Investigation On The Difference Of GJIC And Mechanism In Acute Leukemia Bone Marrow Stromal Cells

Introduction: HIM is the situation of orientation, proliferation, differentiation and development of HSC, of which hematopoietic stromal cell is an important component. Hematopoietic stromal cell regulate the self-renewing, proliferation and differentiation by direct contact hematopotic cells, producing HGF and ECM to supporting the normal hematopoietic ability of body. The research and application on injury and repair of hematopoietic function in the past focus more on HSC, and it is needed to further study of the stromal cells.Gap junction intercellular communication (GJIC), which was mainly composed of connexon, was the principal channel to transmit the signal between adjacent cells, and among all the connexons Cx43 mainly exists in hematopoietic tissue. Recent evidences have demonstrated that the Cx43 protein was expressed and GJIC could be formed between bone marrow stromal cells (BMSCs), which played a key role in the establishment, proliferation, differentiation and maturity of haemopoietic stem cell. Acute leukemia was a common kind of hematopoietic system malignant tumor, and BMSCs derived from leukemia that supported and protected leukemic cells involved in the occurrence and refractory of leukemia. So we suppose whether Cx43 expression level and GJIC function of leukemic BMSCs were different from normal BMSCs? Whether GJIC function of leukemic BMSCs could be transformed by some drugs? Whether capability of supporting hematopoiesis and bionomics of leukemic BMSCs would be affected after the reconstitution of GJIC function? No correlated data was available; therefore in this study we explored the way to resolve above-mentioned problems.Results:1. Evaluated the expression level of Cx43 in normal, primary acute leukemic and complete remission (CR) BMSCs by immunocytochemistry, laser scan confocal microscope (LSCM) technology, flow cytometry, and RT-PCR, and explored the function of GJIC by scrape-loading and dye transfer method (SLDTM) and fluorescence redistribution after photobleaching (FRAP). Result indicated that (1) The expression of Cx43 in ABMSCs, NBMSCs and ABMSCs-CR by immunocytochemistry were (47.2±2.04)%, (86.4±6.23)% and (61.2±8.24)% respective. Expression of Cx43 in ABMSCs was significant lower than it in NBMSCs and ABMSCs-CR (p<0.01). (2) The expression of Cx43 in ABMSCs, NBMSCs and ABMSCs-CR by FCM were (38.75±23.95)%, (76.51±13.15) and (71.98±7.5)% respective. Expression of Cx43 in ABMSCs was significant lower than it in NBMSCs and ABMSCs-CR (p<0.01). (3) The pinel density of ABMSCs, NBMSCs and ABMSCs-CR by LSCM were (34.10±17.91)%, (81.04±8.84)% and (70.33±4.90)% respective. Expression of Cx43 in ABMSCs was significant lower than it in NBMSCs and ABMSCs-CR (p<0.01). (4) mRNA level of Cx43 in ABMSCs was significant lower than it in NBMSCs and ABMSCs-CR (p<0.01). (5) Transmission of LY in NBMSCs and ABMSCs-CR were 10 and 4⁵ cell lines, which was significant higher than it in ABMSCs (p<0.01). (6) CFIRR of ABMSCs was 5.58±1.00(%/min), which was significant lower than it in NBMSCs and ABMSCs-CR (p<0.01).2. To observe whether GJIC function of leukemic BMSCs could be transformed by some drugs, all-trans retinoic acid and amphotericin B were employed in our study. Our finding showed that (1) The expression of Cx43 in ABMSCs, ABMSCs-ATRA and ABMSCs-AB by immunocytochemistry were (47.2±2.04)%, (54.5±5.66)% and (45.3±3.26)% respective. Expression of Cx43 in ABMSCs-ATRA was significant higher than it in ABMSCs (p<0.01), and expression of Cx43 in ABMSCs-AB has no statistics difference with ABMSCs (p > 0.05). (2) The expression of Cx43 in ABMSCs, ABMSCs-ATRA and ABMSCs-AB by FCM were (38.75±23.95)%, (49.5±5.46) and (35.9±4.06)% respective. Expression of Cx43 in ABMSCs-ATRA was significant higher than it in ABMSCs (p<0.01), and expression of Cx43 in ABMSCs-AB has no statistics difference with ABMSCs (p>0.05). (3) The pinel density of ABMSCs, ABMSCs-ATRA and ABMSCs-AB by LSCM were (34.10±17.91)%, (45.25±5.98)% and (32.90±3.98)% respective. Expression of Cx43 in ABMSCs-ATRA was significant higher than it in ABMSCs (p<0.01), and expression of Cx43 in ABMSCs-AB has no statistics difference with ABMSCs (p>0.05). (4) mRNA level of Cx43 in ABMSCs-ATRA was significant lower than it in ABMSCs and ABMSCs-AB (p<0.01). (5) Transmission of LY in ABMSCs and ABMSCs-AB were 1² and 0 cell lines, which was significant lower than it in ABMSCs (p<0.01). (6) CFIRR of ABMSCs-ATRA was 15.03±1.27(%/min), which was significant higher than it in ABMSCs(p<0.05), and ABMSCs-AB (0%/min) was significant lower than it in ABMSCs (p<0.01).3. After interfered by all-trans retinoic acid, the improvement of GJIC function lead to the increased apoptosis rate(5.16±2.35%→9.78±4.88%), delayed multiplication cycle, down-regulated secretion of GM-CSF(10280.0±950→9260.0±123) and SCF(1490.7±188→1327.1±118), enhanced inflow of extracellular Ca2+ in leukemic BMSCs(17.92±3.31nmoL/L→76.54±9.6nmoL/L ) (p<0.01).Conclusions:1. Cx43 expression in leukemic BMSCs was abnormally localized and was significantly lower than in normal and CR BMSCs, and the GJIC function of leukemic BMSCs notably reduced.2. Cx43 expression and GJIC function in leukemic BMSCs was up-regulated by all-trans retinoic acid, however, although Cx43 expression remained lower, GJIC was blocked by amphotericin B.3. The improvement of GJIC function lead to the increased apoptosis rate, delayed multiplication cycle, few secretion of GM-CSF and SCF, enhanced inflow of extracellular Ca²⁺ in leukemic BMSCs.