Expression of phosphatidic acid phosphatase type 2 domain containing 1A in different human colorectal cancer cells / 新乡医学院学报

Objective To investigate the significance of the expression of phosphatidic acid phosphatase type 2 domain containing 1A(PPAPDC1A) in human colorectal cancer cell lines.Methods The high metastatic potential cells LOVO,SW620 and low metastatic potential cells SW480,RKO,HCT116 and DLD-1 were cultured,the expression of PPAPDC1A mRNA and protein in different colorectal cancer cells in logarithmic growth period was detected by real-time quantitative polymerase chain reaction and Western blot.Results There were significant differences in the expressions of PPAPDC1A mRNA and protein among the six human colorectal cancer cells (F =41.213,344.1 16;P ＜ 0.05).The expression of PPAPDC1 A mRNA and protein in highly metastatic potential cells LOVO and SW620 was significantly higher than that in DLD-1,HCT116,RKO and SW480 cells (P ＜0.05).The expression of PPAPDC1A protein in LOVO cells with high metastatic potential was significantly higher than that in SW620 cells(P ＜ 0.05).The expression of PPAPDC1A protein in DLD-1 cells was significantly higher than that in HCT116,RKO and SW480 cells (P ＜0.05).The expression of PPAPDC1 A protein in HCT116 cells with low metastatic potential was significantly higher than that in RKO and SW480 cells (P ＜ 0.05).The expression of PPAPDC1 A protein in RKO cells was significantly higher than that in SW480 cells (P ＜ 0.05).There was no significant difference in the expression of PPAPDC1A mRNA between LOVO and SW620 cells (P ＜ 0.05).There was no significant difference in the expression of PPAPDC1A mRNA between SW480,RKO,HCT116 and DLD-1 cells (P＜ 0.05).Conclusion PPAPDC1A expresses differentially in colorectal cancer cell lines,which may be involved in the invasion and metastasis of colorectal cancer.

Determination of the binding site of hypoxia inducible factor-2a to the MMP-2 promoter / 临床与实验病理学杂志

Purpose To identify the binding of hypoxia in-ducible factor-2a (HIF-2 a) to the hypoxia-response element(HRE) of the matrixmetallo proteinases-2 (MMP-2) gene promoter region and clear the binding site. Methods Electro-phoretic mobility shift assay (EMSA) was used to identify the binding of HIF-2 a to HRE of the MMP-2 gene promoter region in vitro. At the same time, the chromatin immunoprecipitation assay (CHIP) was used to further determine the binding site. Results Successful prediction of two potential HIF-2a binding sites of MMP-2 the promoter region, which were-217～-204 and-1 029 ～-1 007, respectively. Probe test shows that the marked efficiency of sense chain and antisense chain was above 50％, and they could be used for EMSA-electrophoretic mobility shift assay. The results of EMSA showed that there was a binding site of HIF-2 a sense chain and antisense chain moter region int-217～-204. The results of chromatin immuno-precipitation showed that in the experimental group and control group an about 250 bp fragment in MMP-2 promoter containing HRE region was amplified, suggesting that the protein of HIF-2a binded to the HRE in MMP-2 promoter region in vivo. Conclusion HIF-2 a in MMP-2 promoter regionne promoter region in vitro and in vivo.

Construction of granulocyte-macrophage colony stimulating factor gene eukaryotic expressing plasmid and identification of its biological activity / 实用儿科临床杂志

Objective To construct mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) gene eukaryotic expressing plasmid pcDNA3-GM-CSF, to transfect the recombinant into erythroleukemia cell line FBL-3, and identify their biological activity.Methods GM-CSF gene eukaryotic expressing plasmid was constructed by subclone and recombinant was transfected into FBL-3 cells by electroporation. After screening by G418 and cloning by limiting dilution,we obtained positive cell clones(FBL-3-GM-CSF). PCR and RT-PCR were used to identify the integration and stable expression of GM-SF gene in FBL-3-GM-CSF cells. The biological activity was confirmed by the hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. Results Mouse GM-CSF cDNA was amplified from the prokaryotic expressing plasmid PET-30a(+)-GM-CSF by PCR firstly and BamH Ⅰ and EcoRⅠrestriction sites were introduced. The inserted fragment was cut by BamH Ⅰ and EcoR Ⅰ digestion and ligated into pcDNA3 vector. The pcDNA3-GM-CSF eukaryotic expressing plasmid was constructed. The recombinant was cleared with appropriate endoneucleases and sequenced. The findings showed that the orientation of the insert was correct, while no rearrangement or mutation was found. PCR and RT-PCR assay showed that GM-CSF gene had integrated into FBL-3-GM-CSF cells and stably expressed. The hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay demonstrated that the cultured supernatant of FBL-3-GM-CSF cells of expressing GM-CSF should obviously stimulate proliferation of murine marrow mononuclear cells, and could stimulate hematopoietic progenitor cell colony formation. The number of colony formation was 54.67?4.83. The rate of colony formation was 0.547 %.Conclusions GM-CSF gene eukaryotic expressing plasmid is constructed successfully. A cell clone, which can express stably GM-CSF gene and possess biological activity,is obtained. Our studies have founded the base for the preparation of GM-CSF gene-modified vaccine of tumor cell and the study of feasibility of immune therapy of leukemia.