抑瘤素（OSM）对肝源性细胞体外生长的影响
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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。
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 of
each 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.
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。
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 of
each 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.
引文
1． Zarling JM, Shoyab M, Marquardt H et al. Oncostatin M: a growth regulator produced by differentiated histiocytic lymphoma cells.Proc Natl Acad Sci USA, 1986, 83(24) :9739-43．
2． Kasukabe T, Okabe-Kado J, Honma Y et al. Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells.J Biol Chem 1988,263(11) :5431-5．
3． Linsley PS, Bolton-Hanson M, Horn D et al. Identification and characterization of cellular receptors for the growth regulator, oncostatm M.J Biol Chem, 1989, 264(8) :4282-9．
4． Horn D, Fitzpatrick WC, Gompper PT et al. Regulation of cell growth by recombinant oncostatin M. Growth Factors, 1990, 2(2-3) : 157-65．
5． Tanaka M, Hara T, Copeland NG et al. Reconstitution of the functional mouse oncostatin M (OSM) receptor: molecular cloning of the mouse OSM receptor beta subunit. Blood, 1999, 93(3) :804-15．
6． Malik N, Graves D, Shoyab M etal. Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells. DNA Cell Biol, 1992,11(6) :453-9．
7． Klausen P, Pedersen L, Jurlander J et al. Oncostatin M and interleukin 6 inhibit cell cycle progression by prevention of p27kipl degradation in HepG2 cells. Oncogene, 2000,19(32) :3675-83．
8． Li C, Ahlborn TE, Kraemer FB et al. Oncostatin M-induced growth inhibition and morphological changes of MDA-MB231 breast cancer cells are abolished by blocking the MEK/ERK signaling pathway. Breast Cancer Res Treat, 2001, 66(2) :111-21．
9． Lu C, Rak JW, Kobayashi H et al. Increased resistance to oncostatin M-induced growth inhibition of human melanoma cell lines derived from advanced-stage lesions. Cancer Res, 1993, 53(12) :2708-11．
10． Kinoshita T, Sekiguchi T, Xu MJ et al Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci U S A, 1999, 96(13) :7265-70．
11． Halfter H, Lotfi R, Westermann R et al. Inhibition of growth and induction of differentiation of glioma cell lines by oncostatin M (OSM). Growth Factors, 1998;15(2) :135-47．
12． Douglas AM, Grant SL, Goss GA et al. Oncostatin M induces the differentiation of breast cancer cells. Int J Cancer, 1998, 75(1) :64-73．
13． Bruce AG, Hoggatt IH, Rose TM. Oncostatin M is a differentiation factor for myeloid leukemia cells.J Lumunol, 1992,149(4) : 1271-5．
14． Grant SL, Begley CG The oncostatin M signalling pathway: reversing the neoplastic phenotype? Mol Med Today 1999, 5(9) :406-12．
15． Kozlowska K, Zarzeczna M, Cichorek M. Sensitivity of transplantable melanoma cells to cytokines with regard to their spontaneous apoptosis. Pathobiology, 2001;69(5) :249-57．
16． Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells.Nature, 2001,414(6859) : 105-11．
17． Kinoshita T, Tanaka M, Kamiya A et al. Role of Oncostatin M in hematopoiesis and liver development. Cytokine Growth Factor Rev, 2000,11(3) :177-83．
18． Kinoshita T, Sekiguchi T, Xu MJ et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis.Proc Natl Acad Sci U S A, 1999, 96(13) :7265-70．
19． Kinoshita T, Miyajima A. Cytokine regulation of liver development. Biochim Biophys Acta, 2002,1592(3) :303-12．
20． Suzuki A, Iwama A, Miyashita H et al. Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells. Development, 2003, 130(11) :2513-2524．
21． Kamiya A, Kinoshita T, Miyajima A. Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways. FEBS Lett, 2001, 492(1-2) :90-4．
22． Kamiya A, Kinoshita T, Ito Y et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.EMBO, 1999,18(8) :2127-36．
23． Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med,1993, 204(3) :270-9．
24． Tsao MS, Smith JD, Nelson KG et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells. Exp Cell Res, 1984,154(1) :38-52．
25． Cotter TG, Lennon SV, Glynn JG et al. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumour and normal cells. Anticancer Res, 1990,10(5A):1153-9．
