本文選題：杜氏肌營養(yǎng)不良癥 + 臍帶間充質(zhì)干細胞�。� 參考：《昆明醫(yī)科大學(xué)》2016年碩士論文

【摘要】：[目的]分離并鑒定小鼠臍帶間充質(zhì)干細胞(mouse umbilical cord mesenchymal stem cells, mUCMSCs)移植治療X染色體-連鎖肌萎縮(X chromosome-linked muscular dystrophy deficient, mdx)小鼠,觀察臍帶間充質(zhì)干細胞對杜氏肌營養(yǎng)不良癥(Duchenne muscular dystrophy, DMD)的抗炎作用效果,為臨床上干細胞治療DMD的應(yīng)用提供理論依據(jù)和技術(shù)方法。[方法]1. mUCMSCs的分離、鑒定：無菌條件下取妊娠16-17天健康C57BL/6(以下簡稱C57)孕鼠臍帶,組織塊貼壁法分離mUCMSCs,采用形態(tài)學(xué)觀察,免疫表型分析,體外誘導(dǎo)分化等方式對其進行鑒定。2.mdx小鼠的鑒定：采集雜合子子代鼠外周血50-100u1,提取DNA后運用序列特異性引物聚合酶鏈式反應(yīng)(sequence specific primers polymerase chain reaction, PCR-SSP)方法進行基因型鑒定,獲得mdx小鼠用于治療實驗。3. mUCMSCs的標(biāo)記及其在體內(nèi)的分布：將含綠色熒光蛋白(green fluorescence protein,GFP)基因的慢病毒以一定的滴度與mUCMSCs共培養(yǎng),將GFP標(biāo)記的mUCMSCs腹腔移植入mdx小鼠體內(nèi),小動物活體成像儀下觀察細胞分布變化。4. mUCMSCs治療實驗：將mdx小鼠隨機分為3組,分別每次腹腔注射0.4mlmUCMSCs生理鹽水懸液(細胞數(shù)量分別為5×104、5×105、5×106)治療mdx小鼠,每周1次,連續(xù)4次。同時設(shè)置陰性對照和正常對照。5.抗炎治療評估：(1)觀察對比3個移植治療組治療前后mdx小鼠的行為學(xué)變化；(2)連續(xù)接受4次mUCMSCs治療后1周,每只mdx小鼠尾尖采血法采集外周血100u1,分離血清,采用自動生化分析儀測定肌酸激酶(creatine kinase,CK)的含量,并比較分析各組CK值的變化；(3)連續(xù)接受4次mUCMSCs治療后1周,每只mdx小鼠尾尖采血法采集外周血250-300ul,分離血清,采用QAM-INF-1蛋白芯片測定小鼠外周血炎癥因子含量；(4) mUCMSCs治療后1周,采集小鼠腓腸肌組織約10ug,裂解后均質(zhì)機打碎,4℃,14000rpm離心10-15min,取上清液100ul,采用QAM-INF-1蛋白芯片測定小鼠肌肉組織炎癥因子含量；(5)連續(xù)接受4次mUCMSCs治療后1周,取小鼠腓腸肌組織用4%多聚甲醛固定,制作石蠟切片,觀察肌肉組織病理變化；(6)實驗結(jié)束后,解剖并觀察實驗用mdx小鼠腹腔內(nèi)各器官形態(tài)結(jié)構(gòu)的變化。6.統(tǒng)計學(xué)方法：實驗數(shù)據(jù)采用均值±標(biāo)準差表示,用SPSS17.0統(tǒng)計軟件進行分析,采用方差分析、t檢驗、相關(guān)分析等方法完成。P0.05表示有顯著差異,P0.01表示有極顯著差異。[結(jié)果]1.采用組織塊貼壁培養(yǎng)法,使用含有10%胎牛血清的DMEM/F12培養(yǎng)液靜置培養(yǎng)小鼠臍帶組織24小時左右,可在倒置相差顯微鏡下觀察到少數(shù)梭狀貼壁細胞由組織塊邊緣或散在長出,第2-3天可見細胞集落形成,傳代后細胞可快速生長,呈旋渦狀密集排布。取第3代mUCMSCs流式細胞術(shù)檢測,結(jié)果為高表達CD29、 CD90、CD105,低表達CD34；生長曲線呈典型“S”型;在體外可成脂、成骨和成軟骨分化,由此判斷所獲細胞為均一性和活性良好的mUCMSCs。2.采用PCR-SSP方法可以從73只雜合子子代鼠中鑒定出23只mdx小鼠用于本實驗研究。3.將GFP標(biāo)記的mUCMSCs移植入mdx小鼠腹腔,小動物活體成像儀下觀察,移植后3h熒光面積由最初的35.2mm2變?yōu)?3.1mm2,24小時后熒光面積明顯縮小,強度變?nèi)?7天后仍能觀察到綠色熒光在小鼠腹腔的散在分布。