人胚胎干细胞源间充质干细胞分泌的微囊泡对白血病细胞体外抑制作用的研究
发布时间：2019-06-14 06:03
【摘要】：研究背景:最近多项研究表明,人骨髓和脐带来源的间充质干细胞(mesenchymal stem cells,MSCs)通过与白血病细胞直接共培养后可以明显抑制白血病细胞(leukemia cells)的增殖。课题组此前致力于人胚胎干细胞(human embryonic stem cells,h ESCs)的培养及定向分化方面的研究,发现胚胎干细胞在一定条件下可以自发分化为间充质干细胞,我们预期这种由人胚胎干细胞分化而来的间充质干细胞(human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSCs)同样可能抑制白血病细胞的增殖。对此后续的研究表明,h ESC-MSCs确实可以通过间接作用(旁分泌途径)抑制白血病细胞的增殖。而微囊泡(microvesicles,MVs)是细胞分泌至条件培养基中的活性有形成分,是细胞与周围细胞或外界环境进行物质传递和信息交流的物质基础,因此我们推测人胚胎干细胞源间充质干细胞分泌的微囊泡(microvesicles released from human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSC-MVs)在体外也可能抑制白血病细胞的增殖,并对其展开进一步的研究。研究目的:研究人胚胎干细胞源间充质干细胞分泌的微囊泡对白血病细胞的作用及作用机制。研究方法:(1)收集h ESC-MSCs的无血清培养基(条件培养基),运用超速离心法提取微囊泡;在透射电子显微镜以及扫描电子显微镜下对微囊泡的大小和形态进行鉴定。(2)将h ESC-MSCs和h ESC-MSC-MVs分别与白血病细胞株K562和HL60共培养48h后,显微镜下计数肿瘤细胞的数量;用CCK8实验检测肿瘤细胞的活力。(3)在透射电镜下观察共培养前后白血病细胞中的自噬小体的数量;用Western blot技术检测肿瘤细胞自噬相关蛋白LC3及Beclin-1的表达水平。(4)应用Western blot技术检测肿瘤细胞凋亡相关蛋白Bax及Bcl-2的表达水平;流式细胞术检测肿瘤细胞的凋亡。研究结果:(1)h ESC-MSCs和h ESC-MSC-MVs在体外抑制白血病细胞的增殖,并且这种抑制作用呈浓度依赖效应。(2)h ESC-MSC-MVs刺激白血病细胞48h后,透射电镜观察到刺激组较对照组肿瘤细胞中自噬小体明显增多、Beclin-1表达水平增高及LC3II/I比值增高。(3)h ESC-MSC-MVs刺激白血病细胞48h后,肿瘤细胞的凋亡率增高、Bcl-2/Bax比值降低。结论:(1)h ESC-MSC-MVs在体外能抑制白血病细胞株K562和HL60的增殖。(2)h ESC-MSC-MVs促进白血病细胞的自噬。(3)h ESC-MSC-MVs促进白血病细胞的凋亡。
[Abstract]:Background: recent studies have shown that human bone marrow and umbilical cord derived mesenchymal stem cells (mesenchymal stem cells,MSCs) can significantly inhibit the proliferation of leukemia cell line (leukemia cells) by co-culture with leukemia cells. The research group has previously focused on the culture and directional differentiation of human embryonic stem cells (human embryonic stem cells,h ESCs). It has been found that embryonic stem cells can spontaneously differentiate into mesenchymal stem cells under certain conditions. We expect that (human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSCs, which is differentiated from human embryonic stem cells, may also inhibit the proliferation of leukemia cells. Subsequent studies have shown that h ESC-MSCs can inhibit the proliferation of leukemia cells indirectly (paracrine pathway). Microvesicles (microvesicles,MVs) are active and tangible components secreted by cells into conditioned medium, and are the material basis for material transmission and information exchange between cells and surrounding cells or external environment. Therefore, we speculate that microvesicles secreted by human embryonic stem cells (MSCs) may also inhibit the proliferation of leukemic cells in vitro. And carry out further research on it. Objective: to study the effect and mechanism of microvesicles secreted by human embryonic stem cells (MSCs) on leukemia cells. Methods: (1) the serum-free medium (conditional medium) of h ESC-MSCs was collected, and the microvesicles were extracted by ultracentrifugation, and the size and morphology of microvesicles were identified under transmission electron microscope and scanning electron microscope. (2) the number of tumor cells was counted under microscope after h ESC-MSCs and h ESC-MSC-MVs were co-cultured with leukemia cell lines K562 and HL60 for 48 h, respectively. the microvesicles were extracted by ultracentrifugation, and the size and morphology of microvesicles were identified by transmission electron microscope and scanning electron microscope. The activity of tumor cells was detected by CCK8 assay. (3) the number of autophagy bodies in leukemia cells before and after co-culture was observed under transmission electron microscope, the expression of autophagy-related proteins LC3 and Beclin-1 in tumor cells was detected by Western blot. (4) the expression of apoptosis-related proteins Bax and Bcl-2 in tumor cells was detected by Western blot technique, and the apoptosis of tumor cells was detected by flow cytometry. Results: (1) h ESC-MSCs and h ESC-MSC-MVs inhibited the proliferation of leukemic cells in vitro in a concentration-dependent manner. (2) after 48 h ESC-MSC-MVs stimulation of leukemic cells, transmission electron microscope showed that the number of autophagy in the stimulated group was significantly higher than that in the control group. The expression of Beclin-1 and the ratio of LC3II/I increased. (3) the apoptosis rate of leukemic cells increased and the ratio of Bcl-2/Bax decreased after 48 h ESC-MSC-MVs stimulation. Conclusion: (1) h ESC-MSC-MVs can inhibit the proliferation of leukemia cell lines K562 and HL60 in vitro. (2) h ESC-MSC-MVs promotes autophagy of leukemia cells. (3) h ESC-MSC-MVs promotes apoptosis of leukemia cells.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
