Study Of Human Umbilical Cord Blood-derived Stromal Cells Modificated With Cx43on SUP-15Cells In Vitro

Hematopoietic microenvironment (hemopoietic microenvironment, HME) is seemed asthe “soil” to breed hematopoietic stem/progenitor cell and regulate their proliferation anddifferentiation. Leukemia cells are also supported by hematopoietic microenvironment.Bone marrow stromal cells are the main functional components in HME. Resistance tochemical therapy, residual and refractory of leukemia is considered to be caused by thesupportion from bone marrow stromal cells (bone marrow stroma cells, BMSCs). In addtion,a number of studies have indicated that leukemia minimal residual disease (Minimalresidual Disease/leukemia, MRD/MRL) is the key factor lead to the refractory andrelapse of leukmia. Abnormal hematopoietic microenvironment brings the tumer cells asuitable condition for survival. Therefore, to explore the regulation of hematopoieticmicroenvironment on acute leukemia cells, to seek new methods and new targets forremoval of residual leukemic cells in a new perspective is signifigant meanfull.The signal transduction among bone marrow stromal cellsis associated with Gapjunctional intercellular communication (GJIC) which is mainly mediate by Cx43inhematopoietic tissues. Our previous studies have found that up-regulated the expression ofCx43on bone marrow stromal cells can enhance the function of GJIC. And when weco-cultured BMSCs and leukemia cells, the highly expressed Cx43ones can promoteapoptosis in leukemia cells and inhibit their proliferation.Studies have shown that human umbilical cord blood-derived stromal cells(hUCBDSCs) is better than bone marrow stromal cells both in repairing hematopoieticdamage and inhibiting the proliferation of leukemia. Ph+acute lymphoblastic leukemia(Ph+ALL) appears to be less effective with chemical therapy, even for the patients whoreach complete remission,the rate of short-term relapse is still high. In view of the abovereasons, we isolated and culture hUCBDSCs in vitro, transfected hUCBDCs withadenovirus vector carrying Cx43, and then we co-culture hUCBDSCs with SUP-B15cells(a ph+ALL cell line), investigate the effect on SUP-B15and the possible mechanism. Methods1. We isolate hUCBDSCs from human umbilical cord blood, and establish a model ofco-culture (hUCBDSCs and SUP-B15)in vitro. Improved method was adopted toseparateand culture target cells from the healthy maternal umbilical cord blood. SUP-B15cell lines were cultured as described in instruction. Inverted microscope and scanningelectron microscopy were used fot morphology observation. Exploration of appropriateculture conditions. And we have trid to search for a More appropriate culture system.2. The hUCBDSCs were transfected with adenovirus carrying the gene Cx43(Ad-Cx43-GFP) and Airborne virus (Ad-GFP), and the expression of Cx43were tested byRT-PCR, Western-blot and immunofluorescence. Fluorescence recovery afterphotobleaching (FRAP) was used to detect the function of GJIC after transfection.3. Our hUCBDSCs with or without Cx43transfection have been co-cultured withSUP-B15cells for1to6days. CCK-8test have been used to detect the rate of proliferationof SUP-B15. In order to investigate the impaction to SUP-B15caused by up-grating theexpression of Cx43and the function of GJIC, We used Flow cytometry to test the rate ofapopsis and cells cycle after72hrs’co-culture.ResultsThe co-cultured model was established use MSCM. SUP-B15cells appeared to attachto hUCBDSCs cells layer and gather into groups, with lower dioptric power. The formationof filopodia has been observed under scanning electron microscope(SEM).The hUCBDSCs were transfected with Ad-Cx43-GFP and Ad-GFP kept in our lab.Green fluorescence were caught24hours after transfection, and reached the peak at48h, atthe rate of (89.20±3.12)%and (88.62±3.25)%. RT-PCR, Western-blot andimmunofluorescence showed a higher expression of Cx43after Ad-Cx43-GFP transfection,whether in mRNA and protein. Our result of FRAP indicated that the green fluorescencerecovered in40sec after fluorescence quenching in Ad-Cx43-GFP transfection group,however, it would never recover in the rest of groups.The effect of hUCBDSCs on proliferation of SUP-B15in vitro: The hUCBDCs couldinhibite the proliferation of SUP-B15, And it seemed to be more effective after transfectionwith Cx43(p<0.05). The rate of apoptosis was at5.51±0.51％in the Ad-Cx43-GFPtransfection group, which is higher than in the group of hUCBDCs untransfected with Cx43 (2.26±1.41%) and control group(2.97±1.58％)(p<0.05). The data calculation was done bySPSS13.0.ConclusionsA co-culture model with SUP-B15and hUCBDSCs in vitro has been successfullyestablishedCx43up-regulated hUCBDSCs showed a higher function of GJIC.Cx43modified hUCBDCs can inhibit the proliferation of SUP-B15, as well as improvethe rate of apoposis of SUP-B15.