两种不同分离、纯化方法对SD大鼠BMSCs生物学特性影响的研究

发布时间：2018-03-05 20:40

本文选题：骨髓　切入点：间充质干细胞　出处：《泰山医学院》2011年硕士论文　论文类型：学位论文

【摘要】：目的骨髓间充质干细胞(BMSCs)是一种存在骨髓中,来源于中胚层,且具有多向分化潜力的成体干细胞。在特定的诱导条件下可以向中胚层来源的各种细胞,如成骨细胞、脂肪细胞、肌细胞分化。近年来随着对BMSCs的深入研究,发现BMSCs也具有跨胚层分化的潜能,BMSCs移植入活体的中枢神经系统内可以存活并可分化为神经元细胞、神经胶质细胞,体外实验已证实在一定的诱导条件下BMSCs向神经元细胞、神经胶质细胞转化,可不同程度的改善颅脑损伤、脑卒中等疾病的神经功能的缺损状态。因此,BMSCs是一种十分理想的移植治疗神经系统疾病的细胞,具有十分重要的临床应用价值与前景。本研究目的就是探讨通过比较观察使用①密度梯度离心提取+胎牛血清贴壁培养法②全骨髓胎牛血清贴壁培养法这两种方法提取、培养SD大鼠的骨髓间充质干细胞的细胞形态、生物学特性、细胞表型等生长增殖和生物活性各方面有无差异,从而评价两种方法的优势与劣势,以期待寻找一种更好的提取、培养骨髓间充质干细胞的方法。方法在无菌的条件下用密度梯度离心法与全骨髓分别提取4-6周龄的SD大鼠的左、右下肢的BMSCs,继而用含有10%胎牛血清的LG-DMEM(低糖DMEM)培养扩增BMSCs。用密度梯度离心提取出的原代BMSCs再经胎牛血清贴壁培养法传代后可得到较纯化的可大量扩增的BMSCs细胞系(得到的细胞为A组);无菌条件下取全骨髓在胎牛血清中贴壁培养后经换液、传代也可得到大量的BMSCs(得到的细胞为B组)。流式细胞仪检测密度梯度离心法与全骨髓两种方法提取培养的细胞的CD29+,CD44-,CD45-的表达,鉴定所培养的BMSCs,绘制BMSCs的生长曲线,并比较两组BMSCs的细胞形态、冻存复苏BMSCs后存活率和生长状态等。结果 1.A组与B组的BMSCs生物性状皆较稳定,初培养时细胞皆为圆形,传代稳定后细胞呈BMSCs典型的梭形,并以漩涡状排列成长;有典型的特征性的贴壁生长; 2.经流式细胞仪检测A组与B组的BMSCs细胞表面标记物:CD29+CD44-CD45-,结果A组CD29(+93.24%),CD44(-94.90%),CD45(-94.22%);B组CD29(+92.89%),CD44-(96.70%),CD45-(93.10%),均符合BMSCs的特征; 3.A组与B组BMSCs均贴壁生长,原代台盼蓝染色检测细胞活性A组x为89.19%,贴壁法(B组) x为95.27%,并使用统计学软件SPSS16.0进行统计学分析,差异存在统计学意义; 4.A组细胞生长特性:BMSCs贴壁生长,原代培养10天达到90%融合,传代后的BMSCs增殖速度较原代(P0)培养增快,第三代(P3)的BMSCs已基本纯化,BMSCs呈典型的梭形,旋涡状排列生长,随着传代,细胞增殖分化能力减弱;B组细胞不同于A组在于:P0细胞为全骨髓细胞,换液,传代过程是大幅度纯化的主要方法,第四代(P4)细胞镜下观察才可达到基本纯化,但得到的细胞的活性却强于A组。结论 1.密度梯度离心提取和全骨髓胎牛血清贴壁培养这两种方法提取、培养的细胞经流式细胞仪进行表面标志物鉴定符合BMSCs的特征;获得的BMSCs纯度较高,在显微镜下观察两组BMSCs的形态及生长状态相似; 2.密度梯度离心提取间充质干细胞细胞活力低于全骨髓法(存在统计学差异),这种差异可能与过程中的多次离心及洗涤对细胞造成丢失及损伤等原因有关; 3.全骨髓法与密度梯度离心提取法相比操作简单,花费较小,密度梯度离心提取法适于短时间内要求得到纯度较高的间充质干细胞细胞。
[Abstract]:objective
Bone marrow mesenchymal stem cells (BMSCs) is a kind of bone marrow, derived from the mesoderm, and multilineage differentiation potential of adult stem cells. In certain conditions it can induce mesoderm from a variety of cells, such as osteoblasts, adipocytes and muscle cells. In recent years, with in-depth study of BMSCs the BMSCs also has the differentiation potential, BMSCs transplantation into the central nervous system in vivo can survive and can differentiate into neurons and glial cells, in vitro experiments have confirmed that under the condition of certain induction of BMSCs cells into neurons, glial cell transformation, can improve the brain injury in different degree, defect the state of stroke and other neurological diseases. Therefore, BMSCs is a kind of ideal transplantation for the treatment of diseases of the nervous system cells, has clinical application value and prospect is very important. The purpose of this study Is explored through comparative observation using the density gradient centrifugation extraction + fetal bovine serum adherent culture of whole bone marrow adherent culture method for fetal bovine serum of these two kinds of extraction methods, cultivation of SD rat bone marrow mesenchymal stem cells in cell morphology and biological characteristics, there are no differences in cell proliferation and phenotype. The biological activity, so as to evaluate the advantages and disadvantages of the two methods, in order to expect to find a better extraction of cultured bone marrow mesenchymal stem cells.
Method
