大肠癌肿瘤相关成纤维细胞原代培养方法的优化

Optimization of primary culture for colorectal cancer-associated fibroblasts

目的比较大肠癌肿瘤相关成纤维细胞（CCAFs）的不同原代培养方法，筛选最佳培养条件。方法取新鲜直肠癌标本，以高浓度双抗低温处理后进行原代培养；12d细胞计数法比较酶消化法与组织块植入法的CCAFs培养效率；利用6d贴壁率及12d爬出细胞数法比较不同组织块大小（1、8mm^3）、培养基种类（1640、DMEM、F12）、培养瓶材质（玻璃瓶、塑料瓶）与血清浓度（10％、20％）的培养效率；所获CCAFs行免疫荧光鉴定并绘制生长曲线；以SPSS18．0软件进行统计学分析。结果组织块植入法优于酶消化法，12d细胞计数分别为（9．75±0．91）×10^4个与（1．83±1．22）×10^4个（t=13．733，P〈0．01）；12d细胞计数法提示高血清浓度（t=-3．549，P〈0．01），较大组织块（t=-1．115，P〈0．05）可增加细胞培养效率；与F12和DMEM比较，1640培养基具有更高细胞培养效率和性价比，其回归方程为：Y=10．68750—3．96528X22-2．00694X，＋1．03472X4；培养瓶材质不影响细胞数量（t=-0．116，P〉0．05）；6d细胞爬出率与培养瓶和培养基有关，回归方程为：Y=0．81367＋0．20478X．-0．33312X引原代培养的CCAFs表达α-平滑肌肌动蛋白（α—SMA）及波形蛋白（Vimentin）。结论高浓度双抗低温处理组织标本，以含高浓度胎牛血清的1640培养基，采用大组织块植入法是CCAFs原代培养的优化方法，获得的CCAFs具有典型肿瘤相关成纤维细胞（CAFs）特征。

Objective To optimize the conditions for the primary culture of colorectal cancer-associated fibroblasts （CCAFs）. Methods （1） Fresh specimens from rectal carcinoma were pretreated with high concentration antibiotics in low temperature. （2） The collagenase digestion method and tissue explantation method were compared with 12 day cell counts. （3） Different culture conditions, including culture surface （glass or plastics） , medium （PRMI 1640, DMEM, or F12） , fatal bovine serun （FBS） levels （10% or 20% ） and block size （ 1,8 mm3 ）, were assessed with 6-day emigration ratios and 12 day cell counts. （4） CCAFs were identified by immunofluorescence and morphology, the growth curves of CCAFs and normal fibroblasts were compared. （5） Multiple linear regression and t-test were adopted in statistics analysis using SPSS 18.0. Results （ 1 ） Tissue explatation method had more cell yields than eollageuase digestion method [（9.75±0.91） ×10^4 vs. （1.83±1.22）×10^4], t=13.733, P〈0.01. （2） Higher FBS concentration （t = - 3.549, P 〈 0. 01 ）, larger tissue blocks （ t = - 1. 115, P 〈 0. 05 ） and PRMI-1640 medium led to more 12 day cell counts （ regression equation, Y = 10. 687 50 - 3. 965 28X22 - 2. 006 94X3 ＋ 1. 034 72X4 ）, while the texture of culture bottle had no such effects （ t = - 0. 116, P 〉 0. 05 ）. 6-day emigration ratios were related to medium and the culture texture （regression equation, Y= 0.813 67＋ 0.20478X1 - 0. 333 12X22 ）. （3） The harvested cells intensively expressed α-smooth muscle actin （α-SMA） and vimentin, presenting typical characteristics of cancer-associated fibroblasts （CAFs）. Conclusion Pretreatment of surgical specimen with high concentration antibiotics in low temperature, tissue explantation method with large blocks and PRMI 1640 medium with high concentration FBS are the optimized conditions for primary culture of CCAFs.