含精氨酰-甘氨酰-天冬氨酰環(huán)兩親性肽自組裝凝膠與骨髓間充質(zhì)干細(xì)胞相容性研究
發(fā)布時間：2018-10-22 08:09
【摘要】：目的分析骨髓間充質(zhì)干細(xì)胞(BMSCs)與兩親性肽三維凝膠的相容性。方法取3周齡SD健康大鼠3只,分離股骨、脛骨獲取BMSCs,采用流式細(xì)胞術(shù)檢測BMSCs表面抗原;將10mg/mL含精氨酰-甘氨酰-天冬氨酰(RGD)環(huán)兩親性肽溶液加入等體積DMEM/F12培養(yǎng)基數(shù)秒后自組裝成三維凝膠,透射電鏡顯微鏡(TEM)下觀察三維凝膠結(jié)構(gòu);1×10~6 cells/mL BMSCs懸液與RGD環(huán)兩性親肽混合形成三維培養(yǎng)體系,1×10~6 cells/mL BMSCs懸液與多聚賴氨酸混合形成二維培養(yǎng)體系,無血清培養(yǎng);CCK-8法觀察細(xì)胞生長情況,鈣黃綠素乙酰氧基甲酯/碘化丙啶(PI)雙標(biāo)染色,熒光顯微鏡觀察RGD環(huán)兩性親肽對BMSCs增殖的影響。結(jié)果分離培養(yǎng)的細(xì)胞高表達(dá)CD29、CD90,不表達(dá)或低表達(dá)CD34、CD45;TEM顯示凝膠由多空納米纖維構(gòu)成,納米纖維直徑2~5nm,長度100~1 000nm;質(zhì)譜測得合成多肽相對分子質(zhì)量為1 256.37,與理論值一致;高效液相色譜分析兩性親肽純度為95.88%;鈣黃綠素乙酰氧基甲酯/PI雙標(biāo)染色顯示,三維培養(yǎng)體系中,30min后少數(shù)BMSCs出現(xiàn)死亡,12h后細(xì)胞開始增殖,增殖較二維培養(yǎng)活躍,差異有統(tǒng)計學(xué)意義(P0.05);CCK-8細(xì)胞計數(shù)顯示,三維培養(yǎng)體系細(xì)胞增殖活力高于二維培養(yǎng)體系,差異有統(tǒng)計學(xué)意義(P0.05)。結(jié)論 RGD兩性親肽與BMSCs有良好的生物相容性,可能成為組織工程支架材料。
[Abstract]:Objective to analyze the compatibility of bone marrow mesenchymal stem cells (BMSCs) with amphiphilic peptide 3 D gel. Methods three healthy 3-week-old SD rats were used to isolate femur and tibia to obtain BMSCs,. BMSCs surface antigen was detected by flow cytometry. 10mg/mL containing arginyl-glycyl-aspartic (RGD) ring amphiphilic peptide solution was added to the DMEM/F12 medium of equal volume for several seconds and self-assembled into a three-dimensional gel. The three-dimensional gel structure was observed under transmission electron microscope (TEM), 1 脳 10 ~ (6) cells/mL BMSCs suspension and RGD ring amphiphilic peptide were mixed to form a three-dimensional culture system, 1 脳 10 ~ (6) cells/mL BMSCs suspension and polylysine were mixed to form a two-dimensional culture system without serum, and the cell growth was observed by CCK-8 method. The effect of RGD cyclic amphiphilic peptide on the proliferation of BMSCs was observed by double labeling staining of calcium xanthocyanin acetyl methyl ester / propanidine iodide with fluorescence microscope. Results the high expression CD29,CD90, or low expression CD34,CD45;TEM of the cultured cells showed that the gel was composed of multi-empty nanofibers, the diameter of the nanofibers was 2 ~ 5 nm, the length was 100 ~ 1 000 nm, and the relative molecular weight of synthetic polypeptides was 1 256.37, which was consistent with the theoretical values. The purity of amphophilic peptide by HPLC was 95.88.The double labeling staining of calcium xanthophyllin acetyl methyl ester / PI showed that a few BMSCs cells died after 30min, and the cells began to proliferate after 12 hours, and the proliferation was more active than that in two-dimensional culture. The difference was statistically significant (P0.05); CCK-8 cell count showed that the proliferation activity of three-dimensional culture system was higher than that of two-dimensional culture system, the difference was statistically significant (P0.05). Conclusion RGD amphophilic peptide has good biocompatibility with BMSCs and may be used as scaffold material for tissue engineering.
【作者單位】： 南昌大學(xué)第二附屬醫(yī)院骨科;
【基金】：國家自然科學(xué)基金資助項目(81360271,30801158) 江西省教育廳課題(GJJ14054) 江西省自然科學(xué)基金計劃項目(20151BAB205051) 南昌大學(xué)研究生創(chuàng)新專項基金(CX2016326)
【分類號】：R318.08
本文編號：2286602
[Abstract]:Objective to analyze the compatibility of bone marrow mesenchymal stem cells (BMSCs) with amphiphilic peptide 3 D gel. Methods three healthy 3-week-old SD rats were used to isolate femur and tibia to obtain BMSCs,. BMSCs surface antigen was detected by flow cytometry. 10mg/mL containing arginyl-glycyl-aspartic (RGD) ring amphiphilic peptide solution was added to the DMEM/F12 medium of equal volume for several seconds and self-assembled into a three-dimensional gel. The three-dimensional gel structure was observed under transmission electron microscope (TEM), 1 脳 10 ~ (6) cells/mL BMSCs suspension and RGD ring amphiphilic peptide were mixed to form a three-dimensional culture system, 1 脳 10 ~ (6) cells/mL BMSCs suspension and polylysine were mixed to form a two-dimensional culture system without serum, and the cell growth was observed by CCK-8 method. The effect of RGD cyclic amphiphilic peptide on the proliferation of BMSCs was observed by double labeling staining of calcium xanthocyanin acetyl methyl ester / propanidine iodide with fluorescence microscope. Results the high expression CD29,CD90, or low expression CD34,CD45;TEM of the cultured cells showed that the gel was composed of multi-empty nanofibers, the diameter of the nanofibers was 2 ~ 5 nm, the length was 100 ~ 1 000 nm, and the relative molecular weight of synthetic polypeptides was 1 256.37, which was consistent with the theoretical values. The purity of amphophilic peptide by HPLC was 95.88.The double labeling staining of calcium xanthophyllin acetyl methyl ester / PI showed that a few BMSCs cells died after 30min, and the cells began to proliferate after 12 hours, and the proliferation was more active than that in two-dimensional culture. The difference was statistically significant (P0.05); CCK-8 cell count showed that the proliferation activity of three-dimensional culture system was higher than that of two-dimensional culture system, the difference was statistically significant (P0.05). Conclusion RGD amphophilic peptide has good biocompatibility with BMSCs and may be used as scaffold material for tissue engineering.
【作者單位】： 南昌大學(xué)第二附屬醫(yī)院骨科;
【基金】：國家自然科學(xué)基金資助項目(81360271,30801158) 江西省教育廳課題(GJJ14054) 江西省自然科學(xué)基金計劃項目(20151BAB205051) 南昌大學(xué)研究生創(chuàng)新專項基金(CX2016326)
【分類號】：R318.08
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