AIM:The main purpose of this study is to investigate the regulatory role of C/EBP? in mouse vesicular glutamate transporter 2(mVGLUT2) gene expression. METHODS:The promoter region of mVGLUT2 was cloned to PGL3-basic vector. Site-direction mutation was used to identify the CCAAT-enhancer-binding protein ?(C/EBP?) binding site. The promoter activity was observed by luciferase system. The binding between C/EBP? protein and mVGLTU2 promoter region was determined by EMSA. The C/EBP? gene expression was inhibited by its specific siRNA. RESULTS:mVGLUT2 promoter activity decreased about 50% after mutation of C/EBP? binding site. EMSA showed that C/EBP? protein bound onto mVGLUT2 promoter region. Meanwhile,when C/EBP? gene expression was inhibited by its specific siRNA,mVGLUT2 promoter activity,mRNA level and protein level were decreased about 60%,40% and 45%,respectively. CONCLUSION:C/EBP? is involved in the regulation of mVGLUT2 gene expression.

AIM: The main purpose of this study is to investigate the regulatory role of C/EBPα in mouse vesicular glutamate transporter 2(mVGLUT2) gene expression. METHODS: The promoter region of mVGLUT2 was cloned to PGL3-basic vector. Site-direction mutation was used to identify the CCAAT-enhancer-binding protein α(C/EBPα) binding site. The promoter activity was observed by luciferase system. The binding between C/EBPα protein and mVGLTU2 promoter region was determined by EMSA. The C/EBPα gene expression was inhibited by its specific siRNA. RESULTS: mVGLUT2 promoter activity decreased about 50% after mutation of C/EBPα binding site. EMSA showed that C/EBPα protein bound onto mVGLUT2 promoter region. Meanwhile, when C/EBPα gene expression was inhibited by its specific siRNA, mVGLUT2 promoter activity, mRNA level and protein level were decreased about 60%, 40% and 45%, respectively. CONCLUSION: C/EBPα is involved in the regulation of mVGLUT2 gene expression.

To generate a mWAP-hLF hybrid locus that the transcription of human lactoferrin (hLF) genomic sequence is directed by the up & down stream regulatory sequence of murine whey acidic protein (mWAP) gene locus, we describe here a successive three-step 'Gap-repair' method. First, a gap-repair vector based on pBR322 vector backbone by inserting six joint homologous arms was constructed. Then using 'Gap-repair 'method mediated by Red recombination system of lambda-prophage in Escherichia coli, in the first step, the 8 kb 3' flanking region of the mWAP gene was subcloned from the Bacterial artificial chromosome which harbors the mWAP gene locus(mWAP BAC) into the gap-repair vector; in the second step, the 29 kb hLF genomic sequence from the ATG code to the TAA code was subcloned from the hLF BAC; in the third step, the 12 kb 5' flanking region of the mWAP gene was subcloned from the mWAP BAC. Finally, all these three DNA fragments were automatically combined together without any gap in the gap-repair vector, and a 49 kb mWAP-hLF hybrid locus that the hLF genomic sequence was flanked by the 5' & 3' flanking region of mWAP gene locus was constructed. The result was confirmed by PCR, restriction enzyme digestion and sequencing. Our method provide a new way for the construction of large mammary-gland expression vector.
Animals ; Bioreactors ; DNA Repair ; genetics ; Genetic Engineering ; methods ; Humans ; Hybridization, Genetic ; Lactoferrin ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Mice, Transgenic ; Milk Proteins ; genetics

Objective To detect the expression of Beclin1 and Bcl2 in invasive pituitary adenomas and to explore the relationship of Beclin1 and Bci2 in invasive pituitary adenomas and the relativity between the 2 genes.Methods 61 specimens were classified into invasive group (32 cases) and non-invasive group (29 cases) according to the comprehensive evaluation of invasive pituitary adenomas.lmmunofluorescence analysis and RT-PCR were adopted respectively to detect the protein and mRNA expressions of Beclinl and Bcl2.The difference and relativity of Beclin1 and Bcl2 expression in invasive group and non-invasive group were analyzed.Results 32 specimens of pituitary adenoma were invasive and 29 were non-invasive.Beclin1 protein and mRNA expressions were lower in the invasive group than in the non-invasive group (P ＜0.01 ).Bcl2 protein and mRNA expressions were higher in the invasive group than in the non-invasive group (P ＜0.01 ).Pearson related analysis showed that Beclin1 mRNA expression was negtively correlated with Bcl2 mRNA expression in the invasive group ( r =-0.42,P =0.028 ).Conclusions Beclinl expression is decreased in invasive pituitary adenomas.The invasiveness of pituitary adenoma is closely related to the high expression of Bcl2 protein and mRNA,and the low expression of Beclin1 protein and mRNA.The inhibition of the autophagy may lead to the enhancement of the invasiveness of pituitary adenomas and that inhibition may come from the interaction of Beclin1 and Bcl2.

Objective:To explore the role of tripartite motif-containing protein 21(TRIM21)in chronic apical periodontitis(CAP)and its potential mechanism.Methods:Human CAP tissue and normal periodontal tissue were collected.The expression of inflamma-tory factors(IL-1 β,IL-6,TNF-a,TGF-β1),osteoclast related genes(TRAP,RANKL,CTSK),macrophage polarization related genes(CD86,iNOS,CD206,Arg1)and TRIM21 were detected by RT-qPCR.TRIM21 protein was detected by immunohistochemical staining,and the osteoclasts was detected by tartrate resistant acid phosphatase(TRAP)staining.The inflammation cell model was es-tablished by stimulating Raw264.7 cells with lipoteichoic acid(LTA)or lipopolysaccharide(LPS),and the expression of the above factors was detected.The bone marrow-derived macrophages(BMDMs)extracted from wild-type and TRIM21-/-mice stimulated by LPS were used to verify the expression of the above factors by RT-qPCR,the osteoclasts were detected by TRAP staining,and the po-larization of macrophages was detected by immunofluorescence staining.Results:In CAP tissue the expression of inflammatory factors,osteoclast related genes,CD86,iNOS and TRIM21 increased,while CD206 and Arg1 decreased,and osteoclasts were more than that in normal tissue.The stimulation of LTA/LPS promoted the proliferation of Raw264.7 cells,and the expression of these factors in cells was consistent with that in tissues.After LPS stimulation,BMDMs of TRIM21-/-mice had lighter inflammation,lower expression of os-teoclast specific genes,fewer osteoclasts and lower M1 polarization than those of wild type mice.Conclusion:TRIM21 might promote the progress of CAP by promoting M1 polarization of macrophages.