骨髓和静脉来源的间质细胞作为种子细胞的研究

Using bone marrow and vein derived interstitial cells as seed cells of TEHV

目的诱导分化骨髓间充质干细胞(BMSCs),并以大隐静脉来源的间质细胞为对照,优选出构建组织工程瓣膜间质的一种种子细胞。方法分离扩增犬骨髓间充质干细胞,定向诱导分化为成纤维细胞,以静脉间质细胞为对照,比较两种细胞在形态学、增殖能力、合成胶原及在去细胞猪主动脉瓣上的黏附生长能力,以及两种细胞冻存和复苏率。内皮细胞(IC)为对照,比较两者在形态学、增殖能力、合成胶原及在去细胞猪主动脉瓣叶(AVL)上的黏附能力,对种植后AVL作苏木素伊红(HE)、免疫组织化学染色和扫描电镜检测。结果光镜观察,两种细胞均贴壁生长,形态梭形;第3代骨髓来源IC倍增时间为38h、上清液羟脯氨酸含量为(1.518±0.206)mg/L,静脉源IC为36h、(1.432±0.204)mg/L两者差异均无统计学意义(P>0.05);骨髓源IC在AVL上的黏附测得A值为0.497±0.012而静脉源IC为0.348±0.030,两者差异有统计学意义(P<0.05)。HE染色、电镜示两种细胞均能够种植在AVL,免疫组织化学显示:AVL上细胞表达Laminin。结论BMSCs可向瓣膜IC分化,与静脉源IC差异无统计学意义(P<0.05),但黏附生长能力则优于静脉来源者可作为TEHV间质的种子细胞。

Objective To induce born marrow mesenchymal stem cells (BMSCs) differentiating committedly toward valve interstitial cells (IC) and compare to the saphenous vein-derived IC and select an excellent seed cell of tissue engineered heart valves (TEHV).Methods The canine BMSCs were separated,expanded and induced toward valve IC.The induced BMSCs were expanded,purified and investigated in cell morphology,proliferation capability,collagen-synthesized capability and adhesive to AVL compare to venous IC.Then the acellularized AVL was detected by HE staining,immuohistochemistry staining and scanning electron microscope.Results Both cellsadhesively grew uniformly.The double time of bone marrow-derived IC was about 38 h and that of venous wall IC about 36 h;The content of hydroxyproline of bone marrow-derived IC medium was (1.518±0.206) mg/L and that of venous IC (1.432± 0.204) mg/L.There was no significant difference in double time and collagen-synthesized capability,but there was significant difference in adhesion to detergent acellularized porcine valves between two kinds of cells for A value of bone marrow-derived IC was 0.497± 0.012 and that of venous IC 0.348± 0.030.Under scanning electron microscopy and HE staining,it was found that two kinds of cells could be planted on AVL and immunohistochemistry revealed that the expression of Laminin was detected in AVL.Conclusion BMSCs could differentiate toward valve IC and most of features of it had no significant difference from venous IC.However the adherence capability of it was superior to venous wall IC.BMSCs can be used as seed cells to fabricate TEHV.