重组人OSM的制备及诱导肝癌细胞分化的实验研究
|
摘要
抑瘤素M(OSM)能够诱导多种肿瘤细胞,如神经胶质瘤、骨肉瘤、乳腺癌、肺腺癌、白血病细胞发生形态和/或功能上的分化成熟。OSM对胚胎及成体肝脏均具有独特的生物学作用。鉴于此,本研究围绕OSM对肝癌细胞的诱导分化作用进行了如下工作。利用基因重组技术构建了毕赤酵母真核表达载体pPICZαC-hOSM,电转化毕赤酵母X-33,筛选出能够稳定高效分泌表达重组人OSM(rhOSM)的工程菌。经SDS-PAGE、Western Blot、N-末端15个氨基酸残基序列分析、AKTA explorer多功能纯化系统纯化、质谱测定其相对分子质量及体外活性检测,证明应用毕赤酵母表达系统分泌表达的rhOSM与天然OSM具有相同的理化性质和生物学活性。利用80 L发酵罐进行rhOSM中试规模发酵,并对其发酵条件进行优化,rhOSM表达量达到280 mg·L-1。同时建立了一种分离纯化rhOSM的新方法。
利用光镜、透射电镜、MTT、RT-PCR、免疫荧光、免疫细胞化学染色、流式细胞术等方法和技术,体外水平观察rhOSM对人肝癌细胞SMMC-7721生长的抑制作用、细胞形态、超微结构、信号传导分子、细胞周期和凋亡率的影响及诱导肝癌细胞分化的相关生化指标的检测。结果表明rhOSM能够抑制SMMC-7721细胞生长、诱导其分化、促进其凋亡。通过观察rhOSM对BALB/c小鼠肝癌细胞H22皮下移植瘤生长的抑制情况、外周血中AFP水平、瘤组织病理学改变,表明rhOSM对小鼠肝癌细胞具有抑制生长、促进分化的作用。实验结果同时表明,短期腹腔注射rhOSM对小鼠肝脏、脾脏没有造成损伤,但对小鼠胸腺组织具有抑制作用。
本研究创新之处:1)利用80 L发酵罐进行了rhOSM毕赤酵母工程菌的中试规模发酵,对其表达条件进行优化,并建立一种适用于大规模分离纯化rhOSM的方法,证实应用毕赤酵母表达系统表达的rhOSM与天然OSM具有相同的理化性质和生物学活性; 2)从体内、体外水平,全面、系统地研究了rhOSM对肝癌细胞的作用,证实rhOSM能够抑制肝癌细胞增殖、诱导其分化和凋亡。国内外未见相关报道。
To induce tumor cells to differentiate into normal cells is a new branch of oncotherapy and has become more and more concerned in this field. At present, the basic researches and clinical applications of leukemia as the model of differentiation have been performed comprehensively and profoundly. Although there has been a lot of investigations on solid tumor cells and still at the stage of research, the effect was not very satisfactory. Therefore, it is crucial to search for non-toxic and highly efficient substances that can induce the differentiation of solid tumor cells efficiently, which has great practical value and significance and needs further research to resolve.
Oncostatin M (OSM), a glycoprotein monomer of 28,000 Da, was originally isolated and purified from the conditioned media of phorbol 12-myristate 13-acetate (PMA)-stimulated human histiocytic lymphoma U937 cells by Zarling et al and was named by its activity to inhibit the proliferation of A375 melanoma cells. OSM belongs to the Interleukin(IL)-6 subfamily of cytokines, whose members include LIF, G-CSF, CNTF, IL-6, IL-11, IL-31, CT-1, CLC. The main property of the family is its function redundancy. Among the family members, OSM is most closely related to LIF structurally, functionally and genetically. OSM is a multifunctional cellular regulator and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. Furthermore, OSM exhibits many unique biological activities in inflammation, CNS, fetal and adult hematopoiesis, osteogenesis and immune system. OSM can stimulate acute phase protein synthesis in hepatocytes, regulate the tissue metalloproteinases and tissue inhibitors of metalloproteinases, stimulate the proliferation of human adipose tissue-derived mesenchymalstem cells (hATSCs) and inhibit their differentiation.
OSM exhibits unique biological activities in liver. OSM promotes differentiation of hepatic cells and exhibits differentiation markers, which are accompanied by functional and morphological maturation. As the fetal liver matures, it gradually loses hematopoietic potential, and HSCs relocate to the bone marrow. Roles of OSM in adult liver include regeneration, tissue remodeling, regulating lipid metabolism, preventing hepatocytes apoptosis, prevention and treatment of liver injury. There has been previously reported that OSM can induce morphology and/or function differentiation and maturation of many tumor cells, which are as follows: glioma cells, osteosarcoma cells, breast cancer cells, lung adenocarcinoma cells, myeloid leukemia cells. OSM can induce cells to differentiate into hepatocytes from embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, adipose tissue-derived stromal cells (hADSC) and hepatic stem cells in liver with cooperative effects of HGF. According to unique biological activities in liver and induction of differentiation of some solid tumor cells by OSM, we tried to examine the ability of induced differentiation of hepatoma cells by OSM. So, studies were done as follows. And finally we identified that OSM can induce the differentiation of hepatoma cells.
1 Constructing, screening and identificating of Pichia pastoris engineering strains expressing rhOSM steadily at a high level
1.1 Screening of the transformed Pichia pastoris strains
Linearize the expression vector pPICZαC-hOSM after confirmed by sequencing and transform it into Pichia pastoris X-33 via electroporation. 20 single clones were picked from YPD plates containing Zeocin. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the expression primers. Yeast clones tested positively by PCR were proliferated and then rhOSM was induced with 0.5% methanol. The supernatant of fermentation was identified by SDS-PAGE and Western Blot and the highest level strain was screened. The concentration of rhOSM in 50 mL conical tubes can reached 45 mg·L-1.
1.2 Identification of rhOSM
rhOSM was purified by AKTA explorer using Mono S cation exchange and Source TM30 RPC hydrophobic chromatography with a linear gradient elution. The purity was more than 95%. MALDI-TOF-MS showed that molecule weight of rhOSM was 27300.92 which was commensurate with OSM secreted from U937 cells. Amino acid sequencing confirmed that the 15th N-terminal amino acid of rhOSM was identical with natural OSM except for the 6th amino acid which attending the formation of intramolecular disulfide linkage .
2 Studies on pilot-scale fermentation and purification process of rhOSM
2.1 Pilot-scale fermentation of rhOSM in Pichia pastoris
We performed pilot-scale fermentation of rhOSM in an 80 L fermentor. The study was focused mainly on pH value, culture medium, dissolved oxygen, temperature, methanol feeding speed, initial biomass and etc. The results indicated that in the FM21 medium of pH 5.0, when a cell yield of 190 g·L-1 wet weight was achieved, methanol induction phase began. In the fermentation broth of pH5.5, DO above 25% and the supply speed of methanol is 9.3 mL·h-1·L-1 initial fermentation volume, after methanol induction for about 36 h the expression level of rhOSM peaked. The concentration of rhOSM in the broth can reached 280mg·L-1. 2.2 A new method to purify rhOSM
Centrifugate the fermentation product and collect the supernatant. The supernatant was added into ammonium sulphate to reach about 25% (g·L-1), the supernatant was centrifuged again. The supernatant was loaded onto a phenyl Sepharose 6 Fast Flow column (pH7.0) and SP Sepharose XL column (pH4.5). The bound protein was eluted with step-wise elution. The degree of purity could be more than 95% and the yield coefficient was 62%. We acquired rhOSM 6.94 g from 40 L fermentation broth.
2.3 Activity assay of rhOSM
To verify rhOSM produced and purified from P. pastoris is fully active, the biological activity of rhOSM was tested in an in vitro growth inhibition assay on human A375 melanoma cell line with MTT. rhOSM showed a dose-dependent proliferation inhibition effect on human A375 melanoma cell. The purified rhOSM had a specific growth inhibition activity of 6.26×104 RU·μg-1 , which was commensurate with typical values (6.2×10 4 RU·μg-1) obtained with standard OSM. Purified rhOSM showed no significant loss in its activity when stored for more than 6 months at -80℃.
3 Studies on inducing human hepatoma SMMC-7721 cell lines differentiation in vitro by rhOSM
3.1 Investigate OSM receptors in human SMMC-7721 hepatoma cell
We investigated whether gp130、OSMRβ、LIFRα、OSM gene expression in the human hepatoma SMMC-7721 cell using RT-PCR. Our findings demonstrated that SMMC-7721 cells express LIFRα/gp130 and OSMRβ/gp130 but not OSM gene. It was concluded that SMMC-7721 cells did not produce cytokine OSM.
3.2 Growth inhibition assay on human hepatoma SMMC-7721 cell by rhOSM
We measured the growth curves of rhOSM-treated SMMC-7721 cell. The results demonstrated that rhOSM can obviously inhibit the proliferation of SMMC-7721 cell. And 1 ng·mL-1, 10 ng·mL-1 , 100 ng·mL -1 rhOSM showed a time and dose-dependent proliferation inhibition effect on human SMMC-7721 cell. After treatment for 6 days by three doses of rhOSM, the proliferation inhibition rate of SMMC-7721 cell respectively was 27.01%, 54.33%, 81.79%.
3.3 rhOSM-induced cell cycle arrest and apoptosis
After treatment for 5 days by 1 ng·mL-1 , 10 ng·mL -1, 100 ng·mL-1 rhOSM, we examined cell cycle and apoptosis of SMMC-7721 cell by flow cytometry. The results demonstrate that rhOSM induced cell cycle arrest in G0 /G1 phase and apoptosis, the apoptosis ratio gradually rised with the concentrations of rhOSM rising.
3.4 Signal transducer and activator of transcription 3 (STAT3) activation
After treatment of SMMC-7721 cell for 2 hours by 100 ng·mL-1 rhOSM, we examined STAT3 phosphorylation by immunofluorescence staining. The results showed that cell membrane, cytoplasm and nucleus all showed green fluorescein. It demonstrated that OSMR allowed strong activation of STAT3 and then to travel to the nucleus where it acted.
3.5 Morphology and ultra-microstructure observation
The observation under light microscopy and transmission electronic microscopy showed that the SMMC-7721 cell morphology turned to rotundity and oval, and in part rotundity. The volume of cell turned smaller, cytoplasm turned much more abundant, and nucleus turned smaller. The nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular. In the cytoplasm, mitochondria and typical, rough endoplasmic reticulum increased. The degree of glycogen hyperplasia lessen. Those changes demonstrated that rhOSM induced the mature and normality of morphology and ultra-microstructure of SMMC-7721 cell.
