人脱细胞软骨细胞外基质对异种软骨种子细胞的影响

摘要

目的观察人脱细胞软骨细胞外基质（hACAM）对异种兔软骨种子细胞增殖和表型的影响。　　方法将差速梯度离心法制作的人脱细胞软骨细胞外基质，配制成0.5%的浆料，分别铺于6孔细胞培养板和96孔细胞培养板，形成1 mm 厚的薄膜，以此培养板为实验组。空白对照组培养板仅使用单纯培养液培养。在培养板上培养兔关节软骨细胞。通过 hochest33258染色验证人软骨细胞外基质脱细胞完全与否；分别在第5、15天两个时间点，通过倒置显微镜、甲苯胺蓝染色观察两种培养方法的兔软骨细胞的生长形态、细胞表型和增殖情况，用 CCK-8细胞增殖实验比较两种培养方法在第1、3、7、10天的增殖情况。结果通过 hochest33258染色发现差速梯度离心的人软骨细胞外基质脱细胞完全。通过倒置显微镜观察第5和15天的实验组细胞增殖情况优于空白对照组，甲苯胺蓝染色的结果也证明这个观点；CCK-8细胞增殖试验显示第7天hACAM 组在细胞增殖方面明显地优于单纯培养液的对照组（P=0.0298），hACAM 组的软骨细胞与空白组的软骨细胞在第10天的增殖情况相比没有统计学差异。　　结论 hACAM 免疫原性低，无细胞毒性，能够很好地促进异种软骨细胞的增殖。%Objective To observe the proliferation of rabbit chondrocytes on human articular cartilage acellular matrix (hACAM) during in vitro expansion. Methods hACAM was prepared with the differential centrifugation as 0.5%slurry, and then coated the 6-well and 96-well plate to form 1 mm thick film as the experimental group. There was no any process for the control group. Chondrocytes were isolated from articular cartilage of New Zealand white rabbits and cultured on hACAM-coated plates hochest33258 staining was used to verify no cell in the hACAM. The cell proliferation and morphology were analyzed with an inverted microscope, toluidine blue staining, and Cell Counting Kit-8 (CCK-8) assay. Results The cells of hACAM groups grew faster than that of the control groups under the inverted microscope in d5, d15, which was also proved by the results of toluidine blue staining. The cells of hACAM groups proliferated significantly faster than that of the control groups (P=0.0298<0.05) in d7. The experiment groups and the control groups were not statistically significant difference in proliferation (P=0.1322>0.05) by CCK-8 cell proliferation assay in d10. Conclusion hACAM promotes the proliferation of chondrocytes at low immunogenicity and no cytotoxic effects, and then it is used as an ideal biological material.