Two Zn(II)-based Coordination Polymers: Fe3+ Ion and Prevention Activity Detection On Postpartum Hemorrhage By Regulating Anticoagulant Factor Activity.

Two new Zn(II)-based coordination polymers {[Zn3(L1)6(H2O)]∙(H2O)4}n (1, HL1 = 4-(tetrazol-5-yl)phenyl-4,2':6',4″-terpyridine) and [Zn2Cl2(L2)2H2O]n (2, HL2 = 4-([2,2':6',2″'-terpyridin]-4'-yl)benzoic acid) have been successfully prepared using two similar organic ligands with distinct donor groups under similar reaction conditions. The distinct structural features and donor atoms make the two complexes show different water stability, and the complex 1 with good water stability, which can be utilized as the sensor for Fe3+ ion detection in water. The value of Stern-Volmer quenching constant of 1 to the Fe3+ is 5.77 × 104 M- 1, which lies in the top region of the reported CP-based sensors. The mechanism investigation reveals that the energy transfer of resonance from the complex 1 to the Fe3+ ion can account for its fluorescent quenching behavior. The treatment activity of compounds 1 and 2 on the postpartum hemorrhage (PPH) was assessed. First, the cytotoxicity of compounds 1 and 2 on human umbilical vein endothelial cells was assessed with Cell Counting Kit-8 detection kit. Then, to evaluate the prevention of compounds 1 and 2 on the PPH, we conducted the Lowry method and detected the clotting factor IX and anticoagulant factor III contents after the indicated treatment. Finally, the inflammatory response in mice was determined by ELISA method, and the IL-6 and IL-8 levels were determined.

[Construction of recombinant adenovirus of SEA and CD80 genes co-expression regulated by mouse TERT promoter and identification of its expression in hepatoma cells].

AIM: To construct recombinant co-expression adenovirus vector of SEA and CD80 genes regulated by mouse TERT(telomerase reverse transcriptase, TERT) promoter and to observe the expression of SEA and CD80 in the Hepa1-6 cells mediated by it. METHODS: Using AdEasy adenovirus system, the core promoter region of mTERT was subcloned to shuttle plasmid pShuttle2 and Myc-Max response element was inserted upstream of it to regulate the expression of SEA and CD80. The recombinant co-expression adenovirus vector of SEA and CD80 genes was constructed and named as Ad-MMRE-mTERT-BIS. Hepatoma cell line Hepa1-6 and fibroblast cell line NIH3T3 were infected by recombinant adenovirus at MOI(multiplicity of infection)of 100, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining. RESULTS: SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on NIH3T3 cells. CONCLUSION: The recombinant co-expression adenovirus vector of SEA and CD80 gene regulated by mTERT promoter was sucessfully constructed and make targeting-expression of SEA and CD80 on the surface of hepatoma cells, which lays the foundation for further research on application of SEA and CD80 in targeted genetherapy for hepatoma.

Cloning of mouse telomerase reverse transcriptase gene promoter and identification of proximal core promoter sequences essential for the expression of transgenes in cancer cells.

Telomerase is a ribonucleoprotein complex, whose function is to add motif-specific nucleotides to the end of chromosomes. Telomerase consists of three major subunits, the telomerase RNA template (hTR), the telomerase-associated protein (TEP1) and telomerase reverse transcriptase (TERT). TERT is the most important component responsible for the catalytic activity of telomerase and a rate-limiting determinant of the activity. Telomerase activities were at high levels in approximately 90% of mouse cancers or tumor-derived cell lines through TERT transcriptional up-regulation. Unlike human telomerase, telomerase activity exists in colon, liver, ovary and testis but not in brain, heart, stomach and muscle in normal mouse tissues. In this study, we prepared 5' truncations of 1086 bp fragments upstream of the initiating ATG codon of the mTERT gene to construct luciferase reporter gene plasmids, and transfected these plasmids into a normal mouse cell line and several cancer lines to identify the core promoter region essential for transcriptional activation in cancer cells by a luciferase assay. We constructed a eukaryotic expression vector of membrane-expressing staphylococcal endotoxin A (SEA) gene driven by the core promoter region of the mTERT gene and observed if the core promoter region could express the SEA gene in these cancer cells, but not in normal cells following transfection with the construct. The results showed that the transcriptional activities of each fragment of the mTERT gene promoter in the cancer cell lines Hepa1-6, B16 and CT26 were higher than those in NIH3T3 cells, and the proximal 333-bp fragment was the core promoter of the mTERT gene in the cancer cells. The proximal 333-bp fragment was able to make the SEA express on the surface of the cancer cells, but not in NIH3T3 cells. It provides a foundation for cancer targeting gene therapy by using the mTERT gene promoter.