不同层软骨细胞与骨髓间充质干细胞在体内外三维共培养时细胞外基质特点的实验研究

发布时间：2018-04-29 02:43

本文选题：分层软骨 + 分层软骨细胞　；参考：《大连医科大学》2017年博士论文

【摘要】：研究背景:正常的关节软骨是一种不均一的分层组织,这种分层结构对维持关节软骨的结构和功能至关重要。自体软骨移植被认为是治疗软骨缺损最常用而且最有效的手段。但是,自体软骨细胞移植并未将关节软骨不同层之间的天然差异加入考虑。最近越来越多的研究着重于再生分层软骨,而不只是再生均一一致的软骨组织。使用不同层的软骨细胞再生相应的分层是其中一种手段之一。不同层的软骨细胞能够通过组织学水平分离,并且能够在体外维持一定的分层特性。在体外研究中发现使用不同层软骨细胞分别再生相应层的软骨组织能够维持不同层相应的特性。但是不同层的软骨细胞在体外短暂单层培养扩增就失去其原有的分层特性,而且很难再次重新获得这种分层特性。那些使用不同层软骨细胞修复软骨损伤的研究,均采用低代扩增或者未扩增的不同层软骨细胞。然而,临床上原代软骨细胞必须通过几代扩增后才能获得足够的数量。因此,需要一种方法能够使用较少的短暂扩增或者未扩增的不同层软骨细胞来再生分层软骨。软骨细胞和间充质干细胞共培养修复软骨损伤,被证明是一种有望代替单纯软骨细胞修复软骨损伤的手段。从关节非负重区获得的低代扩增或者未扩增的软骨细胞与骨髓间充质干细胞共培养被证明能够产生与单独软骨细胞成软骨相似的效果。因此,这种方法可以减少软骨细胞的需求数量,这样就可以使用短暂扩增甚至不扩增的软骨细胞用来修复软骨损伤,而这些细胞由于没有去分化过程,所以仍维持分层特性。此外,使用共培养技术时,由于可能不需要软骨细胞扩增,所以软骨损伤修复甚至可以变成一步操作。但是,不同层的软骨细胞与骨髓间充质干细胞共培养后,两者是否能够一起维持不同层的特性仍然不明确。在这篇研究中,我们使用一种常用的体外三维培养体系,海藻酸钠水凝胶体外培养体系,用来研究不同层的软骨细胞和骨髓间充质干细胞共培养后,在体内外是否仍能够维持其不同层的特性。第一部分不同层软骨细胞的分离和体外单层培养环境下分层特性的维持目的:1.提取表层和中深层软骨细胞;2.验证原代表层和中深层软骨细胞之间的基因表达、蛋白合成和增殖速度的差异;3.检测传代后的表层和中深层软骨细胞之间基因表达、蛋白合成和增殖速度的差异;4.分离并验证骨髓间充质干细胞。方法:使用显微取皮刀提取1月龄新西兰兔膝关节股骨面表层约100um厚的软骨组织作为表层软骨细胞来源,剩下的软骨组织作为中深层软骨细胞来源。通过二型胶原酶消化获得原代的表层和中深层软骨细胞。抽取1月龄新西兰兔股骨及胫骨内骨髓,利用骨髓间充质干细胞贴壁的特性分离获得骨髓间充质干细胞。贴壁的骨髓间充质干细胞通过多向诱导证明其多项分化能力。对原代表层和中深层软骨细胞(p0代)进行col2a、acan和prg4基因检测,以gapdh作为内参,使用2-ΔΔct方法进行相对比较。并且将原代表层和中深层软骨细胞加到96孔板中进行单层培养细胞增殖实验。第3、5、7、10天,使用cck-8(cellcountingkit-8)检测增殖率。原代表层和中深层软骨细胞长至90%融合时定义为p1代,以此类推,对p1和p2代同样进行基因和蛋白质水平的检测,并进行增殖速度的检测。结果:发现原代表层和中深层软骨细胞之间存在明显差异,即表层软骨细胞表达更高的prg4基因和col2a基因,中深层软骨细胞表达更高的acan基因。体外单层培养增殖实验发现,中深层软骨细胞增殖速度更快。然而体外扩增一代后,增殖速度差异消失,而基因表达和蛋白水平的差异逐渐缩小,p2代时差异消失。抽取的骨髓贴壁的细胞呈现成纤维细胞样。而扩增后到第三代的骨髓间充质干细胞能够被诱导向脂肪细胞、成骨细胞和软骨细胞方向。结论:1.通过组织学分离的p0代表层软骨细胞和中深层软骨细胞呈现不同的特性;2.随着传代,不同层软骨细胞的基因表达、蛋白水平和增殖速度差异逐渐消失。第二部分不同层软骨细胞与骨髓间充质干细胞体外三维共培养环境下细胞外基质成分的变化目的:1.体外使用海藻酸钠水凝胶三维培养体系将表层和中深层软骨细胞分别与骨髓间充质干细胞共培养;2.检测不同时间点(第1、7、21天)时软骨细胞外基质成分的基因表达情况;3.检测不同时间点(第1、7、21天)时软骨细胞外基质主要成分(gag)含量、dna含量变化以及lubricin分泌情况。方法:将p0代表层或中深层软骨细胞单独或与骨髓间充质干细胞以1:2比例混合后,按6×106/ml细胞浓度和2%海藻酸钠混合,使用注射器一滴一滴将海藻酸钠水凝胶滴入到凝固液(102mm氯化钙)中形成凝固的小珠,加入到成软骨诱导培养基中培养。于第1、7、21天时收获小珠。分别检测基因表达情况和gag、dna含量。取培养第1、7、21天的培养24小时的上清用elisa法测量lubricin浓度。结果:基因表达方面第1天和第7天时,中深层软骨细胞共培养组相比于表层软骨细胞共培养组表达更高的acan基因,更低的prg4基因,而col2a水平无明显差异。不同层软骨细胞组之间也有同样的趋势。培养第7天时中深层软骨细胞共培养组和中深层软骨细胞组分别比表层软骨细胞共培养组和表层软骨细胞组沉积更多的gag含量。但是到体外培养的后期(21天),我们所检测的基因表达和gag含量等都无明显差异。尽管单纯分层软骨细胞组相比于相应的共培养组沉积更多的gag含量(以dna标准化),但是当以初始加入的软骨细胞数量标准化后,第7天时,共培养组相比于相应单纯软骨细胞组沉积相似的gag含量。而第21天时,以初始加入的软骨细胞数量标准化后,共培养组相比于相应单纯软骨细胞组反而沉积更多的gag含量。类似的是,早期(第1、7天)表层软骨细胞组和表层软骨细胞共培养组分别相比于中深层软骨细胞组和中深层软骨细胞共培养组分泌更多的lubricin。但是体外培养第21天时,软骨细胞组和软骨细胞共培养组之间的lubricn浓度无明显差异。结论:1.表层和中深层软骨细胞分别与骨髓间充质干细胞共培养后能够在体外三维培养早期(第1、7天)维持不同层软骨细胞的分泌细胞外基质的能力;2.但是随着体外培养时间的延长(21天),这种分泌细胞外基质能力的差异逐渐消失。第三部分不同层软骨细胞与骨髓间充质干细胞三维共培养小珠裸鼠皮下植入后细胞外基质成分的变化目的:1.将海藻酸钠水凝胶三维共培养小珠植入裸鼠皮下异位成软骨;2.分别在第2、8周时,处死裸鼠,进行组织学和生物化学检测(DNA和GAG定量)。方法:将海藻酸钠凝胶小珠分别植入到14只4-6周龄裸鼠皮下。分别于第2周和第8周处死裸鼠,取出海藻酸钠小珠,进行DNA和GAG定量以及免疫荧光等检测。结果:与体外培养相似,体内第2周时,中深层软骨细胞共培养组相比于表层软骨细胞共培养组沉积更多的GAG。以初始加入的软骨细胞数量标准化后,第2周时共培养组相比于相应单纯软骨细胞组反而沉积更多的GAG成分。免疫荧光结果显示,体内第2周时表层软骨细胞组和表层软骨细胞共培养组分别相比于中深层软骨细胞组和中深层软骨细胞共培养组表达更高水平的Lubricin。但是二型胶原免疫荧光染色的荧光信号强度却没有明显差异,这也与体外实验结果相符合。与体外培养不完全一样的是,体内培养后期(第8周),共培养组和单纯软骨细胞组都沉积了相似的GAG成分(以DNA标准化)。而以初始加入的软骨细胞数量标准化后发现,共培养组沉积的GAG含量明显高于单纯软骨细胞组。此外,体内培养第8周时免疫荧光染色结果发现在共培养组之间Lubricin和二型胶原荧光信号无明显差异。同时通过荧光观察发现,体内培养相比于体外培养呈现出更为明显的细胞与细胞之间的直接接触。结论:1.表层软骨细胞共培养组和中深层软骨细胞共培养组在体内成软骨早期(第2周),能够维持细胞外基质分泌能力的差异;2.在体内成软骨后期(第8周),共培养组之间细胞外基质成分分泌能力的差异消失。
