蝎毒多肽对辐射后M-NFS-60细胞的促增殖作用及其机制的研究
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摘要
背景和目的:
放射治疗是三大经典的肿瘤治疗手段之一。放疗在杀伤肿瘤细胞的同时也对患者体内增殖活跃的细胞造成损伤,尤其是对射线极为敏感的造血系统受到严重损伤,使肿瘤患者的造血和免疫系统功能障碍,引发多种并发症,严重影响患者的生活质量,增加了肿瘤治疗的难度。放射损伤后,造血功能障碍的主要原因之一就是射线杀伤了维持造血功能的造血干细胞。造血干细胞的增殖和分化受到细胞因子(造血生长因子)的调控,因此缓解辐射后造血功能抑制的常规方法是给予辐射病人细胞因子治疗,尤其是在放射治疗后立即给予细胞因子治疗以及细胞因子联合持续给药的治疗方法。研究发现,辐射后残存的造血干/祖细胞对细胞因子的增殖反应能力显著下降;持续给予数种细胞因子联合治疗引起炎症和免疫反应以及其他的副作用。能否通过改善造血干/祖细胞对细胞因子的反应性,促进造血系统功能的自然恢复,寻找天然物质替代细胞因子的治疗,减轻治疗的副作用,是血液系统疾病和肿瘤领域研究的热点问题。以往本实验室的研究表明,蝎毒多肽(scorpion venom polypeptide, SVP)不但可高选择性杀伤多种肿瘤细胞,增强带瘤小鼠的免疫力,还可减轻放化疗对骨髓造血功能抑制的副作用,具有保护辐射损伤小鼠造血干细胞及祖细胞,加速其增殖能力恢复的作用。
M-NFS-60细胞为小鼠源性的造血因子依赖粒白血病细胞株,在一定程度上可反映正常造血干细胞的增殖特性。本研究采用该细胞作为实验对象,在前期研究的基础上,从蝎毒多肽的多种组分中筛选促进M-NFS-60细胞增殖作用最强的组分,并进一步研究这些组分对辐射后M-NFS-60细胞的促增殖作用及其可能的机制和信号转导途径。
材料和方法:
1.材料:
1.1细胞株:M-NFS-60细胞,为依赖r-h-MCSF的小鼠粒白血病细胞株,购自美国ATCC公司(CRL-1838?)
1.2 SVP:由广州医学院蛇毒研究所分离纯化赠予,包括粗毒(C)、SVP I1、SVP I2、SVP II1、SVP II2、SVP II3、SVP III1、SVP III2、SVP III3、SVP IV和SVP V(以下简称I1、I2、II1、II2、II3、III1、III2、III3、IV和V);经预实验后,从II3中分离出5个组分:A1、A2、A3、A4、A5,并经高效液相鉴定,其中A4、A5为纯品。
2.方法:
2.1 SVP有效组分的筛选:用AlamarBlueTM摄入法筛选SVP对M-NFS-60细胞有促增殖作用的组分,并确定其有效浓度,作为体外实验中SVP的使用浓度。取对数生长期M-NFS-60细胞用RPMI 1640培养液洗涤3次,台盼蓝染色计数活细胞,用含10%胎牛血清的RPMI 1640培养基(含r-h-M-CSF62ng/ mL)调整细胞浓度为5×104cells/ mL,每孔80μL加入96孔板中;分别加入10μL生理盐水、不同浓度的SVP组分、IL-3(10ng/ mL)、SVP+IL-3;每组设3个平行孔;加入AlamarBlueTM10μL,常规培养48h,于全自动酶标仪上测各组吸光度值,再根据AlamarBlueTM方法既定公式换算出各实验组相对于对照组的细胞增殖率。
. 2.2 SVP对辐射后M-NFS-60细胞促增殖作用的研究:M-NFS-60细胞经60Coγ-射线一次均匀照射,分组及实验方法同上。
2.3 SVP对M-NFS-60细胞周期的影响: SVP处理辐射前或辐射后M-NFS-60细胞,用流式细胞术(FCM)检测其对细胞周期的影响,进一步观察SVP对M-NFS-60细胞的促增殖作用。
2.4 SVP对IL-3R和M-CSFR表达的影响:分别用FCM、免疫荧光和Western blotting方法检测IL-3R和M-CSFR的表达水平。
2.5 SVP对磷酸化蛋白STAT5表达的影响: SVP处理M-NFS-60细胞,分别用FCM和Western blotting方法检测磷酸化STAT5蛋白的表达水平。
结果:
1. SVP处理M-NFS-60细胞48小时后,在(0.5～5 )mg/L的终浓度范围内均可促进细胞的增殖,其中II3、IV 3mg/L浓度的作用最为显著,故选用3mg/L的SVPII3、IV为体外实验处理组分,3mg/L的SVP III3为对照。纯品A4、A5作用强于A1、A2、A3,这是本课题组首次得到的具有促进造血细胞增殖作用的蝎毒多肽纯品。
2. M-NFS-60细胞经60Coγ-射线一次均匀照射后,加入II3、IV、III3(3 mg/L)和IL-3(10ng/ mL)以及II3+IL-3、IV+IL-3、III3+IL-3,作用48h,细胞的增殖率(﹪)分别为121.58±2.62 (II3)、123.39±4.45(III3)、123.51±5.04 (IV),140.12±1.68(IL-3),与空白对照组(100.00±0.01)相比具有显著性差异(P<0.01);II3、III3、IV分别与IL-3联用处理细胞48h后的增殖率分别为163.98±9.20(II3+IL3)、159.89±8.31(IV +IL3)、148.92±9.74(III3+IL3),与SVP处理组比较差异具有显著性(P<0.05)。
3. SVP可增加M-NFS-60细胞S期的百分率,使辐射后细胞阻滞在G2-M期。
4. SVP可上调M-NFS-60细胞IL-3R的表达水平。在足量M-CSF维持下,对细胞MCSFR无明显影响;在1/4浓度M-CSF维持下,可上调MCSFR的表达水平。
5. SVP处理M-NFS-60细胞后,磷酸化蛋白STAT5的表达水平与对照组相比明显增高。
综上所述,SVP可保护辐射损伤M-NFS-60细胞,加速其增殖能力恢复,并且可以上调M-NFS-60细胞IL-3R和磷酸化STAT5蛋白的表达水平;提示SVP促进辐射损伤后造血功能恢复的作用机理可能与细胞因子受体信号转导途径的激活有关。
结论:
1.本论文结果表明,SVPII3、IV、A4、A5等组分具有类细胞因子的作用,可以促进M-NFS-60细胞的增殖,并且与IL-3具有协同促增殖作用。
2. SVP组分促M-NFS-60细胞的增殖作用与其上调IL-3R和M-CSFR的表达有关,并可能通过JAK-STATs通路发挥作用。
3.首次分离得到了纯化的SVPA4、A5,并证实它们具有类细胞因子的作用,为进一步研究蝎毒多肽促进造血重建的作用和机制奠定了很好的基础,SVP单体也具有较好的应用前景。
Background and Objective:
Radiation therapy, one of the three classic methods of tumor therapy can impair normal cells active in proliferation, especially cells of the hematopoietic system which is extremely sensitive to the rays when killing the tumor cells .Consequently,by inducing the dysfunction of the hematopoietic system and immune system,radiotherapy may lead to a variety of complications,that will impact the life quality of patients and increase the difficulties of tumor therapy. One of the main causes of hematopoietic dysfunction after the injury of radiation is that the rays destruct the hematopoietic stem cells that maintain the haematogenesis,.The proliferation and differentiation of the hematopoietic stem cells are regulated by cytokines (hematopoietic growth factor) , and so the conventional method of easing hematopoietic inhibition is to give cytokines in patients, especially give cytokine treatment immediately after radiation therapy as well as continuously administrating a combined cytokines .It has been found that the proliferative response to cytokines of the remnants of hematopoietic