Objective To construct the pcDNA3. 1-Brugia malayi (Bm) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) eukaryotic recombinant plasmid and to study its effect on mouse cellular immunity response. Methods Total RNA was prepared from periodic Bm. The target gene fragments were amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique and then were inserted into the cloning vector. pGEM-T Easy, and sub-cloned into pcDNA3. 1. Purified pcDNA 3. 1-BmGAPDH recombinant plasmid and CpG were injected into the anterior tibial muscle of BALB/c mice in order to induce host immunity response. Mice injected with PBS and mice injected with blank plasmid were prepared as controls. The mouse models were immunized for 3 times with an interval of 2 weeks. RT-PCR was utilized to detect target gene expression in the muscle tissue. MTT method was used to measure the immunized mice T lymphocytes stimulation index, while enzyme-linked immunoassay (ELISA) was used to determine the serum interleukin (IL)-4 and interferon (IFN)-γ level. Means were compared using t test with SPSS software. Results The recombinant plasmid pcDNA3. 1-BmGAPDH was constructed sucessfully. The target gene was 1020 bp long and its homology with known gene sequence in database was 99%. BmGAPDH gene in the injected muscle of the immunized mice was detected by PCR. The proliferation of spleen T lymphocytes was higher in pcDNA3-BmGAPDH group than in the 2 control groups which were 1. 398, 1. 006 and 1. 017,respectively (P< 0. 05). The levels of IFN-γ and IL-4 in serums from the immunized mice were significantly higher than those of the PBS control group and blank plasmid control group which were 163.905, 58.589, 51. 317 and 107. 906, 27.111, 34.627, respectively (P<0. 05). Immune adjuvant CpG could accelerate and boost antigen-specific immune responses induced by vaccine, which presented as significant increase of IFN-γ and lymphocyte proliferation at 4 weeks after immunization.Conclusion The recombinant eukaryotic plasmid pcDNA3. 1-BmGAPDH is constructed and could elicit cellular immune responses in immunized mice.

ObjectiveTo construct the eukaryotic expression plasmids containing cysteine protease inhibitor (CPI) and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) gene from periodic Brugia malayi (Bm),and to observe its cellular immune response in mouse.Methods pcDNA3.1 (+)-BmCPI/BmGAPDH was constructed.The recombinant plasmids were screened and identified by digestion with restriction enzyme.BALB/c mice were injected intramuscularly with a dosage of 100 μg purified recombinant plasmid DNA with GpG oligodeoxynucleotide (CpG ODN) and two same doses were administrated at 2-week intervals.pcDNA3.1 (+) and phosphate buffered solution (PBS) were used as controls.The tissue of muscles at 4 weeks after the third injection was collected and the target gene was detected by reverse transcription-polymerase chain reaction (RTPCR).Two weeks after the third immunization,the stimulation index (SI) of spleen lymphocytes of immunized mice was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method and the serum levels of interleukin (IL)-4 and interferon (IFN)-γ were detected by enzyme-linked immunosorbent assay (ELISA).The data were analyzed by t test.ResultsBmCPI/BmGAPDH gene in the injected muscle of the immunized mice was detected by RT-PCR. At 6 weeks after immunization,the SIot spleen T lymphocytes in pcDNA3.1 (+)-BmCPI/CpG group and pcDNA3.1 (+)-BmCPI/BmGAPDH/CpG group were 1.466 ± 0.635 and 1.610 ± 0.112,respectively,which were both higher than PBS group and pcDNA3.1( +)-CpG group (1.004 ± 0.019 and 1.078 ± 0.129,respectively) (t=64.438,45.318,42.749 and 34.314,respectively; all P＜0.05).At 4 weeks after immunization,the serum levels of IL-4 and IFN-γ of mice in pcDNA3.1 ( + )-BmCPI/BmGAPDH/ CpG group were significantly higher than those in pcDNA3.1 (+)-CpG group (t=288.053 and 76.453,respectively; both P＜0.05),while the serum level of IFN-γ was also higher than that in pcDNA3.1 (+)-BmCPI/CpG group (t=129.642,P＜0.05). ConclusionThe recombinant eukaryotic plasmid pcDNA3.1 (+)-BmCPI/BmGAPDH could be expressed in mice,and could elicit specific cellular immune responses in immunized mice.

OBJECTIVE: To investigate the effect of lactic acid-induced upregulation of PLEKHA4 expression on biological behaviors of glioma cells and the possible molecular mechanism. METHODS: GEO database and GEPIA2 website were used to analyze the relationship between PLEKHA4 expression level and the pathological grade of glioma. A specific PLEKHA4 siRNA was transfected in glioma U251 and T98G cells, and the changes in cell proliferation ability were assessed by real-time cell analysis technology and Edu experiment. The colony-forming ability of the cells was evaluated using plate cloning assay, and cell cycle changes and cell apoptosis were analyzed with flow cytometry. The mRNA expression of PLEKHA4 was detected by PCR in glioma samples and controls and in glioma cells treated with lactic acid and glucose. Xenograft mice in vivo was used to detect tumor formation in nude mice; Western blotting was used to detect the expressions of cyclinD1, CDK2, Bcl2, β-catenin and phosphorylation of the key proteins in the MAPK signaling pathway. RESULTS: The results of GEO database and online website analysis showed that PLEKHA4 was highly expressed in glioma tissues and was associated with poor prognosis; PLEKHA4 knockdown obviously inhibited the proliferation and attenuated the clone-forming ability of the glioma cells (P < 0.05). Flow cytometry showed that PLEKHA4 knockdown caused cell cycle arrest in G1 phase and promoted apoptosis of the cells (P < 0.01). PLEKHA4 gene mRNA expression was increased in glioma samples and glioma cells after lactate and glucose treatment (P < 0.01). PLEKHA4 knockdown, tumor formation ability of nude mice decreased; PLEKHA4 knockdown obviously lowered the expression of cyclinD1, CDK2, Bcl2 and other functional proteins, inhibited the phosphorylation of ERK and p38 and reduced the expression of β-catenin protein (P < 0.01). CONCLUSION PLEKHA4 knockdown inhibited the proliferation of glioma cells and promoted apoptosis by inhibiting the activation of the MAPK signaling pathway and expression of β-catenin. Lactic acid produced by glycolysis upregulates the expression of PLEKHA4 in glioma cells.
Humans ; Animals ; Mice ; Up-Regulation ; beta Catenin/metabolism* ; Mice, Nude ; Brain Neoplasms/pathology* ; Lactic Acid ; Cell Line, Tumor ; Glioma/pathology* ; Cell Proliferation ; Apoptosis ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; RNA, Messenger/genetics* ; Gene Expression Regulation, Neoplastic