The Mechanism Of Engraftment Promotions By Marrow Mesenchymal Induced By Chemokine Receptors In ALLO-HSCT

Introduction:In recent years, the research about bone marrow mesenchymal stem cells revealsthat it hasthe effect of facilitating the engraftment of donor hematopoietic stem cells.Animal experiment and clinical statistics both reveal that bone marrow mesenchymalstem cells and hematopoietic stem cells transplant together could promote theengraftment ofhematopoietic stem cells; but the mechanism of that effect is not clear.One of the research hot points among the hematopoietic stem cells' homing,proliferation and differentiation after allo-hsct is the co-worker of chemokines andtheir receptors. The chemokine receptors are one kind of G protein co-reactedreceptor super family expressed on different kind of cells, which have 7 string acrossthe cell membrane; they participate in the growing, growth, differentiation, apoptosis,tissue distribution of the cells by co-acting with chemokines. They can activatephospholipid kinase, fat kinase, protein kinase, then modulate the consistency ofcalcium ion, then activate JAK-STAT pathway, and then to a series of signalpathways.The recent research already reveals that chemokine and its receptors haveimportant effect on the transferring and homing of hematopoietic stem cells: duringthe homing procedure, lympohocytes adhere to the HEV by the CD62L which isexpressed on the cell membrane, and then CCR7 on lymphocytes combine to SLC, which is expressed on endothelium cells, the co-reaction of CCR7-SLC promote thehoming of T cells to lymphonotes. As for B cells, BLC/CXCR5 could induct thetransferring and homing of mature B lymphocytes, and also affect the development ofmature B cells in the B cells districts of subsidiary lymphonotes. The chemokine andits receptors not only could promote the homing of hematopoietic stem cells, theyalso have important effects on the proliferation and differentiation of hematopoieticstem cells: SDF-1/CXCR4 take part in the proliferation, differentiation, transferringof hematopoietic stem cells, and the growth of the pre-B cells needs the expression ofCXCR4; SDF-1, ELC, SLC, TECK, MDC et al chemokines have different effect ineach period of the differentiation procedure of T cells in the thymus, among these theSLC and ELC together have effect of the distributing of T cells in the subsidiarylympho-organ. They also have significant modulating effects on the co-reaction ofDC to T cells, the activation, polarization and effect of T cells. Based on the effects ofchemokines and its receptors to the homing and proliferation on hematopoietic stemcells, to know the mechanism about the promoting effect of bone marrowmesenchymal cells to the homing of hematopoietic stem cells, we investigate theeffect of bone marrow mesenchymal stem cells on chemokine receptors on T cells, toexplor the immune modulating mechanism of MSC.Methods:1,The research on the culture and biology characteristic of bone marrowmesenchymal stem cells: collect healthy volunteer bone marrow and C57BL/6 mousebone marrow from thigh and shankbone in asepsis condition, then inoculate the cellsin the 25cm² cultivate flask at the density of 2.0×10⁵; then keep the adhesive cells24-48 hours later, after that change the cultivate liquid every 3 days. When the cellsfuse to about 80-90%, cultivate it to the next passage at the rate of 1:5. Collect thebone marrow mesenchymaI stem cells from healthy human and C57BL/6 mouse ofthe first passage (P1), the third passage (P3) and the fifth passage (P5) to draw the growth chart. Take 2×10⁵ cells from the forth and fifth passage, labeled it indouble-labeled way with CD34, CD44, CD166, HLA-DR antibody. Collect the cellsin flow cytometry scan by the CellQuest software and testify the rate of CD34⁺,CD44⁺, C166⁺, HLA-DR⁺ cells.2,The effects of mice bone marrow mesenchymal stem cells on chemokinereceptors of mice spleenocytes after PHA induced transformation: C57BL/6 mouseslpeenocytes is cultured in 24 holes flasks by the density of 1×10⁶/hole, add PHA intothe holes, culture the cells for 72 hours. The cells are divided into three groups: groupA add the MSC by the rate 0.1; group B add the MSC by the rate of 0.01; the controlgroup cultured alone. The whole system is cultured under the condition of 37℃,5%CO₂ for 3 days. Observe the cells every day, collect the suspended spleenocytes 3days later and testify by the flow cytometry scan.3,The animal model of bone marrow mesenchymal stem cells and hematopoieticstem cells intravenously together: take the bone marrow of C57/BL6 mice in asepsiscondition, the 0 day of the BMT BALB/c mice accept ⁶⁰Co TBI (TBI9.0Gy,0.5Gy/min), the BALB/c mice accept BMT after the irradiation within 4 hours. Themice are divided into three groups: group A receive BMC 4×10⁷ and MSC 4×10⁵,group B receive BMC4×10⁷ and brine, group C receive only brine. Observation guideline: 1 the survival time and general situation, 2 the weight change.4,Statistical analysis of data: the data all expressed by average±samplestandard