[Abstract]:Background: normal articular cartilage is an uneven stratified tissue, which is essential to maintain the structure and function of articular cartilage. Autologous cartilage transplantation is considered to be the most commonly used and most effective method for the treatment of cartilage defects. However, autologous chondrocyte transplantation does not make the natural difference between the different layers of articular cartilage. More recently, more and more studies have focused on regeneration of stratified cartilage rather than just homogeneous cartilage tissue. It is one of the means to regenerate the corresponding stratification with different layers of chondrocytes. Chondrocytes in different layers can be separated by histological level and can maintain a certain level of delamination in vitro. In vitro, it is found that cartilage tissue using different layers of chondrocytes to regenerate the corresponding layer of cartilage can maintain the corresponding characteristics of different layers. However, it is difficult to regain the stratification characteristic again, and it is difficult to regain the stratification characteristic again. Studies on the repair of cartilage damage by bone cells use different layers of chondrocytes of low generation or non amplification. However, the clinical primary chondrocytes must be amplified by a few generations to obtain sufficient quantities. Therefore, a method can be used to regenerate the layers of different layers of chondrocytes that are short or not expanded. Cartilage, chondrocytes and mesenchymal stem cells co culture and repair cartilage damage, which has been proved to be a promising alternative to repair cartilage damage by simple chondrocytes. The co culture of low generation or unexpanded chondrocytes from the bone marrow mesenchymal stem cells obtained from the non weight-bearing area of the joint has been proved to be capable of producing chondrocytes with individual chondrocytes. A similar effect of cartilage. Therefore, this method can reduce the demand for cartilage cells, which can be used to repair cartilage damage by using short or even non amplification cartilage cells, which still maintain stratification due to the absence of dedifferentiation. In addition, the use of co culture technology may not require cartilage. When cells expand, the repair of cartilage damage can even become a step. However, it is still unclear whether the two layers of chondrocytes can maintain the characteristics of different layers together after co culture with bone marrow mesenchymal stem cells. In this study, we use a commonly used in vitro three-dimensional culture system, sodium alginate hydrogel. The external culture system is used to study the characteristics of different layers of chondrocytes and bone marrow mesenchymal stem cells in vitro and in vitro. The first part is the separation of different layers of cartilage cells and the maintenance of stratification characteristics in the monolayer culture environment: 1. extraction of surface and middle layer cartilage cells; 2. The gene expression between the representative layer and the medium deep cartilage cells, the difference in the protein synthesis and proliferation rate; 3. the gene expression between the surface and the middle deep cartilage cells after the passage was detected, the difference in the protein synthesis and proliferation rate; and 4. to separate and verify the bone marrow mesenchymal stem cells. Method: the 1 month old New Zealand rabbit knee was extracted with a microscoper. The cartilaginous tissue about 100um thick on the surface of the femur surface is the source of the surface cartilage cells. The remaining cartilage tissue is the source of the medium deep cartilage cells. The original surface and middle deep cartilage cells are obtained through the digestion of type two collagenase. The bone marrow of the femur and tibia in 1 month old New Zealand rabbits is extracted and the properties of the bone marrow mesenchymal stem cells are applied to the wall. Bone marrow mesenchymal stem cells were obtained. The adherent bone marrow mesenchymal stem cells showed multiple differentiation ability through multi