發(fā)布時(shí)間：2018-09-03 18:27

【摘要】：[目的] 驗(yàn)證大鼠骨髓干細(xì)胞(MSCs)體外培養(yǎng)的方法及其生物學(xué)特性，研究MSCs的多項(xiàng)分化潛能以及其在體外定向誘導(dǎo)分化為骨髂肌細(xì)胞，設(shè)計(jì)一組動(dòng)物模型，分別將誘導(dǎo)后、未誘導(dǎo)的MSCs、陽(yáng)性對(duì)照細(xì)胞注射到動(dòng)物模型體內(nèi)，研究MSCs在體內(nèi)的誘導(dǎo)分化情況及其最終的形態(tài)變化；從而，找到肌肉缺損最佳的修復(fù)方法，為臨床治療開拓廣闊的前景。 [方法] 首先培養(yǎng)大鼠骨髓干細(xì)胞(MSCs)、大鼠骨骼肌細(xì)胞(Mb，，主要用于陽(yáng)性對(duì)照)，將體外培養(yǎng)的MSCs第三代用5—氮雜胞苷誘聯(lián)合MyoD、TGF-β1、IGF-1導(dǎo)分化，用免疫組織化學(xué)方法鑒定誘導(dǎo)后的細(xì)胞。制做動(dòng)物模型：取16只10周齡的Wistar大鼠，在其左后小腿脛前肌中部注射無(wú)水乙醇0.2ml，使其脛前肌中部變性壞死。右后小腿脛前肌中部注射無(wú)菌生理鹽水0.2ml。將16只大鼠模型隨機(jī)分成四組，第一組陰性對(duì)照，只注射培養(yǎng)基；第二組實(shí)驗(yàn)組，注射MSCs；第三組實(shí)驗(yàn)組，注射誘導(dǎo)后的MSCs；第四組陽(yáng)性對(duì)照組，注射Mb。分別將第三代細(xì)胞、誘導(dǎo)后的細(xì)胞用BrdU標(biāo)記，然后收集等待注射。動(dòng)物模型建立4h后，就可以注射。注射部位雙小腿脛前肌中部。在注射細(xì)胞后的第3d、6d、9d和12d分別取材進(jìn)行形態(tài)觀察和免疫學(xué)檢查； [結(jié)果] 大鼠MSCs體外培養(yǎng)生長(zhǎng)良好，形態(tài)不規(guī)則，流式細(xì)胞儀檢測(cè)絕大部分細(xì)胞處于G1期(79.4％)，免疫組化檢測(cè)CD44陽(yáng)性，而CD34陰性。大鼠Mb體外培養(yǎng)生長(zhǎng)良好，形態(tài)規(guī)則，流式細(xì)胞儀檢測(cè)絕大部分細(xì)胞處于G1期(79.8％)，免疫組化檢測(cè)Desmine，Myosin均陽(yáng)性。經(jīng)誘導(dǎo)后MSCs，形態(tài)多
[Abstract]:[objective] to verify the method and biological characteristics of rat bone marrow stem cell (MSCs) cultured in vitro, to study the differentiation potential of MSCs and its directional differentiation into osteoiliac muscle cells in vitro, and to design a group of animal models. The uninduced MSCs, positive control cells were injected into the animal model to study the differentiation of MSCs in vivo and its final morphological changes, so as to find the best method of repairing muscle defect and open up a broad prospect for clinical treatment. [methods] cultured rat bone marrow stem cell (MSCs),) rat skeletal muscle cells (Mb, was mainly used as positive control). The third generation of MSCs was induced by 5-azacytidine combined with MyoD,TGF- 尾 1IGF-1. The induced cells were identified by immunohistochemical method. Animal model: sixteen 10-week-old Wistar rats were injected with anhydrous ethanol 0.2 ml in the middle of the anterior tibial muscle of the left posterior leg to make the degeneration and necrosis of the middle part of the anterior tibial muscle. The right posterior leg anterior tibial muscle was injected with sterile saline 0.2 ml. Sixteen rats were randomly divided into four groups, the first group was negative control group, the other group was injected with culture medium, the second group was injected with MSCs;, the third group was injected with MSCs;, the fourth group was injected with MSCs; positive control group, and the second group was injected with Mb.. The third generation cells were labeled with BrdU and then collected for injection. The animal model can be injected 4 hours later. The injection site is the middle of the anterior tibial muscle of both legs. Morphological observation and immunological examination were performed on the 3rd day, 6th day and 12th day after injection. [results] Rat MSCs cultured in vitro grew well and had irregular morphology. Flow cytometry showed that most of the cells were in G1 phase (79.4%). Immunohistochemical staining showed that CD44 was positive, but CD34 was negative. Rat Mb grew well in vitro and its morphology was regular. Flow cytometry showed that most of the cells were in G1 phase (79.8%), and Desmine,Myosin was positive by immunohistochemistry. After induction, the morphology of MSCs, was numerous.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2;R687.2

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