3.6 Examination of expression level of AFP
We examined AFP expression level in the cytoplasm of rhOSM-treated SMMC-7721 cell by SABC-AP immunocytochemistry staining. The result showed that expression level of AFP was down-regulated, the product was blue and less staining, uniformly distributed in the cytoplasm surrounding the nuclear region.
3.7 The investigation of specific biochemical markers of hepatocyte differentiation
After treatment of SMMC-7721 cell for 6 days with 10 ng·mL-1 and 100 ng·mL-1 rhOSM,γ- glutamyltranspeptidase (γ-GT) activity and the secretory amount of alpha-fetoprotein (AFP) were significantly decreased, while alkaline phosphatase (ALP) activity and the secretory amount of albumin (ALB) were significantly increased, which showed a time and dose-dependent manner. Those changes demonstrated that rhOSM induced the mature and normality of function of SMMC-7721 cell.
4 Studies on inducing murine H22 hepatoma cells differentiation in vivo by rhOSM
4.1 Establishment of animal model and animal grouping
50 BALB/c mice were inoculated with murine H22 hepatoma cells (2×106) into the right axillary subcutaneous. About 10 days after inoculation, we could touch a small solid tumor in each mouse. Then the mice were divided into 5 groups, intraperitoneal administration of NaCl, rhOSM (25, 50, 100μg·kg-1·d-1) and ATRA (1 mg·kg-1·d-1).
4.2 Effect of rhOSM on tumor growth and peripheral blood levels of AFP
All treatment groups could inhibit tumor growth. The average tumor volume and weight of the middle and high dose of rhOSM were much lower than NaCl group (P<0.01), high dose of rhOSM was commensurate with ATRA group (P>0.05). There was no significant statistical difference between all treatment groups and NaCl group about body weight (P>0.05). AFP levels of all treatment groups were much lower than NaCl group (P<0.01). AFP levels were significantly decreased with the dose of rhOSM increasing. There was no significant statistical difference between the high dose of rhOSM and ATRA group (P>0.05).
4.3 Effect of rhOSM on thymus index, spleen index and liver index
The thymus indexes of all treatment groups were much lower than NaCl group (P<0.01), which indicated that both rhOSM and ATRA had suppressive effect on mice thymus and suppressive effect became more serious with the dose of rhOSM increasing. The liver index of all treatment groups were much higher than NaCl group (P<0.01), which indicated that rhOSM was not toxic to mice liver in short-term intraperitoneal administration of rhOSM. There was no significant statistical difference between all treatment groups and NaCl group (P>0.05),. which indicated that rhOSM did not impair mice spleen.
4.4 Histopathological changes of tumor tissues
Tumor volumes of all treatment groups were much smaller than NaCl group. Tumor cell density of all treatment groups decreased at different extent. Cell shrinkage, dwindled karyon. Karyon deeply stained and cytoplasm stained red. Tumor tissues gradually differentiated into mature. Interstitial connective tissue in tumor tissue increased.
To sum up, in this study we constructed vector pPICZαC-hOSM, transformed it into Pichia pastoris X-33 and screened the high level and steady expressing Pichia pastoris engineering strains of rhOSM. To the best of our knowledge, this is the first report that we performed the pilot-scale fermentation of rhOSM in an 80 L fermentor and built a new method for large-scale purification of rhOSM. For the first time, we confirmed that rhOSM could induce hepatoma cells differentiation. Our studies built theoretical and experimental foundation for research of treatment of other solid tumors by rhOSM and industry production of rhOSM.
利用光镜、透射电镜、MTT、RT-PCR、免疫荧光、免疫细胞化学染色、流式细胞术等方法和技术,体外水平观察rhOSM对人肝癌细胞SMMC-7721生长的抑制作用、细胞形态、超微结构、信号传导分子、细胞周期和凋亡率的影响及诱导肝癌细胞分化的相关生化指标的检测。结果表明rhOSM能够抑制SMMC-7721细胞生长、诱导其分化、促进其凋亡。通过观察rhOSM对BALB/c小鼠肝癌细胞H22皮下移植瘤生长的抑制情况、外周血中AFP水平、瘤组织病理学改变,表明rhOSM对小鼠肝癌细胞具有抑制生长、促进分化的作用。实验结果同时表明,短期腹腔注射rhOSM对小鼠肝脏、脾脏没有造成损伤,但对小鼠胸腺组织具有抑制作用。
本研究创新之处:1)利用80 L发酵罐进行了rhOSM毕赤酵母工程菌的中试规模发酵,对其表达条件进行优化,并建立一种适用于大规模分离纯化rhOSM的方法,证实应用毕赤酵母表达系统表达的rhOSM与天然OSM具有相同的理化性质和生物学活性; 2)从体内、体外水平,全面、系统地研究了rhOSM对肝癌细胞的作用,证实rhOSM能够抑制肝癌细胞增殖、诱导其分化和凋亡。国内外未见相关报道。
To induce tumor cells to differentiate into normal cells is a new branch of oncotherapy and has become more and more concerned in this field. At present, the basic researches and clinical applications of leukemia as the model of differentiation have been performed comprehensively and profoundly. Although there has been a lot of investigations on solid tumor cells and still at the stage of research, the effect was not very satisfactory. Therefore, it is crucial to search for non-toxic and highly efficient substances that can induce the differentiation of solid tumor cells efficiently, which has great practical value and significance and needs further research to resolve.
Oncostatin M (OSM), a glycoprotein monomer of 28,000 Da, was originally isolated and purified from the conditioned media of phorbol 12-myristate 13-acetate (PMA)-stimulated human histiocytic lymphoma U937 cells by Zarling et al and was named by its activity to inhibit the proliferation of A375 melanoma cells. OSM belongs to the Interleukin(IL)-6 subfamily of cytokines, whose members include LIF, G-CSF, CNTF, IL-6, IL-11, IL-31, CT-1, CLC. The main property of the family is its function redundancy. Among the family members, OSM is most closely related to LIF structurally, functionally and genetically. OSM is a multifunctional cellular regulator and can act on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. Furthermore, OSM exhibits many unique biological activities in inflammation, CNS, fetal and adult hematopoiesis, osteogenesis and immune system. OSM can stimulate acute phase protein synthesis in hepatocytes, regulate the tissue metalloproteinases and tissue inhibitors of metalloproteinases, stimulate the proliferation of human adipose tissue-derived mesenchymalstem cells (hATSCs) and inhibit their differentiation.
OSM exhibits unique biological activities in liver. OSM promotes differentiation of hepatic cells and exhibits differentiation markers, which are accompanied by functional and morphological maturation. As the fetal liver matures, it gradually loses hematopoietic potential, and HSCs relocate to the bone marrow. Roles of OSM in adult liver include regeneration, tissue remodeling, regulating lipid metabolism, preventing hepatocytes apoptosis, prevention and treatment of liver injury. There has been previously reported that OSM can induce morphology and/or function differentiation and maturation of many tumor cells, which are as follows: glioma cells, osteosarcoma cells, breast cancer cells, lung adenocarcinoma cells, myeloid leukemia cells. OSM can induce cells to differentiate into hepatocytes from embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, adipose tissue-derived stromal cells (hADSC) and hepatic stem cells in liver with cooperative effects of HGF. According to unique biological activities in liver and induction of differentiation of some solid tumor cells by OSM, we tried to examine the ability of induced differentiation of hepatoma cells by OSM. So, studies were done as follows. And finally we identified that OSM can induce the differentiation of hepatoma cells.
1 Constructing, screening and identificating of Pichia pastoris engineering strains expressing rhOSM steadily at a high level
1.1 Screening of the transformed Pichia pastoris strains
Linearize the expression vector pPICZαC-hOSM after confirmed by sequencing and transform it into Pichia pastoris X-33 via electroporation. 20 single clones were picked from YPD plates containing Zeocin. Then the genomic DNA of the transformed yeasts were extracted to perform PCR using the expression primers. Yeast clones tested positively by PCR were proliferated and then rhOSM was induced with 0.5% methanol. The supernatant of fermentation was identified by SDS-PAGE and Western Blot and the highest level strain was screened. The concentration of rhOSM in 50 mL conical tubes can reached 45 mg·L-1.
1.2 Identification of rhOSM
rhOSM was purified by AKTA explorer using Mono S cation exchange and Source TM30 RPC hydrophobic chromatography with a linear gradient elution. The purity was more than 95%. MALDI-TOF-MS showed that molecule weight of rhOSM was 27300.92 which was commensurate with OSM secreted from U937 cells. Amino acid sequencing confirmed that the 15th N-terminal amino acid of rhOSM was identical with natural OSM except for the 6th amino acid which attending the formation of intramolecular disulfide linkage .
2 Studies on pilot-scale fermentation and purification process of rhOSM
2.1 Pilot-scale fermentation of rhOSM in Pichia pastoris
We performed pilot-scale fermentation of rhOSM in an 80 L fermentor. The study was focused mainly on pH value, culture medium, dissolved oxygen, temperature, methanol feeding speed, initial biomass and etc. The results indicated that in the FM21 medium of pH 5.0, when a cell yield of 190 g·L-1 wet weight was achieved, methanol induction phase began. In the fermentation broth of pH5.5, DO above 25% and the supply speed of methanol is 9.3 mL·h-1·L-1 initial fermentation volume, after methanol induction for about 36 h the expression level of rhOSM peaked. The concentration of rhOSM in the broth can reached 280mg·L-1. 2.2 A new method to purify rhOSM
Centrifugate the fermentation product and collect the supernatant. The supernatant was added into ammonium sulphate to reach about 25% (g·L-1), the supernatant was centrifuged again. The supernatant was loaded onto a phenyl Sepharose 6 Fast Flow column (pH7.0) and SP Sepharose XL column (pH4.5). The bound protein was eluted with step-wise elution. The degree of purity could be more than 95% and the yield coefficient was 62%. We acquired rhOSM 6.94 g from 40 L fermentation broth.
2.3 Activity assay of rhOSM
To verify rhOSM produced and purified from P. pastoris is fully active, the biological activity of rhOSM was tested in an in vitro growth inhibition assay on human A375 melanoma cell line with MTT. rhOSM showed a dose-dependent proliferation inhibition effect on human A375 melanoma cell. The purified rhOSM had a specific growth inhibition activity of 6.26×104 RU·μg-1 , which was commensurate with typical values (6.2×10 4 RU·μg-1) obtained with standard OSM. Purified rhOSM showed no significant loss in its activity when stored for more than 6 months at -80℃.
3 Studies on inducing human hepatoma SMMC-7721 cell lines differentiation in vitro by rhOSM
3.1 Investigate OSM receptors in human SMMC-7721 hepatoma cell
We investigated whether gp130、OSMRβ、LIFRα、OSM gene expression in the human hepatoma SMMC-7721 cell using RT-PCR. Our findings demonstrated that SMMC-7721 cells express LIFRα/gp130 and OSMRβ/gp130 but not OSM gene. It was concluded that SMMC-7721 cells did not produce cytokine OSM.
