同种异体骨髓间充质干细胞输注治疗大鼠辐射损伤
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摘要
背景与目的
放射治疗是肿瘤治疗中的一项重要手段,随着肿瘤发病率的增高,放射治疗的应用相对以往明显增加,随之出现的放射性损伤也相应增多。自1895年伦琴发现X射线,以及铀、镭等相继发现,揭开了人类利用电离辐射的序幕。然而,电离辐射却是一把双刃剑,它也会对人体健康造成一定的危害。辐射损伤是由于辐射能作用于机体后引起体内一系列的变化,当损伤严重时可使组织器官发生不可逆的破坏甚至导致机体死亡。国内外学者对于辐射损伤的氧自由基学说较为肯定,氧自由基的产生和清除保持一个动态的平衡,辐射损伤可生成大量自由基,引发脂质发生氧化反应,导致细胞膜破坏和细胞死亡,继而引起体内抗氧化物质活力的变化,打破机体的氧化-抗氧化体系的平衡。超氧化物歧化酶(superoxide dismutase,SOD)是体内重要的自由基清除剂,在机体的氧化与抗氧化平衡中发挥重要的作用,能清除自由基,保护细胞膜免受氧化和脂质过氧化损伤。丙二醛(malondialdehyde, MDA)是自由基攻击生物膜引发脂质过氧化的产物,可与蛋白、核酸(DNA、RNA)和磷脂等含有氨基的物质交联,引起这些大分子化合物的分子交联。因此MDA水平可反映机体内脂质过氧化的程度,其在血液的水平可间接反映细胞损伤的程度。本实验研究观察指标之一就是间充质干细胞(mesenchymal stem cells, MSCs)移植后能否改变大鼠体内氧自由基水平。肾脏、睾丸、肺脏、肝脏等机体脏器属于放射高敏感性器官,容易受到辐射损伤,出现器官功能障碍,影响机体代谢、解毒、分解等,最终导致生命危害。本研究模拟临床放射治疗的辐射模式建造损伤模型,并检测相应脏器的功能指标,探索辐射损伤后各脏器结构、功能的变化以及MSCs干预治疗的效果。
MSCs是中胚层来源的具有多向分化能力的干细胞,国内外研究发现MSCs能够在特定条件下分化为骨细胞、软骨细胞、神经细胞等；是组织工程、细胞及基因治疗理想的靶细胞。它不仅取材方便、培养容易、增殖较快,而且具有多向分化潜能,免疫原性也较低的特点,低表达MHCⅠ和不表达MHCⅡ提示在进行同种异体间输注时,不会产生排斥反应。
目前对于辐射损伤的治疗仅仅停留在调节、改善的水平上,未能从根本上解决问题。本实验立题基于MSCs这些特性进行干预治疗,采用同种异基因MSCs进行输注,研究MSCs在辐射损伤大鼠体内修复和改善受损脏器结构和功能。
实验方法
第一部分大鼠间充质干细胞的体外培养与鉴定
实验在无菌条件下分离大鼠肱骨、股骨和胫骨,用PBS缓冲液冲出骨髓,离心后接种含10%胎牛血清(FBS)的L-DMEM培养基中,按2×106／cm2密度接种于培养瓶中,置于孵箱中培养。48h后半量换液,第五天全量换液,待细胞生长至80-90%融合时,0.25%胰酶消化传代。倒置显微镜观察细胞形态,流式细胞术进行细胞周期分析,用流式细胞仪检测细胞表面抗原CD34、CD45、CD29及CD44的表达,进行细胞鉴定,从而为本实验提供大量的同种异基因大鼠骨髓间充质干细胞。
第二部分MSCs治疗辐射损伤效果的研究
选取15只雄性SD大鼠随机分为三组,即对照组、模型组和治疗组。模型组和治疗组大鼠一次性接受直线加速器产生的X线照射,6Gy／周,共3周,总剂量18Gy,对照组不接受照射。其中治疗组大鼠在接受照射24h后尾静脉注射间充质干细胞3×106／只,共三次；模型组和对照组大鼠同期尾静脉注射同等体积的生理盐水。常规饲养大鼠,待模型组大鼠处于濒死状态时,腹腔注射苯巴比妥钠注射液麻醉大鼠,提取大鼠血清应用全自动分析仪及酶标仪检测血清中肌酐(Creatinine, Cr)、尿素(Urea,UA)、丙氨酸氨基转移酶(Alanine aminotransferase,ALT)、天冬门氨酸氨基转移酶(Aspartate Aminotransferase,AST)、乳酸脱氢酶(Lactate Dehydrogenase,LDH)、碱性磷酸酶(Alkaline Phosphatase,ALP)及睾酮(Testosterone, T)的水平；采用硫代巴比妥酸和黄嘌呤氧化法分别检测血清和各个组织中MDA含量和SOD活性；制作各个组织常规石蜡切片,观察三组大鼠各个组织结构的差异,从功能学水平,生化水平及细胞形态学水平研究MSCs是否具有治疗辐射损伤的作用。
统计学方法
实验资料结果用均数士标准差(x±s)表示,显著性标准为P<0.05,采用SPSS13.0统计软件包进行统计分析。组间比较用单因素方差分析(One-Way ANOVA);重复测量数据用重复测量方差分析(Reperted measurement experiment);组间两两显著性比较方差齐采用LSD法,方差不齐用Dunnett T3。相关性采用偏相关分析(Partial correlation analysis)。
结果
第一部分大鼠间充质干细胞的培养与鉴定
①本实验分离的骨髓间充质干细胞于48h后贴壁生长,全量换液后可见细胞呈星形、多边形和不规则形；10天左右时,细胞即可融合近80%。传代培养后细胞形态基本上趋于一致,光镜下见细胞呈梭形,核圆,位于细胞中央,胞质肥大,胞膜清晰；
②流式细胞术证明G0+G1期的细胞约占80%以上,说明MSCs在体外具有只增殖不分化的特点；
③经流式细胞仪检测,Cell Quest软件分析,结果显示第3代MSCs的特异性表面标志物：CD34-/CD45-/CD29+/CD44+,CD29+、阳性率为98.9%,CD44+、阳性率为93.5%,说明本实验培养的细胞为间充质干细胞。
第二部分MSCs移植治疗辐射损伤效果的研究
①观察大鼠的一般状态：对照组大鼠精神状态好,活动自如；模型组大鼠精神萎靡,反应迟钝,活动少,嗜睡,毛发变黄；治疗组大鼠精神状态一般,毛发微黄,反应较模型组灵敏。
②全自动生化分析仪检测血清中肌酐和尿素的含量,结果显示：对照组、模型组及治疗组血清中尿素含量分别为4.90±0.31、19.64±0.75、11.88±1.16mmol/ml;Levene检验方差不齐,F=5.956,P=0.016；采用Dunnett's T3法进行多重比较,P值均<0.05,差异具有统计学意义。肌酐含量分别为44.20±3.56、85.40±4.62、58.40±3.51μmol/ml,Levene检验方差齐,F=0.276,P=0.763；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
③全自动生化分析仪检测血清中ALT、AST、ALP及LDH的含量,结果显示：对照组、模型组及治疗组血清中ALT的含量分别为19.00±6.32、54.20±3.56、36.00±4.74IU/L;Levene检验方差齐,F=0.757,P=0.490；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。AST的含量分别为48.00110.93、187.80137.09、89.80±13.37IU/L;Levene检验方差齐,F=2.486,P=0.125；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。ALP的含量分别为5.40±3.44、342.60153.87、163.40123.87 IU/L;Levene检验方差不齐,F=7.135,P=0.009；采用Dunnett's T3法进行多重比较,P值均<0.05,差异具有统计学意义。LDH的含量分别为133.40±34.27、768.20±27.99、407.60±63.82 IU/L;Levene检验方差齐,F=3.380,P=0.069；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
④酶标仪检测大鼠血清中睾酮(T)的水平,结果显示：对照组、模型组、治疗组分别为6.55±0.55 IU/L、0.76±0.52 IU/L、4.28±0.78 IU/L;Levene检验方差齐,F=0.277,P=0.763；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义；说明MSCs治疗组与模型组相比,能够升高雄性大鼠体内T的含量。
⑤采用硫代巴比妥酸和黄嘌呤氧化法分别检测血清SOD的活性及MDA含量,结果显示：对照组、模型组及治疗组血清SOD的活性分别为110.0959-5.4115U/ml,64.1013±3.6994U/ml,84.2043±3.1574U/ml;Levene检验方差齐,F=1.700,P=0.224；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。MDA含量分别为7.8059±1.3564nmol/ml、50.9283±3.2640nmol/ml、21.6118±0.9332nmol/ml,Levene检验方差齐,F=3.597,P=0.06；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
⑥病理形态学显示：模型组与对照组比较,脑、肝脏、肾脏、脾脏、睾丸的结构破坏明显；MSCs治疗组与模型组相比,各脏器的结构得到明显改善；
结论
1、本实验方法能有效地获得骨髓间充质干细胞,采用贴壁法培养的骨髓细胞经鉴定为间充质干细胞。
2、本实验从功能学水平、形态学水平、生化水平检测,发现MSCs具有修复和改善大鼠脏器辐射损伤的效果,说明同种异体骨髓间充质干细胞输注具有治疗大鼠辐射损伤的作用。
本研究的创新点：本研究从MSCs具有多向分化潜能的干细胞特征入手,研究骨髓间充质干细胞在辐射损伤动物体内调节、修复受损的脏器,能够从根本上解决损伤脏器修复的问题；
