Erythropoietin inhibits angiotensin II induced cardiomyocyte hypertrophy in vitro via activating PI3K/Akt-eNOS pathway / 中华心血管病杂志

<p><b>OBJECTIVE</b>To explore the effect of erythropoietin (EPO) on angiotensin II (AngII) induced neonatal rat cardiomyocyte hypertrophy and the association with PI3K/Akt-eNOS signaling pathway.</p><p><b>METHODS</b>Cardiomyocytes were isolated from new-born Sprague-Dawley rats and stimulated by AngII in vitro. The cell surface area and mRNA expression of atrial natriuretic factor (ANF) of cardiomyocytes were determined in the presence and absence of various concentrations of EPO, phosphatidylinositol 3'-kinase (PI3K) inhibitor LY294002 and nitric oxide synthase (NOS) inhibitor L-NAME. Intracellular signal molecules, such as Akt, phosphorylated Akt, eNOS and phosphorylated eNOS protein expressions were detected by western blot. Nitric oxide (NO) level in the supernatant of cultured cardiomyocytes was assayed by NO assay kit.</p><p><b>RESULTS</b>EPO (20 U/ml) significantly inhibited AngII induced cardiomyocyte hypertrophy as shown by decreased cell surface area and ANF mRNA expression (all P < 0.05). EPO also activated Akt and enhanced the expression of eNOS and its phosphorylation (all P < 0.05), increased the NO production (P < 0.01). These effects could be partially abolished by cotreatment with LY294002 or L-NAME (all P < 0.05).</p><p><b>CONCLUSION</b>EPO attenuates AngII induced cardiomyocytes hypertrophy via activating PI3K-Akt-eNOS pathway and promoting NO production.</p>

Expression of the human papillomavirus type 16L/E7 fusion protein in E. coli and observation of its immunogenicity in mice / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>Many epidemiological and experimental evidences prove that cervical cancers are strongly associated with genital high-risk types of human papillomavirus (HPV). HPV16 is present in 50% of the tumor specimens. Thus, it is important to develop vaccines against HPV16 and cervical cancer. The authors studied the expression of the HPV16 L1DeltaCE7N fusion protein in E. coli and observed its immunogenicity.</p><p><b>METHODS</b>The fragment of HPV16 L1DeltaC gene and the E7N gene were amplified by PCR separately; the fusion gene named L1DeltaCE7N was generated by fusing E7N to the C terminal of L1DeltaC then the chimeric gene was cloned into prokaryotic expression vector pGEX-2T and expressed in E. coli strain JM109. The L1DeltaCE7N protein expressed were detected by Western blot. Finally its immunogenicity was characterized in immunized mice.</p><p><b>RESULTS</b>It was proved that the sequence and open reading frame of fusion gene L1DeltaE7N was correct by sequencing; SDA-PAGE gel analysis showed that HPV16 L1/E7 fusion protein was highly expressed in E. coli; the protein was expressed as soluble form and the molecular weight was about 85 x 10(3). The fusion protein could be purified by affinity chromatography and gel filtration. The ELISA result indicated that L1/E7 could elicit specific antibodies against L1 and E7 in immunized mice. In vivo tumor protection test indicated that tumor formation was retarded or prevented in the mice after vaccination with L1/E7, when C57 BL/6 mice were challenged by syngeneic HVP16E6 and E7 transformed tumor cells.</p><p><b>CONCLUSION</b>HPV16L1/E7 fusion protein was expressed in E. coli, it can be a candidate for prophylactic and therapeutic vaccine for HPV16-associated infection and tumors.</p>

Construction and identification of non-replication recombinant vaccinia virus co-expressing human papillomavirus type 16 L1/L2/E6/E7 proteins / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To generate a human papillomavirus (HPV16) prophylactic and therapeutic vaccine candidate for cervical cancer.</p><p><b>METHODS</b>HPV16 major capsid protein L1 gene/minor capsid protein L2 gene and HPV16 early E6/E7 genes were inserted into a vaccinia virus expression vector. A strain of non-recombinant vaccinia virus containing the sequences was obtained through a homologous recombination and identified.</p><p><b>RESULTS</b>DNA hybridization confirmed that the HPV16L1/L2/E6/E7 genes were integrated into vaccinia virus DNA. Western Blot result showed that full-length L1/L2/E6/E7 proteins were co-expressed in CEF cells infected with the recombinant virus.</p><p><b>CONCLUSION</b>NTVJE6E7CKL1L2 could be taken as a candidate of prophylactic and therapeutic vaccine for HPV-associated tumors and their precancerous transformations.</p>

Gene expression of collagen types IX and X in the lumbar disc / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.</p><p><b>METHODS</b>Fetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.</p><p><b>RESULTS</b>In fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen.</p><p><b>CONCLUSIONS</b>Obvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.</p>

The induction and function study on dendritic cells derived from blasts from patients with acute myelogenous leukemia / 中国实验血液学杂志

To investigate the induction method and function of dendritic cells (DC) derived from acute myelogenous leukemia (AML) blasts in vitro, cytokine-supplemented suspension cultures of leukemia blasts in 25 AML patients were performed. Mononuclear cells were cultured for 8 to 12 days using recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF), recombinant human interleukin-4 (rhIL-4) and recombinant human tumor necrosis factor-alpha (rhTNF-alpha). Morphology, phenotype, cytogenetics, and function of induced cells were studied. The results showed that after culture for 3 days, cells in 20/25 AML cases demonstrated an increase in size with dendritic morphology. After culture for 8 - 9 days, the percentage of such cells reached peak. When cultured for 12 days, the total number of cells and the number of cells with DC morphology decreased greatly. Phenotypic analyses of cells (11/20 cases) were measured by flow cytometry before and after culture. Before culture, cells did not express CD1a, CD80 and CD83, while expressed CD54, CD86 and HLA-DR with low frequency. After culture, cells upregulated CD1a, CD80, CD83, CD54, CD86 and HLA-DR significantly. A marked increase of the T-cell stimulatory capacity could be generated in Allo-MLRs. FISH confirmed the leukemic origin of generated cells. In conclusion, leukemia-derived DC can be generated from AML blasts using cytokine combination (GM-CSF, IL-4, and TNF-alpha) in vitro.

Non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins elicits anti-tumor immunity in mice / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the anti-tumor immunity of the non-replicating recombinant vaccinia virus expressing HPV16 E6 and E7 proteins.</p><p><b>METHODS</b>C57BL/6 mice were immunized by non-replicating recombinant vaccinia virus (NTVJmE6E7), and then specific CTLs were determined. Immune protection effects were evaluated by challenges of different doses of TC-1 tumor cells. Immunotherapeutic effects in form of recurrence were evaluated on the tumor-removed mice.</p><p><b>RESULTS</b>Mice immunized by NTVJmE6E7 could generate TC-1 cell specific cytotoxic T lymphocyte (CTL). Mice boosted with NTVJmE6E7 could tolerate the challenge of 1 x 10(4) TC-1 cells. NTVJmE6E7 could effectively prevent the tumor recurrence in the tumor-removed mice.</p><p><b>CONCLUSION</b>NTVJmE6E7 can be taken as a candidate of therapeutic vaccine for HPV-associated tumors and their precursor lesions.</p>