stem/progenitor cells will significantly decrease after radiation.However, continuous co-administration of several kinds of cytokines will induce inflammation and immune responses as well as other side effects .Nowadays, the hot issues in the field of blood diseases (hematological system diseases) and cancer research are to improve the responsibility of hematopoietic stem / progenitor cells to cytokines , promote the natural recovery of the hematopoietic function, to find substitute cytokine treatment ,and to reduce the side effects of treatment. The previous study of our laboratory has shown that scorpion venom peptide (SVP) can not only destruct a variety of tumor cells with high selectivity and enhance the immunity of the mice with tumors, but also reduce the suppressive effects on the bone marrow haematogenesis by radiotherapy and chemotherapy. By protecting the hematopoietic stem cells and progenitor cells damaged by radiation and accelerating the resumption of their proliferation ability .
M-NFS-60 cell , the mouse-derived promyelocytic leukemia cell line with the feature of hematopoietic factor-dependentcy, can reflect the normal hematopoietic stem cell proliferation characteristics to a certain extent. The objectives of this study is first to choose the strongest component of SVP to promote M-NFS-60 cell proliferation, and secondly, to study the role of these components to the proliferation of irradiated M-NFS-60 cell and its possible mechanisms and signal transduction pathways.
Materials and Methods:
1. Materials
1.1 Cell line: M-NFS-60 cell, the promyelocytic leukemia cells in mice, ordered from ATCC of USA .
1.2 SVP:purificated and presented by Guangzhou Institute of Snake Venom,including crude venom (C) ,SVP I1,SVP I2,SVP II1,SVP II2,SVP II3,SVP III1,SVPIII2,SVPIII3,SVPIVandSVP V(referredto as I1,I2,II1,II2,II3,III1,III2,III3,IV and V).After pre-experiment, A1,A2,A3,A4,A5 were purificated from II3,among which A4,A5 were pures identified by High Performance Liquid Chromatogrophy .
2. Methods
2.1 Screening effective components of SVP :
SVP components which of promote cells proliferation were Screened by Method of AlamarBlueTM Intake , and their effective concentration were determined as the in vitro concentration guide of SVP. The test cells were washed with RPMI 1640 culture medium for 3 times, then the living cells were counted by trypan blue staining. Cell concentration was adjusted to 5×104cells/ml with RPMI 1640 culture medium containing 10% FBS (containing r-h-M-CSF62ng/ml), and 80μl was added per hole in 96-well plates, which was then added, respectively, 10μl of normal saline, or different concentrations of SVP components, or IL-3 (10ng/ml) ,or SVP + IL-3. Each group includes 3 parallel of holes.After adding of 10μl AlamarBlueTM for conventional culture of 48h, the absorbance value of each group (well)in the fully automated microplate reader was measured , and the relative rate of cell proliferation in the experimental group was caculated, according to the established formula of AlamarBlueTMmethod.
2.2 Study of the proliferation-enhancing effect of effective SVP component on the irradiated M-NFS-60 cell: M-Nfs-60 cells were exposed to a uniform 60Coγ-ray irradiation, and theiy following study was similar to that of 2.1.
2.3 Study of the effect of SVP on cell cycle: normal and irradiated M-NFS-60 cells were treated with SVP, and cell cycle was detected by flow cytometry , with a further observation of SVP action on the proliferation 0f M-NFS-60 .
2.4 Study of the effect of SVP on the IL-3R and MCSFR expression: respectively, the expression level of IL-3R and MCSFR were detected by FCM,confocal laser scanning microscopy and Western blotting .
2.5 Study of the effect of SVP on the phosphorylation STAT5 expression: M-NFS-60 cells were treated with SVP,and the expression level of phosphorylation STAT5 was detected by Western blotting .