4. mUCMSCs抗炎治療mdx小鼠情況評估：(1)臨床癥狀觀察：3個移植治療組mdx小鼠接受腹腔注射后,均未觀察到明顯的精神萎靡、活動減少、毛發(fā)散亂、消瘦等癥狀。連續(xù)治療4次后,運動功能改善,靈活度更高。(2)移植治療4次后1周時,一次分別腹腔注射5×104、5×105、5×106mUCMSCs的3個治療組血清CK值依次為434.60±19.39U/L,277.00±20.65 U/L,287.14±42.67 U/L,與未治療組為1374.60±127.13 U/L相比,具有統(tǒng)計學(xué)意義(P0.05)。(3)蛋白芯片技術(shù)檢測分析結(jié)果顯示：連續(xù)接受4次mUCMSCs治療后1周,40個外周血候選炎癥因子中有14個因子的含量在治療前后有明顯變化,其中Etaxin、集落刺激因子(Granulocyte colony-stimulating factor, G-CSF)、粒-單核細胞集落刺激因子(granulocyte-macrophage colony-stimulating factor, GM-CSF)、干擾素-γ (interferon-y, INF-y)、IL-2、IL-4、IL-5、IL-6、IL-17、單核細胞趨化蛋白(monocyte chemoattractant protein, MCP-1γ、血小板因子4 (Platelet Factor 4, PF4)、腫瘤壞死因子受體-Ⅰ(Tumor necrosis factor receptor-I, TNFR-I)治療后降低,而細胞間黏附分子-1 (intercellular adhesion molecule-1, ICAM-1)治療后升高；連續(xù)接受4次mUCMSC治療1周后,mdx小鼠IFN-γ、IL-5、IL-6、TNFR-1含量隨細胞劑量增加而降低；連續(xù)接受4次mUCMSCs治療后1周,mdx小鼠IFN-γ、IL-5、IL-6、TNFR-1含量明顯低于未治療的對照組mdx小鼠；連續(xù)接受4次mUCMSCs治療后1周,可檢測到mdx小鼠后肢肌肉組織CD30L、IL-21、 MIP-1a、PF4含量明顯低于未治療的對照組mdx小鼠。(4)mdx小鼠腓腸肌行HE染色后顯微鏡下觀察,可見mdx小鼠肌肉組織有炎癥細胞浸潤；肌纖維大小不等,部分裂開,輪廓變圓,部分呈均質(zhì)性改變；肌細胞核增多、變大、中心移位；腓腸肌肌纖維間隙稍增寬,少量脂肪、纖維結(jié)締組織增生。連續(xù)接受4.次mUCMSCs治療后1周的三組mdx小鼠病理學(xué)變化有不同程度的改善,表現(xiàn)為炎癥細胞浸潤減輕,肌細胞大小趨于一致,肌細胞間炎性細胞浸潤減輕,細胞大小趨于統(tǒng)一。三個治療組核中心移位纖維(Centrally nucleated fiber, CNF)比例分別為80.37%±7.65、78.17%±6.65、73.81±6.05與未治療組為94.37±11.65相比,CNF較少有統(tǒng)計學(xué)意義(P0.05)。5.細胞移植生物安全性：移植后1周脫頸處死并解剖各組小鼠,3個細胞移植治療組mdx小鼠腹腔及腹壁下及內(nèi)臟均未見異常組織,初步判斷mUCMSCs治療mdx是安全的。[結(jié)論]1.采用組織塊貼壁培養(yǎng)法,使用含有10%血清的DMEM/F12培養(yǎng)液可以從C57小鼠臍帶分離到呈旋渦狀貼壁生長,高表達CD29、CD90、CD105,并可誘導(dǎo)向成脂、成骨和成軟骨分化的臍帶間充質(zhì)干細胞(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個mUCMSC、連續(xù)注射4次的劑量治療mdx小鼠,可以降低mdx小鼠外周血的CK含量,影響IFN-γ、IL-5、IL-6、 TNFR-1等部分炎癥相關(guān)因子的表達,緩解后肢肌肉組織的病理損傷,腹腔注射mUCMSCs治療mdx小鼠的作用機制可能與減輕上述炎癥因子介導(dǎo)的炎癥反應(yīng)有關(guān)。
[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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R746.3

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