【摘要】：目的：觀察兔骨髓間充質干細胞(BMSCs),經(jīng)轉化生長因子-β1(TGF-β1)等向軟骨細胞誘導8d、11d、14d、17d、20d后,與藻酸鈣凝膠復合體外培養(yǎng),細胞在三維條件下增殖情況及共同構建組織工程化軟骨情況。 方法：穿刺新西蘭大耳白兔股骨抽取骨髓,密度梯度離心法分離、培養(yǎng)BMSCs。將傳至第3代的BMSCs予以含TGF-β1、地塞米松等成份的無血清誘導液定向向軟骨細胞誘導。根據(jù)不同的誘導時間,實驗組分成為A、B、C、D、E5組：A組：誘導至第8d;B組：誘導至11d；C組：誘導至14d；D組：誘導至17d：E組：誘導至20d。通過細胞形態(tài)、TB染色、Ⅱ型膠原免疫組化、培養(yǎng)液內多聚蛋白聚糖(AG)含量,比較各實驗組定向誘導成軟骨細胞情況。分別將以上各組成軟骨細胞復合藻酸鈣凝膠,以1×107/ml的密度制作為細胞-藻酸鈣凝膠珠,繼續(xù)體外培養(yǎng)14d。培養(yǎng)過程中觀察細胞在藻酸鈣凝膠中生長、增殖情況,并通過Ⅱ型膠原免疫組化、阿利新藍比色法測定各組Ⅱ型膠原表達和多聚蛋白聚糖(AG)的含量,綜合比較各實驗組構建組織工程化軟骨的情況。 結果：密度梯度離心法分離純化的BMSCs定向成軟骨細胞誘導8d時形態(tài)有較明顯改變,14d時具有明顯的軟骨細胞形態(tài)。誘導培養(yǎng)液在誘導4d時即可檢出少量AG,8d時AG濃度出現(xiàn)明顯升高,并保持緩慢升高至20d。在14d時,誘導的細胞爬片TB染色可見異染顆粒；Ⅱ型膠原免疫組化明顯陽性表現(xiàn)。與藻酸鈣復合后,各組成軟骨細胞在凝膠3D支架中均保持分裂增殖,AO染色見細胞能增殖成“葡萄串”樣。各實驗組材料切片Ⅱ型膠原免疫組化均可見陽性表達,以C組、D組為明顯；AG含量檢測顯示各實驗組、陽性對照組間AG含量具有差異(p0.05),C組、D組均高于陽性對照組,以C組AG含量為最高。 結論：1.采用密度梯度離心法獲取的兔BMSCs,在體外經(jīng)過14d的定向誘導后基本向軟骨細胞分化;2.藻酸鈣凝膠與種子細胞生物相容性良好,可以采用注射方式植入誘導14d后的成軟骨細胞在藻酸鈣凝膠內增殖良好,分泌軟骨細胞外基質功能較強。以BMSCs為種子細胞、藻酸鈣為支架的可注射式軟骨組織工程方法具有可行性。
[Abstract]:Aim: to observe the effect of transforming growth factor 尾 1 (TGF- 尾 1) on rabbit bone marrow mesenchymal stem cells (BMSCs),) induced by transforming growth factor 尾 1 (TGF- 尾 1) into chondrocytes for 8 days, 11 days, 14 days and 17 days, and then cultured in vitro with calcium alginate gel. The proliferation of cells in three-dimensional condition and the construction of tissue engineered cartilage. Methods: bone marrow was extracted from the femur of New Zealand big ear white rabbit, and BMSCs. was isolated by density gradient centrifugation. The third passage of BMSCs was induced to chondrocytes by serum-free medium containing TGF- 尾 1 and dexamethasone. According to the different induction time, the experimental group was divided into two groups: group A: induced to the 8th day: group B: induced to 11 days: group C: induced to 14 days: group D: induced to 17d:E group: induced to 20 days. By means of cell morphology, TB staining, type 鈪，

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