Results:
1. the normal M-NFS-60 cells SVP treated with SVP of 0.5～5 mg / L final concentration range for 48 hours, cell proliferation may be promoted, with 3mg / L concentration of SVP II3 and IV being the most significant , 3 mg/L of SVP II3、IV components were determined for in vitro treatment, and 3mg/L of SVP III3 for the control.The functions of the pures of A4 and A5 were more significant than A1, A2 and A3 .This is the first time that our team gain the pures of SVP that enhancing the proliferation action of hematopoietic cells .
2. M-NFS-60 cells were irradiated by 60Coγ-ray and then were treated with 3mg / L of II3, III3,IV,or IL-3(10ng/ml), or II3+IL3,IV +IL3, III3+IL3 for 48 hours. The cell proliferating rates (%) were 121.58±2.62 (II3) ,123.39±4.45 (III3) ,123.51±5.037 (IV) ,and 140.12±1.68(IL-3).Being significantly increased compared with the blank control group (100.00±.006), (P <0.01).When treated with combined SVP and IL-3, the cell proliferating rates were 163.98±9.20(II3+IL3),159.89±8.31(IV +IL3)and 148.92±9.74(III3+IL3). Compared with SVP treatment group, the difference was significant (P<0.05).
3. Compared with the control group, SVP could significantly promote S phase percentage of normal M-NFS-60 cells.And SVP could promote G2-M phase percentage of irradiated M-NFS-60 cells.
4. SVP can increase the level of IL-3R expression in normal and irradiated M-NFS-60 cell.
5. SVP can increase the expression of phosphorylated STAT5 in M-NFS-60 cell.
SVP can protect irradiated M-NFS-60 cells by accelerating the recovery of the proliferation ability.At the same time, it can up-regulate the level of IL-3R and phosphorylated STAT5 in M-Nfs-60 cell . It is suggested that the mechanism of prompting the recovery of irradiated hematopoietic cells by SVP may be associated with the activation of cytokine receptor signal transduction pathway.
Conclusion:
1.The results show that SVPIV,II3,A4,A5 could promote the proliferation of M-NFS-60 cells, and they have a synergistic effect with IL-3. It is suggested SVP has similer role of cytokine.
2.The function of prompting the proliferation of M-NFS-60 cells by SVP is associated with the up-regulation of IL-3R and M-CSFR, and SVP may be play their roles through JAK-STATs signal transduction pathway.
3. The pures of A4 and A5 were abtained for the first time, and they were confirmed to have similer role of cytokine,which lay the foundation for the study of their function and mechanism. The monomer of SVP has better prospect.
放射治疗是三大经典的肿瘤治疗手段之一。放疗在杀伤肿瘤细胞的同时也对患者体内增殖活跃的细胞造成损伤,尤其是对射线极为敏感的造血系统受到严重损伤,使肿瘤患者的造血和免疫系统功能障碍,引发多种并发症,严重影响患者的生活质量,增加了肿瘤治疗的难度。放射损伤后,造血功能障碍的主要原因之一就是射线杀伤了维持造血功能的造血干细胞。造血干细胞的增殖和分化受到细胞因子(造血生长因子)的调控,因此缓解辐射后造血功能抑制的常规方法是给予辐射病人细胞因子治疗,尤其是在放射治疗后立即给予细胞因子治疗以及细胞因子联合持续给药的治疗方法。研究发现,辐射后残存的造血干/祖细胞对细胞因子的增殖反应能力显著下降;持续给予数种细胞因子联合治疗引起炎症和免疫反应以及其他的副作用。能否通过改善造血干/祖细胞对细胞因子的反应性,促进造血系统功能的自然恢复,寻找天然物质替代细胞因子的治疗,减轻治疗的副作用,是血液系统疾病和肿瘤领域研究的热点问题。以往本实验室的研究表明,蝎毒多肽(scorpion venom polypeptide, SVP)不但可高选择性杀伤多种肿瘤细胞,增强带瘤小鼠的免疫力,还可减轻放化疗对骨髓造血功能抑制的副作用,具有保护辐射损伤小鼠造血干细胞及祖细胞,加速其增殖能力恢复的作用。
M-NFS-60细胞为小鼠源性的造血因子依赖粒白血病细胞株,在一定程度上可反映正常造血干细胞的增殖特性。本研究采用该细胞作为实验对象,在前期研究的基础上,从蝎毒多肽的多种组分中筛选促进M-NFS-60细胞增殖作用最强的组分,并进一步研究这些组分对辐射后M-NFS-60细胞的促增殖作用及其可能的机制和信号转导途径。
材料和方法:
1.材料:
1.1细胞株:M-NFS-60细胞,为依赖r-h-MCSF的小鼠粒白血病细胞株,购自美国ATCC公司(CRL-1838?)
