CBD-PTHrP胶原靶向结合和促BM-MSCs软骨形成的实验研究
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摘要
背景：
近年来，由于软骨组织退行性改变和缺损而引起的诸如腰椎间盘突出症、退行性骨关节炎等在内的退行性骨关节疾病越来越多。关节软骨相关的退化性关节疾病严重影响超过三分之一的世界人口的关节健康。虽然治疗软骨组织缺损的外科技术已得到广泛的研究和发展，但是现存的治疗手段中没有任何一种能从根本上治疗软骨组织缺损。因此，优化关节软骨病变的治疗策略具有很高的社会和经济重要性。随着组织工程技术在骨科领域相关研究的不断深入和发展，为骨科退行性相关疾病的治疗开辟出一条崭新的治疗途径。
目前采用组织工程学技术构建的椎间盘替代物研究尚处于起步阶段，研究主要集中于支架材料、种子细胞和生物活性因子三个方面。骨髓间充质干细胞(Bone marrowmesenchymal stem cells，BM-MSCs)作为一种常用的种子细胞，已广泛应用于组织工程修复中。为克服组织工程中种子细胞不断衰减，传代后种子细胞老化退变，退变的种子细胞生物学功能下降等问题，大量的外源性生长因子加入到支架材料中，以维持种子细胞的生物学功能。但所添加的生长因子由于不具有靶向结合支架材料的能力，不能很好滞留在支架材料中发挥生物学功能，从而不能对种子细胞起到持续性的促进作用。胶原蛋白，尤其是I型胶原蛋白是目前最常用的组织工程支架材料，因此I型胶原是组织工程材料靶向性的重要靶点。
甲状旁腺激素相关蛋白(Parathyroid hormone related protein，PTHrP)是1987年从恶性高血钙肿瘤患者的肿瘤中提取纯化的。它是由141个氯基酸组成，其N-端PTHrP(1-34)与N-端甲状旁腺激素(N-PTH)氨基酸序列具有很高的相同性。PTHrP的生理活性除了类似N-PTH外，还具有其他的生物功能，如促进钙的传递，抑制骨的重吸收，促进骨髓间充质干细胞成软骨分化，同时抑制软骨细胞老化。同时，胶原结合域(collagen-binding domain, CBD)是由七个氨基酸构成的寡肽序列(TKKTLRT)组成，具有与胶原紧密结合的功能，被广泛用于胶原结合相关活性分子的重组表达中。
目的：
在本实验中，我们利用基因重组技术构建了新型生物活性重组蛋白CBD-PTHrP，该重组蛋白具有双重的生物学功能：一方面可以通过其中的PTHrP成分发挥诱导骨髓间充质干细胞成软骨分化和抑制软骨细胞肥大的生物学功能，另一方面该融合蛋白又能够通过CBD序列的偶联，将PTHrP锚定在胶原蛋白支架材料上，从而使PTHrP能对种子细胞起到持续靶向性的作用。因此，利用基因重组技术构建新型生物活性因子解决种子细胞在实验中易老化退变的问题能为构建新型软骨组织工程替代材料奠定基础。
方法：
1、CBD-PTHrP重组融合蛋白的表达、纯化和胶原靶向性结合能力鉴定
将CBD的编码序列构建到引物两端，以cDNA为模板利用PCR的方法获得编码CBD-PTHrP和NAT-PTHrP两种融合蛋白的基因序列，然后连接到pET-32a原核表达载体中。转化TOP10感受态细胞后筛选鉴定出阳性克隆，然后提取质粒转化Rosetta(DE3)感受态细胞，利用IPTG诱导CBD-PTHrP和NAT-PTHrP两种融合蛋白表达，经Ni柱纯化得到目的蛋白。将浓度为0,0.5,1,2,4,8,12,16μm的融合蛋白添加到铺有胶原的96孔板中，利用类ELISA的方法检测两种融合蛋白对胶原的靶向结合能力。
2、人源骨髓间充质干细胞的分离培养和传代鉴定
抽取人骨髓，利用密度梯度离心法分离得到骨髓间充质干细胞，然后利用DMEM/F12+10%FBS培养基进行培养，在细胞传至三代时，收取细胞利用流式细胞术对细胞表面抗原：CD34、CD44、CD45、CD73、CD90、CD105进行检测，对所分BM-MSCs的分子标记进行鉴定。
3、CBD-PTHrP促BM-MSCs向软骨细胞分化和抑制软骨细胞老化的实验研究
收集培养的BM-MSCs细胞，制成细胞小球后利用成软骨细胞诱导培养基进行培养，每2-3天换液一次，两周后用100ng/ml的NAT-PTHrP和CBD-PTHrP蛋白分别处理，并设空白对照组，再诱导培养两周后，收集细胞小球，分别提取RNA和总蛋白，利用Real-time PCR和Western blot检测COL1A1、COL2A1、COL10A1和Sox-9在mRNA和蛋白水平上的表达情况，细胞小球石蜡包埋切片后，利用番红-O染色检测软骨形成情况，免疫组织化学染色检测COL1A1、COL2A1、COL10A1和Sox-9在细胞小球组织中的表达情况。
结果：
1、构建了pET-32a-NAT-PTHrP和pET-32a-CBD-PTHrP重组质粒，并诱导表达和纯化得到了CBD-PTHrP和NAT-PTHrP两种融合蛋白。ELISA检测405nm吸光度结果提示：相比于NAT-PTHrP蛋白，CBD-PTHrP蛋白在胶原材料上的残留量更多，具有更强的胶原结合能力。
2、分离得到了原代人BM-MSCs，免疫标记的鉴定结果为CD34和CD45阴性，排除了所分细胞为造血细胞和内皮细胞的可能性；同时CD44、CD73、CD90和CD105表面抗原为阳性，明确了所分细胞为BM-MSCs。
3、 Real-time PCR和Western blot的结果显示，相比于空白对照组，CBD-PTHrP和NAT-PTHrP蛋白都能显著的诱导COL2A1和Sox-9基因在mRNA和蛋白水平的表达，有效的抑制COL1A1和COL10A1基因在mRNA和蛋白水平的表达。番红-O染色表明相比于空白对照组，添加CBD-PTHrP和NAT-PTHrP两种融合蛋白进行诱导的细胞小球中有更多的软骨细胞形成。免疫组织化学的检测结果也表明CBD-PTHrP和NAT-PTHrP都能诱导COL2A1和Sox-9蛋白的表达和抑制COL1A1和COL10A1蛋白的表达，这与Real-time PCR和Western blot的实验结果是一致的。
结论：
相比于NAT-PTHrP蛋白，在CBD短肽的作用下，CBD-PTHrP蛋白能更好的锚定在I型胶原上，提示CBD-PTHrP蛋白在动物和体外水平上都能更好的结合到胶原支架材料上，对其中的种子细胞起到持续的诱导作用。同时，CBD-PTHrP蛋白具有与NAT-PTHrP蛋白一样的促进BM-MSCs向软骨细胞分化的生物学作用，并能够有效的抑制软骨细胞的肥大，提示在PTHrP的N端加上CBD短肽对其生物学活性没有任何影响，使得PTHrP能通过CBD短肽持续的锚定在胶原支架上，进而持续的诱导胶原支架中的BM-MSCs向软骨细胞分化和抑制软骨细胞肥大。以上实验结果为CBD-PTHrP应用到基于BM-MSCs的软骨组织工程中奠定了坚实的实验基础。
Background:
Recent years, there are more and more degenerative joint diseases such as lumbar discherniation (LDH) and degenerative osteoarthritis caused by articular cartilage degenerativeand defect. These degenerative joint diseases of articular cartilage affect more than a thirdof the world population. Surgical techniques for the treatment of cartilage defects have beenextensive research and development. However, until now, there is no treatment canfundamentally cure the loss of biological function caused by disc degeneration. Thereforeoptimized treatment strategies for articular cartilage lesions are of a high socio-economicimportance. With the research and development of tissue engineering of orthopedics, itoffers a new ideal approach for the treatment of degenerative joint disease.
