上调表达ChM-Ⅰ的大鼠MSCs向软骨细胞分化的研究

发布时间：2018-06-07 21:31

本文选题：软骨调节素-I + 生物因子　；参考：《大连医科大学》2012年硕士论文

【摘要】：研究目的：探索ChM－Ⅰ的上调表达诱导MSCs向软骨细胞方向分化的作用。研究方法： 1.密度梯度离心法获得大鼠MSCs，传代培养并扩增。使用流式细胞仪检测细胞表面抗原（CD90、CD29）。将冻存的稳转pcDNA3.1（+）/ChM－Ⅰ质粒的MSCs和稳转pcDNA3.1（+）质粒的MSCs复苏及扩培。 2.高糖DMEM无血清特定培养诱导液（含10ng/mlTGF-β1、100nmol／L地塞米松、50umol／ml抗坏血酸、1％ITS） 3.实验分六组（1）：实验1组（稳定转染表达载体pcDNA3.1(+)/ChM-Ⅰ的MSCs细胞组）（2）：实验2组（稳定转染表达载体pcDNA3.1(+)/ChM-Ⅰ的MSCs细胞＋诱导液组）(3)：对照1组（正常MSCs组）(4)：对照2组（正常MSCs+诱导液组）(5)：对照3组（稳定转染表达载体pcDNA3.1（+）的MSCs细胞组）(6)：对照4组(稳定转染表达载体pcDNA3.1（+）的MSCs＋诱导液组),细胞培养至7天、14天、21天三个时间点，通过使用倒置显微镜观察细胞形态的改变；甲苯胺蓝染色检测软骨基质的分泌情况；免疫细胞化学检测软骨特异性Ⅱ型胶原的表达情况。通过以上实验方法说明表达ChM-Ⅰ的大鼠MSCs细胞株具有向软骨细胞分化的能力。研究结果： 1. MSCs细胞株的扩增培养及鉴定：单细胞悬液接种48h后换液，倒置显微镜观察并拍照：可于培养皿底部见到呈梭形或三角形的贴壁细胞，培养11～13天后培养皿底部细胞密度达到85%以上。通过传代，细胞在6h内可达80%以上贴壁，，细胞生长速度加快，可见细胞呈成纤维细胞样生长，折光性较强，群落呈鱼群样。流式细胞仪鉴定结果：MSCs阳性表面抗原CD90、CD29的表达率分别为99.79%、98.41%；MSCs阴性表面抗原CD45、CD11b/c的表达率分别为2.77%、2.51%。提示MSCs达到实验要求。 2.冻存的稳定转染表达载体pcDNA3.1(+)/ChM-Ⅰ的MSCs细胞组和稳定转染表达载体pcDNA3.1（+）的MSCs细胞组复苏及扩培：常规复苏后，通过增值、传代，细胞生长旺盛，排列密集整齐，呈编织状、旋涡状。 3.六组细胞甲苯胺蓝染色和Ⅱ型胶原免疫组化结果及分析：实验2组诱导后的细胞和3个对照组的细胞由梭形向多边形转变。甲苯胺蓝染色和Ⅱ型胶原免疫组化结果显示：对照1、3组检测的特异性软骨基质的分泌和软骨Ⅱ型胶原的表达均为阴性；实验1组和对照2、4组均检测存在特异性软骨基质的分泌和软骨型Ⅱ胶原的表达，但实验1组为弱阳性，对照2、4组为阳性，说明ChM-Ⅰ的上调表达可诱导MSCs向软骨细胞分化，但不及TGF-β1的效率；实验2组检测软骨基质的分泌和软骨Ⅱ型胶原的表达呈强阳性，优于对照2、4组；实验2组和实验1组均能检测到特异性软骨基质的分泌和软骨Ⅱ型胶原的表达，且实验2组优于实验1组；说明稳定转染表达载体pcDNA3.1(+)/ChM-Ⅰ的MSCs细胞在TGF-β1的协同作用下向软骨细胞方向转化的能力显著提高。研究结论： 1.本实验证明上调表达ChM-Ⅰ可诱导MSCs向软骨细胞分化。 2.在TGF-β1的协同作用下，上调表达ChM-Ⅰ的MSCs可提高向软骨细胞分化的能力。 3.多重表达载体和多种生物因子联合诱导MSCs可能成为构建组织工程软骨的重要方式。
[Abstract]:Objective: To explore the effect of up regulation of the expression of MSCs on the differentiation of chondrocytes into chondrocytes.
Research methods:
The rat MSCs was obtained by 1. density gradient centrifugation. The cell surface antigen (CD90, CD29) was detected by flow cytometry. The MSCs of the frozen pcDNA3.1 (+) /ChM I plasmid and the MSCs recovery and expansion of the stable pcDNA3.1 (+) plasmid were obtained.
2. high glucose DMEM serum-free specific culture inducing liquid (containing 10ng/mlTGF- beta 1100nmol / L dexamethasone, 50umol / ml ascorbic acid, 1%ITS)
3. the experiment was divided into six groups (1): the 1 group (2) of the stable transfected expression vector pcDNA3.1 (+) /ChM- I (2): the 2 groups (3) of the MSCs cells of the stable transfection vector pcDNA3.1 (+) /ChM- I (3): the control 1 (normal MSCs group) (4): the control 2 group (5): the control 3 group (stable transfection expression vector pcD) NA3.1 (+) MSCs cell group) (6): control 4 groups (stably transfected expression vector pcDNA3.1 (+) MSCs + inducer group), cell culture to 7 days, 14 days, 21 days at three time points, by using inverted microscope to observe cell morphological changes; toluidine blue staining detection of cartilage matrix secretion; immunocytochemical detection of cartilage specificity The expression of type II collagen indicates that the MSCs cell line expressing ChM- I has the ability to differentiate into chondrocytes.
The results of the study:
Amplification and identification of 1. MSCs cell lines:
