机械牵张和间接共培养诱导骨髓间充质干细胞向韧带细胞分化的体外研究
发布时间：2019-07-03 09:30
【摘要】： 研究背景 韧带在维持关节稳定，协调关节运动中发挥着重要作用。外伤、长期反复劳累过度等会造成韧带损伤，但由于韧带组织血供少，损伤后很难完全愈合，最终造成关节功能严重障碍的病例。韧带损伤也使得很多优秀的运动员就此葬送了运动生涯。现代外科的发展已使人类替换病损韧带成为现实，外科使用过的替换物包括异种韧带、同种异体韧带、自体韧带和人工合成材科等，但这些措施的远期效果都不够理想。组织工程学的诞生使体外构建有活性的生物韧带的研制成为可能，为这一疾病的治疗提供了新途径 细胞成分对提高韧带的力学负载的承受力，基因激活和韧带自我更新和调节的能力非常重要。但如何选择理想的种子细胞?种子细胞需要怎样的微环境才能达到预期的功能要求?这些问题都有待进一步研究。 本试验的目的是：探索在体外条件下如何诱导BMSCs向韧带成纤维细胞分化。因此，本实验中我们采用与韧带成纤维细胞间接共培养和力学刺激两种方法，研究体外条件下诱导BMSCs向韧带成纤维细胞转化，试图为体外诱导BMSCs向韧带成纤维细胞转化提供可行的方法，为韧带组织工程学种子细胞的研究提供思路。 研究方法 1．采用Percoll液密度梯度离心法和传代贴壁筛选法相结合分离获得骨髓间充质细胞，建立大鼠BMSCs(rBMSCs)和人BMSCs(hBMSCs)体外培养的方法；通过形态学方法与细胞表面特异抗原流式细胞术检测方法，对在体外所培养的rBMSCs和hBMSCs进行鉴定；采用体外诱导rBMSCs和hBMSCs向成骨细胞和成脂细胞分化的方法，对BMSCs的干细胞生物学功能进行鉴定。 2．实时荧光定量PCR检测第一代到第六代大鼠BMSCsⅠ型胶原蛋白，Ⅲ型胶原蛋白和韧粘素-C mRNA的表达。 3．将大鼠BMSCs和大鼠韧带成纤维细胞间接共培养3、6、12天，用实时荧光定量PCR检测，间接共培养后BMSCs三种韧带特性蛋白(Ⅰ型胶原蛋白，，Ⅲ型胶原蛋白和韧粘素-C)的mRNA表达；竞争放免法和免疫组化的方法，检测细胞这三种蛋白合成。 4．对大鼠BMSCs施以10％，1赫兹的周期性牵张刺激不同时段后，分析细胞的变形和重排；并用实时荧光定量PCR检测牵张不同时间段后，其Ⅰ型、Ⅲ型胶原和韧粘素-C mRNA的表达；用竞争放免法和免疫组化，检测细胞这三种蛋白合成；激光共聚焦显微镜观察力学刺激对细胞骨架的影响。 实验结果 1．成功获得了rBMSCs和hBMSCs两种干细胞。我们获得的细胞具有干细胞的形态学特征(核大浆少)；细胞表面特异性抗原鉴定(rBMSCs CD44、CD90表达阳性而CD34、CD45表达阴性，hBMSCs CD44表达阳性CD14、CD45表达阴性)，且这些特征在第一代到第六代的细胞中稳定；rBMSCs和hBMSCs这两种细胞具有向成骨细胞和脂肪细胞分化的能力。综合上述三个特点，我们认为实验中分离培养的细胞为骨髓间充质干细胞。 2．Ⅰ型胶原、Ⅲ型胶原和韧粘素-C mRNA在第一代到第六代大鼠BMSCs中均稳定表达。 3．间接共培养6天，BMSCsⅠ型胶原和Ⅲ型胶原的mRNA表达分别是对照组的2.0和2.2倍，Ⅰ型胶原和Ⅲ型胶原mRNA与内参照GAPDH的相对表达量在间接共培养6天组分别为：3.9±0.2和1.9±0.2，对照组分别为：1.9±0.3和0.8±0.1，间接共培养组与对照组相比有统计学差异(P＜0.05)而韧粘素-C的表达无明显改变。间接共培养12天，Ⅰ型和Ⅲ型胶原的蛋白含量增加，分别从对照组的12.4±0.8 ng／μg和5.0±0.4 ng／μg增加到间接共培养组的13.6±1.3 ng／μg和5.9±0.5 ng／μg，间接共培养组与对照组相比有统计学差异(P＜0.05)；韧粘素-C mRNA的表达，为对照组的2.0倍，对照组和间接共培养组的相对表达量分别为0.07±0.02和0.14±0.02。 4．力学刺激使细胞胞体较对照组变细长，细胞重新定向排列在远离牵张力方向60～80°和100～130°这两个以90°轴对称的区域。力学刺激12小时后，BMSCsⅠ型胶原和Ⅲ型胶原mRNA的表达与对照组相比增高有显著差异(P＜0.05)；24小时后，两种胶原蛋白的合成有统计学差别。韧粘素-C mRNA的表达在力学作用24h和36h后，分别增高到对照组的2.65±0.03和2.89±0.04倍；细胞骨架在力学刺激作用下亦发生了改变，F-actin随牵张时间增长，含量逐渐减少。 结论 1．我们分离获得的rBMSCs和hBMSCs细胞具有向成骨细胞和脂肪细胞分化的能力，且它们表面特征分子和胶原蛋白，在第一代到第六代的细胞中稳定表达。 2．Ⅰ型胶原、Ⅲ型胶原和韧粘素-C mRNA在第一代到第六代BMSCs中稳定表达。 3．与韧带成纤维细胞间接共培养，可以促进BMSCsⅠ型、Ⅲ型胶原蛋白和韧粘素-C的合成。 4．力学刺激可以促进BMSCs合成Ⅰ型胶原、Ⅲ型胶原和韧粘素-CmRNA的表达及Ⅰ型和Ⅲ型胶原蛋白的合成，使细胞重排，细胞骨架发生改变。
[Abstract]:Study Background The ligament plays a role in maintaining the stability of the joint and coordinating the joint movement. It is important to play an important role in the injury of the ligament, which can cause the damage of the ligament, but due to the small blood supply of the ligament, it is difficult to completely heal after the injury, and finally, the joint function is serious. The case of the disorder. The injury of the ligament also causes many excellent athletes to be buried in this place The development of modern surgery has made it a reality to replace the disease-damaged ligament, and the surgical-used replacement includes the different ligament, the allogenic ligament, the autograft and the synthetic material, but the long-term effect of these measures It is not ideal. The birth of tissue engineering