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目的 :探讨人胰腺星状细胞的两种新分离方法。方法 :通过酶消化-密度梯度离心法进行静止态的人正常胰腺星状细胞的分离,分离获得的细胞在含10%FBS的DMEM/F12继续培养。通过组织块外植法获得激活态的人肿瘤相关胰腺星状细胞(Ca PSCs),分离获得的细胞在含20%FBS的DMEM/F12继续培养。结果:通过方法改良,1 g人正常胰腺组织能够获得(0.5~5.0)×106个星状细胞,细胞活率约为90%。使用新的组织块外植法后,组织块不易掉落,细胞爬出时间显著缩短,约5~10 d即可获得Ca PSCs。所有细胞贴壁良好,细胞倍增时间约为24 h。正常胰腺星状细胞(normal pancreatic stellate cells,NPSCs)培养早期形态符合典型静止态星状细胞特征,细胞为多边形或圆形,内含丰富脂滴,在荧光显微镜下可观察到320nm激发波长下的蓝绿色自发荧光。NPSCs培养晚期和Ca PSCs呈多触角状,胞内无脂滴,细胞增殖速度明显快于静止态细胞。激活态的细胞均表达α-SMA、Desmin、vimentin、GFAP等胰腺星状细胞特征性标记。结论:新的原代培养方法能够满足静止态和激活态两种状态胰腺星状细胞的分离培养,同时能够增加细胞得率、缩短分离培养时间,所获得细胞的活性和纯度符合进一步实验的要求,为胰腺星状细胞的相关研究提供了便利。
Objective: To explore two new methods for the isolation of human pancreatic stellate cells. Methods: Quiescent human normal pancreatic stellate cells were isolated by enzyme digestion - density gradient centrifugation. The isolated cells were cultured in DMEM / F12 containing 10% FBS. Activation of human tumor-associated pancreatic stellate cells (Ca PSCs) was obtained by tissue explant explantation. The isolated cells were further cultured in DMEM / F12 containing 20% FBS. Results: By means of the method improvement, the number of astrocytes (0.5-5.0) × 106 astrocytes was found in 1 g human normal pancreatic tissue, and the cell viability was about 90%. After using the new tissue explant method, the tissue block is not easy to drop, the cell creep time is significantly shortened, and the Ca PSCs can be obtained in about 5 to 10 days. All cells adherent well, cell doubling time is about 24 h. The normal morphology of normal pancreatic stellate cells (NPSCs) was characterized by typical quiescent stellate cells. The cells were polygonal or round in shape and contained abundant lipid droplets. Under the condition of 320 nm excitation wavelength, Cyan autofluorescence. NPSCs cultured in late and Ca PSCs were multi-antennae, intracellular lipid droplets, cell proliferation was significantly faster than quiescent cells. The activated cells expressed the characteristic markers of pancreatic stellate cells such as α-SMA, Desmin, vimentin and GFAP. CONCLUSION: The new primary culture method can be used to separate and culture pancreatic stellate cells in both stationary and active states, and at the same time increase the cell yield and shorten the isolation and culture time. The activity and purity of the obtained cells are in accordance with the requirements of further experiments , For the pancreatic stellate cell related research has provided convenience.