發(fā)布時間：2018-09-05 20:10

【摘要】：目的：應(yīng)用組織塊法體外培養(yǎng)原代成纖維細胞,觀察細胞形態(tài)、生長特點、增殖能力以及細胞與醫(yī)用可吸收明膠海綿體外共培養(yǎng)情況,為進一步研究成纖維細胞在組織工程學(xué)的應(yīng)用打下基礎(chǔ)。 方法：取4-6月齡新西蘭大白兔,取其皮下筋膜組織,組織塊法培養(yǎng)原代成纖維細胞。傳代至第3代。倒置顯微鏡觀察細胞形態(tài),免疫組織學(xué)染色后共聚焦顯微鏡觀察細胞形態(tài),分別應(yīng)用MTT和CCK8試劑盒研究細胞生長增殖規(guī)律,并對兩種方法進行比較,Ⅰ/Ⅲ型膠原蛋白染色觀察其分泌蛋白情況,PKH26標(biāo)記下用掃描電鏡觀察成纖維細胞與明膠海綿體外共培養(yǎng)情況。 結(jié)果：用筋膜組織行組織塊法培養(yǎng)可成功得到大量原代成纖維細胞,此法取材廣泛,過程簡單,成纖維細胞于體外培養(yǎng)時增殖活躍。分別用CCK8與MTT法連續(xù)觀察7天,于細胞周期的第4-5天后細胞出現(xiàn)增殖平臺期(平均4.1±2.0天和4.6±1.7天)。兩種方法比較無明顯統(tǒng)計學(xué)差異(P0.05)。免疫組織學(xué)染色后共聚焦顯微鏡觀察可見細胞為典型的成纖維細胞形態(tài)：大胞體,多突起的紡錘形或星形的扁平細胞,細胞核呈規(guī)則的卵圓形,多核仁。Ⅰ/Ⅲ型膠原蛋白染色陽性。PKH26熒光染料可很好標(biāo)記成纖維細胞細胞壁,染色率100%。掃描電鏡觀察成纖維細胞在明膠海綿支架中生長旺盛,增殖活躍。 結(jié)論：1.皮下筋膜組織塊法培養(yǎng)成纖維細胞是一種簡單易行的方法,可于體外獲得大量成纖維細胞2.明膠海綿是一種良好的組織工程學(xué)韌帶支架,與成纖維細胞貼敷率高,體外共培養(yǎng)良好。
[Abstract]:Objective: to culture primary fibroblasts in vitro by tissue mass method, and observe the morphology, growth characteristics, proliferation ability and co-culture of cells with medical absorbable gelatin in vitro. It will lay a foundation for further research on the application of fibroblasts in tissue engineering. Methods: primary fibroblasts were cultured from 4-6 months old New Zealand white rabbits with subcutaneous fascia tissue. Pass on to the third generation. The morphology of cells was observed by inverted microscope and confocal microscope after immunohistochemical staining. The law of cell growth and proliferation was studied by MTT and CCK8 kit, and the two methods were compared. Type 鈪，

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