本文编号：2499169
[Abstract]:Background: recent studies have shown that human bone marrow and umbilical cord derived mesenchymal stem cells (mesenchymal stem cells,MSCs) can significantly inhibit the proliferation of leukemia cell line (leukemia cells) by co-culture with leukemia cells. The research group has previously focused on the culture and directional differentiation of human embryonic stem cells (human embryonic stem cells,h ESCs). It has been found that embryonic stem cells can spontaneously differentiate into mesenchymal stem cells under certain conditions. We expect that (human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSCs, which is differentiated from human embryonic stem cells, may also inhibit the proliferation of leukemia cells. Subsequent studies have shown that h ESC-MSCs can inhibit the proliferation of leukemia cells indirectly (paracrine pathway). Microvesicles (microvesicles,MVs) are active and tangible components secreted by cells into conditioned medium, and are the material basis for material transmission and information exchange between cells and surrounding cells or external environment. Therefore, we speculate that microvesicles secreted by human embryonic stem cells (MSCs) may also inhibit the proliferation of leukemic cells in vitro. And carry out further research on it. Objective: to study the effect and mechanism of microvesicles secreted by human embryonic stem cells (MSCs) on leukemia cells. Methods: (1) the serum-free medium (conditional medium) of h ESC-MSCs was collected, and the microvesicles were extracted by ultracentrifugation, and the size and morphology of microvesicles were identified under transmission electron microscope and scanning electron microscope. (2) the number of tumor cells was counted under microscope after h ESC-MSCs and h ESC-MSC-MVs were co-cultured with leukemia cell lines K562 and HL60 for 48 h, respectively. the microvesicles were extracted by ultracentrifugation, and the size and morphology of microvesicles were identified by transmission electron microscope and scanning electron microscope. The activity of tumor cells was detected by CCK8 assay. (3) the number of autophagy bodies in leukemia cells before and after co-culture was observed under transmission electron microscope, the expression of autophagy-related proteins LC3 and Beclin-1 in tumor cells was detected by Western blot. (4) the expression of apoptosis-related proteins Bax and Bcl-2 in tumor cells was detected by Western blot technique, and the apoptosis of tumor cells was detected by flow cytometry. Results: (1) h ESC-MSCs and h ESC-MSC-MVs inhibited the proliferation of leukemic cells in vitro in a concentration-dependent manner. (2) after 48 h ESC-MSC-MVs stimulation of leukemic cells, transmission electron microscope showed that the number of autophagy in the stimulated group was significantly higher than that in the control group. The expression of Beclin-1 and the ratio of LC3II/I increased. (3) the apoptosis rate of leukemic cells increased and the ratio of Bcl-2/Bax decreased after 48 h ESC-MSC-MVs stimulation. Conclusion: (1) h ESC-MSC-MVs can inhibit the proliferation of leukemia cell lines K562 and HL60 in vitro. (2) h ESC-MSC-MVs promotes autophagy of leukemia cells. (3) h ESC-MSC-MVs promotes apoptosis of leukemia cells.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R733.7
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