1.2 SVP:由广州医学院蛇毒研究所分离纯化赠予,包括粗毒(C)、SVP I1、SVP I2、SVP II1、SVP II2、SVP II3、SVP III1、SVP III2、SVP III3、SVP IV和SVP V(以下简称I1、I2、II1、II2、II3、III1、III2、III3、IV和V);经预实验后,从II3中分离出5个组分:A1、A2、A3、A4、A5,并经高效液相鉴定,其中A4、A5为纯品。
2.方法:
2.1 SVP有效组分的筛选:用AlamarBlueTM摄入法筛选SVP对M-NFS-60细胞有促增殖作用的组分,并确定其有效浓度,作为体外实验中SVP的使用浓度。取对数生长期M-NFS-60细胞用RPMI 1640培养液洗涤3次,台盼蓝染色计数活细胞,用含10%胎牛血清的RPMI 1640培养基(含r-h-M-CSF62ng/ mL)调整细胞浓度为5×104cells/ mL,每孔80μL加入96孔板中;分别加入10μL生理盐水、不同浓度的SVP组分、IL-3(10ng/ mL)、SVP+IL-3;每组设3个平行孔;加入AlamarBlueTM10μL,常规培养48h,于全自动酶标仪上测各组吸光度值,再根据AlamarBlueTM方法既定公式换算出各实验组相对于对照组的细胞增殖率。
. 2.2 SVP对辐射后M-NFS-60细胞促增殖作用的研究:M-NFS-60细胞经60Coγ-射线一次均匀照射,分组及实验方法同上。
2.3 SVP对M-NFS-60细胞周期的影响: SVP处理辐射前或辐射后M-NFS-60细胞,用流式细胞术(FCM)检测其对细胞周期的影响,进一步观察SVP对M-NFS-60细胞的促增殖作用。
2.4 SVP对IL-3R和M-CSFR表达的影响:分别用FCM、免疫荧光和Western blotting方法检测IL-3R和M-CSFR的表达水平。
2.5 SVP对磷酸化蛋白STAT5表达的影响: SVP处理M-NFS-60细胞,分别用FCM和Western blotting方法检测磷酸化STAT5蛋白的表达水平。
结果:
1. SVP处理M-NFS-60细胞48小时后,在(0.5～5 )mg/L的终浓度范围内均可促进细胞的增殖,其中II3、IV 3mg/L浓度的作用最为显著,故选用3mg/L的SVPII3、IV为体外实验处理组分,3mg/L的SVP III3为对照。纯品A4、A5作用强于A1、A2、A3,这是本课题组首次得到的具有促进造血细胞增殖作用的蝎毒多肽纯品。
2. M-NFS-60细胞经60Coγ-射线一次均匀照射后,加入II3、IV、III3(3 mg/L)和IL-3(10ng/ mL)以及II3+IL-3、IV+IL-3、III3+IL-3,作用48h,细胞的增殖率(﹪)分别为121.58±2.62 (II3)、123.39±4.45(III3)、123.51±5.04 (IV),140.12±1.68(IL-3),与空白对照组(100.00±0.01)相比具有显著性差异(P<0.01);II3、III3、IV分别与IL-3联用处理细胞48h后的增殖率分别为163.98±9.20(II3+IL3)、159.89±8.31(IV +IL3)、148.92±9.74(III3+IL3),与SVP处理组比较差异具有显著性(P<0.05)。
3. SVP可增加M-NFS-60细胞S期的百分率,使辐射后细胞阻滞在G2-M期。
4. SVP可上调M-NFS-60细胞IL-3R的表达水平。在足量M-CSF维持下,对细胞MCSFR无明显影响;在1/4浓度M-CSF维持下,可上调MCSFR的表达水平。
5. SVP处理M-NFS-60细胞后,磷酸化蛋白STAT5的表达水平与对照组相比明显增高。
综上所述,SVP可保护辐射损伤M-NFS-60细胞,加速其增殖能力恢复,并且可以上调M-NFS-60细胞IL-3R和磷酸化STAT5蛋白的表达水平;提示SVP促进辐射损伤后造血功能恢复的作用机理可能与细胞因子受体信号转导途径的激活有关。
结论:
1.本论文结果表明,SVPII3、IV、A4、A5等组分具有类细胞因子的作用,可以促进M-NFS-60细胞的增殖,并且与IL-3具有协同促增殖作用。
2. SVP组分促M-NFS-60细胞的增殖作用与其上调IL-3R和M-CSFR的表达有关,并可能通过JAK-STATs通路发挥作用。
3.首次分离得到了纯化的SVPA4、A5,并证实它们具有类细胞因子的作用,为进一步研究蝎毒多肽促进造血重建的作用和机制奠定了很好的基础,SVP单体也具有较好的应用前景。
Background and Objective:
Radiation therapy, one of the three classic methods of tumor therapy can impair normal cells active in proliferation, especially cells of the hematopoietic system which is extremely sensitive to the rays when killing the tumor cells .Consequently,by inducing the dysfunction of the hematopoietic system and immune system,radiotherapy may lead to a variety of complications,that will impact the life quality of patients and increase the difficulties of tumor therapy. One of the main causes of hematopoietic dysfunction after the injury of radiation is that the rays destruct the hematopoietic stem cells that maintain the haematogenesis,.The proliferation and differentiation of the hematopoietic stem cells are regulated by cytokines (hematopoietic growth factor) , and so the conventional method of easing hematopoietic inhibition is to give cytokines in patients, especially give cytokine treatment immediately after radiation therapy as well as continuously administrating a combined cytokines .It has been found that the proliferative response to cytokines of the remnants of hematopoietic stem/progenitor cells will significantly decrease after radiation.However, continuous co-administration of several kinds of cytokines will induce inflammation and immune responses as well as other side effects .Nowadays, the hot issues in the field of blood diseases (hematological system diseases) and cancer research are to improve the responsibility of hematopoietic stem / progenitor cells to cytokines , promote the natural recovery of the hematopoietic function, to find substitute cytokine treatment ,and to reduce the side effects of treatment. The previous study of our laboratory has shown that scorpion venom peptide (SVP) can not only destruct a variety of tumor cells with high selectivity and enhance the immunity of the mice with tumors, but also reduce the suppressive effects on the bone marrow haematogenesis by radiotherapy and chemotherapy. By protecting the hematopoietic stem cells and progenitor cells damaged by radiation and accelerating the resumption of their proliferation ability .
M-NFS-60 cell , the mouse-derived promyelocytic leukemia cell line with the feature of hematopoietic factor-dependentcy, can reflect the normal hematopoietic stem cell proliferation characteristics to a certain extent. The objectives of this study is first to choose the strongest component of SVP to promote M-NFS-60 cell proliferation, and secondly, to study the role of these components to the proliferation of irradiated M-NFS-60 cell and its possible mechanisms and signal transduction pathways.
