| Objective: The in vitro amplification of hematopoietic stem cells(HSCs)Has always been the research hotspot and difficulty.In recent years,many studies have shown that mesenchymal stem cells can promote the proliferation of hematopoietic stem cells.Our previous study in vivo found MSC promote bone marrow proliferation and also found that blood vessel growth significantly,Previous in vitro studies found that the MSC promote HSC proliferation,Shh help MSC promote HSC proliferation.This study will further explore whether MSC promoting HSC proliferation is related to angiogenic factors,and the mechanism of Shh enhancing MSC promoting HSC proliferation.In addition,This study can not only provide basis for further research of HSC expansion in vitro,but also provide a good application prospect for hematopoietic stem cell transplantation(HSCT)and the therapy of blood system of malignant disease.Method : 1.We extract the Healthy volunteers ' bone marrow,According to the characteristics of adherent growth in the plastic dish,Using the whole bone marrow culture method to expand MSCs to third passage(p3).We use flow cytometry(FCM)and the anti human CD105 – FITC? CD34-ECD ?CD45-PCy7 three antibodiesto Identify the superficial markers of MSC and its purity.2.Humanbone marrow mononuclear cells(MNCs)is isolated by density gradient centrifugation,and then we use the sorter miniMASC magnetic beads to separated CD34 + cells,and through counting board,The purity of CD34+cell is determined by FCM by using the anti human CD34-ECD,CD45-PEcy7 and IgG1-ECD,CD45-PEcy7.3.Using Transwell(micro mesh aperture 0.4 um)to Culture P3 generation of MSC and CD34 + cells.two groups:HSC+MSC and HSC,each group repeats three times,Samples are collected after 3?7?10 days,counting the number of HSCs,Founding that cell proliferationis fastest in 7 days,so we will develop 7 days as the point of this study.4.Co-culture including two groups: HSC+MSC,HSC+MSC+Shh,In the two Co-culture groups,MSCs are cultured in the 24-well plates and CD34+ cell plated on the 0.4 um cell culture inserts.culture alone including four groups:HSC,MSC,HSC+Shh,MSC+Shh,MSC and HSC are cultured in 24 orifice plate respectively.5.We respectively inoculate HSC and MSC in Transwell upper and lower chamber,Containing Shh protein groups,we add Shh protein toTranswell lower chamber,free of Shh protein groups,We do not add it.culture alone groups,We respectively inoculate HSC and MSC in ordinary 24 orifice plate,Containing Shh protein groups,we add Shh protein toTranswell lower chamber,free of Shh protein groups,We do not add it.Samples are collected after7 days.6.Detecting the cell count,RNA content,Q – PCR results: Using count plate to count the number of HSC and MSC,using TRIZOL method to detecte total RNA of HSC and MSC,Then puting each extraction of RNA to reverse transcription into cDNA and put cDNA in-20 ? to save;At last,using fluorescence qRT-PCR method to detectthe mRNA level of Ki67,Tie-2,VEGF,Ang-1,and using 2-??Ct to analysize the Ki67,Tie-2,VEGF,Ang-1 mRNA relative quantitativeexpression.7.All the statistics data is analysized by SPSS17.0 software,and we analysize the proliferation effect of Shh or and MSConHSC by using one-way ANOVA,p<0.05 is statistically significant.Resuts:1.MSC are revealed fibroblast-like morphology and in fusiform shape,arranged in bundles or whorls;MSC in p3 are 52.4% positive for CD105 and show negligible presence for CD45(0.3%)and CD34(2.2%).The purity of MSCs is 52.4% 2.By using MiniMACS to separate the HSC,is circular by Microscopically,FCM reveales that approximately 81.9% of these cells express CD34.3.Culturing 3,7,10 days,the number of HSC: HSC group: 10 days >7 days > 3 days.HSC+MSC group 10 days > 7 days > 3 days.HSC counts were increased.After 7 days of culture,the count of HSC increase fastest,The results found that,the best effect time thatMSC can promote the proliferation of HSC is 7 days.4.The seventh day of culturing,The number of HSC(×104)? :HSC+Shh group and HSC+MSC group have larger numbers than HSC group respectively,HSC+MSC+Shh group has larger numbers than HSC+MSC group,and the values of P<0.05;The number of MSC(×105): MSC+Shh group and MSC+HSC group have larger numbers than MSC group respectively,MSC+HSC+Shh group has larger umbers than MSC+HSC group,and the values of P<0.05;5.The seventh day of culturing,Totel RNA level of HSC: HSC+Shh group and HSC+MSC group have higher than HSC group respectively,HSC+MSC+Shh group has higher than HSC+MSC group,and the values of P<0.05;Totel RNA level of MSC: MSC+Shh group and MSC+HSC group have higher than MSC group respectively,MSC+HSC+Shh group has higher numbers than MSC+HSC group,and the values of P<0.05;6.RQ by Qr T-PCR for Ki67?Tie-2 mRNA in HSC and VEGF ? Ang1 mRNA in MSC.The RQ of Ki67: HSC+Shh group and HSC+MSC group are higher than HSC group respectively,HSC+MSC+Shh group is higher than HSC+MSC group,all the P values<0.05;The RQ of Tie-2: HSC+Shh group and HSC+MSC group are higher than HSC group respectively,HSC+MSC+Shh group is higher than HSC+MSC group,all the P values<0.05;7.The RQ of VEGF:MSC+Shh group and MSC+HSC group are higher than MSC group respectively,MSC+HSC +Shh group is higher than MSC+HSC group,all the P values < 0.05.The RQ of Ang1: MSC+Shh group and MSC+HSC group are higher than MSC group respectively,MSC+HSC +Shh group is higher than MSC+HSC group,all the P values<0.05.Conclusion:1.We culture MSC and sort HSC successfully.2.MSC co-cultured with HSC can promote the proliferation of HSC;Shh can promote the proliferation of HSC;Shh help MSc promote HSCproliferation,This results are in line with previous research;3.the mechanism of MSC promoting the proliferation of HSC is related to angiogenesis factor secretion.4.the mechanism of Shh promote HSC proliferation is related to angiogenesis factor secretion.5.the mechanism of Shh help MSc promote HSC proliferation is related to angiogenesis factor secretion. |
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