xylene and 40 mg·m-3ammonia mixed in the static type canister)and control group(put into the air-filled static type canister)(n=10).Each mouse was weighed at set times every day,exposed for 35 d continuously.The behavior changes of the mice were also observed everyday. The epididymal cauda sperms were collected to detect the sperm density and vitality.The sperm malformation rate was detected by Diff-Quick dyeing method.The apoptotic rate of epididymal cauda sperms was detected by flow cytometry(FCM).Then Western blotting method was used to determine the expression levels of Caspase-9 and Cleaved Caspase-3 proteins in testis tissue of the mice.Results:Four weeks after the exposure to pollutants,the body weight of the mice in experimental group was significantly lower than that in control group(P<0.01).The concentration and vitality of sperm malformation rate of the mice in experimental group were significantly lower than those in control group(P<0.01)35 d after exposure.Compared with control group,the apoptotic rate of sperm of the mice in experimental group was significantly increased(P< 0.01).However,the expression levels of Caspase-9 and Cleaved Caspase-3 in the testis tissue of the mice in experimental group and control group had no significant differences(P>0.05).Conclusion:The main pollutants in indoor decoration can significantly influence the quality of spermatozoa in the epididymis.

Optimization of sulfated modification conditions of polysaccharide from Bupleurum Chinense and comparison of their antioxidant abilities between before and after modification / 吉林大学学报(医学版)

Objective: To investigate the influencing factors of sulfated modification of Bupleurum chinense polysaccharides (BCP),and to elucidate the possible mechanism of improving the antioxidant ability of sulfated BCP (S-BCP).Methods:BCP was sulfated by chlorosulfonic acid-pyridine method.The degree of substitution (DS)of S-BCP was observed by adjusting the volume ratios of chlorosulfonic acid to pyridine (1:2,1:4,and 1:8).The structures of BCP and S-BCP were analyzed by infrared (IR)spectroscopy,the morphology of BCP and S-BCP were observed under scanning electron microscopy (SEM).The antioxidant model was established by using 1, 1-diphenyl-2-picrylhydrazyl (DPPH)free radical scavenging.The experiment was divided into positive control group,BCP group and S-BCP group,and the scavenging rates of DPPH free radical in various groups were compared. Results:When the volume ratio of chlorosulfonic acid to pyridine was 1 : 4,the reaction time was 2 h and the reaction temperature was 60 ℃,the maximum sulfur content percentage of S-BCP was 18.62% and the DS was the highest (DS = 2.32 ).Compared with BCP group, the scavenging rate of DPPH free radical of S-BCP was significantly increased (P <0.05).Conclusion:The volume ratio of chlorosulfonic acid to pyridine can affect the DS of S-BCP.The sulfated modification can increase the anti-oxidant capacity of BCP by changing its physic-chemical characters and spatial conformation.

Construction and eukaryotic expression of recombinant HSA-TP5 fusion gene expression vector / 吉林大学学报(医学版)

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

Construction and eukaryotic expression of recombinant HSA-TP5 fusion gene expression vector / 吉林大学学报(医学版)

Objective:To construct the expression vector of the fusion protein of human serum albumin (HSA) and thymopentin (TP5) and to express it in Pichia pastoris,and to elucidate the biological activity of fusion protein.Methods:The HSA-TP5 fusion gene was constructed by gene recombination and transfected into Pichia pastoris to construct the eukaryotic expression system of HSA-TP5.The recombinant eukaryotic expression plasmid of PPICZα-HSA-TP5 was obtained by agarose gel electrophoresis and purification reagent.The two step fermentation method was used to ferment gene engineering bacteria of HSA-TP5 in high density,and the fermentation supernatant protein was precipitated and concentrated;the purified fusion protein was obtained by cation exchange chromatography and hydrophobic chromatography and analyzed by SDS-polyacrylamide gel electrophoresis.The effect of the fusion protein on the proliferation of lymphocytes was detected by MTT assay.Results:The HSA target gene fragment with length of 1 845 bp was achieved by PCR method.The HSA-TP5-pPICZαC fusion plasmid was identified by restriction endonuclease digestion,and the fragment length was 707 bp.The sequence analysis showed that the HSA and TP5 sequences of the target genes were identical with the gene sequences reported in GenBank and were fused by forward fusion.PCR method confirmed that the eukaryotic recombinant plasmid PPICZ αC-HSA-TP5 was integrated into the yeast genome,and compared with control group,the target gene PCR product length was found to be 1 860 bp.SDS-PAGE analysis showed that the expression level of HSA-TP5 fusion protein was gradually increased with the induction time within 72 h.HSA-TP5 fusion protein was purified by cation exchange chromatography and AKTA multifunctional protein purification system.The MTT assay results showed that HSA-TP5 fusion protein was consistent with TP5 protein in promoting lymphocyte proliferation activity.Conclusion:HSA-TP5 fusion protein can be obtained by constructing the eukaryotic expression system of Pichia pastoris and owns the biological activity.

Expression and purification of Tat-GFP fusion protein and its cell membrane penetrating activity / 吉林大学学报(医学版)

Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.