CAJ | 학술논문

目的应用比较蛋白质组学分析方法筛选组织工程化软骨成熟过程中变化显著的细胞外基质蛋白,通过线性相关分析,研究与软骨力学性能相关的蛋白.方法分离人胎儿软骨细胞培养并扩增,接种到聚羟基乙酸/聚乳酸(PGA/PLA)支架材料,体外培养4周后随机分为3组,每组6例,A组继续体外培养6周、B组回植裸鼠体内培养6周、C组回植裸鼠体内培养12周.取材后进行大体观察、湿重及体积测定、组织学及生物力学等相关检测,比较体内外构建软骨与天然软骨形成质量上的差异.同时进行蛋白质组学分析,选择细胞外基质中比值大于2倍的蛋白与相应各组杨氏模量做相关性分析,根据相关系数筛选与软骨力学性能最相关的蛋白.结果 3组均能在体内外形成透明软骨样组织,C组和B组软骨外观近似天然成人软骨,组织学显示大量成熟软骨陷窝,而A组形成的软骨组织结构较松散,组织学可见不规则肥大软骨陷窝.C组湿重和生物力学检测分别为(372.5±35.4) mg和(8.68±2.65) MPa,明显高于B组的(346±34.5) mg和(3.25±1.24)MPa (P＜0.01),而A组为(184.4±12.28) mg和(0.7±0.23) MPa.应用蛋白质组学技术检测到细胞外基质中共44个差异蛋白,选择胞外基质中C组和A组之间差异最大的6个蛋白,与相应各组杨氏模量进行线性相关分析,发现核心蛋白聚糖(Decorin)、软骨粘附素(Chondroadherin)和纤调蛋白(Fibromodulin)与软骨力学性能变化显著相关(P＜0.05).结论蛋白质组学技术能够提供大量的蛋白质信息,组织构建与比较蛋白质组学技术相结合,有利于进一步分析组织工程化软骨胞外基质蛋白与力学性能的关系.
목적 응용비교단백질조학분석방법사선조직공정화연골성숙과정중변화현저적세포외기질단백,통과선성상관분석,연구여연골역학성능상관적단백.방법 분리인태인연골세포배양병확증,접충도취간기을산/취유산(PGA/PLA)지가재료,체외배양4주후수궤분위3조,매조6례,A조계속체외배양6주、B조회식라서체내배양6주、C조회식라서체내배양12주.취재후진행대체관찰、습중급체적측정、조직학급생물역학등상관검측,비교체내외구건연골여천연연골형성질량상적차이.동시진행단백질조학분석,선택세포외기질중비치대우2배적단백여상응각조양씨모량주상관성분석,근거상관계수사선여연골역학성능최상관적단백.결과 3조균능재체내외형성투명연골양조직,C조화B조연골외관근사천연성인연골,조직학현시대량성숙연골함와,이A조형성적연골조직결구교송산,조직학가견불규칙비대연골함와.C조습중화생물역학검측분별위(372.5±35.4) mg화(8.68±2.65) MPa,명현고우B조적(346±34.5) mg화(3.25±1.24)MPa (P＜0.01),이A조위(184.4±12.28) mg화(0.7±0.23) MPa.응용단백질조학기술검측도세포외기질중공44개차이단백,선택포외기질중C조화A조지간차이최대적6개단백,여상응각조양씨모량진행선성상관분석,발현핵심단백취당(Decorin)、연골점부소(Chondroadherin)화섬조단백(Fibromodulin)여연골역학성능변화현저상관(P＜0.05).결론 단백질조학기술능구제공대량적단백질신식,조직구건여비교단백질조학기술상결합,유리우진일보분석조직공정화연골포외기질단백여역학성능적관계.
Objective To study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.Methods Human chondrocyte,cultured and expanded,were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds.After 4 weeks of culture in vitro,the constructs were divided into three groups.There were 6 specimens in each group.For the regular in vitro culture group (A),the constructs were kept in culture at the original condition for an additional 6 weeks.For in vivo groups,the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C).All specimens were harvested for gross observation,average wet weight and volume measurement,histology,immunohistochemistry and biomechanics to evaluate the results.Meanwhile,comparative proteomic analysis was performed for each group,and those proteins involved in extracellular matrix with at least 2 folds up-regulation were choosen for further exploration.The correlations between Young' s modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.Results All these samples in the three groups eventually formed hyaline-like cartilage structure.Specimens in C and B groups were similar with adult articular cartilage in appearance,and had multiple mature lacuna in histology.However,those specimens in A group had loose texture with irregular hypertrophy lueuna.Specimens implanted for 12 weeks in vivo had better wet weight (372.5 ± 35.4) mg and Young' s modulus (8.68 ± 2.65) MPa than those cultured in vivo for6 weeks (346 ± 34.5) mg,(3.25 ± 1.24) MPa (P ＜ 0.01).In group A,they were (184.4 ± 12.28) mgand (0.7 ± 0.23) MPa.This study had detected 44 proteins in ECM by comparative proteomic analysis,then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups.The correlation results indicated the content of Decorin,Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P ＜ 0.05).Conclusions Comparative proteomic analysis could provide large scale information of associated proteins,making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.