makes it possible to build an active biological ligament in vitro and to treat the disease. The invention provides a new way cell component, which is used for improving the bearing force of the mechanical load of the ligament, the gene activation and the ligament self more, The ability to new and adjust is very important. How to select the ideal seed cells? What micro-environment is needed for seed cells? In order to meet the expected functional requirements? Some questions need to be further studied. The purpose of this test is to explore how in vitro conditions BMSCs were induced to differentiate into the ligament fibroblasts. Therefore, in this experiment we used two methods of indirect co-culture and mechanical stimulation with the ligament fibroblasts, and the transformation of BMSCs into the ligament fibroblasts under the condition of in vitro was studied, and the BMSCs were induced to induce BMSCs in vitro. to provide a viable method for the transformation of the ligament fibroblasts, band-organized man Methods 1. The method of study was provided for the study of seed cells.1. The bone marrow-derived mesenchymal cells were obtained by the combination of Percoll liquid density gradient centrifugation and passage-attached screening method, and the in vitro culture of BMSCs (rBMSCs) and human BMSCs (hBMSCs) in rats was established. The method comprises the following steps of: carrying out identification on the rBMSCs and hBMSCs cultured in vitro by using a morphological method and a cell surface specific antigen flow cytometry detection method; and inducing the rBMSCs and hBMSCs into the osteoblast and the hBMSCs in vitro. the biological function of the stem cells of the BMSCs is identified by the method of the differentiation of the adipocyte,2. the real-time fluorescence quantitative PCR detection first generation to the second generation 3,6 and 12 days of BMSCs and the rat's ligament fibroblasts were co-cultured for 3,6 and 12 days, and the three kinds of ligament characteristic proteins of BMSCs were detected by real-time fluorescence quantitative PCR (RT-PCR) and indirect co-culture. Type I collagen, type III collagen egg 4. The expression of the mRNA of BMSCs in the rat and the method of immunohistochemistry was used to detect the synthesis of the three proteins.4. After 10% and 1-Hz periodic stretch of BMSCs were applied to the rat BMSCs for different periods, the deformation and rearrangement of the cells were analyzed. The Expression of Type I, Type 鈪
本文编号：2509276