本研究的价值：本实验从MSCs多向分化潜能着手,研究同种异体骨髓MSCs输注对大鼠辐射损伤脏器的影响,为治疗辐射损伤提供了有力的实验依据,同时为MSCs的生物细胞治疗打开另一个全新的思路。
Background:
Radiation therapy is an important cancer treatment, with the increased incidence of cancer, radiation therapy significantly increased compare with the past, the result of radiation damage is also a corresponding increase.Since 1895, Roentgen discovered X-rays, as well as uranium, radium and other have found that opening a prelude to human using ionizing radiation. However, ionizing radiation is a double-edged sword, it would also cause some harm to human health. Radiation damage is due to radiant energy acting on the body caused by the body after a series of changes, as the damage tissues and organs can occur in severe irreversible damage to the body and even lead to death. Domestic and foreign scholars are sute of oxygen free radical theory of radiation damage.Free radicals are dynamic equilibrium from produce and clearance in vivo.With radiation damage, the balance is gradually destroyed and resulting in excess free radicals.Excessive free radicals can induce peroxidation of lipid membrane, leading to cell membrane damage and cell death. Superoxide dismutase(SOD)is an important free radical scavenger in vivo, who can protect cell membranes from oxidation and lipid peroxidation. SOD plays an important role in the body in vivo on oxidation and antioxidant balance, can eliminate free radicals and protect cells from damage.Malondialdehyde(MDA)is a product of radical attack on membrane leading to lipid peroxidation. MDA combine with proteins, nucleic acids (DNA, RNA) and phospholipids and other substances containing amino cross-linking, causing these into compounds---the molecular cross-linked macromolecules.Therefore,the level of MDA can reflect the body's level of lipid peroxidation, and its blood level can indirectly reflect the extent of cell damage. One of the indicators of this study is whether MSCs transplantation change the level of free radicals in rats or not.Kidney, testis, lung, liver and other organs of the body belonging to the high radiation sensitivity of organs which are susceptible to radiation damage, leading to organ dysfunction and then affect metabolism, detoxification and decomposition, etc.,eventually leading to life-endangering.This study simulated clinical radiation therapy radiation model to build animal model for the construction of the radiation pattern and detect the corresponding organ of the function parameters.
Domestic and international study found that Bone marrow mesenchymal stem cells(MSCs) have multi-differentiation potential and can differentiate into bone cells, cartilage cells, nerve cells and so on at the appropriate conditions in vitro;MSCs is tissue engineering, cell and gene therapy an ideal cells.It is not only convenient, easy to cultivate,the proliferation of faster, but also has multi-directional differentiation potential,lower immunogenicity and low expression of MHC I and non-expression of allogeneic MHCⅡ.MSCs can not be rejected after inter-infusion, it will not produce rejection. In this study, based on these characteristics of MSCs, we injecte MSCs in the model to study the function of MSCs whether can anti-oxidation or not of rats through detecting SOD and MDA levels and the pathology of various organizations and at the same time to verify the low immunogenicity of the characteristics of MSCs.
Methods
Part I:mesenchymal stem cells (MSCs) were cultured in vitro and identified
MSCs were isolated from the bone marrow of rats'humerus,femjur, tibia under aseptic condition,and then the cells were collected at a desity of 2×106/cm2 after centrifugation. L-DMEM containing 0.1 volume fraction of fetal bovine serum was added in the culture flask for routine culture. Changing half of the medium after 48h and the whole medium was changed on the fifth day. Cells were not digested until the cells reached confluence over 80% after medium changes.Morphologic changes of MSCs were observed by phase contrast microscope and the cell cycle were detected and analyzed by flow cytometry. Identifying of cells using antigen of CD34,CD44,CD29,CD45.
