【摘要】：研究背景:最近多項(xiàng)研究表明,人骨髓和臍帶來源的間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs)通過與白血病細(xì)胞直接共培養(yǎng)后可以明顯抑制白血病細(xì)胞(leukemia cells)的增殖。課題組此前致力于人胚胎干細(xì)胞(human embryonic stem cells,h ESCs)的培養(yǎng)及定向分化方面的研究,發(fā)現(xiàn)胚胎干細(xì)胞在一定條件下可以自發(fā)分化為間充質(zhì)干細(xì)胞,我們預(yù)期這種由人胚胎干細(xì)胞分化而來的間充質(zhì)干細(xì)胞(human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSCs)同樣可能抑制白血病細(xì)胞的增殖。對此后續(xù)的研究表明,h ESC-MSCs確實(shí)可以通過間接作用(旁分泌途徑)抑制白血病細(xì)胞的增殖。而微囊泡(microvesicles,MVs)是細(xì)胞分泌至條件培養(yǎng)基中的活性有形成分,是細(xì)胞與周圍細(xì)胞或外界環(huán)境進(jìn)行物質(zhì)傳遞和信息交流的物質(zhì)基礎(chǔ),因此我們推測人胚胎干細(xì)胞源間充質(zhì)干細(xì)胞分泌的微囊泡(microvesicles released from human embryonic stem cell derived-mesenchymal stem cells,h ESC-MSC-MVs)在體外也可能抑制白血病細(xì)胞的增殖,并對其展開進(jìn)一步的研究。研究目的:研究人胚胎干細(xì)胞源間充質(zhì)干細(xì)胞分泌的微囊泡對白血病細(xì)胞的作用及作用機(jī)制。研究方法:(1)收集h ESC-MSCs的無血清培養(yǎng)基(條件培養(yǎng)基),運(yùn)用超速離心法提取微囊泡;在透射電子顯微鏡以及掃描電子顯微鏡下對微囊泡的大小和形態(tài)進(jìn)行鑒定。(2)將h ESC-MSCs和h ESC-MSC-MVs分別與白血病細(xì)胞株K562和HL60共培養(yǎng)48h后,顯微鏡下計(jì)數(shù)腫瘤細(xì)胞的數(shù)量;用CCK8實(shí)驗(yàn)檢測腫瘤細(xì)胞的活力。(3)在透射電鏡下觀察共培養(yǎng)前后白血病細(xì)胞中的自噬小體的數(shù)量;用Western blot技術(shù)檢測腫瘤細(xì)胞自噬相關(guān)蛋白LC3及Beclin-1的表達(dá)水平。(4)應(yīng)用Western blot技術(shù)檢測腫瘤細(xì)胞凋亡相關(guān)蛋白Bax及Bcl-2的表達(dá)水平;流式細(xì)胞術(shù)檢測腫瘤細(xì)胞的凋亡。研究結(jié)果:(1)h ESC-MSCs和h ESC-MSC-MVs在體外抑制白血病細(xì)胞的增殖,并且這種抑制作用呈濃度依賴效應(yīng)。(2)h ESC-MSC-MVs刺激白血病細(xì)胞48h后,透射電鏡觀察到刺激組較對照組腫瘤細(xì)胞中自噬小體明顯增多、Beclin-1表達(dá)水平增高及LC3II/I比值增高。(3)h ESC-MSC-MVs刺激白血病細(xì)胞48h后,腫瘤細(xì)胞的凋亡率增高、Bcl-2/Bax比值降低。結(jié)論:(1)h ESC-MSC-MVs在體外能抑制白血病細(xì)胞株K562和HL60的增殖。(2)h ESC-MSC-MVs促進(jìn)白血病細(xì)胞的自噬。(3)h ESC-MSC-MVs促進(jìn)白血病細(xì)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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R733.7

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