| Objective(1)To obtain the high expression of Wnt5a protein in bonemarrow-drived mesenchymal stem cells (bMSCs)though recombinatedadenoviral vector encoding Wnt5a(Ad5-Wnt5a-GFP) transducting intobMSCs; To Explore exogenous Wnt5a gene modified bMSCs on inhibitoryproliferation and induced differentiation of HL60cells and the underlyingmolecular mechanisms in HL60cell lines;(2)Though establishing SCIDleukemia mice model and exogenous Wnt5a modified bMSCs transplantedSCID leukemia mice model, to research the levels of continuous functionalexogenous Wnt5a expression in bone marrow of SCID leukemia mice, andto explore the role of exogenous Wnt5a gene modified bMSCs on theprotection of bone marrow and the suppression of tumor cells in SCIDleukemia mice during chemotherapy.Methods(1)Recombinated adenoviral supernatants were harvested andconcentrated by infection each other like Ping-Pong with package cell HEK293, and recombinated adenoviral titer was determined. To getbMSCs by density gradient centrifugation and Differential stick walltraining methods; The growth and morphological characteristics of thebMSCs was observed by optical microscope; The expression level of cellsurface markers CD34, CD44and CD45in the BMSCs were detected byFCM; the ability of potential differentiate in bMSCs were detected byalizarin red staining and ALP staining;(2) To detected the effect of bMSCs modified by Ad5-Wnt5a-GFP:The transducted bMSCs which express green fluorescence protein (GFP)were observed by Fluorescence microscope; the transduction efficiency andscreen the optimal multiplicity of infection(MOI)were detected by FCM;The mRNA and protein expression of exogenous Wnt5a in bMSCs weredetected by RT-PCR and ELISA.And the survival state and proliferation ofbMSCs in different groups were observed by cell counting;(3) Through the methods of exogenous Wnt5a gene modified bMSCsco-cultured with HL60cells, to detect the role of Wnt5a on tumor growthinhibition and induction of differentiation.HL60cells were divided intofour groups, Cells in group A were co-cultured with bMSCs/Wnt5a andserved as the experimental group. Cells in group B were co-cultured withbMSCs/GFP. Cells in group C were co-cultured with bMSCs. Cells ingroup D were cultured alone and served as the blank control group. Thesecells are referred to as, HL60/bMSCs/GFP, HL60/bMSCs, and HL60,respectively. the cell cycle of HL60cells was detected by using FCM, thelevel of proliferation of HL60cells in different group was detected by MTT,The morphological changes of HL60cells in different group were observedby Wrights stain； Changes in HL60cell surface differentiation antigen,CD13, CD14, CD68in all groups were detected by IHC method; (4)From the molecular level and protein level, the expression ofWnt5a receptor of ROR2and FZD5, Wnt signal transduction protein ofβ-Catenin, the downstream target genes of CaMK II and cyclinD1,respectively, were detected by RT-PCR, IHC and Western Blot;(5) SCID mice were total body irradiation with Co60, total dose of2.0Gy. HL60cells were injected by tail vein to establish human SCIDleukemia mice models； Record survival time and the incidence ofleukemia in SCID mice； After injected by HL60cells, white blood cellsand tumor cell morphology and number change in SCID mice wereobserved by Wright-Giemsa staining; The changes of CD33positiveexpression rate in the mice bone marrow cells were detected by FCM;Leukemia Cells in the liver, spleen, lung and kidney of SCID mice weredetected by FCM and HE staining；(6) In the bone marrow of Wnt5a gene modified bMSCs transplantedSCID mice,Wnt5a sustained expression and the expression of the timelimit were detected.24hours after injected HL60cells, exogenous Wnt5amodified bMSCs were injected in SCID leukemia mice by tail vein,transplanted twice, at intervals of24hours, To establish exogenous Wnt5amodified bMSCs transplanted SCID leukemia mouse model; SCID micewere divided into four Group,group A: experimental group (Wnt5amodified bMSCs transplanted SCID leukemia model mice); group B:emptyvector control group(Ad5-GFP transfection bMSCs transplanted SCIDleukemia model mice); group C blank control group1(bMSCs transplantedSCID leukemia model mice); group D blank control group2(the SCIDleukemia model mice); Exogenous Wnt5a gene mRNA and proteinexpression levels in bone marrow cells in each group of SCID leukemiamice were detected by RT-PCR and western blot; (7)21days after the exogenous Wnt5a gene modified bMSCstransplanted to SCID leukemia mice model, Ara-c administeredSubcutaneously twice daily and seven days in a row; SCID mice weredivided into four Group as part5. Record the survival time and survivalrate of SCID leukemia mice; one week after the end of chemotherapy ineach group of SCID leukemia mice,the counts of white blood cells, redblood cells, and platelet were observed by Hematology analyzer; the colonyforming ability of bone marrow hematopoietic cell in each group of SCIDleukemia mice was detected by Semi-solid methylcellulose colony culturemethod; After ended chemotherapy, the counts of CD33positive cells inbone marrow and peripheral blood in each group of SCID leukemia modelmouse were detected by FCM and IHC. Leukemia cells and pathologicalchanges in the liver, spleen, lung, kidney in each group of SCID leukemiamice were observed by HE staining。