目的：异常的骨髓微环境参与白血病的发生与发展，白血病发生过程中也会引起微环境的重塑。在我们前期的研究中发现，急性髓系白血病病人骨髓血浆中血小板生成素（TPO）的水平显著高于正常人，提示骨髓中TPO水平的异常升高可能与急性髓系白血病相关，且有文献报道在白血病的微环境重塑过程中出现TPO的升高。为深入研究异常微环境在白血病细胞干性维持及白血病发生发展中的作用，我们将TPO转染人骨髓间充质干细胞，体外实验探索其在白血病细胞干性中的作用，进一步运用组织工程学的方法在免疫缺陷小鼠体内重建异位人的骨髓微环境，并用这种体内模型研究过表达TPO的异常的骨髓微环境在白血病发生发展中的作用。

方法：构建pCDH-EF1-TPO-T2A-copGFP慢病毒载体，感染人血小板裂解液（HPL）培养的第二代骨髓间充质干细胞后，流式细胞术分选GFP阳性过表达TPO的间充质干细胞以及作为对照感染了空载体的间充质干细胞，WB鉴定两组间充质干细胞内TPO的蛋白表达，免疫荧光共聚焦检测转入的TPO在细胞内定位情况，ELISA检测培养上清中TPO的分泌水平。将急性髓系白血病细胞系TF-1分别与这两组间充质干细胞体外共培养4天后，KI67及7-AAD标记、流式细胞仪检测两组TF-1细胞的G0期细胞比例的差异；Annexin V及PI标记、流式细胞仪检测VPl6对两组TF-1细胞处理24h和48h后的细胞凋亡情况；MTT法检测两组TF-1细胞增殖能力，PI标记、流式细胞仪检测两组TF-1细胞的细胞周期。在免疫缺陷小鼠体内重建异位的人骨髓微环境实验中，通过反复冻融浓缩后血小板获得人血小板裂解液（HPL），以α-MEM作为基础培养基分别添加10% HPL和10%胎牛血清(FBS)培养骨髓来源的间充质干细胞。比较两种添加成分对MSC形态、免疫表型、多向分化能力以及增殖能力的影响；将HPL培养的MSC接种于β-TCP支架材料并移植到免疫缺陷小鼠背部皮下，8-12W后取出移植体做HE染色，观察MSC在体内形成骨以及骨髓结构的能力。将过表达TPO间充质干细胞以及作为对照感染了空载体的间充质干细胞分别接种于β-TCP支架材料并移植到免疫缺陷小鼠背部皮下，8-12W后取出移植体做HE染色，观察MSC在体内形成异常骨髓微环境的情况。

结果：成功构建pCDH-EF1-TPO-T2A-copGFP慢病毒载体，包装病毒，感染间充质干细胞。WB检测到过表达TPO的间充质干细胞胞内有TPO的表达，空载体组也有微弱的内源性的表达，免疫荧光共聚焦检测TPO大部分定位在细胞浆内，ELISA检测过表达TPO的间充质干细胞TPO的分泌水平显著高于空载体组。与过表达TPO的间充质干细胞共培养的TF-1细胞的G0期显著高于对照组；用VP16处理TF-1细胞时，与过表达TPO的间充质干细胞共培养的TF-1细胞在24h和48h细胞凋亡比例均显著低于对照组，表现出更强的抗化疗杀伤；TPO水平的升高对TF-1细胞的增殖能力无明显影响；与表达TPO的间充质干细胞共培养的TF-1细胞的G0/G1期比例高于对照组，S期比例低于对照组。在免疫缺陷小鼠体内重建异位人骨髓微环境实验结果显示，用HPL和FBS培养的MSC均呈现长梭形成纤维样细胞结构，均具有多向分化能力；两种体系培养的MSC免疫表型无明显区别，但用人血小板裂解液培养的MSC具有更强的增殖能力和向成骨分化能力；用人血小板裂解液培养的MSC具有在体内形成骨组织样结构的能力，成功构建小鼠体内的人的小骨模型。

结论：间充质干细胞TPO表达水平的升高可增加白血病细胞G0期比例，维持其静息从而使其更能抵抗化疗药物的杀伤。HPL培养的MSC具有更强的增殖能力以及成骨分化潜能，它能在NOD/SCID鼠上重建异位的人源骨髓微环境。

Objective:  Abnormal BM niche participates in the initiation and development of leukemia, leukemia cells also can remodel the BM niche. In our previous research, we found that the TPO expression level in BM plasma of AML patients was higher than that in normal donors, which suggesting that the abnormal increase of TPO protein level may correlate with AML. In order to further demonstrate the role of abnormal BM niche in the stemness maintenance of leukemia cells and in the initiation and development of leukemia, we transfected mesenchymal stem cells with TPO and studied the role of abnormal BM niche in the stemness maintenance of leukemia cells in vitro. Furthermore, we applied the tissue engineer technology to construct ectopic human bone marrow niche in immunodeficiency mouse. This new model was then used to study the role of TPO mediated abnormal BM niche in the initiation and development of leukemia.