Part II:The study of MSCs'effects on Rats of irradiation-induced injury
15 male SD rats were randomly divided into three groups, the control group, model group and treatment group.Whole body irradiation of 6.0Gy were performed in model group and treatment group each week, a total of three weeks, the cumulative total dose is 18Gy, and the control group does not accept the irradiation. Rats in treatment group receive infusion of MSCs with 3×106 each after the end of 24h radiation treatment; while in model group and control group rats received infusion of normal saline lml by tail vein. Rats were killed in extremis, Intraperitoneal injection of pentobarbital sodium to anesthetize rats.The application of automatic extraction of serum and microplate reader analyzer serum creatinine, urea,alanine aminotransferase (ALT),aspartic acid aminotransferase (AST) and the level of testosterone(T);The content of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and organisms were measured by hydroxylamine and chromatometry methods;the difference of three groups in organizational structure were observed by conventional paraffin sections.we study the level of functional, biochemical and cellular level of the functions of MSCs on radiation damage treatment.
Statistical methods:The results of experimental data with mean±standard deviation(x±s) that significant for the 0.05 standard, using SPSS 13.0 statistical packages for statistical analysis. Group compared with one-way ANOVA (One-Way ANOVA);repeat measurements analysis of variance with repeated measures (Reperted measurement experiment);inter-group comparison was significant homogeneity using LSD method, with variance arrhythmia Dunnett's T3.The relevance of the use of partial correlation analysis (Partial correlation analysis).
Results
Part I:Mesenchymal stem cells'culture and identification
①The experimental separation of bone marrow-derived mesenchymal stem cells can integrate 80% in about 10 days. After cultured cells are basically in line, we can see spindle cells by microscope, the nuclear circle is located in the central of cell.Cytoplasm is hypertrophy and the membrane is clear;
②Flow cytometry proved G0+G1 phase is about 80% of cell.MSCs in vitro differentiation with only the characteristics of non-proliferation;
③by flow cytometry and Cell Quest software analysis, results showed that MSCs in P3 generation-specific surface markers:CD34-negative, CD45-negative, CD29-positive, the positive rate was 98.9%, CD44-positive, the positive rate was 93.5% on cultured cells in this experiment as mesenchymal stem cells.
Part II:The study of MSCs'effect on the radiation damage Rats
④The mice in control group are good state of mind and freedom of movement; Compare with the control group, the mice in model group are inactive, unresponsive, less active, lethargy and hair turning yellow; while the one with treatment group the general state of mind, hair yellow, with higher sensitive response than the model group.
②Automatic biochemical analyzer:serum levels of creatinine and urea showed: control group, model group and treatment group, serum urea were 4.90±0.31,19.64±0.75,11.88±1.16mmol/ml;Inter-group comparison was using Dunnett's T3 with variance arrhythmia, p<0.05,statistically significant differences;Creatinine contents were 44.20±3.56,85.40±4.62,58.40±3.51μmol/ml;Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences;
③Automatic biochemical analyzer:serum ALT、AST、ALP and LDH levels of content, results showed:control group, model group and treatment group, serum ALT levels were 19.00±6.32,54.20±3.56,36.00±4.74IU/L;Inter-group comparison was significant homogeneity using LSD method, p<0.05, statistically significant differences;Serum AST levels were 48.00±10.93, 187.80±37.09,89.80±13.37IU/L;Compare the control group with model group, F=2.91,p<0.05.Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences.Serum ALP levels were 5.40±3.44,342.60±53.87,163.40±23.87IU/L;Inter-group comparison was using Dunnett's T3 with variance arrhythmia, p<0.05,statistically significant differences;Serum LDH levels were 133.40±34.27,768.20±27.99, 407.60±63.82IU/L;Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences.
④Detection microplate reader detection of serum testosterone levels on control group, model group and treatment group showed that:6.55±0.55,0.76±0.52, 4.28±0.78IU/L.Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences;
⑤Using thiobarbituric acid and xanthine oxidase were determined in serum SOD activity and MDA content, results showed:control group, model group and treatment group, serum SOD activity were 110.0959±5.4115,64.1013±3.6994, 84.2043±3.1574 (U/ml); Compare the control group with model group, F=2.91, p<0.05.Compare the model group with treatment group, the control group with model group,statistically significant differences.MDA contents were 7.8059±1.3564,50.9283±3.2640,21.6118±0.9332nmol/ml;Compare the model group with treatment group, the control group with model group,statistically significant differences.
⑥Pathology results:MSCs treatment group compared with the model group, enabling the brain,liver,kidney, testicular structures were obviously improved;
Conclusion
1.The method can effectively enhance rat bone marrow-derived mesenchymal stem cells.
2.This study found that MSCs have the repair effects of radiation damage organs in rats from functional level, morphology and biochemical level.
The innovation of this study:
In this study, we explore mesenchymal stem cells'character of multilineage differentiation in vivo to regulate and repair of injured organs, which fix the problem from a fundamental solution.
The value of this study:
