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Effects of Retinoic Acid Related Gene JWA on Function of Human Bone Marrow Stromal Cells in Vitro

Author: ChangCheng
Tutor: ChenXingHua
School: Third Military Medical University
Course: Internal Medicine
Keywords: bone marrow stromal cells retinoic acid JWA gene hammerhead ribozyme migration adhesion
CLC: R55
Type: PhD thesis
Year: 2006
Downloads: 35
Quote: 0
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Abstract

ObjectiveHematopoietic microenvironment (HME) has been proved to support and regulate the homing, proliferation, differentiation, development and mature of hematopoietic cells. As a major component of HME, bone marrow stromal cells (BMSCs) play a very important role in the function of HME. Impairment of BMSCs followed by the injury of HME delays the hematopoietic reconstitution of hematopoietic stem cell transplantation. It is one of the important ways to restore hematopoietic microenvironment of promoting the functions of BMSCs. It has been shown that retinoic acid (RA), especially all-trans retinoic acid (atRA), regulates the adhesion and migration function of BMSCs by regulating the expression of target genes. Therefore, those retinoic acid associated genes may play an important role in the function regulation of BMSCs. JWA gene is a novel putative cytoskeleton associated and retinoic acid responsible gene, which might be involved in cellular proliferation, differentiation, stress response, amino acid metabolism, intracellular trafficking and nuclear transportation. It is reasonable to believe that cellular proliferation and differentiation induced by atRA are mediated at least in part through regulation of JWA gene expression. So, JWA gene may play a role in the function regulation of BMSCs and hematopoietic reconstitution.In order to evaluate the effects of JWA gene on the regulation of adhesion and migration function of BMSCs, this study was designed to observe the relationship between the changes of BMSCs function and expression of JWA gene after BMSCs were treated with atRA, and detect the changes of adhesion and migration function of BMSCs after JWA gene expression was inhibited by ribozyme.Methods1. BMSCs of 27 patients before and after PBSCT treatment were cultured in vitro. After treated with atRA at 0.01μmol/L, 0.1μmol/L, or 1μmol/L, expression of ICAM-1 protein and VCAM-1 protein were detected by flow cytometric analysis, and soluble ICAM-1 (sICAM-1) protein was determined by radioimmunoassay. Then BMSC was co-cultured with CD34~+ cells, and binding rate of BMSC to CD34~+ cells was measured. 2. BMSCs of patients excluded with malignant diseases were cultured in vitro. After administration of atRA at different doses(0.01μmol/L, 0.1μmol/L, 1μmol/L, 10μmol/L), expression of JWA mRNA in cells was determined by semi-quantitive RT-PCR method. Cell ultrastructure was observed with electron microscopy. Expressions of ICAM-1 and VCAM-1 protein were detected by flow cytometric analysis. Expressions of FN and LN protein were detected by immunohistochemical. Cell migration was measured using modified Boyden’s chamber. The re-organization of F-actin in cells was analyzed by confocal microscopy. Then BMSCs were co-cultured with Jurkat cells, and binding rate of BMSCs to Jurkat cells and proliferation of Jurkat cells were measured. Expression of FAK mRNA and protein of Jurkat cells were detected by sqRT-PCR and Western blotting, respectively. Expressions of VLA-4 and LFA-1 protein of Jurkat cells were detected by flow cytometric analysis. The correlation between expression of JWA mRNA and other indexes about adhesion and migration function was investigated statistically.3.A recombinant JWA-specifichammerheadribozymeretroviralvector was constructed and transferred into cultured human BMSCs. The effect of ribozyme on expression of JWA mRNA was detected by sqRT-PCR.4. Expressions of ICAM-1 and VCAM-1 protein in transfected cells were detected by flow cytometric analysis. Expressions of FN and LN protein in transfected cells were detected by immunohistochemical. Cell migration of transfected cells was measured using modified Boyden’s chamber. The expressions of F-actin andα-tubulinin in transfected cells were analyzed by confocal microscopy. Then transfected cells were co-cultured with Jurkat cells, and binding rate of transfected cells to Jurkat cells and proliferation of Jurkat cells were measured.Results1. After treatment with conditioning redimen for PBSCT, the expressions of ICAM-1 and VCAM-1 proteins in BMSCs and the expression level of sICAM-1 protein in supernatant of BMSCs culture were down-regulated, and the binding rate of BMSCs to CD34~+ cells decreased. After administration of atRA, the expression of ICAM-1 protein in BMSCs, sICAM-1 protein in culture medium and binding rate of BMSCs to CD34~+ cells all increased significantly.2. After treated with atRA, mitochondria and endoplasmic reticulum in BMSCs were found easily, and microfilament was obvious. Expression of JWA mRNA, ICAM-1 protein, VCAM-1 protein, FN protein, LN protein, F-actin protein and the migrating cells of BMSCs increased significantly as the increase of atRA concentration. Binding rate of BMSCs to Jurkat cells increased significantly and expression of VLA-4 protein, LFA-1 protein, FAK protein and FAK mRNA in co-cultured Jurkat cells and proliferation of Jurkat cells increased significantly. Correlation analysis revealed that there was a positive correlation between JWA mRNA expression and atRA concentration, ICAM-1 expression, VCAM-1 expression, LN expression, binding rate of BMSCs to Jurkat cells, and the migrating cells of BMSCs respectively.3. In contrast to non-transfected cells (BMSCs group) and cells transfected only with pLXSN (B-pLXSN group), JWA mRNA expression decreased significantly in the cells transfected with recombinant vector of ribozyme (B-JWARZ group).4. In contrast to BMSCs group and B-pLXSN group, expression of ICAM-1, VCAM-1, FN, LN, F-actin andα-tubulin protein and the number of migrating cells in B-JWARZ group decreased significantly. In B-JWARZ group, the binding rate to Jurkat cells and proliferation of Jurkat cells also decreased significantly.Conclusions1. atRA can partly restore adhesion function of BMSCs injured by pretreatment for PBSCT by upregulating the expression of adhesion molecules.2. atRA maybe regulate the function of BMSCs by upregulating the expression of JWA gene..3. JWA gene and JWA protein may play a role in the regulation of expression of cell adhesion molecules, maintain of cell shape and migration function of BMSCs.This study may provide a new sight of mechanisms research of gene regulation of BMSCs function and contribute to the research of regulation mechanisms of homing, adhesion, differentiation and proliferation of hematopoietic stem cells. These results also contribute to reveal and provide an indirect experimental evidence of the functions of JWA gene. It is a promising idea worthy to be studied that if BMSCs modified with JWA gene could promote the rehabilitation of injured hematopoietic microenvironment.

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