Results(1) The recombinated adenoviral titers of Wnt5a was4.7×109(PFU)/mL； Under an inverted microscope, bMSCs were observed to be fusiformand dividing cells in pairs,which were enhanced refraction and a smallersize; After passaged, the cells were no longer a colony growth,but a evenlydistributed growth; CD44which was bMSCs surface differentiationantigen, the positive rate of was97.77%,and basically did not expressCD34, CD45,which were hematopoietic stem cell marker,the positive rateswere1.07%and0.10%; After bMSCs were induced osteogenesis, Alizarinred staining and ALP activity were detected in cells, found that theinduction group compared with the non-induction group, alizarin redpositive staining was significantly enhanced,ALP activity was significantly higher, the difference was statistically significant (P <0.01);(2)Under the fluorescence microscopy,Ad5-Wnt5a-GFP transfectedBMSCs for72hours, cell growth state was good.Exogenous Wnt5a genesuccessfully transfected bMSCs, when MOI=100and transfected for72h,cell growth state was the best,cell viability was maintained at more than80%, the average transfection rate remained stable at38.68%; The mRNAand protein levels of exogenous Wnt5a in bMSCs were sustained highexpression, compared with the other two control groups, the difference wasstatistically significance (P <0.05)；When exogenous Wnt5a gene modifiedbMSCs for72h, the cells entered in the logarithmic growth phase. When4days to7days, cell counts significantly increased, compared with the othertwo control groups, the difference was statistically significant (P <0.05);(3) Co-cultured three days later, compared with groups B, C, andD,the proliferation of HL60cells in the experimental group significantlyreduced,the difference was statistically significant (P <0.05); the cell cycleof HL60cells in the experimental group compared with groups B, C, and D,the difference in three control groups was no statistically significant (P>0.05); proliferation of the active phase cells decreased,cell cycle arrest inG2phase, the difference was statistically significant (P<0.05)；Under thelight microscope, the HL60cells in group A showed a decrease in thenuclear-cytoplasmic ratio and nuclear depression, compared with groups B,C, and D. The effect of Wnt5a on the expression of CD13, CD14, andCD68in HL60cells to show that HL60cells stained very strongly for thegranulocyte surface marker antigen CD13at the membrane in groups A-D.The staining results are represented as a percentage of positive samples pergroup. Statistical differences were not found between group A and groups B, C, and D.(P>0.05). The expression of the monocytic surface markerantigen CD14,CD68were weak in the HL60cells in groups B-D, but wasstrongly positive in group A. the difference was statistically significant (P<0.05);(4) Changes in the mRNA and protein levels of FZD5, ROR2,β-catenin, CaMKII, and cyclinD1were detected by RT-PCR,IF andWestern blot analysis. The mRNA and protein expression of FZD5exhibited no significant difference between group A and groups B-D (P>0.05). Wnt5a induced a significant increase (P <0.05) in ROR2andCaMKII mRNA and protein expression as compared with the control. ThemRNA and protein levels of β-catenin and cyclinD1were remarkablydecreased in group A than in groups B-D (P＜0.05);(5) Successfully established leukemia mice model, tumor formationrate was100%natural course of the disease was30-42days, solid tumorsin the abdomen and buttocks of mice and hepatosplenomegaly wereobserved; Peripheral blood leukocytes in SCID mice was (3.75±0.65)×109/L, WBC counts were increased to (10.23±1.24)×109/L, and theproportion of HL60cells was (10±2)%, compared with SCID mice whichwere not injected HL60cells, the difference was statistically significant (P<0.01); In SCID mice bone marrow cells, CD33cells positive rate was(0.84±1.24)%, after injected HL60cells, CD33cells positive rate wasincreased to (11.17±0.23)%, the difference was statistically significant (P<0.01);3weeks after injected HL60cells, leukemia cells which expressCD33in the bone marrow, liver, lung, spleen, kidney of SCID mice weredetected by IHC, compared with SCID mice which were not injectedHL60cells, CD33positive cells were