犬骨髓单个核细胞诱导分化为平滑肌样细胞

Canine bone marrow mononuclear cells differentiate into smooth muscle-like cells

目的:探讨体外诱导骨髓单个核细胞分化为平滑肌样细胞的可行性,为构建组织工程器官提供种子细胞。方法:实验于2005-10/2006-12在教育部重点实验室,实验室等级1级的首都医科大学宣武医院外科实验室完成。实验材料:体质量20～30kg成年雌性杂种犬6只,由北京市科宇实验动物养殖中心提供(许可证号为SCXK(京)2000-0015),动物饲养级别1级。实验方法:①骨髓单个核细胞的分离培养:穿刺健康成年实验犬髂前上棘,抽取骨髓10mL,加入肝素抗凝,以等体积PBS充分混匀,4℃条件下转入离心管中,取20mL淋巴细胞分离液,将稀释骨髓按1∶1小心加在分离液的界面上,用水平离心机以1500r/min离心10min后,单个核细胞位于血浆和离心液界面层。吸取界面层单个核细胞层,以含体积分数为0.1胎牛血清的PBS冲洗3次,备用。②骨髓单个核细胞诱导分化为平滑肌样细胞:将收获的犬骨髓单个核细胞以由含体积分数为0.15胎牛血清的DMEM/F12混合培养液、0.5μg/L内皮细胞生长因子、2μg/L碱性成纤维生长因子、10μg/L血小板源性生长因子BB、100μg/L胰岛素样生长因子、1mmol/L磷酸维生素C、0.04mmol/L脯氨酸组成的诱导培养基重悬,以1×109L-1种植于胶原铺底的培养瓶中,置37℃、含50mL/LCO2饱和湿度培养箱中,进行诱导培养。倒置相差显微镜下观察细胞形态特征。在诱导培养后第1,3,5,7,9,11天绘制细胞生长曲线。采用免疫组织化学方法进行细胞表型鉴定,并评价细胞生长增殖情况。结果:①骨髓单个核细胞向平滑肌样细胞诱导的细胞生长曲线:接种第1,3,5,7,9,11天计数每孔细胞数分别为(2.73±0.18)×108,(3.17±0.36)×108,(7.35±1.28)×108,(23.61±1.56)×108,(53.48±3.47)×108,(48.51±5.36)×108L-1。②免疫组织化学方法鉴定细胞表型:原代细胞仅少数α-平滑肌肌动蛋白染色明显阳性,明显阳性细胞约占总数的(60.5±5.9)%,细胞内肌丝样结构不明显,而平滑肌肌球蛋白重链、碱性钙调宁蛋白染色阴性;P3代细胞生长5d,细胞生长汇合后,绝大部分诱导后细胞α-平滑肌肌动蛋白、平滑肌肌球蛋白重链、碱性钙调宁蛋白染色阳性,可见胞浆内清晰的肌丝样结构,明显阳性细胞分别占细胞总数的(86.2±13.5)%,(78.7±10.2)%,(76.63±13.8)%,明显高于原代细胞(P<0.05)。③平滑肌样细胞的亚型分析:原代细胞爬片标本Flk-1染色多数呈阳性,第3代细胞Flk-1染色几乎阴性,阳性细胞胞膜、胞浆着色呈棕黄色。原代及传代细胞Vimentin染色均呈阳性,阳性细胞胞浆着棕黄色。本组细胞Desmin染色阴性。结论:骨髓单个核细胞可以被定向诱导成为平滑肌样细胞,能够满足组织工程种子细胞的要求。

AIM： Bone marrow mononuclear cells （BMMCs） from canine are generally considered to be useful for fabricating tissue engineering, thus this paper examines their capacity to differentiate into smooth muscle-like cells （SMLCs） in vitro. METHODS： The experiment was conducted at the First-Degree Laboratory, the State Key Laboratory of Ministry of Education, the Surgery Laboratory of Xuanwu Hospital of Capital Medical University from October 2005 to December 2006. Materials： Six female mongrel dogs （20-30 kg） were obtained from the Key Laboratory Animal Cultivate Center of Beijing [The number of the license is SCXK（Beijing）2000-0015], animals were at the first degree of breeding. Methods： （1）lsolation and culture of BMMCs： Bone marrow （10 mL for each dog） was aspirated from the anterior superior lilac spine and immediately mixed with hepadn and equal volume of phosphate buffered solution （PBS）. Then 20 mL mixture was centrifuged at 4 % on a same volume of lymphocyte separation medium for 10 minutes at 1500 rpm. BMMCs on the layer between plasma and centrifugate, separated by a gradient centrifugator, were washed three times with PBS containing 10% fetal bovine serum （FBS）.（2）Differentiation of BMMCs into SMLCs： Dog BMMCs were cultured and induced in DMEM/F12 containing 15% FBS, and epidermal growth factor （0.5 μg/L）, basic flbroblast growth factor （2μg/L）, platelet derived growth factor BB （10μg/L）, insulin-like growth factor （100μg/L）, ascorbic acid （1 mmol/L） and praline （0.04 mmol/L） in culture flask coated with collagen. Cells were seeded at a density of 1×10^9 L^-1 and maintained at 37 % in a humidified atmosphere （50 milL CO2）. The morphologic characteristics of the cells were observed under an inverted phase contrast microscope. The growth of the induced cells was recorded on the 1^st, 3^rd, 5^th, 7^th, 9^th, 11^th days of culture. Cell phenotypes were identified by immunohistochemical staining, while the cell growth and proliferation were also evaluated. RESULTS： （1）The numbers of SMLCs that differentiated from BMMCs per well of the culture plate on the 1^st, 3^rd, 5^th, 7^th, 9^th, 11^th days of culture were （2.73±0.18）×10^8, （3.17±0.36）×10^8, （7.35±1.28）×10^8, （23.61±1.56）×10^8, （53.48±3.47）×10^8, （48.51±5.36）×10^8 L^-1, respectively.（2）Some of the primary cells were positive for alpha-smooth muscle actin and negative for calponine and myosin heavy chain. A large proportion （60.5 ±5.9）% of the cells were obviously positive for alpha-smooth muscle actin but the myofilament structure was ambiguous; P3 cells grew to confluence 5 days later, and most of the induced cells were positive for alpha-smooth muscle actin, calponine and myosin heavy chain, accounting for （86.2±13.5）%, （78.7±10.2）%, （76.63±13.8）%, separately. A myofilament structure was clearly evident in the cytoplasm of these P3 cells. The number of positive cells in this population were obviously much more than among the primary cells （P 〈 0.05）. （3）Most of the primary SMLCs expressed FIk-1, but the P3 cells were almost negative. The cell membrane and cytoplasm of the positive cells were stained brown. The primary and passaged cells were both positive for Vimentin and negative for Desmin. The cytoplasma of the Vimentin positive cells were stained brown. CONCLUSION： BMMCs can be oriented to differentiate into SMLCs in vitro. Canine BMMCs may be a useful source of SMLCs for tissue engineering.