【作者】 孔宁；

【导师】 颜炜群；

【作者基本信息】 吉林大学 ， 医学生物工程学， 2009， 博士

【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。更多还原

【Abstract】 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 level1.1 Screening of the transformed Pichia pastoris strainsLinearize 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 rhOSMrhOSM 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 rhOSM2.1 Pilot-scale fermentation of rhOSM in Pichia pastorisWe 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 rhOSMTo 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 rhOSM3.1 Investigate OSM receptors in human SMMC-7721 hepatoma cellWe 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 rhOSMWe 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 apoptosisAfter 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 observationThe 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 AFPWe 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 differentiationAfter 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 rhOSM4.1 Establishment of animal model and animal grouping50 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 AFPAll 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 indexThe 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 tissuesTumor 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.更多还原