In this study,we focus on the differentiation potential of MSCs to discover MSCs' function on the radiation damage organs, providing strong experimental evidences for treatment of radiation damages and opening another new idea for biological treatment of MSCs.
放射治疗是肿瘤治疗中的一项重要手段,随着肿瘤发病率的增高,放射治疗的应用相对以往明显增加,随之出现的放射性损伤也相应增多。自1895年伦琴发现X射线,以及铀、镭等相继发现,揭开了人类利用电离辐射的序幕。然而,电离辐射却是一把双刃剑,它也会对人体健康造成一定的危害。辐射损伤是由于辐射能作用于机体后引起体内一系列的变化,当损伤严重时可使组织器官发生不可逆的破坏甚至导致机体死亡。国内外学者对于辐射损伤的氧自由基学说较为肯定,氧自由基的产生和清除保持一个动态的平衡,辐射损伤可生成大量自由基,引发脂质发生氧化反应,导致细胞膜破坏和细胞死亡,继而引起体内抗氧化物质活力的变化,打破机体的氧化-抗氧化体系的平衡。超氧化物歧化酶(superoxide dismutase,SOD)是体内重要的自由基清除剂,在机体的氧化与抗氧化平衡中发挥重要的作用,能清除自由基,保护细胞膜免受氧化和脂质过氧化损伤。丙二醛(malondialdehyde, MDA)是自由基攻击生物膜引发脂质过氧化的产物,可与蛋白、核酸(DNA、RNA)和磷脂等含有氨基的物质交联,引起这些大分子化合物的分子交联。因此MDA水平可反映机体内脂质过氧化的程度,其在血液的水平可间接反映细胞损伤的程度。本实验研究观察指标之一就是间充质干细胞(mesenchymal stem cells, MSCs)移植后能否改变大鼠体内氧自由基水平。肾脏、睾丸、肺脏、肝脏等机体脏器属于放射高敏感性器官,容易受到辐射损伤,出现器官功能障碍,影响机体代谢、解毒、分解等,最终导致生命危害。本研究模拟临床放射治疗的辐射模式建造损伤模型,并检测相应脏器的功能指标,探索辐射损伤后各脏器结构、功能的变化以及MSCs干预治疗的效果。
MSCs是中胚层来源的具有多向分化能力的干细胞,国内外研究发现MSCs能够在特定条件下分化为骨细胞、软骨细胞、神经细胞等；是组织工程、细胞及基因治疗理想的靶细胞。它不仅取材方便、培养容易、增殖较快,而且具有多向分化潜能,免疫原性也较低的特点,低表达MHCⅠ和不表达MHCⅡ提示在进行同种异体间输注时,不会产生排斥反应。
目前对于辐射损伤的治疗仅仅停留在调节、改善的水平上,未能从根本上解决问题。本实验立题基于MSCs这些特性进行干预治疗,采用同种异基因MSCs进行输注,研究MSCs在辐射损伤大鼠体内修复和改善受损脏器结构和功能。
实验方法
第一部分大鼠间充质干细胞的体外培养与鉴定
实验在无菌条件下分离大鼠肱骨、股骨和胫骨,用PBS缓冲液冲出骨髓,离心后接种含10%胎牛血清(FBS)的L-DMEM培养基中,按2×106／cm2密度接种于培养瓶中,置于孵箱中培养。48h后半量换液,第五天全量换液,待细胞生长至80-90%融合时,0.25%胰酶消化传代。倒置显微镜观察细胞形态,流式细胞术进行细胞周期分析,用流式细胞仪检测细胞表面抗原CD34、CD45、CD29及CD44的表达,进行细胞鉴定,从而为本实验提供大量的同种异基因大鼠骨髓间充质干细胞。
第二部分MSCs治疗辐射损伤效果的研究
选取15只雄性SD大鼠随机分为三组,即对照组、模型组和治疗组。模型组和治疗组大鼠一次性接受直线加速器产生的X线照射,6Gy／周,共3周,总剂量18Gy,对照组不接受照射。其中治疗组大鼠在接受照射24h后尾静脉注射间充质干细胞3×106／只,共三次；模型组和对照组大鼠同期尾静脉注射同等体积的生理盐水。常规饲养大鼠,待模型组大鼠处于濒死状态时,腹腔注射苯巴比妥钠注射液麻醉大鼠,提取大鼠血清应用全自动分析仪及酶标仪检测血清中肌酐(Creatinine, Cr)、尿素(Urea,UA)、丙氨酸氨基转移酶(Alanine aminotransferase,ALT)、天冬门氨酸氨基转移酶(Aspartate Aminotransferase,AST)、乳酸脱氢酶(Lactate Dehydrogenase,LDH)、碱性磷酸酶(Alkaline Phosphatase,ALP)及睾酮(Testosterone, T)的水平；采用硫代巴比妥酸和黄嘌呤氧化法分别检测血清和各个组织中MDA含量和SOD活性；制作各个组织常规石蜡切片,观察三组大鼠各个组织结构的差异,从功能学水平,生化水平及细胞形态学水平研究MSCs是否具有治疗辐射损伤的作用。
统计学方法
实验资料结果用均数士标准差(x±s)表示,显著性标准为P<0.05,采用SPSS13.0统计软件包进行统计分析。组间比较用单因素方差分析(One-Way ANOVA);重复测量数据用重复测量方差分析(Reperted measurement experiment);组间两两显著性比较方差齐采用LSD法,方差不齐用Dunnett T3。相关性采用偏相关分析(Partial correlation analysis)。
结果
第一部分大鼠间充质干细胞的培养与鉴定
①本实验分离的骨髓间充质干细胞于48h后贴壁生长,全量换液后可见细胞呈星形、多边形和不规则形；10天左右时,细胞即可融合近80%。传代培养后细胞形态基本上趋于一致,光镜下见细胞呈梭形,核圆,位于细胞中央,胞质肥大,胞膜清晰；
②流式细胞术证明G0+G1期的细胞约占80%以上,说明MSCs在体外具有只增殖不分化的特点；
③经流式细胞仪检测,Cell Quest软件分析,结果显示第3代MSCs的特异性表面标志物：CD34-/CD45-/CD29+/CD44+,CD29+、阳性率为98.9%,CD44+、阳性率为93.5%,说明本实验培养的细胞为间充质干细胞。
第二部分MSCs移植治疗辐射损伤效果的研究
①观察大鼠的一般状态：对照组大鼠精神状态好,活动自如；模型组大鼠精神萎靡,反应迟钝,活动少,嗜睡,毛发变黄；治疗组大鼠精神状态一般,毛发微黄,反应较模型组灵敏。
②全自动生化分析仪检测血清中肌酐和尿素的含量,结果显示：对照组、模型组及治疗组血清中尿素含量分别为4.90±0.31、19.64±0.75、11.88±1.16mmol/ml;Levene检验方差不齐,F=5.956,P=0.016；采用Dunnett's T3法进行多重比较,P值均<0.05,差异具有统计学意义。肌酐含量分别为44.20±3.56、85.40±4.62、58.40±3.51μmol/ml,Levene检验方差齐,F=0.276,P=0.763；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
③全自动生化分析仪检测血清中ALT、AST、ALP及LDH的含量,结果显示：对照组、模型组及治疗组血清中ALT的含量分别为19.00±6.32、54.20±3.56、36.00±4.74IU/L;Levene检验方差齐,F=0.757,P=0.490；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。AST的含量分别为48.00110.93、187.80137.09、89.80±13.37IU/L;Levene检验方差齐,F=2.486,P=0.125；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。ALP的含量分别为5.40±3.44、342.60153.87、163.40123.87 IU/L;Levene检验方差不齐,F=7.135,P=0.009；采用Dunnett's T3法进行多重比较,P值均<0.05,差异具有统计学意义。LDH的含量分别为133.40±34.27、768.20±27.99、407.60±63.82 IU/L;Levene检验方差齐,F=3.380,P=0.069；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
④酶标仪检测大鼠血清中睾酮(T)的水平,结果显示：对照组、模型组、治疗组分别为6.55±0.55 IU/L、0.76±0.52 IU/L、4.28±0.78 IU/L;Levene检验方差齐,F=0.277,P=0.763；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义；说明MSCs治疗组与模型组相比,能够升高雄性大鼠体内T的含量。
⑤采用硫代巴比妥酸和黄嘌呤氧化法分别检测血清SOD的活性及MDA含量,结果显示：对照组、模型组及治疗组血清SOD的活性分别为110.0959-5.4115U/ml,64.1013±3.6994U/ml,84.2043±3.1574U/ml;Levene检验方差齐,F=1.700,P=0.224；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。MDA含量分别为7.8059±1.3564nmol/ml、50.9283±3.2640nmol/ml、21.6118±0.9332nmol/ml,Levene检验方差齐,F=3.597,P=0.06；采用LSD法进行组间两两比较,P值均<0.05,差异具有统计学意义。
⑥病理形态学显示：模型组与对照组比较,脑、肝脏、肾脏、脾脏、睾丸的结构破坏明显；MSCs治疗组与模型组相比,各脏器的结构得到明显改善；
结论
1、本实验方法能有效地获得骨髓间充质干细胞,采用贴壁法培养的骨髓细胞经鉴定为间充质干细胞。
2、本实验从功能学水平、形态学水平、生化水平检测,发现MSCs具有修复和改善大鼠脏器辐射损伤的效果,说明同种异体骨髓间充质干细胞输注具有治疗大鼠辐射损伤的作用。
本研究的创新点：本研究从MSCs具有多向分化潜能的干细胞特征入手,研究骨髓间充质干细胞在辐射损伤动物体内调节、修复受损的脏器,能够从根本上解决损伤脏器修复的问题；
本研究的价值：本实验从MSCs多向分化潜能着手,研究同种异体骨髓MSCs输注对大鼠辐射损伤脏器的影响,为治疗辐射损伤提供了有力的实验依据,同时为MSCs的生物细胞治疗打开另一个全新的思路。
Background:
Radiation therapy is an important cancer treatment, with the increased incidence of cancer, radiation therapy significantly increased compare with the past, the result of radiation damage is also a corresponding increase.Since 1895, Roentgen discovered X-rays, as well as uranium, radium and other have found that opening a prelude to human using ionizing radiation. However, ionizing radiation is a double-edged sword, it would also cause some harm to human health. Radiation damage is due to radiant energy acting on the body caused by the body after a series of changes, as the damage tissues and organs can occur in severe irreversible damage to the body and even lead to death. Domestic and foreign scholars are sute of oxygen free radical theory of radiation damage.Free radicals are dynamic equilibrium from produce and clearance in vivo.With radiation damage, the balance is gradually destroyed and resulting in excess free radicals.Excessive free radicals can induce peroxidation of lipid membrane, leading to cell membrane damage and cell death. Superoxide dismutase(SOD)is an important free radical scavenger in vivo, who can protect cell membranes from oxidation and lipid peroxidation. SOD plays an important role in the body in vivo on oxidation and antioxidant balance, can eliminate free radicals