摘要
背景:肌源性干细胞是一种成体多能干细胞,已成为基因治疗和细胞介导的组织工程领域内的研究热点,临床应用自体肌源性干细胞注射治疗压力性尿失禁具有广泛前景。目的:探讨大鼠肌源性干细胞体外原代培养的方法,为临床应用自体肌源性干细胞注射治疗压力性尿失禁提供实验依据。设计:重复观察测量。单位:武汉大学人民医院生殖中心与泌尿外科。材料:实验于2003-12/2004-05在武汉大学人民医院泌尿外科实验室完成。选取4～6周龄雌性SD大鼠20只,XI型胶原酶、胰酶、多聚赖氨酸(Sigma公司),dispase酶(Gibco公司),鸡胚提取液(自制)。方法:①将大鼠以质量浓度为10g/L的戊巴比妥钠30g/kg腹腔麻醉后,无菌条件下取腓肠肌,置入冷DMEM培养基中,D-Hank’s液洗涤组织块,除去筋膜、肌腱、神经和血管,剪成1.0～3.0mm3小块,移入离心管。向组织中加入0.2%XI型胶原酶及0.1%的胰酶,采用胶原酶分离大鼠骨骼肌细胞。②应用差速贴壁法大鼠骨骼肌细胞进行纯化。细胞筛网过滤后接种至多聚赖氨酸包被的培养瓶中,37℃、体积分数为0.05的CO2培养箱中孵育1h,未贴壁细胞移入新的培养瓶中,加入新的培养基37℃培养1h后,未贴壁细胞再次转瓶换液,37℃培养过夜,如此反复,每次间隔24h转瓶换液,培养第5～6天贴壁的细胞即肌源性干细胞。③细胞生长到70%融合后,胰蛋白酶消化,以1∶2的比例进行传代。传代细胞消化后接种于放有盖玻片的6孔培养板中,加入生长培养基,24h后制备细胞爬片,采用免疫组织化学方法鉴定所培养细胞上特异性结蛋白抗原和干细胞抗原-1的表达。主要观察指标:肌源性干细胞的形态及其鉴定结果。结果:①肌源性干细胞的形态观察:从骨骼肌组织中分离出来的肌源性干细胞呈球形,折光性强。培养12h后开始贴壁仍为圆形,48h后贴壁完全并开始增生,细胞逐渐延展成梭形或纺锤形,有两极,体积小。随培养时间的延长,细胞相互融合形成成熟的多核肌管。②肌源性干细胞的鉴定结果:细胞特异性结蛋白抗原和干细胞抗原-1染色呈阳性,荧光显微镜可见细胞浆发出红色荧光,阳性率达90%。结论:肌原性干细胞属于成体干细胞,具有取材方便、免疫原性低、移植后存活时间长等优点。高纯度的肌原性干细胞可通过体外原代培养获取,免疫组化技术可鉴定其肌源性和干细胞特性,在组织工程和基因治疗中具有广泛的应用前景。
BACKGROUND： Skeletal muscle-derived stem cells （MDSCs）, another adult pluripetent stem cells, have become a hot topic in the field of gene therapy and cell-based tissue engineering. It has wide prospect in treating stress incontinence by periurethral injection of patients＇ MDSCs. OBJECTIVE： To investigate the method of culturing MDSCs in vitro so as to provide experimental basis for treating stress incotinence by injecting MDSCs. DESIGN： Repeated observation. SETTING： Department of Reproductive Medical Center and Department of Urology, Renmin Hospital of Wuhan University. MATERIALS： This experiment was conducted at the Laboratory of Department of Urology , Renmin Hospital , wuhan University from December 2003 to May 2004. Totally 20 female SD rats , aged 4 to 6 weeks , were involved . Type-XI pancreatin, collgenase , pancreatin ,pelylysine （Sigma）, dispase enzyme （Gibeo） , chick embryo extract （self made）. METHODS： （1） Rats were anesthetized intraperitoneally with 10 g/L pentobarbital sodium （30 g/kg）. Under aspetic condition, gastroteneminius were isolated and immediately put into pre-cooled DMEM media （Gibco） containing antibiotics and then removed into the hood. After washed with D-hank＇ s solution three times, muscle biopsies were removed of fascia,tendon, nerve and blood vessels and minced into small pieces about 1-3 mm^3, and then transferred into a centrifugation tube. 0.2% collgenase-type XI （Sigma） and 0.1% pancreatin were added to the left tissue . Skeletal muscle cells of the rats were isolated with collagenase. （2） Differential attachment ＇was used to purify the skeletal muscle cells of the rats. After screened with cell screen cloth , cell suspension was transferred into a polylysine-coated flask and cultured at 37℃ in a humid atmosphere with 0.05 CO2 in air for 1 hour.All the cells that did not adhere to the flask were then transferred to another flask with new culture medium and cultured for approximately 1 hour at 37℃ .Again,the non-adhering cells were transferred to another flask and were incubated at 37℃ overnight. The technique was carried out for an additional 4-5 days. This operation was repeated every 24 hours until the fifth day. Those cells attached on days 5-6 were MDSCs.（3） After they grew with 70% confluence , the cells were digested with trypsin and passaged at the ratio of 1：2. After digestion, the generative cells were inoculated to the culture plate with 6-well cover glass. Growing culture medium was added and cell slide was prepaared 24 hours later. The expression of specific protein antigen and stem cell antigen-1 of the cultured cells were identified with immunohistochemical staining. MAIN OUTCOME MEASURES： Morphology and identification of MDSCs. RESULTS： （1） Morphology of MDSCs： Cells isolated from skeletal muscle tissue took the form of spheres and had high refraction. When subcultured, they began to adhere at the 12^th hour, at that time they were still round, and complete their attachment at the 48th hour. Then they began to expand and ultimately became fusiformhaped or spindle-shaped, having two poles and small size. As time going, they fused with each other to form mature polyuclear myotubes. （2） Identification results of muscle-derived stem calls ： MDSCs were desmin and stem cell antigen-1 （Sea-1） positive specif- ic for some stem cells. Red fluorescence effluenced from cytoplasm was found under the fluorescence microscope and the positive rate reached 90%. CONCLUSION： MDSC belongs to adult stem cells and has the advan-rages of rich source, low immunogenicity and long survival after transplan- tation. High-purity muscle-derived stem cells can be obtained through primary culture, and immunohistochemical technique can identify muscle-derived and specific characteristics of stem cells, so it has a broad application perspective in tissue engineering and gene therapy.
出处
《中国临床康复》
CSCD
北大核心
2006年第25期164-166,F0003,共4页
Chinese Journal of Clinical Rehabilitation
