本文選題：骨髓間充質(zhì)干細(xì)胞 + 細(xì)胞培養(yǎng)��； 參考：《瀘州醫(yī)學(xué)院》2011年碩士論文

【摘要】：目的：探討改良法分離純化SD大鼠骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)的可行性,并評(píng)價(jià)改良法的分離純化效果,為下一步實(shí)驗(yàn)研究提供高純度的BMSCs。 方法：(1)無菌條件下分離SD大鼠股骨、脛骨,清除附于骨上的肌肉和筋膜,磷酸鹽緩沖液(Phosphate buffered saline, PBS)洗凈后從股骨干和脛骨干中間剪斷,用5ml注射器抽取5ml L-DMEM培養(yǎng)基反復(fù)沖洗骨髓腔,沖洗液經(jīng)200目不銹鋼標(biāo)準(zhǔn)篩過濾后以2000rpm速度離心5 min,棄上清及脂肪層后加入完全培養(yǎng)液反復(fù)吹打制成單細(xì)胞懸液,以106個(gè)/ml密度接種于4個(gè)25cm2細(xì)胞培養(yǎng)瓶中,隨機(jī)分為A、B兩組細(xì)胞,每組2瓶,置于37℃、5%C02細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)；(2)48h后首次換液,以后每3天換液一次；(3)細(xì)胞生長至80%～90%融合時(shí),以0.25%胰蛋白酶1ml消化2～3min后加入5ml含12%胎牛血清(fetal bull serum,FBS)的L-DMEM完全培養(yǎng)液,吹打制成單細(xì)胞懸液后以1：2比例傳代擴(kuò)增；(4)第1代細(xì)胞融合達(dá)80%～90%時(shí),A組細(xì)胞PBS洗滌、0.25%胰蛋白酶1ml消化后制成單細(xì)胞懸液,緩慢加入盛有同樣體積Percoll分離液(密度為1.073)的離心管,以2000rpm速度離心20 min。用吸管小心吸取中間白色絮狀層加入另一離心管,再加入完全培養(yǎng)液,充分吹打洗滌細(xì)胞后離心,棄上清液后加入完全培養(yǎng)液重懸細(xì)胞,調(diào)整細(xì)胞密度為105/ml后接種5ml入25cm2細(xì)胞培養(yǎng)瓶,繼續(xù)置細(xì)胞培養(yǎng)箱內(nèi)培養(yǎng)；B組細(xì)胞PBS洗滌、0.25%胰蛋白酶1ml消化后制成單細(xì)胞懸液,調(diào)整細(xì)胞密度為105/ml后直接接種5ml于25cm2細(xì)胞培養(yǎng)瓶(未經(jīng)Percoll分離液分離),置培養(yǎng)箱內(nèi)培養(yǎng)；(5)每日在倒置相差顯微鏡下觀察兩組各代細(xì)胞形態(tài)變化及生長增殖狀況并拍照記錄,采用MTT法繪制兩組第4、第9代BMSCs的生長曲線；(6)A、B組第3代BMSCs用免疫組織化學(xué)染色鑒定細(xì)胞表型；(7)流式細(xì)胞儀檢測A、B兩組第3代細(xì)胞表達(dá)CD34、CD44、CD45、CD90陽性細(xì)胞的百分比；(8)A、B組第3代大鼠BMSCs進(jìn)行細(xì)胞周期檢測；(9)對(duì)A、B組所得的BMSCs進(jìn)行成骨誘導(dǎo)分化培養(yǎng)及鑒定。 結(jié)果：(1)A、B兩組第4、第9代BMSCs生長曲線顯示兩種分離方法分離純化的BMSCs的增殖能力無明顯差異(P0.05)(2)免疫細(xì)胞化學(xué)染色結(jié)果顯示：A組第3代BMSCs的CD29、CD44、CD90、CD 106檢測均呈陽性表達(dá),細(xì)胞膜和胞漿呈棕褐色,而CD34、CD45呈陰性表達(dá),胞膜及胞漿未著色；B組第3代BMSCs的CD29、CD44、CD90、CD 106檢測呈陽性表達(dá),大部分細(xì)胞CD34、CD45陰性表達(dá),少部分陽性表達(dá),提示A組BMSCs的純度高于B組；(3)用流式細(xì)胞儀分別檢測A、B兩組第3代BMSCs,結(jié)果：A組細(xì)胞均一的陽性表達(dá)CD44、CD90,而幾乎不表達(dá)CD34和CD45,A組細(xì)胞表達(dá)CD44、CD90陽性率明顯高于B組(P0.05),而表達(dá)CD34、CD45陽性率明顯低于B組(P0.05),提示A組BMSCs的純度高于B組；(4)對(duì)A、B兩組第3代細(xì)胞進(jìn)行細(xì)胞周期檢測,結(jié)果顯示大部分細(xì)胞處于靜止期,只有少數(shù)的細(xì)胞處于活躍的增殖期,均符合干細(xì)胞特征；(5)A、B兩組第3代BMSCs經(jīng)成骨誘導(dǎo)培養(yǎng)液培養(yǎng)3周后經(jīng)茜素紅染色均可見大量深紅色鈣結(jié)節(jié),兩種方法分離純化的大鼠間充質(zhì)干細(xì)胞均具有良好的成骨分化潛能,無明顯差異。 結(jié)論：(1)本研究采用改良法分離純化SD大鼠骨髓間充質(zhì)干細(xì)胞,成功在單只大鼠分離獲得大量高純度的BMSCs,解決了大鼠BMSCs研究的同源性、細(xì)胞相容性、排斥性問題,同時(shí)節(jié)約大量骨髓與干細(xì)胞資源；(2)改良法具有操作簡便、快速、不影響細(xì)胞增殖能力的優(yōu)點(diǎn)；(3)改良法分離純化獲得的BMSCs形態(tài)和生物學(xué)穩(wěn)定性好、具有強(qiáng)大的增殖能力和良好的成骨分化潛能；(4)改良法分離純化SD大鼠骨髓間充質(zhì)干細(xì)胞效果優(yōu)于傳統(tǒng)的全骨髓貼壁篩選法。
[Abstract]:Objective: To explore the feasibility of separating and purifying SD rat bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) by the improved method, and to evaluate the purification effect of the modified method and provide a high purity BMSCs. for the next experimental study.