significantly increased, the differencewas statistically significant (P <0.01); (6) The expression levels of Wnt5a mRNA and protein in the bonemarrow cells of group A SCID leukemia mice were in the highest (P <0.01),compared with group B,C,D. the expressions of Wnt5a mRNA and proteinin mice bone marrow cells of group B,C,D SCID leukemia mice wererare,the difference had no statistically significant(P>0.05)；In group ASCID leukemia mice, Wnt5a mRNA expression of bone marrow cells atthe4th week was maximum, compared with the expression at the1th week,2th week,3th week and5th week, the differences were statisticallysignificant (P <0.05); the expression at1th week significantly reduced,compared with the expression at2th week,3th week and5th week,thedifference was statistically significant (P <0.05); The difference among theexpressions at the2th week,3th week and5th week were no statisticallysignificant (P>0.05);(7)In the process of chemotherapy,the activity of each group SCID micedecreased, body weight are different extent lighter than before, and theweight of experimental group SCID mice was heavier than the controlgroup mice, and the difference had statistically significant (P <0.05), thedifference of weight in three control group SCID mice had statisticallysignificant (P>0.05); At the end of chemotherapy a week later, the resultsof survival rate of leukemia mice showed that the survival rate of SCIDleukemia mice in group A compared with the other groups, wassignificantly higher,the difference was statistically significant by Fisherexact analysis (P<0.05)； The detection of peripheral blood in SCIDleukemia mice showed that in group A, cells count remained atWBC5.41×109/L,RBC9.15×1012/L,PLT335.32×109/L, significantly higherthan other three groups, the differences was statistically significant (P <0.0 5)；The results of methyl cellulose semi-solid colony culture assay showedthat the colony forming ability of bone marrow in group A wassignificantly higher than the other three control groups, the difference hadstatistically significant (P <0.05); The counts of CD33positive cells inperipheral blood and bone marrow cells of group A were decreased,compared with other three control groups,the difference had statisticallysignificant (P <0.05), One week after chemotherapy,the liver, spleen, lung,kidney in each group SCID mice all were not observed leukemia cellinvasion, the lung and kidney in three control group SCID mice could beobserved different degree of hemorrhage, and the organs in theexperimental group mice could not be observed hemorrhage.Conclusions:(1) Successfully obtained high purity bMSCs and established a systemof exogenous Wnt5a gene stably transfected bMSCs in vitro;(2)The expression of exogenous Wnt5a in bMSCs were stable andhigh effective, could promote the proliferation of bMSCs in vitro;(3) Exogenous Wnt5a can inhibit the proliferation of HL60cells,arrest the cell cycle in G2phase, induce the differentiation of HL60cells tomature direction;(4) Its possible molecular mechanisms for exogenous Wnt5a effect onHL60cells: exogenous Wnt5a bind to cell surface receptor Ror2,lead tointracellular calcium release, then activated the noncanonical Ca2+dependent signaling pathways in HL60cells,to promote CaMK Ⅱexpression, which inhibit the canonical Wnt signal pathway, β-cateninexpression was inhibited, leading to the expression of cyclinD1wasdownregulation in HL60cells. Wnt5a gene in HL60cells acted as a tumor suppressor gene;(5)Successfully established the SCID mice model of human leukemia(in HL60) and exogenous Wnt5a gene modified bMSCs transplantationmodel of SCID leukemia mice, the success rate in modeling was100%.True represented the characteristics of human clinical leukemia wereinvolving the bone marrow and organs diffuse infiltration. The naturalcourse of leukemia in SCID mice was30-42days;(6) From the gene and protein levels, indicated that the exogenous Wnt5agene modified bMSCs could implanted into bone marrow in the SCIDleukemia mice. Exogenous Wnt5a can express from the2th week in SCIDleukemia mice,At the4th week the expression was in peak, AT the5thweek,the expression level of wnt5a was decreased. All of the data aboveabout exogenous Wnt5a gene expression of bone marrow in SCID miceprovides reliable foundation and guarantee for the further experiments;(7) SCID leukemia mice in the treatment of chemotherapy, exogenousWnt5a gene timely play to promote the hematopoietic function of bonemarrow and inhibit the growth of leukemia cells, can significantly improvethe survival rate of SCID leukemia mice, and make chemotherapy receivebetter outcomes;(8)Exogenous Wnt5a in leukemic mice may play a role as ahematopoietic support factor and a tumor suppressor.
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