and protect cells from damage.Malondialdehyde(MDA)is a product of radical attack on membrane leading to lipid peroxidation. MDA combine with proteins, nucleic acids (DNA, RNA) and phospholipids and other substances containing amino cross-linking, causing these into compounds---the molecular cross-linked macromolecules.Therefore,the level of MDA can reflect the body's level of lipid peroxidation, and its blood level can indirectly reflect the extent of cell damage. One of the indicators of this study is whether MSCs transplantation change the level of free radicals in rats or not.Kidney, testis, lung, liver and other organs of the body belonging to the high radiation sensitivity of organs which are susceptible to radiation damage, leading to organ dysfunction and then affect metabolism, detoxification and decomposition, etc.,eventually leading to life-endangering.This study simulated clinical radiation therapy radiation model to build animal model for the construction of the radiation pattern and detect the corresponding organ of the function parameters.
Domestic and international study found that Bone marrow mesenchymal stem cells(MSCs) have multi-differentiation potential and can differentiate into bone cells, cartilage cells, nerve cells and so on at the appropriate conditions in vitro;MSCs is tissue engineering, cell and gene therapy an ideal cells.It is not only convenient, easy to cultivate,the proliferation of faster, but also has multi-directional differentiation potential,lower immunogenicity and low expression of MHC I and non-expression of allogeneic MHCⅡ.MSCs can not be rejected after inter-infusion, it will not produce rejection. In this study, based on these characteristics of MSCs, we injecte MSCs in the model to study the function of MSCs whether can anti-oxidation or not of rats through detecting SOD and MDA levels and the pathology of various organizations and at the same time to verify the low immunogenicity of the characteristics of MSCs.
Methods
Part I:mesenchymal stem cells (MSCs) were cultured in vitro and identified
MSCs were isolated from the bone marrow of rats'humerus,femjur, tibia under aseptic condition,and then the cells were collected at a desity of 2×106/cm2 after centrifugation. L-DMEM containing 0.1 volume fraction of fetal bovine serum was added in the culture flask for routine culture. Changing half of the medium after 48h and the whole medium was changed on the fifth day. Cells were not digested until the cells reached confluence over 80% after medium changes.Morphologic changes of MSCs were observed by phase contrast microscope and the cell cycle were detected and analyzed by flow cytometry. Identifying of cells using antigen of CD34,CD44,CD29,CD45.
Part II:The study of MSCs'effects on Rats of irradiation-induced injury
15 male SD rats were randomly divided into three groups, the control group, model group and treatment group.Whole body irradiation of 6.0Gy were performed in model group and treatment group each week, a total of three weeks, the cumulative total dose is 18Gy, and the control group does not accept the irradiation. Rats in treatment group receive infusion of MSCs with 3×106 each after the end of 24h radiation treatment; while in model group and control group rats received infusion of normal saline lml by tail vein. Rats were killed in extremis, Intraperitoneal injection of pentobarbital sodium to anesthetize rats.The application of automatic extraction of serum and microplate reader analyzer serum creatinine, urea,alanine aminotransferase (ALT),aspartic acid aminotransferase (AST) and the level of testosterone(T);The content of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and organisms were measured by hydroxylamine and chromatometry methods;the difference of three groups in organizational structure were observed by conventional paraffin sections.we study the level of functional, biochemical and cellular level of the functions of MSCs on radiation damage treatment.
Statistical methods:The results of experimental data with mean±standard deviation(x±s) that significant for the 0.05 standard, using SPSS 13.0 statistical packages for statistical analysis. Group compared with one-way ANOVA (One-Way ANOVA);repeat measurements analysis of variance with repeated measures (Reperted measurement experiment);inter-group comparison was significant homogeneity using LSD method, with variance arrhythmia Dunnett's T3.The relevance of the use of partial correlation analysis (Partial correlation analysis).
Results
Part I:Mesenchymal stem cells'culture and identification
①The experimental separation of bone marrow-derived mesenchymal stem cells can integrate 80% in about 10 days. After cultured cells are basically in line, we can see spindle cells by microscope, the nuclear circle is located in the central of cell.Cytoplasm is hypertrophy and the membrane is clear;
②Flow cytometry proved G0+G1 phase is about 80% of cell.MSCs in vitro differentiation with only the characteristics of non-proliferation;
③by flow cytometry and Cell Quest software analysis, results showed that MSCs in P3 generation-specific surface markers:CD34-negative, CD45-negative, CD29-positive, the positive rate was 98.9%, CD44-positive, the positive rate was 93.5% on cultured cells in this experiment as mesenchymal stem cells.
Part II:The study of MSCs'effect on the radiation damage Rats
④The mice in control group are good state of mind and freedom of movement; Compare with the control group, the mice in model group are inactive, unresponsive, less active, lethargy and hair turning yellow; while the one with treatment group the general state of mind, hair yellow, with higher sensitive response than the model group.
