Study Of The Regulative Effect Of Cx43 Gene Modified Leukemic Bone Marrow Stroma Cells On Jurkat Cells In Vitro

Hematopoietic inductive microenvironment (HIM), which was mainly composed of bone marrow stromal cells (BMSCs), acted as the soil in regulating the growth and development of hematopoietic precursor cell (HPC). The development and proliferation of leukemic cells were dependent on the abnormal HIM. The BMSCs derived from leukemia that supported and protected leukemic cells involved in the occurrence and refractory of leukemia. Therefore, it is of great importance and necessary to investigate the difference of normal BMSCs and leukemic BMSCs, so as to explore novel therapeutic target for leukemia.BMSCs have been shown to involve in the hematopoietic regulation through the intercellular communication between BMSCs, as well as between BMSCs and hematopoietic cells. Gap junction intercellular communication (GJIC) was the direct communication mode which generally existed between adjacent cells. Previous evidences have demonstrated that the connexin 43 (Cx43)-mediated GJIC mainly exists between BMSCs, and is essential to BMSCs regulateding normal hematopoiesis, however it is unknown that whether Cx43-mediated GJIC contributed to the intercellular communication between BMSCs and hematopoietic cells as well as between hematopoietic cells. It is reported that the mechanism of benzene and its metabolin leading to leukemia and aplastic anemia is concerned with the lower expression of Cx43 and the miopragia of GJIC, and normal BMSCs can inhibit the proliferation of leukemic cells through GJIC mediated by Cx43. On account of the above studies, it showed that Cx43-mediated GJIC plays a key role in BMSCs regulating normal hematopoiesis, and we suppose that the down-regulation or absence of Cx43 expression and the miopragia of GJIC probably induce the abnormal hematopoietic regulation of leukemic BMSCs, which accordingly affect the proliferation, differentiation and apoptosis of leukemic cells and at last result in the occurrence and progress of leukemia.Based on the background above, in this study, normal BMSCs and leukemic BMSCs were separated and cultured in vitro, and the expression of Cx43 and GJIC function were detected. To up-regulate and stabilize the expression of Cx43, an adenovirus vector with Cx43 gene was constructed and transfected into leukemic BMSCs, with which leukemic cells (Jurkat cells) were co-cultured, then the proliferation, differentiation, apoptosis and drug sensitivity of these Jurkat cells were observed. The aim is to provide theoretical and experimental evidence for the therapy of leukemia from the point of transforming HIM.1. Contents and methods:1.1 Seperated and cultured the normal and leukemic human BMSCs in virto, evaluated the expression of Cx43 mRNA and protein by RT-PCR, immunocytochemistry and laser confocal microscope scan (LCSM) technique, and explored the function of GJIC by scrape-loading and dye transfer method (SLDTM).1.2 By the DNA recombination, the target gene Cx43 was cloned into the shuttle plasmid which expressed the report gene GFP. After the shuttle plasmid and adenovirus genome were homologously recombined in BJ5183 cells, recombinant adenovirus finished the package and reproduction in 293 cells, and obtained recombinant adenovirus Ad-Cx43-GFP and Ad-GFP, respectively. Then, the expression of GFP and Cx43 protein in 293 cells was detected by fluorescent inverted microscope and Western blot.1.3 Used Ad-Cx43-GFP and Ad-GFP to infect the leukemic BMSCs, respectively, and detected the change of Cx43 expression in these BMSCs by RT-PCR, immunocyto- chemistry and LCSM technique, then explored the functional change of GJIC between these BMSCs by SLDTM.1.4 By establishing co-cultured model of leukemic BMSCs and Jurkat cells, we observed the phase relationship bewteen Jurkat cells and BMSCs. Then, Jurkat cells'growth curve and doubling time (DT) was assayed by MTT method, and cell cycle, rate of apoptosis and death of Jurkat cells were detected by flow