小鼠脐带间充质干细胞的分离、鉴定及其抗炎治疗mdx小鼠的研究

发布时间：2018-07-09 21:32

本文选题：杜氏肌营养不良症 + 脐带间充质干细胞　；参考：《昆明医科大学》2016年硕士论文

【摘要】：[目的]分离并鉴定小鼠脐带间充质干细胞(mouse umbilical cord mesenchymal stem cells, mUCMSCs)移植治疗X染色体-连锁肌萎缩(X chromosome-linked muscular dystrophy deficient, mdx)小鼠,观察脐带间充质干细胞对杜氏肌营养不良症(Duchenne muscular dystrophy, DMD)的抗炎作用效果,为临床上干细胞治疗DMD的应用提供理论依据和技术方法。[方法]1. mUCMSCs的分离、鉴定：无菌条件下取妊娠16-17天健康C57BL/6(以下简称C57)孕鼠脐带,组织块贴壁法分离mUCMSCs,采用形态学观察,免疫表型分析,体外诱导分化等方式对其进行鉴定。2.mdx小鼠的鉴定：采集杂合子子代鼠外周血50-100u1,提取DNA后运用序列特异性引物聚合酶链式反应(sequence specific primers polymerase chain reaction, PCR-SSP)方法进行基因型鉴定,获得mdx小鼠用于治疗实验。3. mUCMSCs的标记及其在体内的分布：将含绿色荧光蛋白(green fluorescence protein,GFP)基因的慢病毒以一定的滴度与mUCMSCs共培养,将GFP标记的mUCMSCs腹腔移植入mdx小鼠体内,小动物活体成像仪下观察细胞分布变化。4. mUCMSCs治疗实验：将mdx小鼠随机分为3组,分别每次腹腔注射0.4mlmUCMSCs生理盐水悬液(细胞数量分别为5×104、5×105、5×106)治疗mdx小鼠,每周1次,连续4次。同时设置阴性对照和正常对照。5.抗炎治疗评估：(1)观察对比3个移植治疗组治疗前后mdx小鼠的行为学变化；(2)连续接受4次mUCMSCs治疗后1周,每只mdx小鼠尾尖采血法采集外周血100u1,分离血清,采用自动生化分析仪测定肌酸激酶(creatine kinase,CK)的含量,并比较分析各组CK值的变化；(3)连续接受4次mUCMSCs治疗后1周,每只mdx小鼠尾尖采血法采集外周血250-300ul,分离血清,采用QAM-INF-1蛋白芯片测定小鼠外周血炎症因子含量；(4) mUCMSCs治疗后1周,采集小鼠腓肠肌组织约10ug,裂解后均质机打碎,4℃,14000rpm离心10-15min,取上清液100ul,采用QAM-INF-1蛋白芯片测定小鼠肌肉组织炎症因子含量；(5)连续接受4次mUCMSCs治疗后1周,取小鼠腓肠肌组织用4%多聚甲醛固定,制作石蜡切片,观察肌肉组织病理变化；(6)实验结束后,解剖并观察实验用mdx小鼠腹腔内各器官形态结构的变化。6.统计学方法：实验数据采用均值±标准差表示,用SPSS17.0统计软件进行分析,采用方差分析、t检验、相关分析等方法完成。P0.05表示有显著差异,P0.01表示有极显著差异。[结果]1.采用组织块贴壁培养法,使用含有10%胎牛血清的DMEM/F12培养液静置培养小鼠脐带组织24小时左右,可在倒置相差显微镜下观察到少数梭状贴壁细胞由组织块边缘或散在长出,第2-3天可见细胞集落形成,传代后细胞可快速生长,呈旋涡状密集排布。取第3代mUCMSCs流式细胞术检测,结果为高表达CD29、 CD90、CD105,低表达CD34；生长曲线呈典型“S”型;在体外可成脂、成骨和成软骨分化,由此判断所获细胞为均一性和活性良好的mUCMSCs。2.采用PCR-SSP方法可以从73只杂合子子代鼠中鉴定出23只mdx小鼠用于本实验研究。3.将GFP标记的mUCMSCs移植入mdx小鼠腹腔,小动物活体成像仪下观察,移植后3h荧光面积由最初的35.2mm2变为33.1mm2,24小时后荧光面积明显缩小,强度变弱,7天后仍能观察到绿色荧光在小鼠腹腔的散在分布。4. mUCMSCs抗炎治疗mdx小鼠情况评估：(1)临床症状观察：3个移植治疗组mdx小鼠接受腹腔注射后,均未观察到明显的精神萎靡、活动减少、毛发散乱、消瘦等症状。连续治疗4次后,运动功能改善,灵活度更高。(2)移植治疗4次后1周时,一次分别腹腔注射5×104、5×105、5×106mUCMSCs的3个治疗组血清CK值依次为434.60±19.39U/L,277.00±20.65 U/L,287.14±42.67 U/L,与未治疗组为1374.60±127.13 U/L相比,具有统计学意义(P0.05)。(3)蛋白芯片技术检测分析结果显示：连续接受4次mUCMSCs治疗后1周,40个外周血候选炎症因子中有14个因子的含量在治疗前后有明显变化,其中Etaxin、集落刺激因子(Granulocyte colony-stimulating factor, G-CSF)、粒-单核细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF)、干扰素-γ (interferon-y, INF-y)、IL-2、IL-4、IL-5、IL-6、IL-17、单核细胞趋化蛋白(monocyte chemoattractant protein, MCP-1γ、血小板因子4 (Platelet Factor 4, PF4)、肿瘤坏死因子受体-Ⅰ(Tumor necrosis factor receptor-I, TNFR-I)治疗后降低,而细胞间黏附分子-1 (intercellular adhesion molecule-1, ICAM-1)治疗后升高；连续接受4次mUCMSC治疗1周后,mdx小鼠IFN-γ、IL-5、IL-6、TNFR-1含量随细胞剂量增加而降低；连续接受4次mUCMSCs治疗后1周,mdx小鼠IFN-γ、IL-5、IL-6、TNFR-1含量明显低于未治疗的对照组mdx小鼠；连续接受4次mUCMSCs治疗后1周,可检测到mdx小鼠后肢肌肉组织CD30L、IL-21、 MIP-1a、PF4含量明显低于未治疗的对照组mdx小鼠。(4)mdx小鼠腓肠肌行HE染色后显微镜下观察,可见mdx小鼠肌肉组织有炎症细胞浸润；肌纤维大小不等,部分裂开,轮廓变圆,部分呈均质性改变；肌细胞核增多、变大、中心移位；腓肠肌肌纤维间隙稍增宽,少量脂肪、纤维结缔组织增生。连续接受4.次mUCMSCs治疗后1周的三组mdx小鼠病理学变化有不同程度的改善,表现为炎症细胞浸润减轻,肌细胞大小趋于一致,肌细胞间炎性细胞浸润减轻,细胞大小趋于统一。三个治疗组核中心移位纤维(Centrally nucleated fiber, CNF)比例分别为80.37%±7.65、78.17%±6.65、73.81±6.05与未治疗组为94.37±11.65相比,CNF较少有统计学意义(P0.05)。5.细胞移植生物安全性：移植后1周脱颈处死并解剖各组小鼠,3个细胞移植治疗组mdx小鼠腹腔及腹壁下及内脏均未见异常组织,初步判断mUCMSCs治疗mdx是安全的。[结论]1.采用组织块贴壁培养法,使用含有10%血清的DMEM/F12培养液可以从C57小鼠脐带分离到呈旋涡状贴壁生长,高表达CD29、CD90、CD105,并可诱导向成脂、成骨和成软骨分化的脐带间充质干细胞(mUCMSCs)。2.注射到mdx小鼠腹腔的C57小鼠UCMSCs,可在小鼠腹腔存在至少7天以上。3.按照每周一次腹腔注射5×104-5x106个mUCMSC、连续注射4次的剂量治疗mdx小鼠,可以降低mdx小鼠外周血的CK含量,影响IFN-γ、IL-5、IL-6、 TNFR-1等部分炎症相关因子的表达,缓解后肢肌肉组织的病理损伤,腹腔注射mUCMSCs治疗mdx小鼠的作用机制可能与减轻上述炎症因子介导的炎症反应有关。