3.2 Growth inhibition assay on human hepatoma SMMC-7721 cell by rhOSM
We measured the growth curves of rhOSM-treated SMMC-7721 cell. The results demonstrated that rhOSM can obviously inhibit the proliferation of SMMC-7721 cell. And 1 ng·mL-1, 10 ng·mL-1 , 100 ng·mL -1 rhOSM showed a time and dose-dependent proliferation inhibition effect on human SMMC-7721 cell. After treatment for 6 days by three doses of rhOSM, the proliferation inhibition rate of SMMC-7721 cell respectively was 27.01%, 54.33%, 81.79%.
3.3 rhOSM-induced cell cycle arrest and apoptosis
After treatment for 5 days by 1 ng·mL-1 , 10 ng·mL -1, 100 ng·mL-1 rhOSM, we examined cell cycle and apoptosis of SMMC-7721 cell by flow cytometry. The results demonstrate that rhOSM induced cell cycle arrest in G0 /G1 phase and apoptosis, the apoptosis ratio gradually rised with the concentrations of rhOSM rising.
3.4 Signal transducer and activator of transcription 3 (STAT3) activation
After treatment of SMMC-7721 cell for 2 hours by 100 ng·mL-1 rhOSM, we examined STAT3 phosphorylation by immunofluorescence staining. The results showed that cell membrane, cytoplasm and nucleus all showed green fluorescein. It demonstrated that OSMR allowed strong activation of STAT3 and then to travel to the nucleus where it acted.
3.5 Morphology and ultra-microstructure observation
The observation under light microscopy and transmission electronic microscopy showed that the SMMC-7721 cell morphology turned to rotundity and oval, and in part rotundity. The volume of cell turned smaller, cytoplasm turned much more abundant, and nucleus turned smaller. The nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular. In the cytoplasm, mitochondria and typical, rough endoplasmic reticulum increased. The degree of glycogen hyperplasia lessen. Those changes demonstrated that rhOSM induced the mature and normality of morphology and ultra-microstructure of SMMC-7721 cell.
3.6 Examination of expression level of AFP
We examined AFP expression level in the cytoplasm of rhOSM-treated SMMC-7721 cell by SABC-AP immunocytochemistry staining. The result showed that expression level of AFP was down-regulated, the product was blue and less staining, uniformly distributed in the cytoplasm surrounding the nuclear region.
3.7 The investigation of specific biochemical markers of hepatocyte differentiation
After treatment of SMMC-7721 cell for 6 days with 10 ng·mL-1 and 100 ng·mL-1 rhOSM,γ- glutamyltranspeptidase (γ-GT) activity and the secretory amount of alpha-fetoprotein (AFP) were significantly decreased, while alkaline phosphatase (ALP) activity and the secretory amount of albumin (ALB) were significantly increased, which showed a time and dose-dependent manner. Those changes demonstrated that rhOSM induced the mature and normality of function of SMMC-7721 cell.
4 Studies on inducing murine H22 hepatoma cells differentiation in vivo by rhOSM
4.1 Establishment of animal model and animal grouping
50 BALB/c mice were inoculated with murine H22 hepatoma cells (2×106) into the right axillary subcutaneous. About 10 days after inoculation, we could touch a small solid tumor in each mouse. Then the mice were divided into 5 groups, intraperitoneal administration of NaCl, rhOSM (25, 50, 100μg·kg-1·d-1) and ATRA (1 mg·kg-1·d-1).
4.2 Effect of rhOSM on tumor growth and peripheral blood levels of AFP
All treatment groups could inhibit tumor growth. The average tumor volume and weight of the middle and high dose of rhOSM were much lower than NaCl group (P<0.01), high dose of rhOSM was commensurate with ATRA group (P>0.05). There was no significant statistical difference between all treatment groups and NaCl group about body weight (P>0.05). AFP levels of all treatment groups were much lower than NaCl group (P<0.01). AFP levels were significantly decreased with the dose of rhOSM increasing. There was no significant statistical difference between the high dose of rhOSM and ATRA group (P>0.05).
4.3 Effect of rhOSM on thymus index, spleen index and liver index
The thymus indexes of all treatment groups were much lower than NaCl group (P<0.01), which indicated that both rhOSM and ATRA had suppressive effect on mice thymus and suppressive effect became more serious with the dose of rhOSM increasing. The liver index of all treatment groups were much higher than NaCl group (P<0.01), which indicated that rhOSM was not toxic to mice liver in short-term intraperitoneal administration of rhOSM. There was no significant statistical difference between all treatment groups and NaCl group (P>0.05),. which indicated that rhOSM did not impair mice spleen.
4.4 Histopathological changes of tumor tissues
Tumor volumes of all treatment groups were much smaller than NaCl group. Tumor cell density of all treatment groups decreased at different extent. Cell shrinkage, dwindled karyon. Karyon deeply stained and cytoplasm stained red. Tumor tissues gradually differentiated into mature. Interstitial connective tissue in tumor tissue increased.
To sum up, in this study we constructed vector pPICZαC-hOSM, transformed it into Pichia pastoris X-33 and screened the high level and steady expressing Pichia pastoris engineering strains of rhOSM. To the best of our knowledge, this is the first report that we performed the pilot-scale fermentation of rhOSM in an 80 L fermentor and built a new method for large-scale purification of rhOSM. For the first time, we confirmed that rhOSM could induce hepatoma cells differentiation. Our studies built theoretical and experimental foundation for research of treatment of other solid tumors by rhOSM and industry production of rhOSM.
引文
[1]汤钊猷.从肝癌看癌症临床研究[J].肿瘤, 2009, 29(1):1-4.
[2] Pierce JN, Stein S. Multiple diversity with nonindependent fading[J]. Proc of the IRE, 1960, 48:89-104.
[3] Friend C, Scher W, Holland JG, et al. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro:Stimulation of erythroid differentiation by dimethyl sulfoxide[J]. Proc Natl Acad Sci, 1971, 68(2):378-382.
[4] Sachs L.The differentiation of myeloid leukaemia cells: New possibilities for therapy[J].Br J Haematol, 1978, 40(4):509-517.
[5] Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid[J]. Proc Natl Acad Sci, 1980, 77(5): 2936-2940.
[6] Collins SJ, Ruscetti FW, Gallagher RE, et al. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds[J]. Proc Nati Acad Sci USA, 1978,75(5):2458-2462.
[7] Huang ME, Ye YC, Chen SR, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia[J]. Blood, 1988, 72(2):567-572.
[8] Freemantle SJ, Dragnev KH, Dmitrovsky E. The retinoic acid paradox in cancer chemoprevention[J]. J Natl Cancer Inst, 2006, 98(7):426-427.
[9] Simoni D, Tolomeo M. Retinoids,apoptosis and cancer[J]. Current Pharmaceutical Design, 2001, 7(17):1823-1837.
[10] Estey E, Koller C, Tsimberidou AM, et al. Potential curability of newly diagnosed acute promyelocytic leukemia without use of chemotherapy: the example of liposomal all-trans retinoic acid[J]. Blood, 2005, 105(3):1366-1367.
[11] Wang ZY,Chen Z,Huang W, et al. Problems existing in differentiation therapy of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA)[J]. Blood Cells, 1993, 19(3):633-641.
[12]黄牛,屈凌波,朱七庆,等.维甲类化合物构效关系研究维甲酸核受体与选择性配体相互作用的分子模拟[J].药学学报, 1999, 34(5):102-107.
[13] Kiyokawa H, Richon VM, Rifkind RA, et al. Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells[J]. Mol Cell Biol, 1994,14(11):7195-7203.
[14] Himori T, Ohnuma T, Wakui A. Differential cell growth inhibition and terminal differentiation induction by chemotherapeutic agents of a human acute myelogenous leukemia cell line (HL-60) in vitro[J].Gan To Kagaku Ryoho,1984,11(8):1605-1611.
[15] Baker SJ, Pawlita M, Leutz A, et al. Essential role of c-myc in ara-C-induced differentiation of human erythroleukemia cells[J]. Leukemia, 1994, 8(8):1309-1317.
[16] Varjas T, Nowrasteh G, Budán F, et al. Chemopreventive effect of Panax ginseng[J]. Phytother Res, 2009 , [Epub ahead of print]
[17] Zeng XL, Tu ZG. In vitro induction of differentiation by ginsenoside Rh2 in SMMC-7721 hepatocarcinoma cell line[J]. Pharmacol Toxicol, 2003, 93(6):275-283.
[18] Yuan SL, Wei YQ, Wang XJ, et al. Growth inhibition and apoptosis induction of tanshinone II-A on human hepatocellular carcinoma cells[J]. World J Gastroenterol, 2004,10(14):2024- 2028.
[19]张鹏,王树叶,胡龙虎,等.三氧化二砷治疗急性粒细胞白血病七年总结——附242例分析[J].中华血液学杂志, 2000, 2(12):67-70.
[20] Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells[J].Blood, 1997, 89(9):3345-3353.
[21] Monneret C. Histone deacetylase inhibitors for epigenetic therapy of cancer[J]. Anticancer Drugs, 2007, 18(4):363-370.
[22] Gold MG, Lygren B, Dokurno P, et al. Molecular basis of AKAP specificity for PKA regulatory subunits[J]. Molecular cell, 2006, 24(3):383-395.
[23] Kim SN, Ahn YH, Kim SG, et al. 8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells[J]. Int J Cancer, 2001,93(1):33-41.
[24]吴楠,查锡良.肿瘤细胞的诱导分化[J].生命的化学, 2004, 24(6):496-499.
[25] Hudgins WR, Skack S, Myers CE, et al. Cytostatic activity of phenylacetate and derivatives against tumor cells[J]. Biochem Pharmacol, 1995, 50(8):1273-1279.
[26] Pineau T, Hudgins WR, Liu L, et al. Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs[J]. Biochem pharmacol, 1996, 52(4):659-667.
[27] Faderl S, Talpaz M, Estrov Z, et al. Chronic Myelogenous Leukemia: Biology and Therapy[J]. Ann Intern Med, 1999, 131(3):207-219.
[28]张玉芳,吴重阳,张连生,等.细胞因子体外诱导慢性髓细胞性白血病树突状细胞分化的研究[J].中国实验血液学杂志, 2006, 14(1):137-141.
[29] Glasow A, Prodromou N, Xu K, et al. Retinoids and myelomonocytic growth factors cooperatively activate RARA and induce human myeloid leukemia cell differentiation via MAP kinase pathways[J]. Blood, 2005, 105(1):341-349.
