Ex vivo Culture System of Single Human Hematopoietic Stem Cell Used to Screan the Small Molecular Compounds / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore an efficient, stable system and method to verify the regulation effect of small molecule compounds on human hematopoietic stem cells (hHSC).</p><p><b>METHODS</b>By using combination of flow cytometry with study results of surface markers on hHSC, and optimation of sorting process for further studying the effect of small molecular compounds on stem property of hHSC, the single hHSC was treated with published small molecular compounds such as SR1 and UM171 which possess the expansion effect. After treating with hHSC for 14 d, the flow cytometric analysis of cell phenotypes and cell morphologic observation were performed, at the same time the hematopoietic function of cultured hHSC was verified by colony-forming cell (CFC) test and cobblestone area forming cell (CAFC) test.</p><p><b>RESULTS</b>The effects of SR1 and UM171 and their compositions in multi-cell culture were consistent with the published data, therefore the useful concentration of compounds were obtained. The results of multiparameter sorting of single cell (CD34+ CD38- CD45RA- CD90+ CD49f+) and ex vivo culture were consistent with the results of bulk cell culture. The results of cell phenotype analysis was in accordance with flow cytometric results. In addition, CFC test and CAFC test revealed that the colony-forming ability in treated group was significantly higher than that in control group (P<0.05).</p><p><b>CONCLUSION</b>The rapid, efficient stably amplified and short-time culture system for single hHSC and method for varifying the effect of small molecular compounds are established, which provides platform for screening small molecular compounds and lays the foundation for further study of hHSC expansion.</p>

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhIL-24) on pancreatic cancer.</p><p><b>METHODS</b>Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n = 10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD).</p><p><b>RESULTS</b>The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0 x 10(10) pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05).</p><p><b>CONCLUSION</b>The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.</p>

Effect of endothelial PAS domain protein 1 and hypoxia inducible factor 1alpha on vascular endothelial growth factor expression in human pancreatic carcinoma / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Transcription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma.</p><p><b>METHODS</b>Specimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed.</p><p><b>RESULTS</b>Highly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour.</p><p><b>CONCLUSIONS</b>EPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.</p>