【摘要】：[目的]體外獲得培養(yǎng)、擴(kuò)增及鑒定滇南小耳豬骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)的方法,為組織工程膀胱的構(gòu)建提供一種新的種子細(xì)胞來源。 [方法]用骨穿針抽取滇南小耳豬的骨髓液,采用骨髓液離心加貼壁培養(yǎng)法培養(yǎng)。觀察細(xì)胞形態(tài)、生長及擴(kuò)增情況,并采用MTT法繪制生長曲線,流式細(xì)胞儀檢測第一代、第二代BMSCs的表面標(biāo)志物CD29、CD44、CD45。 [結(jié)果]細(xì)胞形態(tài)均為梭形生長,呈成纖維細(xì)胞樣形態(tài)。流式細(xì)胞檢測結(jié)果：P1CD29陽性率為99.44%,P1CD44陽性率為99.96%,P1CD45陰性率為99.55%。P2CD29陽性率為100%,P2CD44陽性率為100%,P2CD45陰性率為99.89%。 [結(jié)論]采用骨髓液離心加貼壁法能有效地培養(yǎng)及擴(kuò)增出滇南小耳豬BMSCs。通過流式細(xì)胞檢測,對第一代及第二代BMSCs陽性率的比較,第二代的BMSCs純度高,可作為種子細(xì)胞的來源。 [目的]探討豬膀胱平滑肌組織和尿路上皮組織在體外原代培養(yǎng)、擴(kuò)增及鑒定的方法,為構(gòu)建組織工程膀胱提供種子細(xì)胞來源。 [方法]在無菌條件下通過外科手術(shù)切取滇南小耳豬膀胱組織,采用機(jī)械分離法分離出膀胱粘膜層和膀胱平滑肌層,用胰蛋白酶和膠原酶I聯(lián)合消化膀胱平滑肌層獲取膀胱平滑肌細(xì)胞,用中性蛋白酶4℃冷消化膀胱組織,機(jī)械分離法獲取獲取膀胱粘膜層,酶消化法獲取尿路上皮細(xì)胞。用含10%FBS的DMEM/F12培養(yǎng)基培養(yǎng)膀胱平滑肌細(xì)胞,DK-SFM(含表皮生長因子)培養(yǎng)基培養(yǎng)尿路上皮細(xì)胞,再分別觀察兩者細(xì)胞的形態(tài)變化,生長及增殖過程,并通過H-E染色和免疫組織化學(xué)染色鑒定細(xì)胞。 [結(jié)果]平滑肌細(xì)胞原代培養(yǎng)12h后可見大量細(xì)胞貼壁,培養(yǎng)2-3天后細(xì)胞融合達(dá)90%以上。尿路上皮細(xì)胞原代培養(yǎng)18h后可見大量細(xì)胞貼壁并發(fā)生形態(tài)改變,原代細(xì)胞培養(yǎng)4-5天后,融合至80%-90%。在倒置相差顯微鏡下觀察平滑肌細(xì)胞為長梭形,并且表現(xiàn)出典型的“谷和峰”形態(tài),尿路上皮細(xì)胞表現(xiàn)出典型的“鋪路石”樣結(jié)構(gòu)。通過蘇木素—伊紅染色觀察其符合平滑肌細(xì)胞和尿路上皮細(xì)胞形態(tài)特征,分別用平滑肌肌動蛋白抗體和角蛋白AE1/AE3單克隆抗體鑒定兩者細(xì)胞結(jié)果都呈陽性。 [結(jié)論]采用該方法能夠有效地建立起體外培養(yǎng)豬膀胱平滑肌細(xì)胞和尿路上皮細(xì)胞的培養(yǎng)體系：所培養(yǎng)出來的細(xì)胞純度高、活力好,可作為構(gòu)建組織工程膀胱的種子細(xì)胞。 [目的]探討體外誘導(dǎo)骨髓間充質(zhì)干細(xì)胞(BMSCs)向膀胱平滑肌細(xì)胞和尿路上皮細(xì)胞分化的方法,為組織工程膀胱的建立提供一種新的種子細(xì)胞來源。 [方法]選取穩(wěn)定培養(yǎng)系中的第三或四代BMSCs、尿路上皮細(xì)胞和平滑肌細(xì)胞。按照模擬機(jī)體內(nèi)環(huán)境的方法,將實(shí)驗(yàn)分成實(shí)驗(yàn)組和對照組。實(shí)驗(yàn)組：A組：BMSCs+尿路上皮細(xì)胞+表皮細(xì)胞生長因子B：BMSCs+尿路上皮細(xì)胞條件培養(yǎng)液+表皮細(xì)胞生長因子.C組:BMSCs+平滑肌細(xì)胞+堿性成纖維生長因子.D組：BMSCs+平滑肌細(xì)胞條件培養(yǎng)液+堿性成纖維生長因子.對照組：E:BMSCs+表皮細(xì)胞生長因子.F組：BMSCs+堿性成纖維生長因子.G組：BMSCs2ml。共培養(yǎng)誘導(dǎo)的時間為10天左右,分別于3、5、7、10天觀察細(xì)胞形態(tài)的變化并記錄下來。用平滑肌肌動蛋白抗體和角蛋白AE1/AE3單克隆抗體鑒定實(shí)驗(yàn)組和對照組。 [結(jié)果]實(shí)驗(yàn)組于5天左右開始出現(xiàn)細(xì)胞形態(tài)的變化,7天后細(xì)胞形態(tài)明顯改變,10天后90%以上的BMSCs向誘導(dǎo)分化的目的細(xì)胞形態(tài)轉(zhuǎn)變。對照組細(xì)胞形態(tài)變化不明顯。角蛋白AE1/AE3單克隆抗體鑒定實(shí)驗(yàn)組A、B組和對照組E、G組,平滑肌肌動蛋白抗體鑒定實(shí)驗(yàn)組C、D組和對照組F、G組,實(shí)驗(yàn)組結(jié)果均為陽性,對照組結(jié)果均為陰性。 [結(jié)論]骨髓間充質(zhì)千細(xì)胞具有多向分化的能力,在模擬體內(nèi)的微環(huán)境的條件下能誘導(dǎo)BMSCs向尿路上皮細(xì)胞和平滑肌細(xì)胞的分化.可為組織工程膀胱的建立提供一種新的種子細(xì)胞來源。