[30] Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia[J]. Blood, 1997, 89(4):1133-1142.
[31] Tsiftsoglou AS, Pappas IS, Vizirianakis IS. Mechanisms involved in the induced differentiation of leukemia cells[J]. Pharmacol ther, 2003, 100(3):257-290.
[32] Gra?a X, Reddy EP. Cell cycle control in mammalian cells:Role of cyclins,cyclin dependent kinases(CDKs),growth sup-pressor genes and cyclin-dependent kinase inhibitors(CKIs)[J]. Oncogene,1995,11(2):211-219.
[33] Gamboa-Dominguez A, Seidl S, Reyes-Gutierrez E, et al. Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E-cadherin expression in gastric cancer[J]. J Clin Pathol, 2007, 60(7):756-761.
[34] Mata-Greenwood E, Cuendet M, Sher D, et al. Brusatol-mediated induction of leukemic cell differentiation and G1 arrest is associated with down-regulation of c-myc[J]. Leukemia, 2002, 16(11):2275-2284.
[35] Appierto V, Villani MG, Cavadini E, et al. Involvement of c-Fos in fenretinide-induced apoptosis in human ovarian carcinoma cells[J]. Cell Death Differ, 2004,11(3):270-279.
[36] Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases[J]. J Biol Chem, 1995, 270(28): 16483-16486.
[37] Heerssen HM, Segal RA. Location, location, location: a spatial view of neurotrophin signal transduction[J]. Trends Neurosci, 2002, 25(3): 160-165.
[38] Kataoka T, Holler N, Micheau O, et al. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension[J]. J Biol Chem, 2001, 276(22):19548-19554.
[39]高山,雷道德,艾中立.肝癌细胞分化诱导作用的研究进展[J].临床外科杂志, 2004, 12(9):571-573.
[40] Rhyu MS. Telomeres, telomerase, and immortality[J]. J Natl Cancer Inst,1995,87(12):884-894.
[41] Zhang P, Xu Q, Chen WT, et al. Synergistic down-regulation of telomerase by all-trans retinoic acid and antisense oligonucleotide in oral squamous cell carcinoma cell line (Tca8113)[J]. Oral Oncology, 2005, 41(9):909-915.
[42]吴涛,窦科峰,李开宗.全反式维甲酸和苯乙酸钠联合对人肝癌细胞的诱导分化作用[J].第四军医大学学报, 2002, 23 (9) : 783-785.
[43]于鼎,王子慧,蔡广玲.三氧化二砷对人肝母细胞瘤系(HepG2)的作用[J].中华实验外科杂志[J]. 2003, 20(5): 421-422.
[44]高山,艾中立,张力,等.丁酸钠诱导人肝癌SMMC-7721细胞分化及其作用机理的初步探讨[J].数理医药学杂志, 2005, 18(1): 28-30.
[45]郭霞,郭昱.黄芩甙体外对人肝癌细胞BEL-7402的诱导分化作用[J].世界华人消化杂志, 2008, 16(10):1119-1123.
[46]方建林,郑传胜,冯敢生,等.全反式维甲酸对Walker-256肝癌细胞分化及凋亡的诱导[J].世界华人消化杂志, 2008,16(26):2929-2934.
[47] Yamashita Y, Shimada M, Harimoto N, et al. Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells[J]. Int J Cancer, 2003, 103(5):572-576.
[48] Ouyang GL, Cai QF, Liu M, et al. Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide[J]. World J Gastroenterol, 2004, 10(7): 954-958.
[49] Rui-Chuan C, J in-Hua S, Gao-Liang O, et al. Induction of differentiation in human hepatocarcinoma cells by isoverbascoside[J]. Planta Med, 2002, 68(4):370-372.
[50] Mann CD, Neal CP, Garcea G, et al. Prognostic molecular markers in hepatocellular carcinoma : a systematic review[J]. Eur J Cancer, 2007, 43(6):979-992.
[51]彭安,陈敏珍,袁劲松.葛根提取物诱导人肝癌细胞分化的研究[J].现代中西医结合杂志. 2002, 11(16): 1533-1534.
[52]李祺福,欧阳高亮,刘庆榕,等.中国鲎鲎素诱导人肝癌SMMC-7721细胞分化的观察[J].癌症, 2002, 21(5):480-483.
[53] Simon D, K?rber C, Krausch M, et al. Clinical impact of retinoids in redifferentiation therapy of advanced thyroid cancer: final results of a pilot study[J]. Eur J Nucl Med Mol Imaging, 2002, 29(6):775-782.
[54] Camacho LH, Olson J, Tong WP, et al. Phase I dose escalation clinical trial of phenylbutyrate sodium administered twice daily to patients with advanced solid tumors[J]. Investigational New Drugs, 2007, 25(2):131-138.
[55] Zarling JM, Shoyab M, Marquardt H, et al. Oncostatin M: A growth regulator produced by differentiated histiocytic lymphoma cells[J]. Proc Natl Acad Sci USA, 1986, 83(12):9739-9743.
[56] Tanaka M, Miyajima A. Oncostatin M, a multifunctional cytokine[J]. Rev Physiol Biochem Pharmacol, 2003, 149:39-52.
[57] Heinrich PC, Behrmann I, Haan S, et al. Principles of interleukin (IL)-6-type cytokine signaling and its regulation[J]. Biochem J, 2003,374 (Pt 1):1-20.
[58] Malik N, Kallestad JC, Gunderson NL, et al. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M[J]. Mol Cell Biol, 1989, 9(7):2847-2853.
[59] Linsley PS, Kallestad J, Ochs V, et al. Cleavage of a hydrophilic c-terminal domain increase growth-inhibitory activity of oncostatin M[J]. Mol Cell Biol, 1990, 10(5):1882-1890.
[60] Malik N, Graves D, Shoyab M, et al. Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells[J]. DNA Cell Biol, 1992,11(6):453-459.
[61] Jeffery E, Price V, Gearing DP. Close proximity of the genes for leukemia inhibitory factor and oncostatin M[J]. Cytokine, 1993, 5(2):107-111.
[62] Bruce AG, Linsley PS, Rose TM. Oncostatin M[J]. Prog Growth Factor Res, 1992, 4(2): 157- 170.
[63] Gearing DP, Bruce AG. Oncostatin M binds the high-affinity leukemia inhibitory factor receptor[J]. New Biol, 1992, 4(1):61-65.
[64] Liu J, Modrell B, Aruffo A, et al. Interleukin-6 signal transducer gp130 mediates oncostatin M signaling[J]. J Biol Chem, 1992, 267(24):16763-16766.
[65] Nair BC, DeVico AL, Nakamura S, et al. Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M[J]. Science, 1992, 255(5050):1430-1432.
[66] Cichy J,Rose-John S, Pure E. Regulation of the type II oncostatin M receptor expression in lung-derived epithelial cells[J]. FEBS Lett, 1998, 429(3):412-416.
[67] Kisseleva T, Bhattacharya S, Braunstein J, et al. Signaling through the JAK/STAT pathway, recent advances and future challenges[J]. Gene, 2002, 285(1-2):1-24.
[68] Kishimoto T, Taga T, Akira S. Cytokine signal transduction[J]. Cell, 1994, 76(2):253-262.
[69] B?ing I, Stross C, Radtke S, et al. Oncostatin M-induced activation of stress-activated MAP kinases depends on tyrosine 861 in the OSM receptor and requires Jak1 but not Src kinases[J]. Cell Signal, 2006, 18(1):50-61.
[70] Horn D, Fitzpatrick WC, Gompper PT, et al. Regulation of cell growth by recombinant oncostatin M[J]. Growth Factors, 1990,2(2-3):157-165.
[71] Liu J, Clegg CH, Shoyab M. Regulation of EGR-1, c-jun, and c-myc gene expression by oncostatin M[J]. Cell Growth Differ, 1992, 3(5):307-313.
[72] Halfter H, Friedrich M, Resch A, et al. Oncostatin M induces growth arrest by inhibition of Skp2, Cks1, and cyclin A expression and induced p21 expression[J]. Cancer Res, 2006, 66(13):6530-6539.
[73] Bruce AG, Hoggatt IH, Rose TM. Oncostatin M is a differentiation factor for myeloid leukemia cells[J]. J Immunol, 1992, 149(4):1271-1275.
[74] Douglas AM, Grant SL, Goss GA, et al. Oncostatin M induces the differentiation of breast cancer cells[J]. Int J Cancer, 1998, 75(1):64-73.
[75] Halfter H, Lotfi R, Westermann R, et al. Inhibition of growth and induction of differentiation of glioma cell lines by oncostatin M (OSM)[J]. Growth Factors, 1998, 15(2):135-147.
[76] Brounais B, David E, Chipoy C, et al. Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells[J]. Bone, 2009, 44(5):830-839.
[77] Bellido T, Borba VZ, Roberson P, et al. Activation of the Janus Kinase/STAT (signa transducer and activator of transcription) signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation[J]. Endocrinology, 1997, 138(9):3666-3676.
[78] McCormick C, Freshney RI . Activity of growth factors in the IL-6 group in the differentiation of human lung adenocarcinoma[J]. Br J Cancer, 2000, 82(4):881-890.
[79] Rose TM, Weiford DM, Gunderson NL, et al. Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro[J]. Cytokine, 1994, 6(1):48-54.
[80] Vassilieva S, Guan K, Pich U, et al. Establishment of SSEA-1 and Oct-4 expressing rat embryonic stem–like cell lines and effects of cytokines of the IL-6 family on clonal growth[J]. Exp Cell Res, 2000, 258(2):361-373.
[81] Raz R, Lee CK, Cannizzaro LA, et al. Essential role of STAT3 for embryonic stem cell pluripotency[J]. Proc Natl Acad Sci USA, 1999, 96(6):2846-2851.
[82] Kinoshita T, Miyajima A. Cytokine regulation of liver development[J]. Biochim Biophys Acta, 2002, 1592(3):303-312.
[83] Ito Y, Matsui T, Kamiya A, et al. Retroviral gene transfer of signaling molecules into murine fetal hepatocytes defines distinct roles for the STAT3 and ras pathways during hepatic development[J]. Hepatology, 2000, 32(6):1370-1376.
[84] Kamiya A, Kinoshita T, Ito Y, et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer[J]. EMBO J, 1999, 18(8):2127-2136.
[85] Kinoshita T, Sekiguchi T, Xu MJ, et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis[J]. Proc Natl Acad Sci USA, 1999, 96(13):7265-7270.
[86] Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differentiation of hepatic stem cells during liver development[J]. Front Biosci, 2006, 11:1302-1310.
[87] Nishikawa M, Tahara T, Hinohara A, et al. Role of the microenvironment of the embryonic aorta-gonad-mesonephros region in hematopoiesis[J]. Ann N Y Acad Sci, 2001, 938(1):109-116.