Materials and Methods:
1. Materials
1.1 Cell line: M-NFS-60 cell, the promyelocytic leukemia cells in mice, ordered from ATCC of USA .
1.2 SVP:purificated and presented by Guangzhou Institute of Snake Venom,including crude venom (C) ,SVP I1,SVP I2,SVP II1,SVP II2,SVP II3,SVP III1,SVPIII2,SVPIII3,SVPIVandSVP V(referredto as I1,I2,II1,II2,II3,III1,III2,III3,IV and V).After pre-experiment, A1,A2,A3,A4,A5 were purificated from II3,among which A4,A5 were pures identified by High Performance Liquid Chromatogrophy .
2. Methods
2.1 Screening effective components of SVP :
SVP components which of promote cells proliferation were Screened by Method of AlamarBlueTM Intake , and their effective concentration were determined as the in vitro concentration guide of SVP. The test cells were washed with RPMI 1640 culture medium for 3 times, then the living cells were counted by trypan blue staining. Cell concentration was adjusted to 5×104cells/ml with RPMI 1640 culture medium containing 10% FBS (containing r-h-M-CSF62ng/ml), and 80μl was added per hole in 96-well plates, which was then added, respectively, 10μl of normal saline, or different concentrations of SVP components, or IL-3 (10ng/ml) ,or SVP + IL-3. Each group includes 3 parallel of holes.After adding of 10μl AlamarBlueTM for conventional culture of 48h, the absorbance value of each group (well)in the fully automated microplate reader was measured , and the relative rate of cell proliferation in the experimental group was caculated, according to the established formula of AlamarBlueTMmethod.
2.2 Study of the proliferation-enhancing effect of effective SVP component on the irradiated M-NFS-60 cell: M-Nfs-60 cells were exposed to a uniform 60Coγ-ray irradiation, and theiy following study was similar to that of 2.1.
2.3 Study of the effect of SVP on cell cycle: normal and irradiated M-NFS-60 cells were treated with SVP, and cell cycle was detected by flow cytometry , with a further observation of SVP action on the proliferation 0f M-NFS-60 .
2.4 Study of the effect of SVP on the IL-3R and MCSFR expression: respectively, the expression level of IL-3R and MCSFR were detected by FCM,confocal laser scanning microscopy and Western blotting .
2.5 Study of the effect of SVP on the phosphorylation STAT5 expression: M-NFS-60 cells were treated with SVP,and the expression level of phosphorylation STAT5 was detected by Western blotting .
Results:
1. the normal M-NFS-60 cells SVP treated with SVP of 0.5～5 mg / L final concentration range for 48 hours, cell proliferation may be promoted, with 3mg / L concentration of SVP II3 and IV being the most significant , 3 mg/L of SVP II3、IV components were determined for in vitro treatment, and 3mg/L of SVP III3 for the control.The functions of the pures of A4 and A5 were more significant than A1, A2 and A3 .This is the first time that our team gain the pures of SVP that enhancing the proliferation action of hematopoietic cells .
2. M-NFS-60 cells were irradiated by 60Coγ-ray and then were treated with 3mg / L of II3, III3,IV,or IL-3(10ng/ml), or II3+IL3,IV +IL3, III3+IL3 for 48 hours. The cell proliferating rates (%) were 121.58±2.62 (II3) ,123.39±4.45 (III3) ,123.51±5.037 (IV) ,and 140.12±1.68(IL-3).Being significantly increased compared with the blank control group (100.00±.006), (P <0.01).When treated with combined SVP and IL-3, the cell proliferating rates were 163.98±9.20(II3+IL3),159.89±8.31(IV +IL3)and 148.92±9.74(III3+IL3). Compared with SVP treatment group, the difference was significant (P<0.05).
3. Compared with the control group, SVP could significantly promote S phase percentage of normal M-NFS-60 cells.And SVP could promote G2-M phase percentage of irradiated M-NFS-60 cells.
4. SVP can increase the level of IL-3R expression in normal and irradiated M-NFS-60 cell.
5. SVP can increase the expression of phosphorylated STAT5 in M-NFS-60 cell.
SVP can protect irradiated M-NFS-60 cells by accelerating the recovery of the proliferation ability.At the same time, it can up-regulate the level of IL-3R and phosphorylated STAT5 in M-Nfs-60 cell . It is suggested that the mechanism of prompting the recovery of irradiated hematopoietic cells by SVP may be associated with the activation of cytokine receptor signal transduction pathway.
Conclusion:
1.The results show that SVPIV,II3,A4,A5 could promote the proliferation of M-NFS-60 cells, and they have a synergistic effect with IL-3. It is suggested SVP has similer role of cytokine.
2.The function of prompting the proliferation of M-NFS-60 cells by SVP is associated with the up-regulation of IL-3R and M-CSFR, and SVP may be play their roles through JAK-STATs signal transduction pathway.
3. The pures of A4 and A5 were abtained for the first time, and they were confirmed to have similer role of cytokine,which lay the foundation for the study of their function and mechanism. The monomer of SVP has better prospect.
引文
1.从玉文,陈家佩,邵源.辐射后造血细胞对造血因子增殖反应性变化的研究.军事医学科学院院刊,1999 , 23 (2) :119 -122
2. Herodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol. 2005 Oct; 33(10):1071-80.
3. Herodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol. 2005 Oct; 33(10):1071-80.
4. Negrin RS. Clinical applications of hematopoietic growth factors. In: Thomson AW, ed. The Cytokine Handbook. San Diego, CA: Academic Press; 1998. 803–822.