[Abstract]:[Objective] to isolate and identify mouse umbilical cord mesenchymal stem cells (mouse umbilical cord mesenchymal stem cells, mUCMSCs) transplantation in the treatment of X chromosome linkage muscle atrophy (X chromosome-linked muscular dystrophy) mice, and observe the effect of umbilical cord mesenchymal stem cells on Duchenne muscular dystrophy. The effect of anti-inflammatory action provides a theoretical basis and technical method for the application of clinical stem cells in the treatment of DMD. [method]1. mUCMSCs separation, identification: under aseptic conditions, the umbilical cord of pregnant rats was taken at 16-17 days of pregnancy C57BL/6 (hereinafter referred to as C57), tissue block adherence method was used to separate mUCMSCs, morphological observation, immunophenotype analysis and induced differentiation in vitro. The identification of.2.mdx mice was carried out in the same way: the peripheral blood 50-100u1 of heterozygote offspring was collected, and DNA was extracted by the method of sequence specific primer polymerase chain reaction (sequence specific primers polymerase chain reaction, PCR-SSP) to identify the genotype, and to obtain the markers for the mdx mice used for the treatment of experimental.3.. Its distribution in the body: the lentivirus containing green fluorescence protein (GFP) gene was co cultured with mUCMSCs, and the GFP labelled mUCMSCs was transplanted into mdx mice. The.4. mUCMSCs treatment experiment was observed under the living imaging instrument of small animals. The mdx mice were randomly divided into 3 groups. Do not intraperitoneal injection of 0.4mlmUCMSCs saline suspension (the number of cells were 5 x 104,5 x 105,5 x 106 respectively) to treat mdx mice 1 times a week, 4 times a week. Meanwhile, negative control and normal control.5. were set up for anti-inflammatory treatment. (1) the behavior changes of mdx mice were observed and compared before and after treatment in 3 transplantation groups; (2) 4 consecutive mUCMSCs were accepted. 1 weeks after treatment, each mdx mouse tail tip blood sampling method was used to collect peripheral blood 100u1, separate serum, determine the content of creatine kinase (creatine kinase, CK) by automatic biochemical analyzer, and compare the changes of CK value in each group. (3) 1 weeks after 4 consecutive mUCMSCs treatments, each mdx mouse tail tip blood sampling method is used to collect peripheral blood 250-300ul and separate the serum, QAM-INF-1 protein chip was used to determine the content of peripheral blood inflammatory factors in mice; (4) 1 weeks after mUCMSCs treatment, the gastrocnemius muscle tissue of mice was collected about 10ug, the homogenate was broken after cracking, 4, 14000rpm centrifuged 10-15min, 100ul of the supernatant, and QAM-INF-1 protein chip was used to determine the content of inflammatory factors in the muscle tissue of mice; (5) 4 consecutive mUCMSCs was accepted. 