[88] Miyajima A, Kinoshita T, Tanaka M, et al. Role of Oncostatin M in hematopoiesis and liver development[J]. Cytokine Growth Factor Rev, 2000, 11(3):177-183.
[89] Okaya A, Kitanaka J, Kitanaka N, et al. Oncostatin M inhibits proliferation of rat oval cells, OC15-5, inducing differentiation into hepatocytes[J]. Am J Pathol, 2005, 166(3):709-719.
[90] Hamada T, Sato A, Hirano T, et al. Oncostatin M gene therapy attenuates liver damage induced by dimethylnitrosamine in rats[J]. Am J Pathol, 2007, 171(3):872-881.
[91] Grove RL, Mazzucco C, Allegretto N, et al. Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells[J]. J Lipid Res, 1991,32(12):1889-1897.
[92] Kong WJ, Abidi P, Kraemer FB, et al. In vivo activities of cytokine oncostatin M in regulation of plasma lipid levels[J]. J Lipid Res, 2005, 46(6):1163-1171.
[93] Richards CD, Brown TJ, Shoyab M, et al. Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro[J]. J Immunol, 1992, 148(6):1731-1736.
[94] Kovalovich K, DeAngelis RA, Li W, et al. Increased toxin-induced liver injury and fibrosis in interleukin-6-deficient mice[J]. Hepatology, 2000, 31(1):149-159.
[95] Modur V, Feldhaus MJ, Weyrich AS, et al. Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules[J]. J Clin Invest, 1997, 100(1):158-168.
[96] Cross A, Edwards SW, Bucknall RC, et al. Secretion of oncostatin M by neutrophils in rheumatoid arthritis[J]. Arthritis Rheum, 2004, 50(5):1430-1436.
[97] Li WQ, Dehnade F, Zafarullah M. Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway[J]. J Immunol, 2001, 166(5):3491-3498.
[98] Cichy J, John SR, Travis J. Oncostatin M, leukaemia-inhibitory factor and interleukin 6 trigger different effects on alpha1-proteinase inhibitor synthesis in human lung-derived epithelial cells[J]. Biochem J, 1998, 329(Pt 2): 335-339.
[99] Clegg CH, Rulffes JT, Wallace PM, et al. Regulation of an extrathymic T-cell development pathway by oncostatin M[J]. Nature, 1996, 384(6606):261-263.
[100]Clegg CH, Haugen HS, Rulffes JT, et al. Oncostatin M transforms lymphoid tissue function in transgenic mice by sitmulating lymph node T-cell development and thymus autoantibody production[J]. J Exp Hematol, 1999, 27(4):715-725.
[101]Akita S, Malkin J, Melmed S. Disrupted murine leukemia inhibitory factor (LIF) gene attenuates adrenocorticotropic hormone (ACTH) secretion[J]. Endocrinology, 1996, 137(7): 3140-3143.
[102]Heymann D, Godard A, Raher S, et al. Human interleukin for DA cells/leukemia inhibitory factor and oncostatin M enhance membrane expression of intercellular adhesion molecule-1 on melanoma cells but not the shedding of its soluble form[J]. Cytokine, 1995, 7(2):111-117.
[103]Morikawa Y. Oncostatin M in the development of the nervous system[J]. Anat Sci Int, 2005, 80(1):53-59.
[104]Weiss TW, Samson AL, Niego B, et al. Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo[J]. FASEB J, 2006, 20(13):2369-2371.
[105]Larrea E, Aldabe R, Gonzalez I, et al. Oncostatin M enhances the antiviral effects of type I interferon and activates immunostimulatory functions in liver epithelial cells[J]. J Virol, 2009,83(7):3298-3311.
[106]Sporeno E, Barbato G, Graziani R, et al. Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli[J]. Cytokine,1994, 6(3) 255-264.
[107]叶建新,盛伟华,马丽丽,等. hOSM在COS-7细胞中表达及对人375黑色素瘤细胞的生长抑制作用[J].临床肿瘤学杂志, 2006,11(2):128-132.
[108]胡朝全,孙诚谊,孙连生,等.重组腺病毒载体介导HOSM基因对肝癌细胞体外增殖的抑制作用[J].世界华人消化杂志, 2007, 15(7):737-740.
[109]孔宁,牟旭鹏,韩冬,等.人抑瘤素M在毕赤酵母中的高效分泌表达[J].吉林大学学报:医学版, 2008, 34(1): 42-45.
[110]汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2000.
[111]杨松成.质谱技术丛书有机质谱在生物医药中的应用[M].北京:化学工业出版社, 2009.
[112]路萍,吕星,邢瑞云.抑瘤素M在毕赤酵母中的表达及活性鉴定[J].军事医学科学院院刊, 2001, 25(1):39-41.
[113]Giard DJ, Aaronson SA, Todaro GJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors[J]. J natl Cancer Inst, 1973, 51(5): 1417-1423.
[114] Cregg JM. Pichia Protocols[M]. 2nd ed. New Jersey: Humana Press, 2007.
[115]国家药典委员会编.中华人民共和国药典[S].第3版.北京:化学工业出版社, 2005.
[116]Cregg JM, Cereghino JL, Shi J, et al. Recombinant protein expression in Pichia pastoris[J]. Mol Biotechnol, 2000, 16(1):23-52.
[117]唐元家,余柏松.巴斯德毕赤酵母表达系统[J].国外医药:抗生素分册, 2002, 23(6): 246-250.
[118]Clare JJ, Rayment FB, Ballantine SP, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene[J]. Biotechnology(N Y), 1991, 9(5):455-460.
[119]Hasslacher M, Schall M, Hayn M, et al. High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expr Purif, 1997, 11(1):61-71.
[120]Rosenfeld SA, Nadeau D, Tirado J, et al. Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris[J]. Protein Expr Purif, 1996, 8(4):476-482.
[121]朱剑昆,许志祥,黄伟达,等.人重组白细胞介素Ⅱ在毕赤酵母系统中的表达及纯化[J].中国医学科学院学报, 2001,23(2):127-131.
[122]Li Z, Xiong F, Lin Q, et al. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris[J]. Protein Expr Purif, 2001, 21(3): 438-445.
[123]Kobayashi K, Kuwae S, Ohya T, et al. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J]. J Biosci Bioeng, 2000, 89(1): 55-61.
[124]Choi BK, Jiménez-Flores R. Expression and purification of glycosylated bovine beta casein (L70S/P71S) in Pichia pastoris[J]. J Agric Food Chem, 2001, 49(4): 1761-1766.
[125]Sue MP, Mariana L, Fazenda, et al. Heterologous protein production using the Pichia pastoris expression system[J]. Yeast, 2005, 22(4): 249-270.
[126]董荣春,周荣华,吕发度,等. SMMC-7721人体肝癌细胞株的建立及其生物学特性的初步观察[J].第二军医大学学报, 1980, 1(l):5-9.
[127]司徒镇强,吴军正.细胞培养[M].第2版.北京:世界图书出版公司, 2007.
[128]Stonāns I, Stonāne E, Russwurm S, et al. Wiederhold M, J?ger L, Reinhart K. HepG2 human hepatoma cells express multiple cytokine genes[J]. Cytokine, 1999, 11(2):151-156.
[129]姜圣亮,谭江平,石林祥,等. MTT法检测肝癌细胞化疗敏感性方法学探讨[J].同济大学学报:医学版, 2002, 23 (4): 296-298.
[130]Son SH, Yu E, Choi EK, et al. Promyelocytic leukemia protein-induced growth suppression and cell death in liver cancer cell[J]. Cancer Gene Therapy, 2005,12 (1):1-11.
[131]Kim H, Jo C, Jang BG, et al. Oncostatin M induces growth arrest of skeletal muscle cells in G1 phase by regulating cyclin D1 protein level[J]. Cell Signal, 2008, 20 (1):120-129.
[132]Auguste P, Guillet C, Fourcin M, et al. Signaling of TypeⅡOncostatin M Receptor[J]. J Biol Chem, 1997, 272 (25):15760-15764.
[133]Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus [J]. J Recept Signal Transduct Res, 1999, 19 (1-4):75-120.
[134]Kermorgant S, Parker PJ. Receptor trafficking controls weak signal delivery: a strategy used by c-Met for STAT3 nuclear accumulation[J]. J Cell Biol, 2008, 182(5):855-863.
[135]Cheson BD, Jasperse DM, Chun HG, et al. Differentiating agents in the treatment of human malignancies[J]. Cancer Treat Rev, 1986, 13(3):129-145.
[136]Ghadially, Feroze N. Ultrastructural Pathology of the Cell and Matrix[M]. 3rd Edition. London: Butterworth & Co, 1988.
[137]Bennett DC. Mechanisms of differentiation in melanoma cells and melanocytes[J]. Environ Health Persp, 1989, 80: 49-59.
[138]Han G, Chang B, Connor MJ, et al Enhanced potency of 9-cis versus all-trans-retinoic acid to induce the differentiation of human neuroblastoma cells[J]. Differentiation, 1995, 59(1): 61-69.
[139]Lollini PL, De Giovanni C, Del Re B, et al. Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs[J]. Cancer Res, 1989, 49(13): 3631-3636.
[140]Orchel A, Dzierzewicz Z, Parfiniewicz B, et al. Butyrate-Induced Differentiation of Colon Cancer Cells Is PKC and JNK Dependent[J]. Dig Dis Sci, 2005, 50(3): 490-498.
[141]Atencia R, Garcin-Sanz M, Unda F, et al. Apoptosis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells[J]. Exp Cell Res, 1994, 214(2): 663-667.
[142]Lu Y, Lu Q, Chen HL. Diagnosis of primary liver cancer using lectin affinity chromatography of serum alkaline phosphatase[J]. J Exp Clin Cancer Res, 1997, 16 (1):75-80.
[143]Yao DF, Dong ZZ, Yao M. Specific molecular markers in hepatocellular carcinoma[J]. Hepatobiliary Pancreat Dis Int, 2007, 6(3):241-247.
[144]Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications[J]. Clin Chim Acta, 2008, 395 (1-2):19-26.
[145]Li MS, Li PF, He SP, et al. The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line[J]. World J Gastroenterol, 2002, 8(3):469-475.
[146]徐静,李旭.肝癌动物模型的建立[J].实用肝脏病杂志, 2005,8(2):116-118.
[147]胡卫,陈涛.鼠移植性肝癌模型研究进展[J].河南肿瘤学杂志, 2003,16(6):463-466.
[148]肖建兵,林乐岷,李宝馨,等.三氧化二砷联合全反式维甲酸对小鼠肝癌细胞H22及移植瘤的抑制作用[J].哈尔滨医科大学学报, 2006, 40(2):113-115.