②Automatic biochemical analyzer:serum levels of creatinine and urea showed: control group, model group and treatment group, serum urea were 4.90±0.31,19.64±0.75,11.88±1.16mmol/ml;Inter-group comparison was using Dunnett's T3 with variance arrhythmia, p<0.05,statistically significant differences;Creatinine contents were 44.20±3.56,85.40±4.62,58.40±3.51μmol/ml;Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences;
③Automatic biochemical analyzer:serum ALT、AST、ALP and LDH levels of content, results showed:control group, model group and treatment group, serum ALT levels were 19.00±6.32,54.20±3.56,36.00±4.74IU/L;Inter-group comparison was significant homogeneity using LSD method, p<0.05, statistically significant differences;Serum AST levels were 48.00±10.93, 187.80±37.09,89.80±13.37IU/L;Compare the control group with model group, F=2.91,p<0.05.Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences.Serum ALP levels were 5.40±3.44,342.60±53.87,163.40±23.87IU/L;Inter-group comparison was using Dunnett's T3 with variance arrhythmia, p<0.05,statistically significant differences;Serum LDH levels were 133.40±34.27,768.20±27.99, 407.60±63.82IU/L;Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences.
④Detection microplate reader detection of serum testosterone levels on control group, model group and treatment group showed that:6.55±0.55,0.76±0.52, 4.28±0.78IU/L.Inter-group comparison was significant homogeneity using LSD method, p<0.05,statistically significant differences;
⑤Using thiobarbituric acid and xanthine oxidase were determined in serum SOD activity and MDA content, results showed:control group, model group and treatment group, serum SOD activity were 110.0959±5.4115,64.1013±3.6994, 84.2043±3.1574 (U/ml); Compare the control group with model group, F=2.91, p<0.05.Compare the model group with treatment group, the control group with model group,statistically significant differences.MDA contents were 7.8059±1.3564,50.9283±3.2640,21.6118±0.9332nmol/ml;Compare the model group with treatment group, the control group with model group,statistically significant differences.
⑥Pathology results:MSCs treatment group compared with the model group, enabling the brain,liver,kidney, testicular structures were obviously improved;
Conclusion
1.The method can effectively enhance rat bone marrow-derived mesenchymal stem cells.
2.This study found that MSCs have the repair effects of radiation damage organs in rats from functional level, morphology and biochemical level.
The innovation of this study:
In this study, we explore mesenchymal stem cells'character of multilineage differentiation in vivo to regulate and repair of injured organs, which fix the problem from a fundamental solution.
The value of this study:
In this study,we focus on the differentiation potential of MSCs to discover MSCs' function on the radiation damage organs, providing strong experimental evidences for treatment of radiation damages and opening another new idea for biological treatment of MSCs.
引文
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[11].李艳菊,李宁,胡亮杉,胡海燕,郭坤元.间充质干细胞的培养方法的改进[J].组织工程与临床康复.2009,13(1)：71-74.
[12].Dehne T, Tschirschmann M, Lauster R, et al Regenerative potential of human adult precursor cells:Cell therapy-an option for treating cartilage defects?.Z Rheumatol.2009; 22:3942-61.
[13].Rho GJ, Kumar BM, Balasubramanian SS.Porcine mesenchymal stem cells--current technological status and future perspective.Front Biosci. 2009;14:3942-61.
[14].Pittenger MF,Mackay AM, Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284:1432147.
[15].Niemeyer P,Kornacker M,Mehlhorn A,et al.Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenicdifferentiation in vitro.Tissue Eng.2007,13(1): 111-21.
[16].Le Blanc K, Ringden O.Immunomodulation by mesenchymal stem cells and clinical experience.J Intern Med.2007,262(5):509-25.
[17].Tse, W.T.,et al.,Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation. Transplantation,2003. 75(3):p.389-97.
[18].杨光,范东艳,陈强等。贴壁法培养不同龄小鼠骨髓间充质干细胞的生物 学特点,中国实验诊断学,2007,1：13-15
[19].Kuo CK,Tuan RS.Tissue engineering with mensenchymal stem cells.IEEE Eng Med Bio Mag,2003,22(5):51
[20].Shin M,Yoshimoto H,Vacanti JP.In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nano fibrous scaffold.Tissue Eng,2004,10(1-2):33。
[21].叶鑫生,许田,汤锡芳等主编.干细胞和发育生物学[M].第二版.北京：军事医学科学出版社,2000.4
[22].Woodbury D,Schwarz EJ,Prockop DJ,et al.Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neurosci Res,2000,61(4):364-370.
[1].Berezovs'kyi Via, Ianko RV.Effect of alimentary deprivation on PHysiological regeneration of the liver parenchyma in young and mature rats. Fiziol Zh.2008; 54(6):66-71.
[2].Bellinger DL, Silva D, Millar AB, et al. Sympathetic nervous system and lymPHocyte proliferation in the Fischer 344 rat spleen:a longitudinal study. Neuroimmunomodulation.2008;15(4-6):260-71.
[3].Littli JB.Radiation induced genetic instability, ntj.1998;74(6):663-667
[4].Ullrich RL,Ponnaiya B.Radiation-induced instability and its relation to radiation carcinogenesis. Int J Radiat Biol.1998;74(6):747-754.
[5].HARMAN D.Aging:a theory based on free radical and radiation chemistry. J Gerontol.1956;11(3):298-300.
[6].方允中,杨胜,伍国耀.自由基、抗氧化剂、营养素与健康的关系[J].营养学报,2003(04)：43-47.
[7].Shiu C T, Lee T M. Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata.[J].J Exp Bot, 2005,56(421):2851-2865.
[8].张成武,曾昭琪,张媛贞等.钝顶螺旋藻多糖和藻蓝蛋白对小鼠急性放射病的防护作用[J].营养学报,1996(03)：123-26.
[9].Kobayashi S,Hoshi M, Inagaki I,et al.The urinary protein to creatinine ratio and urinary sediments are useful for the screening of chronic kidney disease.Rinsho Byori.2009 Mar;57(3):213-20.
[10].Zhu LH, Li C,Wu JA, et al.Bacteriocins from lactic acid bacteria increases tumor necrosis factor-alPHa expression in a rat kidney model of chronic rejection. Transplant Proc.2008;40(10):3746-7.
[11].Al Akhras MA,Darmani H,Elbetieha A,et al.Influence of 50 Hz magnetic field on sex hormones and other fertility parameters of adult male rats [J].Bioelectromagnetics,2006,27(2):127-131.
[12].Brenner C,Kroemer G.Apop t osis:mitochondria the death signal integrators.Science,2000;289(5):1150-1162.
[13].Vergouwen RP, Huiskamp R, Bas RJ, et al.Radiosensitivity of testicular cells in the p repubertal mouse.Radiat Res,1994;139(3):316-321.
