| BACKGROUND:For acute leukemia hematopoietic stem cell transplantation is an effective treatment.Autologous hematopoietic stem cell transplantation has the advantages of a wide range of application,low cost and safety,but the recurrence rate of patients after transplantation is high and mainly caused by Minimal residual disease(microresidual disease,MRD).The important factor leading to leukemia MRD is the effect of hrematopoietic microenvironment on the growth,support and harboring of leukemia.Stromal cells are the main components of hematopoietic microenvironment and play an important role in regulating the growth of leukemia cells,mediating leukemia residues and drug resistance.Gap junction Intercellular communication(GJIC)is a direct communication between adjacent cells.Connexin43(CX43)mediated GJIC is widely present in bone marrow stromal cells,stromal cells and hematopoietic cells,are also prerequisite for bone marrow stromal involved in hematopoietic regulation.In acute leukemia patients,GJIC function was significantly reduced in accordance with CX43 expression in bone marrow stromal cells Long term of elow GJIC function lever in patients taken autologous hematopoietic stem cell transplantation weaken in the inhibition on leukemia from bone marrow microenvironment,which may be a factor leading to the recurrence of leukemia.Human umbilical cord blood stromal cells(hUCSCs)can inhibit proliferation and promotion of apoptosis in leukemia cell lines in vitro,They repair the hematopoietic microenvironment by homing to bone marrow in vivo.This study investigates if hUCSC can be gene carrier to inhibit the effect of leukemia cells,transfecting new stem cells into MRD mouse,shows that stem cell MDR reconstruction can help purify the MDR from the disease.This project was funded by the National Natural Science Foundation of China(No.81070388)which was titled " The purification effect and mechanism research of CX43 modified human umbilical cord blood-derived stromal cell transplantation on AHSCT residual leukemia ".Tried to affect the proliferation and differentiation process of leukemia cells by changing the GJIC function in hematopoietic microenvironment of leukemia,which could inhibit disease deterioration and help the accumulation of theory and experimental basis on exploring new methods for leukemia treatment.OBJECTIVE : Observe the colonization and repair effect on bone marrow microenvironment of mice co-transplanted with Cx43-modified human umbilical cord blood-derived stromal cell and hematopoietic stem cells,and purify the residual leukemia in transplanted mice by repairing and enhancing the GJIC function to improve hematopoietic regulation of bone marrow microenvironment.Methods:1.CX43 recombinant adenovirus vector(Ad-Cx43-GFP)transfected human umbilical cord blood-derived stromal cellsCells were isolated from umbilical cord blood of healthy pregnant women and then were cultured by improved methods of our department.Ad-Cx43-GFP recombinant adenovirus vector was constructed and then be transfected into human umbilical cord blood-derived stromal cells by routine method.The expression of Cx43 was detected by immunofluorescence,PCR and Western-Blot.The function of GJIC between hUCSCs was detected by fluorescence recovery after photobleaching(FRAP).2.Construct co-culture system of human umbilical cord blood-derived stromal cells and L615 cells to observe the regulation effect of human umbilical cord blood-derived stromal cells on L615 leukemia cells before and after CX43 transfection.1)The experiment was divided into three groups:A: negative control group: L615 cells were cultured separatelyB: hUCSCs / L615 group: co-culture cells group of human umbilical cord blood-derived stromal cells and L615C: Ad-Cx43-hUCSCs/L615 group: co-culture cells group of Ad-Cx43-EGFP transfected human umbilical cord blood-derived stromal cells and L615 cells2)The cell growth curve of L615 cells was measured by CCK8 method,and then their proliferation rates were used for comparison between groups.Flow cytometry was used to detect the apoptosis rate and cell cycle of L615 cells in co-culture system.The flow cytometry was used to detect the proliferation of L615 cells.The expression of caspase3,6,7 in L615 cells from co-culture system was detected by Western-Blot.3.Observe the inhibitory effect of transfected human umbilical cord blood-derived stromal cells on residual leukemia by transplantation of human umbilical cord blood-derived stromal cells combined with autologous hematopoietic stem cells in L615 leukemia MRD mice.1)The L615 cells were injected in L615 homozygous mice via tail vein,and then injected intraperitoneally with cyclophosphamide 200 mg / kg three days later to obtain L615 leukemia MRD mice model.2)The experiment was divided into two groups:A: BM group: bone marrow hematopoietic stem cell transplantation groupB: Ad-Cx43-hUCSCs + BM group: co-transplantation of Ad-Cx43-EGFP transfected human umbilical cord blood-derived stromal cells and bone marrow hematopoietic stem cell3)The residual disease leukemia mice were used as recipients and normal leukemia L615 mice were used as donors.The recipient mice were treated with 6.0 Gy 60 Co γ-ray TBI on day 0;6 h after irradiation,each of them in BM group injected with 2×106 of bone marrow mononuclear cells from donor mice and each of them in Ad-Cx43-hUCSCs + BM group injected with both 1×106 of Dil-labeled Cx43-modified human umbilical cord blood-derived stromal cells and 1×106 of bone marrow mononuclear cells by tail vein.The hematopoietic reconstitution was exanimated by tail vein complete blood court for mice with transplantation in different groups.The bone marrow of transplanted mice was examined by immunohistochemistry.The distribution of Ad-Cx43-hUCSCs in transplanted mice was tracked by fluorescent labeling.HE staining for tissues from liver,spleen and lungs of transplanted mice at different post-transplantation time points.The recovery function of GJIC between bone marrow stromal cells of transplanted mice was detected by FRAP.The mice