骨髓间充质干细胞向淋巴管内皮细胞分化潜能的研究
发布时间：2018-09-13 15:37
【摘要】：研究背景：骨髓间充质干细胞(Mesenchymal stem cells, MSCs)作为骨髓中一类非造血类干细胞,具有自我更新和多向分化潜能,最近发现还具有低免疫原性的特点,因此受到临床医学研究领域的广泛关注。目前,MSCs已被广泛应用于研究和治疗各种缺血或损伤性疾病以及器官移植抗免疫排斥反应等,并取得了初步效果,干细胞替代治疗有望成为临床上一种新的治疗手段。 乳腺癌术后患侧上肢淋巴水肿是一种慢性进行性疾病,主要原因是由于在腋窝淋巴结切除或前哨淋巴结活检中损伤了引流上肢的淋巴管,导致淋巴回流不畅,淋巴液淤积于皮下,病情晚期上肢肿胀,功能丧失,给患者造成巨大的痛苦,严重影响患者生存质量。治疗上目前以保守治疗为主,但是效果不令人满意,促进淋巴管的再生被认为是治疗淋巴水肿最直接和有效的方法,本文设想能不能找到一种方法促进淋巴管生成来治疗乳腺癌术后上肢淋巴水肿。 目的：从成人骨髓中分离培养骨髓间充质干细胞并在体外分化诱导成淋巴管内皮细胞。检测分化的淋巴管内皮细胞在体外形成淋巴管的功能。 方法：在髂后上棘采集健康成人骨髓,采用直接贴壁法分离成人骨髓间充质干细胞,用L-DMEM培养基培养扩增至第3-4代,应用流式细胞术检测细胞表面标志CD90、CD105、CD14、CD34、HLA-DR的表达率。把细胞分成两组,一组用EGM-2培养基培养并加入VEGFC-C(156s),另一组只用EGM-2培养基培养,培养7天后,在相差显微镜下观察细胞的形态变化,应用流式细胞术检测淋巴管内皮细胞的特异性标志物Podoplanin、VEGFR-2和VEGFR-3,并应用RT-PCR检测相应基因的表达。用细胞成管实验分别检测实验组和对照组淋巴管内皮细胞形成淋巴管的功能。 结果：贴壁培养的骨髓间充质细胞形态上为纤维细胞样,细胞培养纯化三代后经流式细胞术测定细胞的CD90、CD105的表达率分别为90.03%和97.86%,而CD14、CD34、HLA-DR的阳性率分别为2.00%、2.18%和1.56%。实验组经EGM-2和VEGF-C(156s)诱导刺激后,分离纯化的骨髓间充质干细胞呈鹅卵石样外观,Podoplanin、VEGFR-2和VEGFR-3的表达率分别为26.53%、2.53%和8.55%,而对照组Podoplanin、VEGFR-2和VEGFR-3的表达率分别为2.84%、1.61%和2.3%,两组相比Podoplanin和VEGFR-3具有统计学意义(P0.01),两组间VEGFR-2无统计学意义(P0.05)。RT-PCR结果,试验组可见podoplanin和vegfr-3特异性的条带,未见vegfr-2的特异性条带,对照组只显示了模糊的podoplanin,而未见vegfr-2和vegfr-3的特异性条带。细胞成管实验显示诱导后的淋巴管内皮细胞具有初步形成管状结构的功能。 结论： 1.成功从成人骨髓中分离培养出骨髓间充质干细胞。 2.分离培养的骨髓间充质干细胞在内皮细胞培养基EGM-2和VEGF-C(156s)的诱导下,可分化为淋巴管内皮细胞。 3.诱导后的淋巴管内皮细胞具有初步形成淋巴管的功能。 4.骨髓间充质干细胞在促进淋巴管生成方面有很好的应用前景。
[Abstract]:BACKGROUND: Bone marrow mesenchymal stem cells (MSCs), as a kind of non-hematopoietic stem cells in bone marrow, have the potential of self-renewal and multi-directional differentiation. Recently, they have been found to have low immunogenicity, so they have attracted wide attention in the field of clinical medicine. At present, MSCs have been widely used in research and treatment. Various ischemic or injury diseases and organ transplantation have achieved preliminary results, and stem cell replacement therapy is expected to become a new clinical treatment.
Lymphedema of the affected upper extremity after breast cancer surgery is a chronic progressive disease. The main reason is that lymphatic drainage of the upper extremity is impaired during axillary lymph node resection or sentinel lymph node biopsy, resulting in lymphatic reflux obstruction, lymph accumulation in the subcutaneous, late stage of the disease, swelling of the upper extremity, loss of function, causing great pain to the patient. At present, conservative treatment is the main treatment, but the effect is unsatisfactory. Promoting lymphatic regeneration is considered to be the most direct and effective method to treat lymphedema. This article envisages that we can find a way to promote lymphangiogenesis to treat upper limb lymphedema after breast cancer surgery.
AIM: To isolate and culture bone marrow mesenchymal stem cells (BMSCs) from adult bone marrow and differentiate into lymphatic endothelial cells in vitro.
Methods: Adult bone marrow mesenchymal stem cells (BMSCs) were isolated from the posterior superior iliac spine by direct adherence method. The expression of CD90, CD105, CD14, CD34 and HLA-DR on the cell surface was detected by flow cytometry. The cells were divided into two groups. One group was cultured in EGM-2 medium and added with VEGF. C-C (156 s), another group was cultured in EGM-2 medium for 7 days. The morphological changes of the cells were observed under phase contrast microscope. The specific markers of lymphatic endothelial cells, Podoplanin, VEGFR-2 and VEGFR-3, were detected by flow cytometry, and the expression of the corresponding genes was detected by RT-PCR. Lymphatic endothelial cells were used to form lymphatic vessels.