5.肖桂元.蝎毒抗肿瘤研究进展中国肿瘤生物治疗杂志,1999,6(2):l6l-3
6.余茂耘,韦传宝.蝎毒的生理活性成分及临床应用.中国临床康复(2004,8(9):1754-5
7.杨志华,刘培光,温本,孔天翰.蝎毒抗癌多肽对X射线照射小鼠免疫功能的影响.中华放射医学与防护杂志, 2005, 25(2):134-136.
8.贺艳杰,孔天翰,董伟华.蝎毒多肽对辐射损伤小鼠骨髓造血干细胞及祖细胞的作用.中华生物医学工程杂志, 2007, 13(5): 272-275.
9. Li J, Yang C, Xia Y, Bertino A, Glaspy J, Roberts M, Kuter DJ. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood. 2001, 98:3241-3248.
10. Negrin RS. Clinical applications of hematopoietic growth factors. In: Thomson AW, editor. The Cytokine Handbook. San Diego, CA:Academic Press; p. 803-822, 1998.
11.王永奎,段萍,李国玲,韩雪飞,董伟华.蝎毒及其分离成分对60Coγ-射线照射后骨髓粒-单系祖细胞的影响.中国组织工程研究与临床康复, 2007,11(11):2098-2099.
12. Parfienczyk A, Kiersnowska-Rogowska B, Rogowski F. Study of interleukins and their soluble receptors in B-cell chronic lymphatic leukemia. Pol Merkur Lekarski. 2004 Jan; 16(91):22-5.
13. Mainou-Fowler T, Proctor SJ, Miller S, et al. Expression and production of interleukin 4 in B-cell chronic lymphocytic leukaemia. Leuk Lymphoma. 2001Aug,42(4):689-98.
14.孔天翰,魏玲,韩雪飞,董伟华.蝎毒抗癌多肽对放疗后肝癌小鼠瘤重和一般状况影响的初步观察.中华放射医学与防护杂志, 2000,20(5):313-316.
15. Winters Z E. P53 Pathways Involving G2 Checkpoint Reg-ulators and the Role of their Subcellular Localisation. J .R. Coll. Surg. Edinb. ,2002. 47(4) :591.
16. Moutoussamy S., Kelly P A. and Finidori J. Growth-hormone-receptor and cytokine-receptor-family signaling. Eur. J. Biochem., 1998, 255: 1-11.
17.张新海.白介素3与疾病的关系.国外医学生理、病理科学与临床分册1997,17(3)226-228.
18.从玉文陈家佩邵源等.辐射后NFS - 60细胞G- CSF受体特性的研究.辐射研究与辐射工艺学报,2002,20(4)275-279.
19.朱红.造血生长因子在造血系统中的研究进展.中华实用医学,2004,6(6)46-48.
20. Zardini DM,Heuschling Ph,Gallois A,et a1.Human umbi1ical cord blood-derived eosinophils cultured in the presence of IL-3 an d IL-5 respond to FMLP with[Ca ]variat ion and o2 production.Immunol Methods,1997,205(1):1.
21. Milatovich A,Kitamura T,Miyajima A,et a1.Gene for the alpha-subunit of the human interleukin-3 receptor(IL3RA)localized to the X-Y pseudoautosomal region[J].Am J Hum Genet,1993,53(5):l146-1153.
22. MartinezM, Margarita PhD, Huston, et al. Biology of common beta recep tor-signaling cytokines: IL-3, IL-5, and GM-CSF. J Allergy Clin Immunol,2003, 112 , 653～665.
23. Heim M H. The JAK-STAT pathway : specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest , 1996 , 26 (1) : 1-12
24. IbAe JN,Wittuhn BA,quelle FW,et a1.Singnaling by the cytokine receptor superfamily:JAKs and STATs[J].Trends Biochem Sci,1994, 19(5):222-227.
25. Yu wM,Hawley TS,Hawlet RG,et a1.Catalytic-dependent and -independemt roles of SHP-2 tyrosin phosphatase in interleuk-3 signaling [J].Oncogene,2003,22(38):5995-6004.
1. Ohno R.Oranulocyte colony—stimulating factor,granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor in the treatment of acute myeloid leukemia and acute lymphoblastiC leukemia.Leuk Res,1998,22(12):l143.
2.雷建蓉.人类重组红细胞生成素作用的研究进展[J].重庆医学,2000,29(5):469—470.
3.侯佩,蔡广研.人类重组促红细胞生成素临床应用进展[J].国外医学泌尿系统分册,2000,20(2):92—94.
4孙强明.人粒细胞一巨噬细胞集落因子研究进展[J] .国外医学·免疫学分册,2000,23(2):86—89.
5. Dong F.The C—terminal cytoplasmic region of the granulocyte colony-stimulating factor receptor mediates apoptosis in maturation incompetent murine Cell s.Exp Hematol,1996,24:214.
6. Taylor EM , Fear AL , Bohm A , et al. Structure2function studies on recombinant human macrophage colony2stimulating factor (M-CSF) . J Biol Chem , 1994 ,269 :31171231177.
7.朱红.造血生长因子在造血系统中的研究进展.中华实用医学,2004,6(6)46-48
8. Zardini DM,Heuschling Ph,Gallois A,et a1.Human umbi1ical cord blood—derived eosinophils cultured in the presence of IL一3 an d IL一5 respond to FMLP with[Ca ]variat ion and o2 production.Immunol Methods,1997,205(1):1.