direction induction. Col2a, acan and PRG4 genes were detected for the original and middle deep cartilage cells (P0 generation). GAPDH was used as the internal parameter and 2- delta delta CT method was used to compare them. The original representative layer and the middle and deep cartilage were compared. Cells were added to the 96 orifice plate for monolayer cell proliferation experiment. On day 3,5,7,10, CCK-8 (cellcountingkit-8) was used to detect the proliferation rate. The original representative layer and the medium deep cartilage cells were defined as the P1 generation when the fusion was long to 90%. By this analogy, the same gene and protein levels were detected for the P1 and P2 generations, and the results of the proliferation rate were detected. It was found that there were obvious differences between the primary and middle layer cartilage cells, that is, the higher expression of PRG4 gene and col2a gene in the surface chondrocytes, and the higher expression of acan gene in the medium deep cartilage cells. The difference in gene expression and protein level gradually diminished and the difference in P2 generation disappeared. The extracted bone marrow cells were fibroblast like cells, and the third generations of bone marrow mesenchymal stem cells could be induced to adipocytes, osteoblasts and chondrocytes. Conclusion: 1. by histologically separated P0 representative layer soft. Bone cells and medium deep cartilage cells showed different characteristics; 2. with the generation, the gene expression of different layers of chondrocytes, protein level and proliferation velocity difference gradually disappeared. Second the changes of extracellular matrix components of different layers of chondrocytes and bone marrow mesenchymal stem cells in vitro: 1. use the sea in vitro The three dimensional culture system of sodium alginate hydrogel co cultured the surface and middle depth chondrocytes with bone marrow mesenchymal stem cells respectively. 2. the expression of gene expression in the extracellular matrix of cartilage was detected at different time points (day 1,7,21). 3. the content of the main component of cartilage extracellular matrix (GAG) at different time points (day 1,7,21) and the content of DNA were changed. Method: after mixing the P0 representative or middle - deep cartilage cells alone or with bone marrow mesenchymal stem cells in the 1:2 ratio, the concentration of 6 x 106/ml cells was mixed with 2% sodium alginate, and the sodium alginate hydrogel was dripped by a drop of syringe into the coagulated droplet in 102mm calcium chloride (102mm calcium chloride). In the culture medium of cartilage induced. The gene expression and gag, DNA content were detected at day 1,7,21. The lubricin concentration was measured by ELISA method for 24 hours culture of culture day 1,7,21. Results: in the first day and seventh day of gene expression, the medium depth chondrocyte co culture group was compared with the surface cartilage. The co culture group expressed a higher acan gene and a lower PRG4 gene, but there was no significant difference in the level of col2a. The same trend was also found between the different layers of chondrocytes. The medium depth chondrocyte co culture group and the medium depth chondrocyte group deposited more GA than the surface chondrocyte co culture group and the surface chondrocyte group at seventh days. G content. But there was no significant difference in gene expression and GAG content in the later period of culture (21 days). Although the simple stratified chondrocyte group had more GAG content than the corresponding co culture group (DNA Standardization), the co culture group was at seventh days when the number of initial chondrocytes was standardized. Compared to