Under sterile conditions by density gradient centrifugation and whole bone marrow were extracted from SD rats aged 4-6 weeks left and right lower limb BMSCs, then with LG-DMEM containing 10% fetal bovine serum (low glucose DMEM) culturing BMSCs. extracted by density gradient centrifugation after primary BMSCs fetal bovine serum adherent after passage culture method can get more purified BMSCs cells can be greatly amplified (the obtained cells were A group); under sterile conditions from whole bone marrow in fetal bovine serum in adherent culture after the change of liquid passage, can also get a lot of BMSCs (the B cell group) by flow cytometry. The detection method of density gradient centrifugation and two different extraction methods of whole bone marrow cells cultured in CD29+, CD44-, CD45- expression and identification of the cultured BMSCs, draw the growth curve of BMSCs, and compared between the two groups of BMSCs cells, cryopreservation and recovery after BMSCs survival rate and growth state.
Result
The BMSCs biological characteristics of 1.A group and B group were all stable. The cells in initial culture were all round. After passage, the cells were BMSCs typical spindle shape and arranged in a whirlpool pattern.
2., flow cytometry was used to detect the surface markers of BMSCs cells in group A and group B: CD29+CD44-CD45-, results A group CD29 (+93.24%), CD44 (-94.90%), CD45 (-94.22%), and -94.90% group, (96.70%), (93.10%) were all consistent with the characteristics of "Qi".
In group 3.A and group B, BMSCs was adhered to growth. Primary trypan blue staining was used to detect cell viability. A group X was 89.19%, adherence method (B group) x was 95.27%, and statistical software SPSS16.0 was used for statistical analysis, the difference was statistically significant.
The growth characteristics of 4.A group: BMSCs cells adherent growth, cultured for 10 days to reach the 90% fusion, the proliferation rate of BMSCs after passage than the primary (P0) develop faster, third generation (P3) BMSCs has been purified, BMSCs showed typical spindle shape, spiral arrangement growth, along with the passage, weakened the proliferation and differentiation of cells; A group is different from the B group cells: P0 cells for bone marrow cells was changed, the process is the main passage substantially purified
Methods the basic purification was achieved under the fourth generation (P4) cell microscope, but the activity of the obtained cells was stronger than that of the A group.
conclusion
1. density gradient centrifugation extraction and whole bone marrow adherent culture of fetal bovine serum from these two methods, the cultured cells by flow cytometry for surface markers with the characteristics of BMSCs; BMSCs get high purity, observe the morphology and growth state of two groups are similar to that of BMSCs under the microscope;
The cell viability of mesenchymal stem cells extracted by 2. density gradient centrifugation is lower than that of the whole bone marrow method.
3., compared with the density gradient centrifugation, the whole bone marrow extraction method is simpler and less expensive. The density gradient centrifugation method is suitable for obtaining high purity mesenchymal stem cells in a short time.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

【参考文献】