[Growth and differentiation effect on HL60 cells by adenovirus-mediated exogenous wnt5a gene modification on human bone marrow mesenchymal stem cells].

OBJECTIVE: To study the effect of human bone marrow mesenchymal stem cell (bMSCs) modified by the adenovirus-mediated exogenous wnt5a gene on the hematopoietic function of bone marrow and the inhibition of the growth of HL60 leukemia cells. METHODS: BMSCs were identified through flow cytometry and modified by Ad5-wnt5a, The transfection rate of wnt5a gene in bMSCs was detected by RT-PCR. The cell growth curves were detected in different groups (bMSCs group, Ad5-GFP group, Ad5-wnt5a group). Ad5-wnt5a-bMSCs and HL60 cells were co-cultured, the surface differentiation antigen (CD13, CD14, CD68) and cell cycles were detected by immunocytochemical and flow cytometry in different groups (HL60 cell group, HL60+bMSCs group, HL60+ Ad5-GFP group, HL60+ Ad5-wnt5a group), respectively. RESULTS: Adenovirus-mediated exogenous wnt5a gene was transfected into bMSCs. The expression of differentiation antigens of CD14, CD68 in HL60+Ad5-wnt5a group were higher than those in control group (P < 0.05), the expression of differentiation antigen CD13 were not significant difference in different groups (P > 0.05). Compared with control group, the cell cycle in HL60+ Ad5-wnt5a group was blocked at G2 phase in the fourth day. CONCLUSION: Exogenous wnt5a gene can promote the growth of bMSCs, and induce HL60 cells to differentiation and maturation.

[Clinical significance of HA117 expression in children with acute leukemia].

OBJECTIVE: To explore the role of the expression of HA117 gene in bone marrow mononuclear cells (BMMNC) with acute leukemia and multidrug resistance. METHODS: HA117 gene expressions in 36 children with acute leukemia and 10 children with Idiopathic thrombocytopenic purpura (ITP) were tested using semi quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique. RESULTS: The HA117 gene was expressed in 75% of children with acute leukemia. There was no significant difference in HA117 gene expression between children with acute lymphoblastic leukemia (ALL, 69.57%) and children with acute myeloid leukemia (AML, 91.67%). But the semi-quantitative expression of HA117/beta-actin in AML childern was significantly higher than in ALL children (q=4.5852, P<0.01). The expressions of HA117 gene and HA117/beta-actin in both ALL and AML children were significantly higher than in ITP children chi2=5.05, 8.81; q=4.4612, 6.9695; P<0.05). The remission patients had lower expression of HA117/beta-actin and similar expression of HA117 compared with initially diagnosed patients. The remission patients had higher expression of HA117 gene and similar expression of HA117/beta-actin compared with patients with ITP. The non-remission patients had higher expression of HA117/beta-actin than remission patients and ITP patients (q=3.1705, 4.4102, P<0.05), but no significant difference from initially diagnosed patients (q=0.5470, P>0.05). CONCLUSION: The expression of HA117 gene is high in the BMMNC of initially diagnosed and non-remission patients with AL. But the remission patients have similar semi-quantitative expression of HA117 as patients with ITP, which indicates that a quantitative testing is more important. The expression of HA117 gene decreases with the improvement of the illness. HA117 is one of the factors that may affect the clinical remission of AML. The new gene HA117 may also be associated with multidrug resistance of leukemia.

Screening and cloning of multi-drug resistant genes in HL-60/MDR cells.

OBJECTIVE: To screen and clone multi-drug resistance (MDR) related genes in MDR acute myeloid leukemia cells (HL-60/MDR). METHODS: HL-60/MDR was established using All-Trans Retinoic Acid. With the HL-60 cells as "tester" and HL60/MDR as "driver", the cDNA library of HL-60/MDR was established by suppression subtractive hybridization. Then 12 of the resulting subtracted cDNA clones were selected for DNA sequencing and homology analysis. The obtained expressed sequence tags (ESTs) were analyzed with the GenBank BLASTN program to identify sequence homologies to known genes. RESULTS: The HL-60/MDR cells had different multi-drug resistance to six kinds of chemotherapeutic drugs. The 211 positive gene clones in differential cDNA library of HL-60/MDR cells were amplified with PCR and 46 gene clones exhibited differential expression (ratio >3). Twelve gene clones with significant differential expression (ratio >5) were screened out to homology analysis. Of these, 11 matched known genes and the rest 1 showed no significant homology to human or non-human known sequences. It was named as gene clone HA117. CONCLUSIONS: This effort provides the partial list of genes differential expressed in HL-60/MDR cells and a novel gene HA117 was found to be related to MDR. Identification of these genes contributes to our understanding of MDR development, and potentially provides candidate target genes to overcome MDR.

[Effect of siRNA targeting VEGF on cell apoptosis and the expression of survivin in K562 cells.].

OBJECTIVE: To study the effect of adenovirus vector-mediated siRNA targeting vascular endothelial growth factor(VEGF) on apoptosis and the expression of survivin in K562 cells. METHODS: K562 cells were infected with recombinant adenovirus Ad5-VEGFsi for 72 hours as experimental group (K562/Ad5-VEGFsi), and empty vector group (K562/Ad5) and blank control group (K562) as controls. VEGF mRNA and survivin mRNA expression were determined by RT-PCR. The protein levels of VEGF and survivin were measured by ELISA and Western blot, respectively. The apoptosis of K562 cells was detected by flow cytometry. RESULTS: The levels of VEGF and survivin mRNA expression in experimental group cells were significantly decreased (P < 0.01). The protein concentration of VEGF in experimental group supernatant was (1121 +/- 15) pg/ml, being lower than that in empty vector group \[(1290 +/- 28) pg/ml\] and black control group \[(1303 +/- 28) pg/ml\] (P < 0.01), and the level of survivin protein in experimental group (0.26 +/- 0.11) was significantly reduced compared with that in blank control group (0.74 +/- 0.10) (P < 0.01). The apoptosis rate of K562/Ad5-VEGFsi cells (16.45 +/- 0.14)% was higher than those of K562/Ad5 cells (3.54 +/- 0.17)% and K562 cells (2.56 +/- 0.20)% (P < 0.01). CONCLUSIONS: VEGF can up-regulate the expression of survivin. After inhibition of VEGF by RNAi, the expression of survivin is decreased subsequently and the rates of cell apoptosis are increased.