Results: The adherent cultured bone marrow mesenchymal cells were fibroblast-like in morphology. The expression of CD90, CD105 and CD14, CD34, HLA-DR were 90.03%, 97.86% and 2.00%, 2.18% and 1.56% respectively by flow cytometry after three generations of cell culture and purification. After stimulation by EGM-2 and VEGF-C (156 s), the experimental group was isolated and purified. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 were 26.53%, 2.53% and 8.55% respectively. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 in control group were 2.84%, 1.61% and 2.3% respectively. There was no significant difference between the two groups (P The results of RT-PCR showed that there were specific bands of Podoplanin and VEGFR-3 in the experimental group, but no specific bands of VEGFR-2 in the experimental group. The control group showed only vague bands of podoplanin, but no specific bands of VEGFR-2 and VEGFR-3 in the control group.
Conclusion:
1. bone marrow mesenchymal stem cells were successfully isolated from adult bone marrow.
2. Bone marrow mesenchymal stem cells can differentiate into lymphatic endothelial cells induced by EGM-2 and VEGF-C (156 s).
3. the lymphatic endothelial cells after induction have the function of forming lymphatic vessels preliminarily.
4. bone marrow mesenchymal stem cells have a good application prospect in promoting lymphangiogenesis.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
本文编号：2241595
[Abstract]:BACKGROUND: Bone marrow mesenchymal stem cells (MSCs), as a kind of non-hematopoietic stem cells in bone marrow, have the potential of self-renewal and multi-directional differentiation. Recently, they have been found to have low immunogenicity, so they have attracted wide attention in the field of clinical medicine. At present, MSCs have been widely used in research and treatment. Various ischemic or injury diseases and organ transplantation have achieved preliminary results, and stem cell replacement therapy is expected to become a new clinical treatment.
Lymphedema of the affected upper extremity after breast cancer surgery is a chronic progressive disease. The main reason is that lymphatic drainage of the upper extremity is impaired during axillary lymph node resection or sentinel lymph node biopsy, resulting in lymphatic reflux obstruction, lymph accumulation in the subcutaneous, late stage of the disease, swelling of the upper extremity, loss of function, causing great pain to the patient. At present, conservative treatment is the main treatment, but the effect is unsatisfactory. Promoting lymphatic regeneration is considered to be the most direct and effective method to treat lymphedema. This article envisages that we can find a way to promote lymphangiogenesis to treat upper limb lymphedema after breast cancer surgery.
AIM: To isolate and culture bone marrow mesenchymal stem cells (BMSCs) from adult bone marrow and differentiate into lymphatic endothelial cells in vitro.
Methods: Adult bone marrow mesenchymal stem cells (BMSCs) were isolated from the posterior superior iliac spine by direct adherence method. The expression of CD90, CD105, CD14, CD34 and HLA-DR on the cell surface was detected by flow cytometry. The cells were divided into two groups. One group was cultured in EGM-2 medium and added with VEGF. C-C (156 s), another group was cultured in EGM-2 medium for 7 days. The morphological changes of the cells were observed under phase contrast microscope. The specific markers of lymphatic endothelial cells, Podoplanin, VEGFR-2 and VEGFR-3, were detected by flow cytometry, and the expression of the corresponding genes was detected by RT-PCR. Lymphatic endothelial cells were used to form lymphatic vessels.
Results: The adherent cultured bone marrow mesenchymal cells were fibroblast-like in morphology. The expression of CD90, CD105 and CD14, CD34, HLA-DR were 90.03%, 97.86% and 2.00%, 2.18% and 1.56% respectively by flow cytometry after three generations of cell culture and purification. After stimulation by EGM-2 and VEGF-C (156 s), the experimental group was isolated and purified. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 were 26.53%, 2.53% and 8.55% respectively. The expression rates of Podoplanin, VEGFR-2 and VEGFR-3 in control group were 2.84%, 1.61% and 2.3% respectively. There was no significant difference between the two groups (P The results of RT-PCR showed that there were specific bands of Podoplanin and VEGFR-3 in the experimental group, but no specific bands of VEGFR-2 in the experimental group. The control group showed only vague bands of podoplanin, but no specific bands of VEGFR-2 and VEGFR-3 in the control group.
Conclusion:
1. bone marrow mesenchymal stem cells were successfully isolated from adult bone marrow.
2. Bone marrow mesenchymal stem cells can differentiate into lymphatic endothelial cells induced by EGM-2 and VEGF-C (156 s).
3. the lymphatic endothelial cells after induction have the function of forming lymphatic vessels preliminarily.
4. bone marrow mesenchymal stem cells have a good application prospect in promoting lymphangiogenesis.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329
【参考文献】
相关期刊论文 前3条
1 路艳蒙,傅文玉,朴英杰,乔东访,安连兵;人骨髓间充质干细胞的超微结构[J];电子显微学报;2002年04期
2 梁艳红;张肇林;田铧;王长明;王世坤;李鑫;宋涛;;单核细胞向淋巴管内皮细胞诱导分化的潜能(英文)[J];中国组织工程研究与临床康复;2010年10期
3 柯金勇;林艳娟;;骨髓间充质干细胞生物学特性的研究[J];医学综述;2008年15期
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