1 weeks after the treatment, the gastrocnemius tissue was fixed with 4% polyformaldehyde, and the paraffin section was made to observe the pathological changes of the muscle tissue. (6) after the experiment, the changes of the morphological structure of various organs in the abdominal cavity of mdx mice were observed and observed by.6. statistical methods: the mean standard deviation of the experimental data was expressed with the mean standard deviation of the experimental data, and the SPSS17.0 statistical software was used. Analysis, the use of variance analysis, t test, correlation analysis and other methods to complete.P0.05 showed significant differences, P0.01 showed very significant differences. [results]1. using tissue block wall culture method, using DMEM/F12 culture medium containing 10% fetal bovine serum for 24 hours in mouse umbilical cord tissue, can be observed under the inverted phase contrast microscope for a few. The spindle adherent cells were formed on the edge of the tissue block or spread out, and the cell colony formed on the 2-3 day. After the passage, the cells could grow rapidly and have a vortex dense arrangement. The third generation mUCMSCs flow cytometry was used to detect high expression of CD29, CD90, CD105, low expression CD34; the growth curve was a typical "S" type; in vitro, it could be made into fat, osteogenesis and softer. Bone differentiation, thus judging the homogeneity and good activity of the cells obtained by mUCMSCs.2., 23 mdx mice can be identified from 73 heterozygote offspring by PCR-SSP method. The GFP labeled mUCMSCs was transplanted into the abdominal cavity of the mdx mice by.3., and the small animal living imaging apparatus was observed. The fluorescence area of 3h after transplantation was from the original 35.2mm2. After 33.1mm2,24 hours, the fluorescence area was obviously reduced and the intensity was weaker. 7 days later, the distribution of green fluorescence in mice abdominal cavity was still observed in the distribution of.4. mUCMSCs in the anti inflammatory treatment of mdx mice. (1) the clinical symptoms were observed: after receiving intraperitoneal injection of mdx mice in 3 transplantation groups, no obvious mental malaise and decreased activity were observed. After 4 times of continuous treatment, the exercise function improved and the flexibility was higher. (2) at 1 weeks after 4 times of transplantation, the serum CK values of the 3 treatment groups, respectively, were 434.60 + 19.39U/L, 277 + 20.65 U/L and 287.14 + U/L, respectively, and compared with those in the untreated group of 1374.60 + 127.13 U/L, respectively, at 4 times after 4 times of transplantation. Statistical significance (P0.05). (3) protein chip technology detection analysis showed that 1 weeks after 4 consecutive mUCMSCs treatment, 14 factors in 40 peripheral blood candidate inflammatory factors were significantly changed before and after treatment, including Etaxin, colony stimulating factor (Granulocyte colony-stimulating factor, G-CSF), and granulocyte mononuclear cells Granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon - gamma (interferon-y, INF-y), IL-2, IL-4, IL-5, IL-6, IL-17, monocyte chemoattractant protein (monocyte), platelet factor 4, tumor necrosis factor receptor - I Eceptor-I, TNFR-I) decreased after treatment, and the intercellular adhesion molecule -1 (intercellular adhesion molecule-1, ICAM-1) increased after treatment. After 1 weeks of 4 consecutive mUCMSC treatment, mdx mice IFN- gamma, IL-5, IL-6, decreased with the increase of cell dose; 1 weeks after 4 consecutive treatments. The content of mdx mice in the control group was significantly lower than that of the untreated control group. 1 weeks after 4 consecutive mUCMSCs treatment, the CD30L, IL-21, MIP-1a, PF4 content of the hind limbs of mdx mice was significantly lower than that of the untreated control group mdx mice. (4) the gastrocnemius muscle of the mdx mice was observed under microscope after HE, and the inflammatory cells in the mdx mouse muscle tissue were soaked. Muscle fibers were different in size, partial disintegration, contour round, and partial homogeneity change; muscle nuclei increased, increased, central displacement, the gastrocnemius muscle fiber gap slightly widened, a small amount of fat, and fibrous connective tissue proliferated. The pathological changes in three groups of mdx mice after 4. consecutive weeks of mUCMSCs treatment were improved in varying degrees, manifested in different degrees. The infiltration of inflammatory cells decreased, the size of muscle cells tended to be consistent, the infiltration of inflammatory cells in myocytes was reduced and the size of cells tended to be unified. The proportion of Centrally nucleated fiber (CNF) in the three treatment groups was 80.37% + 7.65,78.17% + 6.05, respectively, compared with 94.37 + 11.65 in the untreated group, and there was less statistical significance in CNF. (P0.05).5. cell transplantation biological safety: 1 weeks after transplantation and dissecting the neck and dissecting each group of mice, 3 cell transplantation group mdx mice abdominal and abdominal wall and viscera no abnormal tissue, preliminary judgment mUCMSCs treatment mdx is safe. [conclusion]1. using tissue block adherence culture, the use of 10% serum DMEM/F12 culture solution can be used. C57 mouse umbilical cord was isolated from the umbilical cord to the wall of vortexlike growth, high expression of CD29, CD90, CD105, and induced into fat, osteogenic and chondrogenic umbilical cord mesenchymal stem cells (mUCMSCs).2. injected into the C57 mice of the mdx mouse abdominal cavity, and at least 7 days in the mouse abdominal cavity, 5 * 104-5x106 mUCMSC was injected into the abdominal cavity once a week. The dose treatment of mdx mice for 4 consecutive doses can reduce the CK content in peripheral blood of mdx mice, affect the expression of some inflammatory related factors such as IFN- gamma, IL-5, IL-6, TNFR-1, and alleviate the pathological damage of the muscle tissue of the hind limbs. The action of intraperitoneal injection of mUCMSCs in the treatment of mdx mice may be associated with the reduction of the inflammatory reaction mediated by the above-mentioned inflammatory factors. Close.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R746.3

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