【作者】 李晓利；

【导师】 尉承泽； 赵士富；

【作者基本信息】 中国人民解放军军事医学科学院 ， 肿瘤学， 2003， 硕士

【摘要】 抑瘤素(oncostatin M，OSM)具有抑制多种肿瘤细胞增殖和诱导其分化的生物学功能，OSM诱导后，肿瘤细胞表现出生长抑制，并向成熟细胞形态分化，出现一些正常细胞的生理功能。同时，OSM又能够促进胎肝时期肝脏实质细胞的成熟分化。鉴于OSM在肝脏发育中的重要地位以及OSM对肿瘤生长的调控作用，我们建立了表达OSM的CHO-OSM-EGFP细胞株，以大鼠肝脏来源的干细胞株WB-F344(功能上类似于肝卵圆细胞)与肿瘤细胞株HTC为研究对象，对OSM对肝源性干细胞和肿瘤细胞体外生命活动的影响进行了初步探讨。 本实验中，构建了表达人OSM蛋白基因的真核表达载体pNI-OSM-EGFP。通过双酶切将完整的OSM ORF从pEO质粒中切出，克隆到带绿色荧光蛋白的真核表达载体pEGFP-N1中，转染中国仓鼠卵巢细胞(CHO)，G418筛选后获得多个携带有绿色荧光蛋白的阳性克隆。在荧光镜下对阳性克隆进行标记，无菌操作下，经二次挑选出细胞克隆，并进行扩大培养，在流式细胞术鉴定CHO-OSM-EGFP细胞纯度，RT-PCR鉴定OSM基因在mRNA水平表达的基础上，证实获得了OSM稳定表达细胞株CHO-OSM-EGFP。 分别收集CHO、CHO-EGFP，CHO-OSM-EGFP的细胞上清，取不同浓度的上述细胞上清与大鼠肝癌细胞株HTC和Fischer大鼠肝脏来源的干细胞系WB-F344(功能上类似于肝卵原细胞)共培养，4～7天左右，流式细胞术(FACS)分析结果显示CHO-OSM-EGFP上清在体外能够诱导HTC发生凋亡，对HTC细胞周期各时相细胞分布无影响；当在HTC细胞培养体系中加入终浓度为15％～30％CHO-OSM-EGFP上清时，凋亡比例随着CHO-OSM-EGFP上清浓度的增加而增加，当CHO-OSM-EGFP上清终浓度增大军事医学科学院硕士学位论文至40％，凋亡比例反而下降，同时细胞死亡明显多于对照组。与之相反的是CHO－OSM－EGFP上清能使大鼠肝脏干细胞系阳一F344细胞阻滞于GO－GI期，处于S期和M期细胞比例下调，同时向成熟细胞形态分化，而不产生凋亡作用。本研究结果提示：低剂量OSM可以HTC肿瘤细胞的凋亡，高剂量0训将导致mC细胞死亡；而0规不引起肝脏干细胞阳一四时的凋亡，可诱导其向成熟细胞分化。OSM对肝脏来源干细胞与肿瘤细胞的生物学作用及机制有待于进一步深入研究。更多还原

【Abstract】 Cytokine oncostatin M (OSM) has profound effects on proliferation and differentiation of cell lines from tumor types. OSM treated cells show reduced growth rate and differentiation phenotypes. Meanwhile, OSM is capable of stimulating maturation of hepatic parenchymal cells in embryonic liver. But, the mechanisms underlying tumorigenesis of liver carcinoma has not been fully elucidated. A clear understanding OSM regulates the growth and differentiation of tumor and stem cell lines from liver would provide insights into this complicated disease.In this study, the expression plasmid pNl-OSM-EGFP constructed by the insertion of the complete ORF of human OSM into pEGFP-Nl vector. By transfecting Chinese hamster ovary (CHO) cell line. The stable cell line CHO-OSM-EGFP was obtained after screening with G418, and single colony was picked up under the fluorescence microscope. The clonality of the cells were checked by fluorescence activated cell sorting (FACS), the mRNA expression was examined by RT-PCR using OSM primers. In order to demonstrate the effect of OSM on the growth of liver-derived stem cells and tumor cells, the cultured supernatant of cells transfected with pOSM-EGFP-Nl or pEGFP-Nl were collected and added to the HTC or WB-F344 culture plates with the different final concentrations. The cells after 4 ~ 7 days cultivation were analyzed by FACS. Our primary results showed that the cultured supernatant collected from pOSM-EGFP-Nl transfected cells could induce apoptosis of HTC cells, but no influence on the distribution ofeach phases of the cell cycle; In contrast to the above, the same supernatant arrested WB-F344 stem cells at GO-G1 phase of cell cycle, cell proportion in the M and S phase decreased but no apoptosis could be observed. And, the growth arrest caused by the supernatant was accompanied by cellular phenotypic changes. The mechanism of different effect of OSM on liver-derived normal and malignant cells should be further explored, which may open new avenues for developing alternative treatment of liver and other cancers.更多还原