不同来源间充质干细胞生物学特征的比较

Comparison on biological characteristics of mesenchymal stem cells from different tissues

目的:比较脐静脉、成人骨髓及胎儿骨髓3种不同来源的间充质干细胞体外生长特性及分化潜能。方法:①实验于2004-01/2006-06在苏州大学附属第二医院中心实验室完成。孕39～40周健康产妇正常分娩2h的脐带以及3～4月龄流产胎儿均由苏州大学附属第二医院妇产科提供,产妇及其家属均知情同意。正常成人骨髓由苏州大学附属第二医院血液科研究生捐献。本实验经医学伦理委员会批准。②脐带用含青、链霉素的磷酸盐缓冲液冲净静脉腔内残血,夹闭两端,经37℃预热的1.5g/L胶原酶消化,重悬于DMEM-LG(含体积分数为0.1的新生牛血清、20mmol/LHEPES、2mmol/L L-谷氨酰胺、1mmol/L丙酮酸钠),以108L-1密度接种于25cm2培养瓶常规培养,48h后弃上清,以后每两三天半量换液,3周后用5g/L胰酶消化传代。③抽取正常成人骨髓5～10mL,ficoll分离单个核细胞,收取白雾层及以上部分细胞,采用上述培养基按相同条件进行培养,约14～18d胰酶消化传代。④取流产胎儿,无菌条件下分离股骨,冲洗干净后采用两种方法分离间充质干细胞:直接将胎骨剪成约1mm3大小的碎片,磷酸盐缓冲液洗涤后加入少量上述培养基,置10cm培养皿培养;剪去股骨两端后,用上述培养基冲出骨髓腔内细胞,直接培养。⑤观察连续传代对3种不同来源间充质干细胞增殖的影响;免疫荧光及免疫组化分析所得细胞的表型;观察3种不同来源间充质干细胞在诱导培养条件下向成骨及成脂细胞分化的潜能。结果:①不同来源间充质干细胞的体外增殖特性:脐带、成人及胎儿骨髓内均存在间充质干细胞,且细胞形态相似。成人骨髓间充质干细胞的体外增殖能力较差,传至10～12代基本失去增殖能力。脐静脉间充质干细胞的增殖能力强于成人骨髓间充质干细胞,可体外连续传代15～18次。而胎儿来源的间充质干细胞增殖能力及体外传代更新能力均明显强于其他两种间充质干细胞,传至20代时基本保持原有的特性。②不同来源间充质干细胞免疫表型:3种间充质干细胞均高表达CD29、CD44、CD54及HLA-ABC分子,成人骨髓源间充质干细胞还表达中等量的CD106及少量HLA-DR。3种间充质干细胞均不表达造血细胞标志CD34和CD45,其中脐静脉来源的间充质干细胞αSMA呈阳性,vWF呈阴性。③不同来源间充质干细胞的分化潜能:在成脂或成骨条件培养基中,3种间充质干细胞均可向脂肪及成骨细胞分化,油红O及von Kossa染色呈阳性。结论:①脐带、成人及胎儿骨髓内均存在数量不等的间充质干细胞,其形态、生长特性及表面标志具有相似性。②脐静脉源间充质干细胞的增殖能力优于骨髓间充质干细胞,尽管略弱于胎源性间充质干细胞,但其来源广泛、分离方便、间充质干细胞得率高,且没有伦理限制,是一种较好的间充质干细胞来源。

AIM： To compare in vitro growth pattern and differentiation potential of mesenchymal stem cells （MSCs） from umbilical vein, fetal and adult bone marrow. METHODS： ①From January 2004 to June 2006, the experiment was carried out in Central Laboratory of Second Affiliated Hospital of Soochow University. The umbilical cords were obtained from Department of Obstetrics and Gynecology of Second Affiliated Hospital of Soochow University after informed consent of the healthy puerperant and family members. Umbilical cords （gestational ages, 39-40 weeks） within 2 hours after normal deliveries and 3-4 month aborted fetus were collected. Bone marrow from normal adult was donated from a postgraduate of Department of Hematology of Second Affiliated Hospital of Soochow University. The experiment was authorized by Medical Ethics Committee. ②The umbilical cords were internally washed with phosphate-buffered saline （PBS） containing penicillin and streptomycin, and then two ends of umbilical cores were occluded. Umbilical cords were digested with 1.5 g/L collagenase that were preheated at 37℃, re-suspended in DMEM-LG containing new-bom calf serum of 0.1 volume fraction, 20 mmol/L HEPES, 2 mmol/L L-glutamine and 1 mmol/L sodium pyruvate and inoculated in 25 cm^2 culture flask at the density of 10^8 L^-1 for routine culture. Supernatant was discarded 48 hours later, and then half liquid was changed with fresh medium every two or three days. 3 weeks later, it was digested with 5 g/L trypsin for passage. ③5-10 mL bone marrow cells were extracted from normal adults. Mononuclear cells （MNC） were separated by FicolI-Hypaque density gradient centdfugation. Cells in white smog layer and above were collected. The cells were cultured in same condition as mentioned above and would be treated with trypsin at days 14-18. ④Aborted fetus was obtained and femur was harvested sterilely. MSCs were isolated by two methods after washing the femur, namely fetal bone was cut into 1 mm^3 pieces, washed with PBS, and cultured in 10 cm culture dish after adding above-mentioned medium. Cells were washed out of medullary canal by above-mentioned medium for direct culture after cutting out two ends of femur. ⑤ Influence of successive passage on proliferation of MSCs from 3 different tissues was observed. Immunofluorescence and immunohistochemical analysis were employed to determine the phenotype of those cells. The differentiation potential of MSCs from 3 different tissues into osteogenesis and lipoblasts was observed. RESULTS： ①in vitro proliferative characteristics of MSCs from different tissues： MSCs could be isolated from umbilical cores, fetus and adult bone marrow with similar characteristics in morphology. The MSCs derived from adult bone marrow lost their ability to proliferate or passage after 10-12 passages in vitro. The growth and proliferative abilities of MSCs from umbilical vein, which showed 15-18 successive passages in vitro, was better than those of adult MSCs. However, MSCs isolated from fetus bone marrow had the highest potential of proliferation and passage in vitro among those three cells and maintained their biological characteristics after 20 passages.②phenotype of MSCs from different tissues： All of those three MSCs expressed highly CD29, CD44, CD54 and HLA-ABC. MSCs derived from adult bone marrow also expressed intermediate level of CD106 and low level of HLA-DR, respectively. All of those MSCs did not express the markers such as CD34 and CEN5, which belonged to hematopoietic stem cells. ~SMA was positive for MSCs derived from umbilical vein, but they were negative for vWF. ③differentiation potential of MSCs from different tissues： MSCs isolated from different tissues could differentiate into osteogenesis and lipoblasts in condition medium as determined by oil red and von Kossa staining. CONCLUSION： ①MSCs of different volume can be isolated from umbilical cores, fetus and adult bone marrow with similar morphology, proliferative characteristics and phenotype. ②Proliferative ability of MSCs from umbilical veins is better than that of MSCs from bone marrow. Although proliferative ability of MSCs derived from umbilical veins is lower than those derived from fetus bone marrow, it seems to be a better source of MSCs for its wide sources, easy to be collected, high harvesting rate and without ethical restrictions.