cytometry and transmission electron microscope, respectively. Besides, the expression of bcl-2, bax and p53 of Jurkat cells was detected by immunocytochemistry. At last, the effect of 5-FU, HHT and As₂O₃ on the rate of survival and apoptosis of Jurkat cells was assayed by MTT and flow cytometry.2. Results: 2.1 Comparison of Cx43 expression and GJIC function between the normal and leukemic human BMSCs: The RT-PCR results showed that Cx43 mRNA of leukemic BMSCs was lower than that of normal BMSCs (P<0.05). The results from immunocyto- chemistry and LCSM technique indicated that Cx43 proteins of leukemic BMSCs were significantly lower than that of normal BMSCs (P<0.01), and Cx43 was maily expressed on the cytoplasmic membrane of normal BMSCs but on the cytoplasm of leukemic BMSCs, and neither of them expressed on the nucleus. The Comparison of GJIC fuction suggested that dye spended 1-2mins passing through the normal BMSCs, however spended 8mins passing through leukemic BMSCs, and dye transfer intensity of leukemic BMSCs were significantly lower than that of normal BMSCs (P<0.01).2.2 Construction of recombinant adenovirus Ad-Cx43-GFP and Ad-GFP: Cx43 gene was amplified from pcDNA3.0-Cx43 plasmid by PCR and was cloned into the shuttle plasmid expressing the report gene GFP. After linearized recombinant and un-recombinant shuttle plasmid were homologously recombined with pAdEasy-1, respectively, recombinant plasmids were identified by single digestion with restriction endonucleases Pac I, and results suggested that recombinant plasmids pAd-GFP and pAd-Cx43-GFP were successful- ly constructed. Then, these recombinant plasmids were transfected into 293 cells via liposome finishing the package and reproduction, and the protein expression in 293 cells transfected with these recombinant plasmids was detected by Western blot, which showed a conspicuous strap in 43KDa in 293 cells transfected with pAd-Cx43-GFP, however, 293 cells untransfected and transfected with pAd-GFP protein only showed thin expression.2.3 The green fluorescence could be observed in BMSCs about 24h after the recombinant adenovirus Ad-Cx43-GFP and Ad-GFP were transfected into leukemic BMSCs, and the efficiency of transfection were (82.43±3.00)% and (82.29±3.27)%, respectively. By semiquantitative RT-PCR, immunocytochemistry and LCSM technique, the Cx43 expression of leukemic BMSCs transfected with Ad-Cx43-GFP was confirmed to be significantly higher than those transfected with Ad-GFP. The results from LCSM technique indicated that Cx43 expression was significantly increased on the cytoplasmic membrane of BMSCs transfected with Ad-Cx43-GFP. The comparison of GJIC fuction between leukemic BMSCs transfected with Ad-Cx43-GFP and Ad-GFP showed that the dye transfer intensity of Ad-Cx43-GFP group BMSCs was higher than Ad-GFP BMSCs (P<0.01), and the speed of dye transfer was faster than Ad-GFP BMSCs.2.4 The regulative effect of Cx43 gene modified leukemic BMSCs on Jurkat cells:Groups: Jurkat group-Jurkat cells; BMSCs/Jurkat group-Jurkat cells co-cultured with leukemic BMSCs; Ad-BMSCs/Jurkat group-Jurkat cells co-cultured with leukemic BMSCs transfected with Ad-GFP; Ad-Cx43-BMSCs/Jurkat group Jurkat cells co-cultured with leukemic BMSCs transfected with Ad-Cx43-GFP.â‘ Observed by light microscope, Jurkat cells grew in suspension fashion, and was round and mostly dispersed. After co-cultured with leukemic BMSCs, Jurkat cells were adhere to leukemic BMSCs through their pseudopodia by scanning electron microscope.â‘¡The proliferation speed of Jurkats cells derived from Ad-Cx43-BMSCs/Jurkat group (DT: 28.85h) was faster than those from BMSCs/Jurkat group (DT: 32.42h) and Ad-BMSCs/Jurkat group (DT: 31.81h) (P<0.01), but was slower than those from Jurkat group (DT: 26.38h) (P<0.01).