9. Chang MS,McNinch J,Basu R,et a1.Cloning and characterization of the human ⅢegakaryOcyte growth and development factor (MGDF) gene.J Bioi Chem,1995,270:511
10.Petzer AL,Zandstra PW,Piret JM,et a1.Differential cytokine effects on primitive (CD34 /CD38) human hematopoietic cells:Novel response to F1t3—1igand and thrombopoietin.J Exp Med,1996,183:2551
11.罗云萍造血生长因子受体的信号传导与血液系统恶性疾病国外医学临床生物化学与检验学分册2001,22(1)44-45
12. Heim M H. The JAK-STAT pathway : specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest , 1996 , 26 (1) : 1～12
13. David w。Sternberg D,Gary Gililand. I1le Role of Signal Transdu -cer and Activator of Transcription Factors in Leukemogenesis. Journal of Clinical Oncology,2004,22(2):361—371
14. BOLEN BJ,BRUGGE JS.Leukocyte protein tyrosine kinases:Potentail targets for drug discovery[J].Annu Rev Immunol,1997,15:371～404.
15. IHLE JN,WITTHUHN BA,QUELLE FW,et al.Signal through the hematopoietic cytokine receptors[J].Annu Rev Immunol,1995,13:369～398.
16. Jason S.Rawlings,Kristin M.Rosier,et a1.The JAK-STAT signaling pathway.J Cell Sci,2004,1(17):1281—1283
17. Lisa Hendry,Susan John.Regulation of STAT signalling by prote -olytic processing.Eur J Biochem,2004,27(1):4613-4620
18. PELLEGRINI S,DUSANTER-FI.The structure,re-gulation and function of the Janus kinase(JAKs)and the signal transducers and activators of transcriptions(STATs)[J].Eur J Biochem,1997,248:615～633.
19. BONNETT MA,TANG LT,SUGIMOTO S,et al.Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptorβto Ras[J].Proc natl Acad Sci USA,1994,91:7335～7340.
20. TAGA T,KISHIMOTO T.Gp130 and the Interlukin-6 family of cytokine[sJ].Annu Rev Immunol,1997,15:797～819.
21. Kanwar VS,Witthuho B.Compana A,et al. lack of constitutive activation of Janus Kinases. and activation of transcriptio factors in philadelphia chromosome- positiveacute lymphoblastic leukemia[J]. Blood, 1996,87(11):4911-4912.
22. Meydan N.Grunberger T,Dadi H,et al. Inhibition of acute lymphob Lastic leukaemia by a Jak-2 inhibitor [J]. Nature, 1996,379(6566):645-648.
23. Hoefsloot LH,Van Amelsvoort MP,Broeders.LC,et al, Erythropo–etin-inducedactivation of STATs is impaired in themyelody splastic syndrome [J],Blood,1997,89(5):1690-1700.
24. Ward Ac,Loeb DM,soede-bobok AA,et al,Regulation of granulopo–iesis by transcription factors and cytokme signals[J]. leukemia,2000,14(6):973-990.
2. Herodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol. 2005 Oct; 33(10):1071-80.
3. Herodin F, Drouet M. Cytokine-based treatment of accidentally irradiated victims and new approaches. Exp Hematol. 2005 Oct; 33(10):1071-80.
4. Negrin RS. Clinical applications of hematopoietic growth factors. In: Thomson AW, ed. The Cytokine Handbook. San Diego, CA: Academic Press; 1998. 803–822.
5.肖桂元.蝎毒抗肿瘤研究进展中国肿瘤生物治疗杂志,1999,6(2):l6l-3
6.余茂耘,韦传宝.蝎毒的生理活性成分及临床应用.中国临床康复(2004,8(9):1754-5
7.杨志华,刘培光,温本,孔天翰.蝎毒抗癌多肽对X射线照射小鼠免疫功能的影响.中华放射医学与防护杂志, 2005, 25(2):134-136.
8.贺艳杰,孔天翰,董伟华.蝎毒多肽对辐射损伤小鼠骨髓造血干细胞及祖细胞的作用.中华生物医学工程杂志, 2007, 13(5): 272-275.
9. Li J, Yang C, Xia Y, Bertino A, Glaspy J, Roberts M, Kuter DJ. Thrombocytopenia caused by the development of antibodies to thrombopoietin. Blood. 2001, 98:3241-3248.
10. Negrin RS. Clinical applications of hematopoietic growth factors. In: Thomson AW, editor. The Cytokine Handbook. San Diego, CA:Academic Press; p. 803-822, 1998.
11.王永奎,段萍,李国玲,韩雪飞,董伟华.蝎毒及其分离成分对60Coγ-射线照射后骨髓粒-单系祖细胞的影响.中国组织工程研究与临床康复, 2007,11(11):2098-2099.
12. Parfienczyk A, Kiersnowska-Rogowska B, Rogowski F. Study of interleukins and their soluble receptors in B-cell chronic lymphatic leukemia. Pol Merkur Lekarski. 2004 Jan; 16(91):22-5.
13. Mainou-Fowler T, Proctor SJ, Miller S, et al. Expression and production of interleukin 4 in B-cell chronic lymphocytic leukaemia. Leuk Lymphoma. 2001Aug,42(4):689-98.
14.孔天翰,魏玲,韩雪飞,董伟华.蝎毒抗癌多肽对放疗后肝癌小鼠瘤重和一般状况影响的初步观察.中华放射医学与防护杂志, 2000,20(5):313-316.
15. Winters Z E. P53 Pathways Involving G2 Checkpoint Reg-ulators and the Role of their Subcellular Localisation. J .R. Coll. Surg. Edinb. ,2002. 47(4) :591.
16. Moutoussamy S., Kelly P A. and Finidori J. Growth-hormone-receptor and cytokine-receptor-family signaling. Eur. J. Biochem., 1998, 255: 1-11.
17.张新海.白介素3与疾病的关系.国外医学生理、病理科学与临床分册1997,17(3)226-228.
18.从玉文陈家佩邵源等.辐射后NFS - 60细胞G- CSF受体特性的研究.辐射研究与辐射工艺学报,2002,20(4)275-279.
19.朱红.造血生长因子在造血系统中的研究进展.中华实用医学,2004,6(6)46-48.
