| Objective: Hematopoietic stem cells resided in the bone marrow niche have the capacity to self-renew and the potential to differentiate into all kinds of the mature blood cell types.Niche has complex structure and regulates the physiological function of HSCs importantly,which consists of stromal cells, cytokine and extracellular matrix.Mensenchymal stromal cells can differentiate into kinds of stromal cells. Osteogenic niche and epithelial niche are the mainly niches in bone marrow, and the former plays a role in the maintanance for HSC,while the latter regulate its proliferation, differentiation,migration and so on.Bone morphogenetic protein(BMP)-2 is one member of TGF-β family with multiple fuction.As a key molecule in bone regeneration,it can induce the undifferentiated MSC differentiating into osteoblast,which is the mainly components of osteogenic niche.It can also promote angiogenesis,but reserches about its function in epithelia niche is rare.Nowadays,scientists have identified it can regulate hematopoiesis,especially in embryo stage,but its function on adult hematopoiesis is rare.Many studies have shown that MSCs can promote HSCs proliferation,but whether BMP-2 could enhance such an effect on HSCs or not is not well known.In this research we use 24-well transwell to co-culture HSCs and MSCs, and interfere with rh BMP-2.After 3days,we test HSC proliferation.It can not only help to explore whether MSCs or and BMP-2 will influence HSCs proliferation or not,but also lay foundation for the next step to explore the associated mechanism.In addition,it will be helpful for CD34 + cell expansion in vitro and could promote the development of HSCT and hematological malignant therapy. Method: 1. Adherent culture of the whole bone marrow in the plastic dish to expand MSCs to third passage(p3).In the P3,we use FCM to assess the superficial markers of MSC and its purity;2. Human bone marrow MNCs are isolated by density gradient centrifugation. CD34+cells are isolated by MACS Miltenyi Biotec. The purity of CD34+cell is determined by FCM; 3. Co-culture including four groups: HSC,HSC+BMP2,HSC+MSC,HSC+MSC+BMP2, each group repeats four times. 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. Samples are collected after 72 hours; 4. Testing HSC proliferation : counting the number of HSCs, detecting total RNA, using fluorescence q RT-PCR method to detect the m RNA level of Ki67,and using 2-△△Ct to analysize the Ki67 m RNA relative quantitative expression;5. All the statistics data is analysized by SPSS17.0 software, and we analysize the proliferation effect of BMP-2 or and MSC on HSC by using one-way ANOVA, p<0.05 is statistically significant. Resuts: ①BMMSCs are revealed fibroblast-like morphology and in fusiform shape, arranged in bundles or whorls; MSCs in p3 are 52.4% positive for CD105 and show negligible presence for CD45(0.3%) and CD34(2.2%). ②After positiveselection of BMMNCs by mini MACS, FCM reveales that approximately 81.9% of these cells express CD34. ③The number of HSCs(×104):co-culture g r o u p( 3. 0 3 ± 0. 5 0) a n d H S C + B M P 2 g r o u p( 1. 7 0 ± 0. 5 1) h a v e l a r g e r numbers than HSC group(1.2±0.43) respectively,co-culture+BMP2 group(5.29±0.20) has larger number than co-culture group(3.03±0.50),and the values of P<0.05;④Totel RNA level(×50) in different HSC groups:HSC+BMP2 group(138±3.464) and co-culture group(163±3.559)are higher than HSC group(113±2.944) respectively,co-culture+BMP2 group(241±3.651) is higher than co-culture group(163±3.559); ⑤RQ by q RT-PCR for Ki67 m RNA in HSC: co-culture group(3.27±0.08) and HSC+BMP2 group(2.44±0.26) are higher than HSC group(1.00±0.13) respectively,co-culture+BMP2 group(5.08±0.20) is higher than co-culture group(3.27±1.47),all the P values<0.05; Conclusion:①we cuture MSCs and sort HSCs successfully;②MSCs and BMP-2 can promote the proliferation of bone marrow CD34+cells respectively in vitro; ③BMP-2 could enhance MSCs proliferation effect on HSCs in vitro. |
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