[14].Blanco-Rodriguez J, Martinez-Garcia C.Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis:re-evaluation of cell types that exhibit the ability to die during spermatogenesis.Cell Prolif.1996; 29(1):13-31.
[15].Moreno SG, Dutrillaux B,Coffigny H.High sensitivity of rat foetal germ cells to low dose-rate irradiation. Int J Radiat Biol.2001;77(4):529-538.
[16].Gong SL.Guowai Yixue:Fangshe Yixue Heyixue Fence.1999;23(5):227-230.龚守良.生精细胞凋亡与电离辐射[J].国外医学：放射医学核医学分册,1999,23(5)：227-230.
[17].Vergouwen RP, Huiskamp R, Bas RJ, et al.Radiosensitivity of testicular cells in the prepubertal mouse.Radiat Res.1994;139(3):316-326.
[18].Chung NP, Mruk D, Mo MY, et al.A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.Biol Reprod.2001;65(5):1340-1351.
[19].秦绪军,海春旭,何伟梁等.复方中药抗氧化剂对局部-(60)Co照射荷瘤大鼠肝氧化损伤的保护作用[J].世界华人消化杂志,2004(09)：227-32.
[20].汪朝晖,张贤康,缪明永等.牛磺酸对大鼠肝线粒体氧自由基损伤的保护作用[J].安徽医科大学学报,2000(02)：56-60.
[21].Platis O,Anagnostopoulos G, Farmaki K, Posantzis M, Gotsis E, Tolis G. Glucose metabolism disorders improvement in patients with thalassaemia major after 24-36 months of intensive chelation therapy. Pediatr Endocrinol Rev.2004;2 Suppl 2:279-281.
[22].Wanachiwanawin W, Luengrojanakul P, Sirangkapracha P, Leowattana W, Fucharoen S.Prevalence and clinical significance of hepatitis C virus infection in Thai patients with thalassemia. Int J Hematol.2003;78:374-378.
[23].Theal RM,Scott K. Evaluating asymptomatic patients with abnormal liver function test results. Am Fam Physician.1996;53:2111-2119.
[24].Jurczyk K, Wawrzynowicz-Syczewska M, Boron-Kaczmarska A, Sych Z. Serum iron parameters in patients with alcoholic and chronic cirrhosis and hepatitis. Med Sci Monit.2001;7:962-965.
[25].Zhao JD,Jiang GL,Hu WG, et al.Hepatocyte regeneration after partial liver irradiation in rats.Exp Toxicol Pathol.2009;15.):81-902.
[26].丁爱石,王福庄,赵桂玲.缺氧对新生大鼠海马培养细胞的影响.细胞生物学杂志,1993：15：167-169。
[27].Kiray M, Bagriyanik HA, Ergur BU, et al.Antioxidant and antiapoptotic activities of deprenyl and estradiol co-administration in aged rat kidney. Acta Biol Hung.2009;60(1):69-77.
[28].Bellinger DL,Silva D, Millar AB,et al.Sympathetic nervous system and lymPHocyte proliferation in the Fischer 344 rat spleen:a longitudinal study. Neuroimmunomodulation.2008;15(4-6):260-71.
[29].Zhu W.,Chen J.,Cong X.,Hu S., Chen X.,Stem Cells,2006, (24),416-425.
[30].Chen J.,Baydoun A.R., Xu R.,Deng L.,Liu X.,Zhu W.,Shi L.,CongX.,Hu S.,Chen X., Stem Cells,2008,(26),135-145.
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[1].Ladage D,Brixius K,Steingen C,et al.Mesenchymal stem cells induce endothelial activation via paracine mechanisms.Endothelium.2007(2):53-63.
[2].Alexanian AR, Maiman DJ, Kurpad SN, et al.In vitro and in vivo Characterization of Neurally Modified Mesenchymal Stem Cells Induced by Epigenetic Modifiers and Neural Stem Cell Environment. Stem Cells Dev. 2008(17):1231-1252.
[3].Deng X, Chen YX, Zhang X, et al. Hepatic stellate cells modulate the differentiation of bone marrow mesenchymal stem cells into hepatocyte-like cells. J Cell PHysiol.2008(15):56-67.
[4].Zhao D, Wu H, Li F, et al.Electromagnetic field change the expression of osteogenesis genes in murine bone marrow mesenchymal stem cells.J Univ Sci Technolog Med Sci.2008,28(2):152-5.
[5].Dezawa M. Systematic neuronal and muscle induction systems in bone marrow stromal cells:the potential for tissue reconstruction in neurodegenerative and muscle degenerative diseases.Med Mol MorPHol.2008,41(1):14-9.
[6].Mok PL, Cheong SK, Leong CF, et al.In vitro expression of erythropoietin by transfected human mesenchymal stromal cells.Cytotherapy.2008;0(2):116-24.
[7].Gao F, He T, Wang H, et al.A promising strategy for the treatment of ischemic heart disease:Mesenchymal stem cell-mediated vascular endothelial growth factor gene transfer in rats.Can J Cardiol.2007,23(11):891-8.
[8].Colletti EJ, Airey JA, Liu W, et al.Generation of tissue-specific cells from MSC does not require fusion or donor-to-host mitochondrial/membrane transfer.Stem Cell Res.2009;2(2):125-38.
[9].Uchibori R, Okada T, Ito T, et al.Retroviral vector-producing mesenchymal stem cells for targeted suicide cancer gene therapy. J Gene Med.2009;11(5):373-381.
[10].Pan D.In situ (in vivo) gene transfer into murine bone marrow stem cells.Methods Mol Biol.2009;506:159-69.
[11].李艳菊,李宁,胡亮杉,胡海燕,郭坤元.间充质干细胞的培养方法的改进[J].组织工程与临床康复.2009,13(1)：71-74.
[12].Dehne T, Tschirschmann M, Lauster R, et al Regenerative potential of human adult precursor cells:Cell therapy-an option for treating cartilage defects?.Z Rheumatol.2009; 22:3942-61.
[13].Rho GJ, Kumar BM, Balasubramanian SS.Porcine mesenchymal stem cells--current technological status and future perspective.Front Biosci. 2009;14:3942-61.
[14].Pittenger MF,Mackay AM, Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284:1432147.
[15].Niemeyer P,Kornacker M,Mehlhorn A,et al.Comparison of immunological properties of bone marrow stromal cells and adipose tissue-derived stem cells before and after osteogenicdifferentiation in vitro.Tissue Eng.2007,13(1): 111-21.
[16].Le Blanc K, Ringden O.Immunomodulation by mesenchymal stem cells and clinical experience.J Intern Med.2007,262(5):509-25.
[17].Tse, W.T.,et al.,Suppression of allogeneic T-cell proliferation by human marrow stromal cells:implications in transplantation. Transplantation,2003. 75(3):p.389-97.