[Abstract]:[Objective] To obtain the method of culture, amplification and identification of bone marrow mesenchymal stem cells (BMSCs) in small ear pigs in southern Yunnan to provide a new source of seed cells for the construction of bladder.[Methods] Bone marrow fluid from the small ear pig of southern Yunnan was extracted with bone penetrating needle, and the bone marrow liquid was used to centrifuge the bone marrow liquid. The cell morphology, growth and amplification were observed, and the growth curve was drawn by MTT method. The surface markers CD29, CD44, CD of the first generation and the second generation BMSCs were detected by flow cytometry. 45.[Results] The morphology of the cells was shuttle-like growth, which was in the form of fibers. The positive rate of PCD29 was 99. 96%, the positive rate of PCD45 was 99. 55%, the positive rate of P2CD29 was 100%, the positive rate of P2CD44 was 100%, and the negative rate of P2CD45 was 9. 9. 89%.[Conclusion] Bone marrow fluid centrifugation and adherence method can be used to effectively cultivate and amplify the southern Yunnan province. BMSCs were detected by flow cytometry. Compared with the first generation and the second generation BMSCs, the second generation BMSCs were high in purity and could be used as the second generation BMSCs. Objective: To investigate the methods of primary culture, amplification and identification of porcine bladder smooth muscle tissue and upper urinary tract epithelial cells in vitro. The bladder tissue of the small ear pig of southern Yunnan was cut off by surgical technique under aseptic conditions. The bladder mucosa and bladder smooth muscle layer were separated by mechanical separation, and digested with trypsin and collagenase I. Bladder smooth muscle cells were obtained from smooth muscle layer, bladder tissue was digested with neutral protease at 4 鈩，

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