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目的 :研究人中期胚胎脐血间质干细胞生物学特性 ,探讨其在自体宫内基因转移 /治疗 (IUGT)领域的应用前景。方法 :采用L -DMEM完全培养液培养中期胚胎脐血间质干细胞 (MSC) ;流式细胞仪技术检测细胞表面标记 ;地塞米松 /胰岛素等作为脂肪细胞诱导液 ,β -甘油磷酸钠 /抗坏血酸等作为成骨细胞诱导液分别诱导细胞分化 ;将携带有绿色荧光蛋白的重组腺病毒转染MSC ,荧光显微镜下检测MSC表达转基因特性 ;F344新生大鼠肝内注射MSC ,免疫荧光检测 6周后不同组织器官中人MSC存在 ;非肥胖型糖尿病 /重症联合免疫缺陷性小鼠 (NOD/SCID)皮下注射MSC ,病理切片检测第 30d细胞体内成瘤性。结果 :3mL人中期胚胎脐血可以分离、培养出MSC ,细胞均一地表达CD2 9、CD4 4、CD5 9、CD10 5、CD16 6 ,不表达CD34、CD4 5、CD80、CD86、CD4 2a、HLA -DR分子 ,体外培养中具有成脂、成骨能力 ;细胞表达转基因产物绿色荧光蛋白阳性率高达 5 6 32 %± 3 2 8% ;在新生大鼠体内 ,人中期胚胎脐血来源的MSC能向多个不同组织器官迁移 ,在NOD/SCID小鼠内不具有成瘤性。结论 :人中期胚胎脐血MSC适合中、晚期胚胎自体IUGT靶细胞。
OBJECTIVE: To study the biological characteristics of human umbilical cord blood mesenchymal stem cells in mid-term embryos and to explore their potential application in the field of intrauterine genetic transfer / therapy (IUGT). Methods: MSCs were cultured in complete medium of L-DMEM, and cell surface markers were detected by flow cytometry. Dexamethasone / insulin was used as the inducer of adipocytes, β-glycerophosphate / ascorbate Were used as inducers of osteoblasts to induce cell differentiation respectively. MSCs transfected with green fluorescent protein (GFP) were transfected into MSCs. The transgeneic characteristics of MSCs were detected by fluorescence microscope. MSCs were injected into liver of F344 neonatal rats. After 6 weeks of immunofluorescence, MSCs were found in different tissues and organs; MSCs were injected subcutaneously into non-obese diabetic / severe combined immunodeficiency mice (NOD / SCID), and the tumorigenicity in vivo was detected on the 30th day. Results: The cord blood of 3 mL human embryos could be isolated and cultured. The cells expressed CD2 9, CD4 4, CD5 9, CD10 5 and CD16 6 uniformly without expression of CD34, CD4 5, CD80, CD86, CD4 2a and HLA - DR molecules in vitro culture with adipogenic, osteogenic ability; cell expression of transgenic products green fluorescent protein positive rate as high as 5 6 32% ± 32 28%; in neonatal rats, human mid-embryonic cord-derived MSC can be to Multiple different tissues and organs migrate and do not have tumorigenicity in NOD / SCID mice. Conclusion: Human umbilical cord blood MSC of mid-stage embryos is suitable for target IUGT cells of middle and late embryos.