26． Voutsinas G Mutagenesis, apoptosis, basic relation to carcinogenic models. Folia Histochem Cytobiol, 2001, Suppl 2:56-7．
2． Kasukabe T, Okabe-Kado J, Honma Y et al. Purification of a novel growth inhibitory factor for partially differentiated myeloid leukemic cells.J Biol Chem 1988,263(11) :5431-5．
3． Linsley PS, Bolton-Hanson M, Horn D et al. Identification and characterization of cellular receptors for the growth regulator, oncostatm M.J Biol Chem, 1989, 264(8) :4282-9．
4． Horn D, Fitzpatrick WC, Gompper PT et al. Regulation of cell growth by recombinant oncostatin M. Growth Factors, 1990, 2(2-3) : 157-65．
5． Tanaka M, Hara T, Copeland NG et al. Reconstitution of the functional mouse oncostatin M (OSM) receptor: molecular cloning of the mouse OSM receptor beta subunit. Blood, 1999, 93(3) :804-15．
6． Malik N, Graves D, Shoyab M etal. Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells. DNA Cell Biol, 1992,11(6) :453-9．
7． Klausen P, Pedersen L, Jurlander J et al. Oncostatin M and interleukin 6 inhibit cell cycle progression by prevention of p27kipl degradation in HepG2 cells. Oncogene, 2000,19(32) :3675-83．
8． Li C, Ahlborn TE, Kraemer FB et al. Oncostatin M-induced growth inhibition and morphological changes of MDA-MB231 breast cancer cells are abolished by blocking the MEK/ERK signaling pathway. Breast Cancer Res Treat, 2001, 66(2) :111-21．
9． Lu C, Rak JW, Kobayashi H et al. Increased resistance to oncostatin M-induced growth inhibition of human melanoma cell lines derived from advanced-stage lesions. Cancer Res, 1993, 53(12) :2708-11．
10． Kinoshita T, Sekiguchi T, Xu MJ et al Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis. Proc Natl Acad Sci U S A, 1999, 96(13) :7265-70．
11． Halfter H, Lotfi R, Westermann R et al. Inhibition of growth and induction of differentiation of glioma cell lines by oncostatin M (OSM). Growth Factors, 1998;15(2) :135-47．
12． Douglas AM, Grant SL, Goss GA et al. Oncostatin M induces the differentiation of breast cancer cells. Int J Cancer, 1998, 75(1) :64-73．
13． Bruce AG, Hoggatt IH, Rose TM. Oncostatin M is a differentiation factor for myeloid leukemia cells.J Lumunol, 1992,149(4) : 1271-5．
14． Grant SL, Begley CG The oncostatin M signalling pathway: reversing the neoplastic phenotype? Mol Med Today 1999, 5(9) :406-12．
15． Kozlowska K, Zarzeczna M, Cichorek M. Sensitivity of transplantable melanoma cells to cytokines with regard to their spontaneous apoptosis. Pathobiology, 2001;69(5) :249-57．
16． Reya T, Morrison SJ, Clarke MF et al. Stem cells, cancer, and cancer stem cells.Nature, 2001,414(6859) : 105-11．
17． Kinoshita T, Tanaka M, Kamiya A et al. Role of Oncostatin M in hematopoiesis and liver development. Cytokine Growth Factor Rev, 2000,11(3) :177-83．
18． Kinoshita T, Sekiguchi T, Xu MJ et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis.Proc Natl Acad Sci U S A, 1999, 96(13) :7265-70．
19． Kinoshita T, Miyajima A. Cytokine regulation of liver development. Biochim Biophys Acta, 2002,1592(3) :303-12．
20． Suzuki A, Iwama A, Miyashita H et al. Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells. Development, 2003, 130(11) :2513-2524．
21． Kamiya A, Kinoshita T, Miyajima A. Oncostatin M and hepatocyte growth factor induce hepatic maturation via distinct signaling pathways. FEBS Lett, 2001, 492(1-2) :90-4．
22． Kamiya A, Kinoshita T, Ito Y et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer.EMBO, 1999,18(8) :2127-36．
23． Grisham JW, Coleman WB, Smith GJ. Isolation, culture, and transplantation of rat hepatocytic precursor (stem-like) cells. Proc Soc Exp Biol Med,1993, 204(3) :270-9．
24． Tsao MS, Smith JD, Nelson KG et al. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells. Exp Cell Res, 1984,154(1) :38-52．
25． Cotter TG, Lennon SV, Glynn JG et al. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumour and normal cells. Anticancer Res, 1990,10(5A):1153-9．
26． Voutsinas G Mutagenesis, apoptosis, basic relation to carcinogenic models. Folia Histochem Cytobiol, 2001, Suppl 2:56-7．