The single cell suspension was inoculated with 48h after inoculation, and the inverted microscope was observed and photographed: the cell density of the cell at the bottom of the culture dish could be seen at the bottom of the culture dish. The cell density at the bottom of the culture dish reached over 85% after 11~13 days. Through the passage, the cells could be more than 80% in the 6h, and the cell growth speed was quicker and the cells were fibroblasts. MSCs positive surface antigen CD90, CD29 expression rate was 99.79%, 98.41%, MSCs negative surface antigen CD45, CD11b/c expression rate was 2.77% respectively, 2.51%. suggesting MSCs reached the experimental requirements.
2. cryopreserved transfected expression vector pcDNA3.1 (+) /ChM- I MSCs cell group and stable transfection expression vector pcDNA3.1 (+) MSCs cell group resuscitation and expansion:
After regular resuscitation, the cells grew vigorously and arranged densely and neatly, and knitted and whirlpool shaped.
3. six groups of toluidine blue staining and type II collagen immunohistochemical results and analysis: the cells in the 2 groups and the 3 control groups were transformed from spindle to polygon. The results of toluidine blue staining and type II collagen immunohistochemical staining showed that the specific cartilage matrix secretion and cartilage type II collagen expression in the control group were detected in the control group 1,3. Both of the 1 groups and the control 2,4 groups detected the secretion of specific cartilage matrix and the expression of cartilage type II collagen, but the 1 groups were weak positive and the control group 2,4 was positive, indicating that the up-regulated expression of ChM- I could induce the differentiation of MSCs into chondrocytes, but not the efficiency of TGF- beta 1; the 2 groups of experiments detected the secretion and soft tissue of cartilage matrix. The expression of type II collagen was strongly positive, superior to the control group 2,4, and the 2 and 1 experimental groups were able to detect the secretion of the specific cartilage matrix and the expression of collagen type II collagen, and the 2 groups were superior to the experimental group 1, indicating that the stable transfected expression vector, pcDNA3.1 (+) /ChM- I, was directed to the cartilage cell side under the synergistic effect of TGF- beta 1. The ability to convert to transformation is significantly improved.
The conclusions are as follows:
1. this experiment showed that the expression of ChM- I could induce MSCs to differentiate into chondrocytes.
2. under the synergistic effect of TGF- beta 1, up regulation of ChM- I MSCs can enhance the ability to differentiate into chondrocytes.
3. the combined expression of multiple expression vectors and various biological factors can induce MSCs to become an important way to construct tissue-engineered cartilage.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08

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