[149]朱颖,章永平,张学军,等.全反式维甲酸对胰腺癌裸鼠移植瘤的生长抑制作用[J].上海交通大学学报:医学版, 2006, 26(10):1154-1157.
[150]路太英,樊青霞,杨成梁,等.全反式维甲酸对EC9706荷瘤裸鼠抑瘤作用的实验研究[J].肿瘤, 2007, 27(8):599-601.
[151]Sempowski GD, Hale LP, Sundy JS, et al. Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is Associated with thymic atrophy[J]. J Immunol, 2000, 164(4): 2180-2187.
[152]陈慰峰.医学免疫学[M].第4版,北京:人民卫生出版社, 2004.
[153]Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation[J]. Nature,1980,284(5756):555-556.
[154]Grove RI, Mazzucco CE, Radka SF, et al. Oncostatin M up-regulates low density lipoproteinreceptors in HepG2 cells by a novel mechanism[J]. J Biol Chem, 1991,266(27):18194-18199.
[155]Davies PJ, Basilion JP, Chiocca EA, et al. Retinoids as generalized regulators of cellular growth and differentiation[J]. Am J Med Sci, 1988, 296(3):164-170.
[156]Fenaux P, Chevret S, Guerci A, et al. Long-term follow-up confirms the benefit of all-trans retinoic acid in acute promyelocytic leukemia[J]. Leukemia, 2000, 14(8):1371-1377.
[2] Pierce JN, Stein S. Multiple diversity with nonindependent fading[J]. Proc of the IRE, 1960, 48:89-104.
[3] Friend C, Scher W, Holland JG, et al. Hemoglobin synthesis in murine virus-induced leukemic cells in vitro:Stimulation of erythroid differentiation by dimethyl sulfoxide[J]. Proc Natl Acad Sci, 1971, 68(2):378-382.
[4] Sachs L.The differentiation of myeloid leukaemia cells: New possibilities for therapy[J].Br J Haematol, 1978, 40(4):509-517.
[5] Breitman TR, Selonick SE, Collins SJ. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid[J]. Proc Natl Acad Sci, 1980, 77(5): 2936-2940.
[6] Collins SJ, Ruscetti FW, Gallagher RE, et al. Terminal differentiation of human promyelocytic leukemia cells induced by dimethyl sulfoxide and other polar compounds[J]. Proc Nati Acad Sci USA, 1978,75(5):2458-2462.
[7] Huang ME, Ye YC, Chen SR, et al. Use of all-trans retinoic acid in the treatment of acute promyelocytic leukemia[J]. Blood, 1988, 72(2):567-572.
[8] Freemantle SJ, Dragnev KH, Dmitrovsky E. The retinoic acid paradox in cancer chemoprevention[J]. J Natl Cancer Inst, 2006, 98(7):426-427.
[9] Simoni D, Tolomeo M. Retinoids,apoptosis and cancer[J]. Current Pharmaceutical Design, 2001, 7(17):1823-1837.
[10] Estey E, Koller C, Tsimberidou AM, et al. Potential curability of newly diagnosed acute promyelocytic leukemia without use of chemotherapy: the example of liposomal all-trans retinoic acid[J]. Blood, 2005, 105(3):1366-1367.
[11] Wang ZY,Chen Z,Huang W, et al. Problems existing in differentiation therapy of acute promyelocytic leukemia (APL) with all-trans retinoic acid (ATRA)[J]. Blood Cells, 1993, 19(3):633-641.
[12]黄牛,屈凌波,朱七庆,等.维甲类化合物构效关系研究维甲酸核受体与选择性配体相互作用的分子模拟[J].药学学报, 1999, 34(5):102-107.
[13] Kiyokawa H, Richon VM, Rifkind RA, et al. Suppression of cyclin-dependent kinase 4 during induced differentiation of erythroleukemia cells[J]. Mol Cell Biol, 1994,14(11):7195-7203.
[14] Himori T, Ohnuma T, Wakui A. Differential cell growth inhibition and terminal differentiation induction by chemotherapeutic agents of a human acute myelogenous leukemia cell line (HL-60) in vitro[J].Gan To Kagaku Ryoho,1984,11(8):1605-1611.
[15] Baker SJ, Pawlita M, Leutz A, et al. Essential role of c-myc in ara-C-induced differentiation of human erythroleukemia cells[J]. Leukemia, 1994, 8(8):1309-1317.
[16] Varjas T, Nowrasteh G, Budán F, et al. Chemopreventive effect of Panax ginseng[J]. Phytother Res, 2009 , [Epub ahead of print]
[17] Zeng XL, Tu ZG. In vitro induction of differentiation by ginsenoside Rh2 in SMMC-7721 hepatocarcinoma cell line[J]. Pharmacol Toxicol, 2003, 93(6):275-283.
[18] Yuan SL, Wei YQ, Wang XJ, et al. Growth inhibition and apoptosis induction of tanshinone II-A on human hepatocellular carcinoma cells[J]. World J Gastroenterol, 2004,10(14):2024- 2028.
[19]张鹏,王树叶,胡龙虎,等.三氧化二砷治疗急性粒细胞白血病七年总结——附242例分析[J].中华血液学杂志, 2000, 2(12):67-70.
[20] Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells[J].Blood, 1997, 89(9):3345-3353.
[21] Monneret C. Histone deacetylase inhibitors for epigenetic therapy of cancer[J]. Anticancer Drugs, 2007, 18(4):363-370.
[22] Gold MG, Lygren B, Dokurno P, et al. Molecular basis of AKAP specificity for PKA regulatory subunits[J]. Molecular cell, 2006, 24(3):383-395.
[23] Kim SN, Ahn YH, Kim SG, et al. 8-Cl-cAMP induces cell cycle-specific apoptosis in human cancer cells[J]. Int J Cancer, 2001,93(1):33-41.
[24]吴楠,查锡良.肿瘤细胞的诱导分化[J].生命的化学, 2004, 24(6):496-499.
[25] Hudgins WR, Skack S, Myers CE, et al. Cytostatic activity of phenylacetate and derivatives against tumor cells[J]. Biochem Pharmacol, 1995, 50(8):1273-1279.
[26] Pineau T, Hudgins WR, Liu L, et al. Activation of a human peroxisome proliferator-activated receptor by the antitumor agent phenylacetate and its analogs[J]. Biochem pharmacol, 1996, 52(4):659-667.
[27] Faderl S, Talpaz M, Estrov Z, et al. Chronic Myelogenous Leukemia: Biology and Therapy[J]. Ann Intern Med, 1999, 131(3):207-219.
[28]张玉芳,吴重阳,张连生,等.细胞因子体外诱导慢性髓细胞性白血病树突状细胞分化的研究[J].中国实验血液学杂志, 2006, 14(1):137-141.
[29] Glasow A, Prodromou N, Xu K, et al. Retinoids and myelomonocytic growth factors cooperatively activate RARA and induce human myeloid leukemia cell differentiation via MAP kinase pathways[J]. Blood, 2005, 105(1):341-349.
[30] Choudhury A, Gajewski JL, Liang JC, et al. Use of leukemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukemia[J]. Blood, 1997, 89(4):1133-1142.
[31] Tsiftsoglou AS, Pappas IS, Vizirianakis IS. Mechanisms involved in the induced differentiation of leukemia cells[J]. Pharmacol ther, 2003, 100(3):257-290.
[32] Gra?a X, Reddy EP. Cell cycle control in mammalian cells:Role of cyclins,cyclin dependent kinases(CDKs),growth sup-pressor genes and cyclin-dependent kinase inhibitors(CKIs)[J]. Oncogene,1995,11(2):211-219.
[33] Gamboa-Dominguez A, Seidl S, Reyes-Gutierrez E, et al. Prognostic significance of p21WAF1/CIP1, p27Kip1, p53 and E-cadherin expression in gastric cancer[J]. J Clin Pathol, 2007, 60(7):756-761.
[34] Mata-Greenwood E, Cuendet M, Sher D, et al. Brusatol-mediated induction of leukemic cell differentiation and G1 arrest is associated with down-regulation of c-myc[J]. Leukemia, 2002, 16(11):2275-2284.
[35] Appierto V, Villani MG, Cavadini E, et al. Involvement of c-Fos in fenretinide-induced apoptosis in human ovarian carcinoma cells[J]. Cell Death Differ, 2004,11(3):270-279.
[36] Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases[J]. J Biol Chem, 1995, 270(28): 16483-16486.
[37] Heerssen HM, Segal RA. Location, location, location: a spatial view of neurotrophin signal transduction[J]. Trends Neurosci, 2002, 25(3): 160-165.
[38] Kataoka T, Holler N, Micheau O, et al. Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension[J]. J Biol Chem, 2001, 276(22):19548-19554.
[39]高山,雷道德,艾中立.肝癌细胞分化诱导作用的研究进展[J].临床外科杂志, 2004, 12(9):571-573.
[40] Rhyu MS. Telomeres, telomerase, and immortality[J]. J Natl Cancer Inst,1995,87(12):884-894.
[41] Zhang P, Xu Q, Chen WT, et al. Synergistic down-regulation of telomerase by all-trans retinoic acid and antisense oligonucleotide in oral squamous cell carcinoma cell line (Tca8113)[J]. Oral Oncology, 2005, 41(9):909-915.
[42]吴涛,窦科峰,李开宗.全反式维甲酸和苯乙酸钠联合对人肝癌细胞的诱导分化作用[J].第四军医大学学报, 2002, 23 (9) : 783-785.
[43]于鼎,王子慧,蔡广玲.三氧化二砷对人肝母细胞瘤系(HepG2)的作用[J].中华实验外科杂志[J]. 2003, 20(5): 421-422.
[44]高山,艾中立,张力,等.丁酸钠诱导人肝癌SMMC-7721细胞分化及其作用机理的初步探讨[J].数理医药学杂志, 2005, 18(1): 28-30.
[45]郭霞,郭昱.黄芩甙体外对人肝癌细胞BEL-7402的诱导分化作用[J].世界华人消化杂志, 2008, 16(10):1119-1123.
[46]方建林,郑传胜,冯敢生,等.全反式维甲酸对Walker-256肝癌细胞分化及凋亡的诱导[J].世界华人消化杂志, 2008,16(26):2929-2934.
[47] Yamashita Y, Shimada M, Harimoto N, et al. Histone deacetylase inhibitor trichostatin A induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells[J]. Int J Cancer, 2003, 103(5):572-576.
[48] Ouyang GL, Cai QF, Liu M, et al. Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide[J]. World J Gastroenterol, 2004, 10(7): 954-958.