deviation (x±s) and percentage(%). One-way anova is analysis by SPSS12.0 software of Windows, the analysis between each groups is by LSD-test.Results:1. After centrifugate by the lymphocyte separation liquid the clinical originalhealthy human bone marrow can get white cell layer. This layer of cells receivedre-centrifugate and abstersion, the C57BL/6 mouse bone marrow single mixture arecultured respectively after 24-48 hours. Keep the adhibit cells. The adhibit bone marrow mesenchymal stem cells are distributed dividedly, shaped in spindle shape,fiat shape and small round shape. About 3-4 days later the adhibit cells proliferatesharply, 6 days later bigger clone could be seen, 10 days later the cells merge to above80%. The cells tend to distributed as spindle shape and burble shape. After passing tothe next generation the cells merge to 80-90% in 5 days; the self-dieing comes upafter the tenth generation, originaled as block fall offand spread to the relative district.The bone marrow mesenchymal stem cells from C57BL/6 mouse are smallercompared to healthy human bone marrow mesenchymal stem cells; the ambit isclearer. The original generations of the healthy human bone marrow mesenchymalstem cells could be divided into spindle shape, fiat shape and small round shape, cellsgrow faster after passageing than the original generation; and the cells could merge to80-90% within 7 days after passageing. The new generation cells tend to be spindleshape and burble shape. After observing the growth chart of P1, P2, P3 passage cellsthere comes up some character: the latent period of passage culture is 24-36 hours,logarithm proliferation period is 2-3 days, ftated- period comes up in the fifth days oflogarithm proliferation period. There's no significant difference between differentoriginal bone marrow mesenchymal stem cells. Flow cytometry scan shows that thethird passage of hMSC is CD44, CD166 positive, CD34, HLA-DR negative.2. The change of spleenocytes express CCR5, CCR7 and CXCR3 chemokinereceptors after PHA simulation and co-cultured with MSC: the expression of CCR5,CCR7 and CXCR3 all could be tested, in the MSC co-culture system, the expressionof chemokine receptors was affected by MSC and the change of expression wasrelated to the concentration of MSC (graph 1, chart 2). The results revealed that, bonemarrow mesenchymal stem cells could elevate the expression of chemokine receptorsin CCR5, CCR7 and CXCR3 when co-cultured with CD3 spleenocytes in certainratio. To the former two receptors, in the experimental rang, the higher MSC concentration was with, the higher the expression level of chemokine receptors wasdetected.3,The expression of chemokine receptors on T lymphocytes' subsets afterPHA simulation and co-cultured with MSC: in CXCR3 positive cells, onlyCD3⁺CD8⁺ cells could be tested that the expression of chemokine receptors elevatedaccording to the MSC concentration. In the CD3⁺CD8^- subset, the chemokinereceptors expression among different groups there's no significant difference could beobserved. In CCR5 positive cells, the expression of chemokine receptors elevatedaccording to the rising of MSC concentration mainly in CD3⁺ CD8^- cells, and inCD3⁺CD8⁺ subset, only when MSC concentration is 10%, the chemokine receptors ishigher than control groups. In CCR7 positive cells, both CD3⁺ CD8^- and CD3⁺CD8⁺subsets could be observed that the expression of chemokine receptors elevatedaccording to the rising of MSC concentration.4. Until+5 days of BMT the mouse died 66.7% in group C, the left mouse allsurvive more than 30 days. In every group the mouse all express hair losing andshaking, the expression is almost of the same degree. It's considered to be un-specialsyndrome of radiation. Weight average value chart shows that the weight of mouse ingroup A raise slowly, weight of mouse in group B raise and then fall down, weight ofmouse in group C decrease slowly.Conclusion:1. The grow rules of bone marrow mesenchymal stem cells is similar toliterature reports. It helps to know the cell growth circus under the situation of ourlaboratory, and help to develop the next step of the experiment.2,Flow cytometry scan shows that the third passage of hMSC is CD44, CD166positive, CD34, HLA-DR negative. It's accord with the literature reports.3,MSC could up-regulated the expression of chemokine receptors CCR5,CCR7, CXCR3 in spleenocytes when MSCs were co-cultured with splenocytes in certain dose; it's dose depended in the former two chemokine receptors of CCR5,CCR7. The chemokine receptors express differently in the two lymphocytes subsetsof CD3⁺ CD8^- and CD3⁺CD8⁺. The research reveals that: bone marrow mesenchymalstem cells could elevate the expression of chemokine receptors on T cells in certainrates, which may help lymphocytes transferring and homing to lympho-organ aiderBMT under the effect of chemokines, then facilitate the implantation ofhematopoietic stem cells. It may be one of the mechanism of bone marrowmesenchymal cells promoting the implantation of allo-hematopoietic stem cells.