â‘¢The Jurkat cell cycle distribution and proliferation activity detected by flow cytometry showed no difference between BMSCs/Jurkat and Ad-BMSCs/Jurkat group (P>0.05). The DI value of Jurkat cells in four groups ranged from 1.9 to 2.1, which indicated Jurkat cells in our study were heteroploid cells. Among four groups, Jurkat cells in Ad-Cx43-BMSCs/Jurkat group were characterized as the highest S phase cell rate, diploid DNA rate, SPF and PI value, and the lowest G0/G1 phase cell rate, heteroploid DNA rate (P<0.01), but inversely, Jurkat cells in BMSCs/Jurkat and Ad-BMSCs/Jurkat group were characterized as the lowest S phase cell rate, diploid DNA rate, SPF and PI value, and the highest G0/G1 phase cell rate, heteroploid DNA rate (P<0.01). Moreover, an apoptotic hypodiploid peak was observed before G0/G1 phase in each group.â‘£Flow cytometry assay showed that a certain rate of spontaneous apoptosis and death Jurkat cells existed in each group. Among four groups, the apoptosis and death rate of Jurkat cells were highest in Ad-Cx43-BMSCs/Jurkat group (P<0.01), and were lowest in BMSCs/Jurkat and Ad-BMSCs/Jurkat group which showed no difference between these two groups. Moreover, it was further confirmed that apoptotic cells existed in each group by transmission electron microscope, and immunocytochemical assay indicated, the bcl-2 and p53 protein expressed were lower in Jurkat cells of Ad-Cx43-BMSCs/Jurkat group than in those of Jurkat and BMSCs/Jurkat group (P<0.01), and were highest in those of BMSCs/Jurkat group (P<0.01), besides, the bax protein expressed were higher in Jurkat cells of Ad-Cx43-BMSCs/Jurkat group than in those of Jurkat and BMSCs/Jurkat group (P<0.01), and were lowest in those of BMSCs/Jurkat group (P<0.01).⑤After treated with different concentration of 5-FU and As₂O₃, the survival rate of Jurkat cells in each group descended, and it was lowest in Ad-Cx43-BMSCs/Jurkat group (P<0.01), but was highest in BMSCs/Jurkat group (P<0.01). In addition, the IC50 value of Jurkat cells in Ad-Cx43-BMSCs/Jurkat group was 2 times lower than that in BMSCs/Jurkat group. After treated with different concentration of HHA, the survival rate of Jurkat cells in each group descended too, but it was no different between Ad-Cx43-BMSCs/Jurkat group and Jurkat group (P>0.05), however, the survival rate in both of these two groups were lower than that in BMSCs/Jurkat group (P<0.01). The IC50 value of Jurkat cells in Ad-Cx43-BMSCs/Jurkat and Jurkat group was 2.5 times lower than that in BMSCs/Jurkat group.5-FU, As₂O₃ and HHT could induce the apoptosis of Jurkat cells, and with the raise of drug concentration, the apoptosis rate of Jurkat cells increased gradually, which appeared obvious increasing in Jurkat cells of Ad-Cx43-BMSCs/Jurkat group (P<0.01). But, when the concentration of HHT achieved 0.2mg/L, no marked change in the apoptosis rate of Jurkat group and Ad-Cx43-BMSCs/Jurkat group was detected even if continuously increasing the concentration of HHT, and the same condition had happened in BMSCs/ Jurkat group as the concentration of HHT achieved 0.5mg/L. Furthermore, linear correlation statistical analysis showed that inverse correlation existed between the survival rate and apoptosis rate after treated with different concentration of three kinds of drugs (P<0.01).3. Conclusions:3.1 Cx43 expression in leukemic BMSCs was abnormally localized and was significantly lower than that in normal BMSCs. Comparied with normal BMSCs, the GJIC function of leukemic BMSCs notably degraded.3.2 Succefully constructed the recombinant adenovirus vector Ad-Cx43-GFP which highly expressed the Cx43 gene. The function of GJIC between leukemic BMSCs modified by Cx43 gene was up-regulated.3.3 Co-cultured model of leukemic BMSCs and Jurkat cells was succefully constructed. The proliferation and spontaneous apoptosis of Jurkat cells were elevated after co-cultured with the Cx43 gene modified leukemic BMSCs, besides these Jurkat cells highly expressed bax protein and lowly expressed bcl-2 and p53 protein.3.4 Rebuilding the GJIC function of leukemic BMSCs made co-cultured Jurkat cells more sensitive to 5-FU, HHT and As₂O₃, and increased the rate of drug induced Jurkat cells apoptosis. But, apoptosis was probably not mainly responsible for the growth inhibiting of Jurkat cells induced by HHA.