Methods:  The lentivirus vector carrying cDNA of TPO gene and the empty lentivirus vector were constructed and infected into HPL cultured mesenchymal stem cells respectively. Then GFP⁺ cells were sorted by flow cytometry. Western blot was applied to determine the expression level of TPO, confocal immunofluorescence was used to detect the location of TPO in MSC, TPO protein level in culture supernatant of mesenchymal stem cells was detected by ELISA. TF-1 cells were cocultured with mesenchymal stem cells infected with TPO lentivirus or empty lentivirus for 4 days. G0 phase ratios in these two groups of TF-1 cells were then detected by flow cytometry. After these two groups of TF-1 cells were treated with VP16 for 24h or 48h, cell apoptosis was detected by flow cytometry. Cell proliferation capacity was measured by MTT, cell cycle in these two groups of TF-1 cells was analyzed by flow cytometry after TF-1 cells were stained with PI. In the construction of ectopic human bone marrow niche in immunodeficiency mouse, Human Platelet Lysate (HPL) was used as a substitute of fetal bovine serum(FBS) in the culture of MSC. Human Platelet Lysate (HPL) was obtained by repeatedly freezing and thawing of concentrated platelet. Bone marrow derived mesenchymal stem cells were cultured in a-minimal essential medium (a-MEM) containing 10% HPL or 10% FBS. We then compared the morphology, cell phenotype, multilineage differentiation potential in vitro and proliferation capacity between the mesenchymal stem cells cultured with HPL or FBS. To determine the human bone marrow formation capacity of HPL cultured MSC，MSC was seeded on β-TCP scaffolds for 12h, then MSC-coated scaffolds were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12W, the scaffolds were harvested from the mice, fixed, paraffin-embedded, and stained for HE. TPO transfected MSC were seeded on β-TCP scaffolds, then MSC-coated scaffolds were implanted in a subcutaneous pocket on the dorsum of NOD/SCID mice. After 8-12W, the scaffolds were harvested from the mice, human bone marrow formation capacity was determined by HE staining.

Results: pCDH-EF1-TPO-T2A-copGFP lentivirus vector was constructed successfully and mesenchymal stem cells were infected. Mesenchymal stem cells infected with TPO lentivirus have higher TPO express level and secretion level than control cells, which was determined through Western blot and ELISA technology respectively. Most of TPO molecules were distributed in cytoplasm. TF-1 cocultured with mesenchymal stem cells infected with TPO lentivirus have higher G0 phase ratio, higher drug resistance capacity , higer G0/G1 phase ratio but lower S phase ratio than mesenchymal stem cells infected with empty lentivirus. MTT analysis showed no obvious difference in cell proliferation capacity of TF-1 cells．In the construction of ectopic human bone marrow niche in immunodeficiency mouse, it showed that whether cultured in the presence of HPL or FBS, the MSC all displayed a spindle-shaped fibroblast-like morphology. Flow Cytometry analysis revealed no obvious differences in cell immunophenotype in these two groups, they all have the ability to differentiate towards osteoblasts, adipocytes, and chondrocytes in vitro. But mesenchymal stem cells cultured with HPL-contained medium showed stronger proliferation capacity and higher activity to differentiate towards osteoblasts. Mesenchymal stem cells cultured with HPL still have in vivo bone-forming capacity, so we successfully constructed ectopic human bone marrow niche in  NOD/SCID mice.

Conclusion: Overexpression of TPO in Mesenchymal stem cells increases the G0 phase ratio of TF-1 leukemia cells and promotes leukemia cells in quiescence, thus endows TF-1 cells stronger drug resistance capacity. HPL cultured MSC have higher proliferation capacity and potential of differentiate towards osteoblasts, it can reconstruct ectopic human bone marrow niche in the NOD/SCID mice.