[18].杨光,范东艳,陈强等。贴壁法培养不同龄小鼠骨髓间充质干细胞的生物 学特点,中国实验诊断学,2007,1：13-15
[19].Kuo CK,Tuan RS.Tissue engineering with mensenchymal stem cells.IEEE Eng Med Bio Mag,2003,22(5):51
[20].Shin M,Yoshimoto H,Vacanti JP.In vivo bone tissue engineering using mesenchymal stem cells on a novel electrospun nano fibrous scaffold.Tissue Eng,2004,10(1-2):33。
[21].叶鑫生,许田,汤锡芳等主编.干细胞和发育生物学[M].第二版.北京：军事医学科学出版社,2000.4
[22].Woodbury D,Schwarz EJ,Prockop DJ,et al.Adult rat and human bone marrow stromal cells differentiate into neurons[J]. J Neurosci Res,2000,61(4):364-370.
[1].Berezovs'kyi Via, Ianko RV.Effect of alimentary deprivation on PHysiological regeneration of the liver parenchyma in young and mature rats. Fiziol Zh.2008; 54(6):66-71.
[2].Bellinger DL, Silva D, Millar AB, et al. Sympathetic nervous system and lymPHocyte proliferation in the Fischer 344 rat spleen:a longitudinal study. Neuroimmunomodulation.2008;15(4-6):260-71.
[3].Littli JB.Radiation induced genetic instability, ntj.1998;74(6):663-667
[4].Ullrich RL,Ponnaiya B.Radiation-induced instability and its relation to radiation carcinogenesis. Int J Radiat Biol.1998;74(6):747-754.
[5].HARMAN D.Aging:a theory based on free radical and radiation chemistry. J Gerontol.1956;11(3):298-300.
[6].方允中,杨胜,伍国耀.自由基、抗氧化剂、营养素与健康的关系[J].营养学报,2003(04)：43-47.
[7].Shiu C T, Lee T M. Ultraviolet-B-induced oxidative stress and responses of the ascorbate-glutathione cycle in a marine macroalga Ulva fasciata.[J].J Exp Bot, 2005,56(421):2851-2865.
[8].张成武,曾昭琪,张媛贞等.钝顶螺旋藻多糖和藻蓝蛋白对小鼠急性放射病的防护作用[J].营养学报,1996(03)：123-26.
[9].Kobayashi S,Hoshi M, Inagaki I,et al.The urinary protein to creatinine ratio and urinary sediments are useful for the screening of chronic kidney disease.Rinsho Byori.2009 Mar;57(3):213-20.
[10].Zhu LH, Li C,Wu JA, et al.Bacteriocins from lactic acid bacteria increases tumor necrosis factor-alPHa expression in a rat kidney model of chronic rejection. Transplant Proc.2008;40(10):3746-7.
[11].Al Akhras MA,Darmani H,Elbetieha A,et al.Influence of 50 Hz magnetic field on sex hormones and other fertility parameters of adult male rats [J].Bioelectromagnetics,2006,27(2):127-131.
[12].Brenner C,Kroemer G.Apop t osis:mitochondria the death signal integrators.Science,2000;289(5):1150-1162.
[13].Vergouwen RP, Huiskamp R, Bas RJ, et al.Radiosensitivity of testicular cells in the p repubertal mouse.Radiat Res,1994;139(3):316-321.
[14].Blanco-Rodriguez J, Martinez-Garcia C.Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis:re-evaluation of cell types that exhibit the ability to die during spermatogenesis.Cell Prolif.1996; 29(1):13-31.
[15].Moreno SG, Dutrillaux B,Coffigny H.High sensitivity of rat foetal germ cells to low dose-rate irradiation. Int J Radiat Biol.2001;77(4):529-538.
[16].Gong SL.Guowai Yixue:Fangshe Yixue Heyixue Fence.1999;23(5):227-230.龚守良.生精细胞凋亡与电离辐射[J].国外医学：放射医学核医学分册,1999,23(5)：227-230.
[17].Vergouwen RP, Huiskamp R, Bas RJ, et al.Radiosensitivity of testicular cells in the prepubertal mouse.Radiat Res.1994;139(3):316-326.
[18].Chung NP, Mruk D, Mo MY, et al.A 22-amino acid synthetic peptide corresponding to the second extracellular loop of rat occludin perturbs the blood-testis barrier and disrupts spermatogenesis reversibly in vivo.Biol Reprod.2001;65(5):1340-1351.
[19].秦绪军,海春旭,何伟梁等.复方中药抗氧化剂对局部-(60)Co照射荷瘤大鼠肝氧化损伤的保护作用[J].世界华人消化杂志,2004(09)：227-32.
[20].汪朝晖,张贤康,缪明永等.牛磺酸对大鼠肝线粒体氧自由基损伤的保护作用[J].安徽医科大学学报,2000(02)：56-60.
[21].Platis O,Anagnostopoulos G, Farmaki K, Posantzis M, Gotsis E, Tolis G. Glucose metabolism disorders improvement in patients with thalassaemia major after 24-36 months of intensive chelation therapy. Pediatr Endocrinol Rev.2004;2 Suppl 2:279-281.
[22].Wanachiwanawin W, Luengrojanakul P, Sirangkapracha P, Leowattana W, Fucharoen S.Prevalence and clinical significance of hepatitis C virus infection in Thai patients with thalassemia. Int J Hematol.2003;78:374-378.
[23].Theal RM,Scott K. Evaluating asymptomatic patients with abnormal liver function test results. Am Fam Physician.1996;53:2111-2119.
[24].Jurczyk K, Wawrzynowicz-Syczewska M, Boron-Kaczmarska A, Sych Z. Serum iron parameters in patients with alcoholic and chronic cirrhosis and hepatitis. Med Sci Monit.2001;7:962-965.
[25].Zhao JD,Jiang GL,Hu WG, et al.Hepatocyte regeneration after partial liver irradiation in rats.Exp Toxicol Pathol.2009;15.):81-902.
[26].丁爱石,王福庄,赵桂玲.缺氧对新生大鼠海马培养细胞的影响.细胞生物学杂志,1993：15：167-169。
[27].Kiray M, Bagriyanik HA, Ergur BU, et al.Antioxidant and antiapoptotic activities of deprenyl and estradiol co-administration in aged rat kidney. Acta Biol Hung.2009;60(1):69-77.
[28].Bellinger DL,Silva D, Millar AB,et al.Sympathetic nervous system and lymPHocyte proliferation in the Fischer 344 rat spleen:a longitudinal study. Neuroimmunomodulation.2008;15(4-6):260-71.
[29].Zhu W.,Chen J.,Cong X.,Hu S., Chen X.,Stem Cells,2006, (24),416-425.
[30].Chen J.,Baydoun A.R., Xu R.,Deng L.,Liu X.,Zhu W.,Shi L.,CongX.,Hu S.,Chen X., Stem Cells,2008,(26),135-145.