[Abstract]:Study Background The ligament plays a role in maintaining the stability of the joint and coordinating the joint movement. It is important to play an important role in the injury of the ligament, which can cause the damage of the ligament, but due to the small blood supply of the ligament, it is difficult to completely heal after the injury, and finally, the joint function is serious. The case of the disorder. The injury of the ligament also causes many excellent athletes to be buried in this place The development of modern surgery has made it a reality to replace the disease-damaged ligament, and the surgical-used replacement includes the different ligament, the allogenic ligament, the autograft and the synthetic material, but the long-term effect of these measures It is not ideal. The birth of tissue engineering makes it possible to build an active biological ligament in vitro and to treat the disease. The invention provides a new way cell component, which is used for improving the bearing force of the mechanical load of the ligament, the gene activation and the ligament self more, The ability to new and adjust is very important. How to select the ideal seed cells? What micro-environment is needed for seed cells? In order to meet the expected functional requirements? Some questions need to be further studied. The purpose of this test is to explore how in vitro conditions BMSCs were induced to differentiate into the ligament fibroblasts. Therefore, in this experiment we used two methods of indirect co-culture and mechanical stimulation with the ligament fibroblasts, and the transformation of BMSCs into the ligament fibroblasts under the condition of in vitro was studied, and the BMSCs were induced to induce BMSCs in vitro. to provide a viable method for the transformation of the ligament fibroblasts, band-organized man Methods 1. The method of study was provided for the study of seed cells.1. The bone marrow-derived mesenchymal cells were obtained by the combination of Percoll liquid density gradient centrifugation and passage-attached screening method, and the in vitro culture of BMSCs (rBMSCs) and human BMSCs (hBMSCs) in rats was established. The method comprises the following steps of: carrying out identification on the rBMSCs and hBMSCs cultured in vitro by using a morphological method and a cell surface specific antigen flow cytometry detection method; and inducing the rBMSCs and hBMSCs into the osteoblast and the hBMSCs in vitro. the biological function of the stem cells of the BMSCs is identified by the method of the differentiation of the adipocyte,2. the real-time fluorescence quantitative PCR detection first generation to the second generation 3,6 and 12 days of BMSCs and the rat's ligament fibroblasts were co-cultured for 3,6 and 12 days, and the three kinds of ligament characteristic proteins of BMSCs were detected by real-time fluorescence quantitative PCR (RT-PCR) and indirect co-culture. Type I collagen, type III collagen egg 4. The expression of the mRNA of BMSCs in the rat and the method of immunohistochemistry was used to detect the synthesis of the three proteins.4. After 10% and 1-Hz periodic stretch of BMSCs were applied to the rat BMSCs for different periods, the deformation and rearrangement of the cells were analyzed. The Expression of Type I, Type 鈪
本文编号：2509276
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