20. Zardini DM,Heuschling Ph,Gallois A,et a1.Human umbi1ical cord blood-derived eosinophils cultured in the presence of IL-3 an d IL-5 respond to FMLP with[Ca ]variat ion and o2 production.Immunol Methods,1997,205(1):1.
21. Milatovich A,Kitamura T,Miyajima A,et a1.Gene for the alpha-subunit of the human interleukin-3 receptor(IL3RA)localized to the X-Y pseudoautosomal region[J].Am J Hum Genet,1993,53(5):l146-1153.
22. MartinezM, Margarita PhD, Huston, et al. Biology of common beta recep tor-signaling cytokines: IL-3, IL-5, and GM-CSF. J Allergy Clin Immunol,2003, 112 , 653～665.
23. Heim M H. The JAK-STAT pathway : specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest , 1996 , 26 (1) : 1-12
24. IbAe JN,Wittuhn BA,quelle FW,et a1.Singnaling by the cytokine receptor superfamily:JAKs and STATs[J].Trends Biochem Sci,1994, 19(5):222-227.
25. Yu wM,Hawley TS,Hawlet RG,et a1.Catalytic-dependent and -independemt roles of SHP-2 tyrosin phosphatase in interleuk-3 signaling [J].Oncogene,2003,22(38):5995-6004.
1. Ohno R.Oranulocyte colony—stimulating factor,granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor in the treatment of acute myeloid leukemia and acute lymphoblastiC leukemia.Leuk Res,1998,22(12):l143.
2.雷建蓉.人类重组红细胞生成素作用的研究进展[J].重庆医学,2000,29(5):469—470.
3.侯佩,蔡广研.人类重组促红细胞生成素临床应用进展[J].国外医学泌尿系统分册,2000,20(2):92—94.
4孙强明.人粒细胞一巨噬细胞集落因子研究进展[J] .国外医学·免疫学分册,2000,23(2):86—89.
5. Dong F.The C—terminal cytoplasmic region of the granulocyte colony-stimulating factor receptor mediates apoptosis in maturation incompetent murine Cell s.Exp Hematol,1996,24:214.
6. Taylor EM , Fear AL , Bohm A , et al. Structure2function studies on recombinant human macrophage colony2stimulating factor (M-CSF) . J Biol Chem , 1994 ,269 :31171231177.
7.朱红.造血生长因子在造血系统中的研究进展.中华实用医学,2004,6(6)46-48
8. Zardini DM,Heuschling Ph,Gallois A,et a1.Human umbi1ical cord blood—derived eosinophils cultured in the presence of IL一3 an d IL一5 respond to FMLP with[Ca ]variat ion and o2 production.Immunol Methods,1997,205(1):1.
9. Chang MS,McNinch J,Basu R,et a1.Cloning and characterization of the human ⅢegakaryOcyte growth and development factor (MGDF) gene.J Bioi Chem,1995,270:511
10.Petzer AL,Zandstra PW,Piret JM,et a1.Differential cytokine effects on primitive (CD34 /CD38) human hematopoietic cells:Novel response to F1t3—1igand and thrombopoietin.J Exp Med,1996,183:2551
11.罗云萍造血生长因子受体的信号传导与血液系统恶性疾病国外医学临床生物化学与检验学分册2001,22(1)44-45
12. Heim M H. The JAK-STAT pathway : specific signal transduction from the cell membrane to the nucleus. Eur J Clin Invest , 1996 , 26 (1) : 1～12
13. David w。Sternberg D,Gary Gililand. I1le Role of Signal Transdu -cer and Activator of Transcription Factors in Leukemogenesis. Journal of Clinical Oncology,2004,22(2):361—371
14. BOLEN BJ,BRUGGE JS.Leukocyte protein tyrosine kinases:Potentail targets for drug discovery[J].Annu Rev Immunol,1997,15:371～404.
15. IHLE JN,WITTHUHN BA,QUELLE FW,et al.Signal through the hematopoietic cytokine receptors[J].Annu Rev Immunol,1995,13:369～398.
16. Jason S.Rawlings,Kristin M.Rosier,et a1.The JAK-STAT signaling pathway.J Cell Sci,2004,1(17):1281—1283
17. Lisa Hendry,Susan John.Regulation of STAT signalling by prote -olytic processing.Eur J Biochem,2004,27(1):4613-4620
18. PELLEGRINI S,DUSANTER-FI.The structure,re-gulation and function of the Janus kinase(JAKs)and the signal transducers and activators of transcriptions(STATs)[J].Eur J Biochem,1997,248:615～633.
19. BONNETT MA,TANG LT,SUGIMOTO S,et al.Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptorβto Ras[J].Proc natl Acad Sci USA,1994,91:7335～7340.
20. TAGA T,KISHIMOTO T.Gp130 and the Interlukin-6 family of cytokine[sJ].Annu Rev Immunol,1997,15:797～819.
21. Kanwar VS,Witthuho B.Compana A,et al. lack of constitutive activation of Janus Kinases. and activation of transcriptio factors in philadelphia chromosome- positiveacute lymphoblastic leukemia[J]. Blood, 1996,87(11):4911-4912.
22. Meydan N.Grunberger T,Dadi H,et al. Inhibition of acute lymphob Lastic leukaemia by a Jak-2 inhibitor [J]. Nature, 1996,379(6566):645-648.
23. Hoefsloot LH,Van Amelsvoort MP,Broeders.LC,et al, Erythropo–etin-inducedactivation of STATs is impaired in themyelody splastic syndrome [J],Blood,1997,89(5):1690-1700.
24. Ward Ac,Loeb DM,soede-bobok AA,et al,Regulation of granulopo–iesis by transcription factors and cytokme signals[J]. leukemia,2000,14(6):973-990.