[49] Rui-Chuan C, J in-Hua S, Gao-Liang O, et al. Induction of differentiation in human hepatocarcinoma cells by isoverbascoside[J]. Planta Med, 2002, 68(4):370-372.
[50] Mann CD, Neal CP, Garcea G, et al. Prognostic molecular markers in hepatocellular carcinoma : a systematic review[J]. Eur J Cancer, 2007, 43(6):979-992.
[51]彭安,陈敏珍,袁劲松.葛根提取物诱导人肝癌细胞分化的研究[J].现代中西医结合杂志. 2002, 11(16): 1533-1534.
[52]李祺福,欧阳高亮,刘庆榕,等.中国鲎鲎素诱导人肝癌SMMC-7721细胞分化的观察[J].癌症, 2002, 21(5):480-483.
[53] Simon D, K?rber C, Krausch M, et al. Clinical impact of retinoids in redifferentiation therapy of advanced thyroid cancer: final results of a pilot study[J]. Eur J Nucl Med Mol Imaging, 2002, 29(6):775-782.
[54] Camacho LH, Olson J, Tong WP, et al. Phase I dose escalation clinical trial of phenylbutyrate sodium administered twice daily to patients with advanced solid tumors[J]. Investigational New Drugs, 2007, 25(2):131-138.
[55] Zarling JM, Shoyab M, Marquardt H, et al. Oncostatin M: A growth regulator produced by differentiated histiocytic lymphoma cells[J]. Proc Natl Acad Sci USA, 1986, 83(12):9739-9743.
[56] Tanaka M, Miyajima A. Oncostatin M, a multifunctional cytokine[J]. Rev Physiol Biochem Pharmacol, 2003, 149:39-52.
[57] Heinrich PC, Behrmann I, Haan S, et al. Principles of interleukin (IL)-6-type cytokine signaling and its regulation[J]. Biochem J, 2003,374 (Pt 1):1-20.
[58] Malik N, Kallestad JC, Gunderson NL, et al. Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M[J]. Mol Cell Biol, 1989, 9(7):2847-2853.
[59] Linsley PS, Kallestad J, Ochs V, et al. Cleavage of a hydrophilic c-terminal domain increase growth-inhibitory activity of oncostatin M[J]. Mol Cell Biol, 1990, 10(5):1882-1890.
[60] Malik N, Graves D, Shoyab M, et al. Amplification and expression of heterologous oncostatin M in Chinese hamster ovary cells[J]. DNA Cell Biol, 1992,11(6):453-459.
[61] Jeffery E, Price V, Gearing DP. Close proximity of the genes for leukemia inhibitory factor and oncostatin M[J]. Cytokine, 1993, 5(2):107-111.
[62] Bruce AG, Linsley PS, Rose TM. Oncostatin M[J]. Prog Growth Factor Res, 1992, 4(2): 157- 170.
[63] Gearing DP, Bruce AG. Oncostatin M binds the high-affinity leukemia inhibitory factor receptor[J]. New Biol, 1992, 4(1):61-65.
[64] Liu J, Modrell B, Aruffo A, et al. Interleukin-6 signal transducer gp130 mediates oncostatin M signaling[J]. J Biol Chem, 1992, 267(24):16763-16766.
[65] Nair BC, DeVico AL, Nakamura S, et al. Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M[J]. Science, 1992, 255(5050):1430-1432.
[66] Cichy J,Rose-John S, Pure E. Regulation of the type II oncostatin M receptor expression in lung-derived epithelial cells[J]. FEBS Lett, 1998, 429(3):412-416.
[67] Kisseleva T, Bhattacharya S, Braunstein J, et al. Signaling through the JAK/STAT pathway, recent advances and future challenges[J]. Gene, 2002, 285(1-2):1-24.
[68] Kishimoto T, Taga T, Akira S. Cytokine signal transduction[J]. Cell, 1994, 76(2):253-262.
[69] B?ing I, Stross C, Radtke S, et al. Oncostatin M-induced activation of stress-activated MAP kinases depends on tyrosine 861 in the OSM receptor and requires Jak1 but not Src kinases[J]. Cell Signal, 2006, 18(1):50-61.
[70] Horn D, Fitzpatrick WC, Gompper PT, et al. Regulation of cell growth by recombinant oncostatin M[J]. Growth Factors, 1990,2(2-3):157-165.
[71] Liu J, Clegg CH, Shoyab M. Regulation of EGR-1, c-jun, and c-myc gene expression by oncostatin M[J]. Cell Growth Differ, 1992, 3(5):307-313.
[72] Halfter H, Friedrich M, Resch A, et al. Oncostatin M induces growth arrest by inhibition of Skp2, Cks1, and cyclin A expression and induced p21 expression[J]. Cancer Res, 2006, 66(13):6530-6539.
[73] Bruce AG, Hoggatt IH, Rose TM. Oncostatin M is a differentiation factor for myeloid leukemia cells[J]. J Immunol, 1992, 149(4):1271-1275.
[74] Douglas AM, Grant SL, Goss GA, et al. Oncostatin M induces the differentiation of breast cancer cells[J]. Int J Cancer, 1998, 75(1):64-73.
[75] Halfter H, Lotfi R, Westermann R, et al. Inhibition of growth and induction of differentiation of glioma cell lines by oncostatin M (OSM)[J]. Growth Factors, 1998, 15(2):135-147.
[76] Brounais B, David E, Chipoy C, et al. Long term oncostatin M treatment induces an osteocyte-like differentiation on osteosarcoma and calvaria cells[J]. Bone, 2009, 44(5):830-839.
[77] Bellido T, Borba VZ, Roberson P, et al. Activation of the Janus Kinase/STAT (signa transducer and activator of transcription) signal transduction pathway by interleukin-6-type cytokines promotes osteoblast differentiation[J]. Endocrinology, 1997, 138(9):3666-3676.
[78] McCormick C, Freshney RI . Activity of growth factors in the IL-6 group in the differentiation of human lung adenocarcinoma[J]. Br J Cancer, 2000, 82(4):881-890.
[79] Rose TM, Weiford DM, Gunderson NL, et al. Oncostatin M (OSM) inhibits the differentiation of pluripotent embryonic stem cells in vitro[J]. Cytokine, 1994, 6(1):48-54.
[80] Vassilieva S, Guan K, Pich U, et al. Establishment of SSEA-1 and Oct-4 expressing rat embryonic stem–like cell lines and effects of cytokines of the IL-6 family on clonal growth[J]. Exp Cell Res, 2000, 258(2):361-373.
[81] Raz R, Lee CK, Cannizzaro LA, et al. Essential role of STAT3 for embryonic stem cell pluripotency[J]. Proc Natl Acad Sci USA, 1999, 96(6):2846-2851.
[82] Kinoshita T, Miyajima A. Cytokine regulation of liver development[J]. Biochim Biophys Acta, 2002, 1592(3):303-312.
[83] Ito Y, Matsui T, Kamiya A, et al. Retroviral gene transfer of signaling molecules into murine fetal hepatocytes defines distinct roles for the STAT3 and ras pathways during hepatic development[J]. Hepatology, 2000, 32(6):1370-1376.
[84] Kamiya A, Kinoshita T, Ito Y, et al. Fetal liver development requires a paracrine action of oncostatin M through the gp130 signal transducer[J]. EMBO J, 1999, 18(8):2127-2136.
[85] Kinoshita T, Sekiguchi T, Xu MJ, et al. Hepatic differentiation induced by oncostatin M attenuates fetal liver hematopoiesis[J]. Proc Natl Acad Sci USA, 1999, 96(13):7265-7270.
[86] Kamiya A, Gonzalez FJ, Nakauchi H. Identification and differentiation of hepatic stem cells during liver development[J]. Front Biosci, 2006, 11:1302-1310.
[87] Nishikawa M, Tahara T, Hinohara A, et al. Role of the microenvironment of the embryonic aorta-gonad-mesonephros region in hematopoiesis[J]. Ann N Y Acad Sci, 2001, 938(1):109-116.
[88] Miyajima A, Kinoshita T, Tanaka M, et al. Role of Oncostatin M in hematopoiesis and liver development[J]. Cytokine Growth Factor Rev, 2000, 11(3):177-183.
[89] Okaya A, Kitanaka J, Kitanaka N, et al. Oncostatin M inhibits proliferation of rat oval cells, OC15-5, inducing differentiation into hepatocytes[J]. Am J Pathol, 2005, 166(3):709-719.
[90] Hamada T, Sato A, Hirano T, et al. Oncostatin M gene therapy attenuates liver damage induced by dimethylnitrosamine in rats[J]. Am J Pathol, 2007, 171(3):872-881.
[91] Grove RL, Mazzucco C, Allegretto N, et al. Macrophage-derived factors increase low density lipoprotein uptake and receptor number in cultured human liver cells[J]. J Lipid Res, 1991,32(12):1889-1897.
[92] Kong WJ, Abidi P, Kraemer FB, et al. In vivo activities of cytokine oncostatin M in regulation of plasma lipid levels[J]. J Lipid Res, 2005, 46(6):1163-1171.
[93] Richards CD, Brown TJ, Shoyab M, et al. Recombinant oncostatin M stimulates the production of acute phase proteins in HepG2 cells and rat primary hepatocytes in vitro[J]. J Immunol, 1992, 148(6):1731-1736.
[94] Kovalovich K, DeAngelis RA, Li W, et al. Increased toxin-induced liver injury and fibrosis in interleukin-6-deficient mice[J]. Hepatology, 2000, 31(1):149-159.
[95] Modur V, Feldhaus MJ, Weyrich AS, et al. Oncostatin M is a proinflammatory mediator. In vivo effects correlate with endothelial cell expression of inflammatory cytokines and adhesion molecules[J]. J Clin Invest, 1997, 100(1):158-168.
[96] Cross A, Edwards SW, Bucknall RC, et al. Secretion of oncostatin M by neutrophils in rheumatoid arthritis[J]. Arthritis Rheum, 2004, 50(5):1430-1436.
[97] Li WQ, Dehnade F, Zafarullah M. Oncostatin M-induced matrix metalloproteinase and tissue inhibitor of metalloproteinase-3 genes expression in chondrocytes requires Janus kinase/STAT signaling pathway[J]. J Immunol, 2001, 166(5):3491-3498.
[98] Cichy J, John SR, Travis J. Oncostatin M, leukaemia-inhibitory factor and interleukin 6 trigger different effects on alpha1-proteinase inhibitor synthesis in human lung-derived epithelial cells[J]. Biochem J, 1998, 329(Pt 2): 335-339.
[99] Clegg CH, Rulffes JT, Wallace PM, et al. Regulation of an extrathymic T-cell development pathway by oncostatin M[J]. Nature, 1996, 384(6606):261-263.