the corresponding simple chondrocyte group, the content of gag was similar to that of the corresponding chondrocyte group. On the twenty-first day, the co culture group deposited more GAG content than the corresponding chondrocyte group when the number of initial chondrocytes was standardized. Similarly, the early (day 1,7) surface chondrocyte and surface chondrocyte co culture group compared with the coculture group. More lubricin. was secreted in the medium depth chondrocyte group and the medium depth chondrocyte co culture group, but there was no significant difference in the concentration of lubricn between the chondrocyte and chondrocyte co culture groups for twenty-first days in vitro culture. Conclusion: the 1. surface and middle deep cartilage cells co culture with bone marrow mesenchymal stem cells can be in vitro three after the co culture of bone marrow mesenchymal stem cells. The ability to secrete the extracellular matrix of different layers of chondrocytes at early stage (day 1,7); 2. but with the extension of the time of culture in vitro (21 days), the difference between the extracellular matrix capacity of the secretory cells gradually disappeared. Third parts of different layers of chondrocytes and bone marrow mesenchymal stem cells were co cultured with subcutaneous implanted cells after subcutaneous implantation of nude mice. Changes in the components of the external matrix: 1. the three dimensional co cultured beads of sodium alginate hydrogel were implanted subcutaneously into the subcutaneous subcutaneous cartilage in nude mice; 2. the nude mice were killed at week 2,8, and the tissue and biochemical tests (DNA and GAG quantitative) were performed. Methods: the sodium alginate gel beads were subdivided into 14 4-6 weeks old nude mice. Eighth weeks of death, the nude mice were killed, sodium alginate beads were removed, DNA and GAG quantitative and immunofluorescence were detected. Results: similar to in vitro culture, at second weeks in the body, the medium and deep cartilage cell co culture group deposited more GAG. than the surface chondrocyte co culture group, and after the initial addition of chondrocytes, the co culture was co cultured at second weeks. More GAG components were deposited in the group compared with the corresponding simple chondrocyte group. The immunofluorescence results showed that the surface chondrocyte group and the surface chondrocyte co culture group at second weeks showed a higher level of Lubricin. but two collagen immunofluorescence compared with the medium deep cartilage cell group and the medium deep cartilage cell co culture group. The fluorescence intensity of the light staining was not significantly different, which was also in accordance with the experimental results in vitro. The similar GAG component (DNA Standardization) was deposited in the co culture group and the simple chondrocyte group in the late culture (eighth weeks). The content of GAG in the culture group was significantly higher than that in the simple chondrocyte group. In addition, the immunofluorescence staining results showed that there was no significant difference between Lubricin and type two collagen fluorescent signals between the co culture groups at eighth weeks. Conclusion: 1. the co culture group and the medium deep cartilage cell co culture group and the middle depth chondrocyte co culture group can maintain the difference in the secretion of extracellular matrix in the early stage (second weeks), and 2. in the late stage of the cartilage (eighth weeks), the difference of the secretory ability of the extracellular matrix between the co culture groups disappeared.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R68

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