survival rate after transplantation in each group was observed.Results:1.Ad-Cx43-EGFP recombinant adenovirus vector was successfully constructed and be transfected into human umbilical cord blood-derived stromal cells:1)The expression of green fluorescence was observed at 24 h post transfection,and it reached the peak at 48 h post transfection according with 89.30 ± 3.12% of transfection efficiency.2)Immunofluorescence,RT-PCR and Western-Blot confirmed that the expression for Cx43 was significantly increased in CX43-hUCSCs transfected with pAd-Cx43-GFP(CX43 mRNA increased by 2 times,expression of CX43 protein increased by 3 times).However,pAd-GFP transfection group and negative control group did not show similar results.Detecting the GJIC function in hUCSCs showed that the fluorescence intensity of CX43-hUCSCs almost returned to its former level after fluorescence quenching,and pAd-GFP transfection group and negative control group did not show similar results.2.The effect of CX43 gene modified human umbilical cord blood-derived stromal cells on L615 leukemia cells:1)DMEM medium containing 20% fetal bovine serum is used for co-culturing of two kinds of cells in vitro.After72 h co-culture,direct contact of two kind of cells could be observed under microscope.Stromal cells adhesion and wrapped leukemia cells could be observed by scanning electron microscopy.2)CCK8 method was used to detect cell proliferation: At 48 h post co-culture,the proliferation of L615 cells group transfected with Cx43 was significantly inhibited.OD value of Cx43 transfection group was significantly lower than other groups with significant difference statistically(p <0.05).3)Detection of apoptosis by flow cytometry: The apoptotic rate of L615 cells transfected with CX43 group was 9.70 ± 0.83%,which was significantly higher than that in un-transfected group(7.33 ± 0.74%)and negative control group(2.50 ± 0.85%)by showing statistically significant difference(p <0.05).4)Detection of cell cycle by Flow cytometry: The cells percentage of G0 / G1 phase in Cx43 transfection group was about 80.43%,which was lower than that in negative control group(84.43%)(P <0.05).The cells ratio of S phase in Cx43 transfection group was 10.42 %,which increased(P <0.05 when compared that with negative control group(6.38%).The cells ratio of G2 / M phase in Cx43 transfection group(8.52%)was close to that from separate culture group(8.15%)(P> 0.05).6)The detection of expression of caspase3,6,7 in L615 from co-culture group by Western blotting: The expression of caspase 3 in Cx43 transfection group was 1.8 times higher than that in control group(P <0.05).The expression of caspase7 in Cx43 transfection group was 7 times higher than that in control group(P <0.05).There was no significant difference in the expression of caspase6 between groups(P> 0.05).3.Inhibition of Ad-Cx43-hUCSCs on residual leukemia from autologous hematopoietic stem cell transplanted mice:Leukemia cells in peripheral blood in 5 days and more leukemia cells in bone marrow,live,spleen an day in 8 days after that the L615 homozygous mice was injected with 2×106 per each of L615 cells by tail vein on day 0,which showed 11± 1.71 days of average survival time.After intraperitoneal injection 200 mg / kg of cyclophosphamide,white blood cells of mice declined to minimum value in 5 days,then began to increase in 7 days and close to normal in 17 days.None of leukemia cells were found in bone marrow,live,spleen an day on +17 days.The average survival time was 27.33 ± 2.49 days after chemotherapy.The survival time of L615 model mice could be prolonged after taken treatment.1)The distribution of Ad-Cx43-hUCSCs in vivo: CM-Dil labeled Ad-Cx43-hUCSCs were detected in bone marrow,liver,spleen and lung of mice in 24 h after transplantation.the number of Ad-Cx43-hUCSCs increased in bone marrow,liver,spleen but decreased in lung in 7 days after transplantation.We can not detecte redfluorescence any more in 14 days after transplantation.2)The hemogram changing in transplanted mice:The increasing speed of white blood cells and platelets in Ad-Cx43-hUCSCs + BM group were significantly faster than those in BM group after transplantation(P <0.05).3)Detect GJIC function in co-culture system by FRAP: The fluorescence intensity of BMSC in L615 + BM-Ad-Cx43-hUCSCs co-culture group recovered 69.33 ± 1.25% within 5min after quenching and that increased profoundly when compared that with L615 + BM co-culture group(51.67 ± 1.7%)showing significantly significant difference(P <0.05).4)Changes of smear and pathological sections for bone marrow transplanted with human umbilical cord blood-derived stromal cells:there are many leukemia cells were detected in BM group compared with on in Cx43 + hUCSCs + BM group;HE staining showed that the trabecula bone was reduced,the cell morphology was juicy,clustered.5)Survival rate of mice within 28 days after transplantation in groups: The average survival time of transplanted mice in Ad-Cx43-hUCSCs + BM group was 26.9 ± 1.52 days,which was longer than that in BM group(23.70 ± 1.99 days)(p <0.05).The mortality of transplanted mice in Ad-Cx43-hUCSCs + BM group was 20%,which was lower than that in BM group(p <0.05).Conclusions:1.CX43 adenovirus can be successfully transfected to hUCSCs and the expression level of Cx43 in Ad-Cx43-hUCSCs CX43 were up-regulated.2.Co-culture system of human umbilical cord blood-derived stromal cells and L615 cells was successfully established.The co-culture of Ad-Cx43-hUCSCs and L615 cells could inhibit the proliferation of L615 cells and up-regulate the expression of caspase3 and 7 in L615 cells which leading to the apoptosis.GJIC function of L615 cells could be enhanced by co-culture of Ad-Cx43-hUCSCs + BM and L615 cells.3.Ad-Cx43-hUCSCs quickly home to the bone marrow,spleen,liver,lung and other organs by co-transplantation with autologous hematopoietic stem cells,and that also could prolong the survival of transplanted mice through promoting the mouse hematopoietic reconstruction and help to remove residual leukemia. |
PDF Full Text Request