[100]Clegg CH, Haugen HS, Rulffes JT, et al. Oncostatin M transforms lymphoid tissue function in transgenic mice by sitmulating lymph node T-cell development and thymus autoantibody production[J]. J Exp Hematol, 1999, 27(4):715-725.
[101]Akita S, Malkin J, Melmed S. Disrupted murine leukemia inhibitory factor (LIF) gene attenuates adrenocorticotropic hormone (ACTH) secretion[J]. Endocrinology, 1996, 137(7): 3140-3143.
[102]Heymann D, Godard A, Raher S, et al. Human interleukin for DA cells/leukemia inhibitory factor and oncostatin M enhance membrane expression of intercellular adhesion molecule-1 on melanoma cells but not the shedding of its soluble form[J]. Cytokine, 1995, 7(2):111-117.
[103]Morikawa Y. Oncostatin M in the development of the nervous system[J]. Anat Sci Int, 2005, 80(1):53-59.
[104]Weiss TW, Samson AL, Niego B, et al. Oncostatin M is a neuroprotective cytokine that inhibits excitotoxic injury in vitro and in vivo[J]. FASEB J, 2006, 20(13):2369-2371.
[105]Larrea E, Aldabe R, Gonzalez I, et al. Oncostatin M enhances the antiviral effects of type I interferon and activates immunostimulatory functions in liver epithelial cells[J]. J Virol, 2009,83(7):3298-3311.
[106]Sporeno E, Barbato G, Graziani R, et al. Production and structural characterization of amino terminally histidine tagged human oncostatin M in E. coli[J]. Cytokine,1994, 6(3) 255-264.
[107]叶建新,盛伟华,马丽丽,等. hOSM在COS-7细胞中表达及对人375黑色素瘤细胞的生长抑制作用[J].临床肿瘤学杂志, 2006,11(2):128-132.
[108]胡朝全,孙诚谊,孙连生,等.重组腺病毒载体介导HOSM基因对肝癌细胞体外增殖的抑制作用[J].世界华人消化杂志, 2007, 15(7):737-740.
[109]孔宁,牟旭鹏,韩冬,等.人抑瘤素M在毕赤酵母中的高效分泌表达[J].吉林大学学报:医学版, 2008, 34(1): 42-45.
[110]汪家政,范明.蛋白质技术手册[M].北京:科学出版社, 2000.
[111]杨松成.质谱技术丛书有机质谱在生物医药中的应用[M].北京:化学工业出版社, 2009.
[112]路萍,吕星,邢瑞云.抑瘤素M在毕赤酵母中的表达及活性鉴定[J].军事医学科学院院刊, 2001, 25(1):39-41.
[113]Giard DJ, Aaronson SA, Todaro GJ, et al. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors[J]. J natl Cancer Inst, 1973, 51(5): 1417-1423.
[114] Cregg JM. Pichia Protocols[M]. 2nd ed. New Jersey: Humana Press, 2007.
[115]国家药典委员会编.中华人民共和国药典[S].第3版.北京:化学工业出版社, 2005.
[116]Cregg JM, Cereghino JL, Shi J, et al. Recombinant protein expression in Pichia pastoris[J]. Mol Biotechnol, 2000, 16(1):23-52.
[117]唐元家,余柏松.巴斯德毕赤酵母表达系统[J].国外医药:抗生素分册, 2002, 23(6): 246-250.
[118]Clare JJ, Rayment FB, Ballantine SP, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene[J]. Biotechnology(N Y), 1991, 9(5):455-460.
[119]Hasslacher M, Schall M, Hayn M, et al. High-level intracellular expression of hydroxynitrile lyase from the tropical rubber tree Hevea brasiliensis in microbial hosts[J]. Protein Expr Purif, 1997, 11(1):61-71.
[120]Rosenfeld SA, Nadeau D, Tirado J, et al. Production and purification of recombinant hirudin expressed in the methylotrophic yeast Pichia pastoris[J]. Protein Expr Purif, 1996, 8(4):476-482.
[121]朱剑昆,许志祥,黄伟达,等.人重组白细胞介素Ⅱ在毕赤酵母系统中的表达及纯化[J].中国医学科学院学报, 2001,23(2):127-131.
[122]Li Z, Xiong F, Lin Q, et al. Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris[J]. Protein Expr Purif, 2001, 21(3): 438-445.
[123]Kobayashi K, Kuwae S, Ohya T, et al. High-level expression of recombinant human serum albumin from the methylotrophic yeast Pichia pastoris with minimal protease production and activation[J]. J Biosci Bioeng, 2000, 89(1): 55-61.
[124]Choi BK, Jiménez-Flores R. Expression and purification of glycosylated bovine beta casein (L70S/P71S) in Pichia pastoris[J]. J Agric Food Chem, 2001, 49(4): 1761-1766.
[125]Sue MP, Mariana L, Fazenda, et al. Heterologous protein production using the Pichia pastoris expression system[J]. Yeast, 2005, 22(4): 249-270.
[126]董荣春,周荣华,吕发度,等. SMMC-7721人体肝癌细胞株的建立及其生物学特性的初步观察[J].第二军医大学学报, 1980, 1(l):5-9.
[127]司徒镇强,吴军正.细胞培养[M].第2版.北京:世界图书出版公司, 2007.
[128]Stonāns I, Stonāne E, Russwurm S, et al. Wiederhold M, J?ger L, Reinhart K. HepG2 human hepatoma cells express multiple cytokine genes[J]. Cytokine, 1999, 11(2):151-156.
[129]姜圣亮,谭江平,石林祥,等. MTT法检测肝癌细胞化疗敏感性方法学探讨[J].同济大学学报:医学版, 2002, 23 (4): 296-298.
[130]Son SH, Yu E, Choi EK, et al. Promyelocytic leukemia protein-induced growth suppression and cell death in liver cancer cell[J]. Cancer Gene Therapy, 2005,12 (1):1-11.
[131]Kim H, Jo C, Jang BG, et al. Oncostatin M induces growth arrest of skeletal muscle cells in G1 phase by regulating cyclin D1 protein level[J]. Cell Signal, 2008, 20 (1):120-129.
[132]Auguste P, Guillet C, Fourcin M, et al. Signaling of TypeⅡOncostatin M Receptor[J]. J Biol Chem, 1997, 272 (25):15760-15764.
[133]Heim MH. The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus [J]. J Recept Signal Transduct Res, 1999, 19 (1-4):75-120.
[134]Kermorgant S, Parker PJ. Receptor trafficking controls weak signal delivery: a strategy used by c-Met for STAT3 nuclear accumulation[J]. J Cell Biol, 2008, 182(5):855-863.
[135]Cheson BD, Jasperse DM, Chun HG, et al. Differentiating agents in the treatment of human malignancies[J]. Cancer Treat Rev, 1986, 13(3):129-145.
[136]Ghadially, Feroze N. Ultrastructural Pathology of the Cell and Matrix[M]. 3rd Edition. London: Butterworth & Co, 1988.
[137]Bennett DC. Mechanisms of differentiation in melanoma cells and melanocytes[J]. Environ Health Persp, 1989, 80: 49-59.
[138]Han G, Chang B, Connor MJ, et al Enhanced potency of 9-cis versus all-trans-retinoic acid to induce the differentiation of human neuroblastoma cells[J]. Differentiation, 1995, 59(1): 61-69.
[139]Lollini PL, De Giovanni C, Del Re B, et al. Myogenic differentiation of human rhabdomyosarcoma cells induced in vitro by antineoplastic drugs[J]. Cancer Res, 1989, 49(13): 3631-3636.
[140]Orchel A, Dzierzewicz Z, Parfiniewicz B, et al. Butyrate-Induced Differentiation of Colon Cancer Cells Is PKC and JNK Dependent[J]. Dig Dis Sci, 2005, 50(3): 490-498.
[141]Atencia R, Garcin-Sanz M, Unda F, et al. Apoptosis during retinoic acid-induced differentiation of F9 embryonal carcinoma cells[J]. Exp Cell Res, 1994, 214(2): 663-667.
[142]Lu Y, Lu Q, Chen HL. Diagnosis of primary liver cancer using lectin affinity chromatography of serum alkaline phosphatase[J]. J Exp Clin Cancer Res, 1997, 16 (1):75-80.
[143]Yao DF, Dong ZZ, Yao M. Specific molecular markers in hepatocellular carcinoma[J]. Hepatobiliary Pancreat Dis Int, 2007, 6(3):241-247.
[144]Debruyne EN, Delanghe JR. Diagnosing and monitoring hepatocellular carcinoma with alpha-fetoprotein: new aspects and applications[J]. Clin Chim Acta, 2008, 395 (1-2):19-26.
[145]Li MS, Li PF, He SP, et al. The promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 cell line[J]. World J Gastroenterol, 2002, 8(3):469-475.
[146]徐静,李旭.肝癌动物模型的建立[J].实用肝脏病杂志, 2005,8(2):116-118.
[147]胡卫,陈涛.鼠移植性肝癌模型研究进展[J].河南肿瘤学杂志, 2003,16(6):463-466.
[148]肖建兵,林乐岷,李宝馨,等.三氧化二砷联合全反式维甲酸对小鼠肝癌细胞H22及移植瘤的抑制作用[J].哈尔滨医科大学学报, 2006, 40(2):113-115.
[149]朱颖,章永平,张学军,等.全反式维甲酸对胰腺癌裸鼠移植瘤的生长抑制作用[J].上海交通大学学报:医学版, 2006, 26(10):1154-1157.
[150]路太英,樊青霞,杨成梁,等.全反式维甲酸对EC9706荷瘤裸鼠抑瘤作用的实验研究[J].肿瘤, 2007, 27(8):599-601.
[151]Sempowski GD, Hale LP, Sundy JS, et al. Leukemia inhibitory factor, oncostatin M, IL-6, and stem cell factor mRNA expression in human thymus increases with age and is Associated with thymic atrophy[J]. J Immunol, 2000, 164(4): 2180-2187.
[152]陈慰峰.医学免疫学[M].第4版,北京:人民卫生出版社, 2004.
[153]Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation[J]. Nature,1980,284(5756):555-556.
[154]Grove RI, Mazzucco CE, Radka SF, et al. Oncostatin M up-regulates low density lipoproteinreceptors in HepG2 cells by a novel mechanism[J]. J Biol Chem, 1991,266(27):18194-18199.
[155]Davies PJ, Basilion JP, Chiocca EA, et al. Retinoids as generalized regulators of cellular growth and differentiation[J]. Am J Med Sci, 1988, 296(3):164-170.
[156]Fenaux P, Chevret S, Guerci A, et al. Long-term follow-up confirms the benefit of all-trans retinoic acid in acute promyelocytic leukemia[J]. Leukemia, 2000, 14(8):1371-1377.