Methods: (1) the femur and tibia of SD rats were separated from the femur and tibia under aseptic conditions, and the muscles and fasciae attached to the bone were removed. The phosphate buffer solution (Phosphate buffered saline, PBS) was washed from the middle of the femoral shaft and tibial shaft, and the bone marrow cavity was washed by the 5ml syringe 5ml L-DMEM medium. The rinse solution was filtered through the 200 mesh stainless steel standard sieve to 2. 000rpm speed centrifugation 5 min, discard the supernatant and fat layer after adding complete culture fluid and repeatedly blow into a single cell suspension, 106 /ml density inoculated in 4 25cm2 cell culture bottles, randomly divided into A, B two groups, each group of 2 bottles, at 37, 5%C02 cell culture box culture; (2) 48h after the first liquid, and then every 3 days to change liquid once; (3) fine. (3) fine (3) fine When the cell growth was 80% ~ 90% fusion, 0.25% trypsin 1ml was digested for 2 ~ 3min and added to the L-DMEM complete culture of 5ml containing 12% fetal bovine serum (fetal bull serum, FBS). After being blown into a single cell suspension, it was amplified in a 1:2 proportion. (4) when the first generation of cell fusion reached 80% to 90%, the A group cells PBS washing and 0.25% trypsin digested after digestion. The single cell suspension was slowly added to the centrifuge tube with the same volume Percoll separation liquid (density 1.073). At the 2000rpm speed centrifuge, the white floc layer was carefully absorbed into the other centrifuge tube with a sucker, and the complete culture fluid was added to the centrifuge. The centrifuge was fully blown after the washing cells were blown, and the supernatant was abandoned after the supernatant was discarded, and the suspension cells were added and adjusted. After the cell density was 105/ml, 5ml was inoculated into 25cm2 cell culture bottle and continued to be cultured in cell culture box; B group cells were washed by PBS, 0.25% trypsin 1ml was digested to make single cell suspension, and the cell density was 105/ml, and 5ml was directly inoculated with 5ml in 25cm2 cell culture bottle (not separated by Percoll separation solution) and incubated in culture box; (5) daily in the culture box. In the inverted phase contrast microscope, the morphological changes and growth and proliferation of two groups of cells were observed and recorded. The growth curves of two groups of fourth, ninth generation BMSCs were drawn by MTT; (6) A, B group third BMSCs was used to identify the cell phenotype by immunohistochemical staining; (7) flow cytometry was used to detect A, B two and third generation cells expressed CD34, CD44, CD45, CD90 Yang The percentage of sex cells; (8) A, B group third generation rats BMSCs cell cycle detection; (9) A, B group BMSCs induced osteogenic differentiation, culture and identification.
Results: (1) A, B two groups fourth, ninth generation BMSCs growth curve showed that two separate methods of separation and purification of BMSCs proliferation capacity of no significant difference (P0.05) (2) immunocytochemical staining results showed that the third generation of BMSCs CD29, CD44, CD90, CD 106 of the third generation of A group were positive expression, cell membrane and cytoplasm were brown, CD34, negative expression, The cell membrane and cytoplasm were not stained, and the CD29, CD44, CD90, CD 106 of the third generation of B group showed positive expression. Most of the cells were expressed in CD34, CD45 negative and few positive expressions, suggesting that the purity of BMSCs in the A group was higher than that in the B group; (3) the A was detected by flow cytometry and two groups and third generations respectively. No expression of CD34 and CD45, A group cells expressed CD44, CD90 positive rate was significantly higher than that of B group (P0.05), but the expression of CD34, CD45 positive rate was significantly lower than B group (P0.05), suggesting that the purity of the A group was higher than that of the group. (4) the cell cycle detection of the two groups third generation cells showed that most of the cells were in the stationary phase, only a few cells were active. The proliferation period was in line with the characteristics of stem cells. (5) A, B two group third generation of third generation of third generations after culture medium culture 3 weeks after the culture of alizarin red stained with a large number of dark red calcium nodules, two methods of purified rat mesenchymal stem cells have good osteogenic differentiation potential, no significant difference.
Conclusions: (1) the modified method was used to separate and purify SD rat bone marrow mesenchymal stem cells, and successfully isolated a large number of high purity BMSCs in a single rat, and solved the homology, cytocompatibility and rejection of the rat BMSCs study, and saved a large number of bone marrow and stem cell resources. (2) the improved method was easy to operate, fast and no shadow. The advantages of the proliferation ability of ringing cells; (3) the BMSCs morphology and biological stability obtained by the modified method were good, with strong proliferation ability and good osteogenic differentiation potential, and (4) the effect of the modified method for the separation and purification of bone marrow mesenchymal stem cells in SD rats was better than that of the traditional bone marrow adherence screening method.

【學(xué)位授予單位】：瀘州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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