Intervertebral disc tissue engineering is still in its infancy, and the exploration of disctissue engineering mainly focuses on three parts: seeding cells, scaffolds and growth factors.Bone marrow derived mesenchymal stem cells (Bone marrow mesenchymal stem cells,BM-MSCs) has been widely used as a common seed cells in tissue engineering. Duringtissue engineering, the decay of seed cells, degenerating of seed cells with passaged, theloss of biology function of degeneration seed cells are the major difficulties. In order toovercome these difficulties, a large number of exogenous growth factors have been added tothe scaffolds to maintain the seed cell biology function. However in practice, factor simplydelivered in solution is difficult to be retained in the scoffolds due to its rapid diffusion inextracellular fluids and cannot promote the seed cells persistence. Collagen, especially typeI collagen is the most commonly used tissue engineering scaffolds, so it is an importanttarget of tissue engineering materials targeting.
Parathyroid hormone-related protein (Parathyroid hormone related protein, PTHrP) is identified and purified as a factor involved in human hypocalcaemia of malignancy in1987.It is composed by141amino acid chloride. There is a high homology between itsN-terminal PTHrP (1-34) and the N-terminal parathyroid hormone (N-PTH) amino acidsequence. Apart from similar physiological activity of N-PTH, PTHrP also has otherbiological functions, such as proliferating chondrocytes and inhibits chondrocytedifferentiation toward hypertrophy in the growth plate through the PTHrP-Indian hedgehog(IHH) axis. Meanwhile, the collagen binding domain (collagen-binding domain, CBD),which consists of a sequence of seven amino acids (TKKTLRT),and takes a role of collagenbinding, has been widely used for binding the associated active molecules to collagen.
Objectives:
In this study, we build a new biologically active recombinant protein CBD-PTHrP byrecombinant DNA technology, it has a dual biological function: on the one hand, PTHrPcould induce bone marrow mesenchymal stem cells into cartilage differentiation and inhibitchondrocyte differentiation toward hypertrophy, on the other hand CBD sequence couldbind to the collagen scaffold material, so that the PTHrP play a lasting effect on the seedcells. Therefore, new bioactive factor CBD-PTHrP, building by recombinant DNAtechnology, is used to solve aging of seed cells in the experiment degeneration, and lay thefoundation for building a new type of intervertebral disc.
Methods:
1. The expression and purification of CBD-PTHrP recombinant protein, and theidentification of collagen target capacity.
CBD coding sequence was constructed to the both ends of the primers, and cDNA wasused as a template. The coding gene sequence of CBD-PTHrP and NAT-PTHrP protein wasacquired by PCR, and then inserted into the prokaryotic expression vector pET-32a. Therecombinant plasmid was transformed into TOP10competent cells and the positive cloneswere screened and identified, and then the positive plasmid was transformed into theRosetta (DE3) competent cells. IPTG was used to induce CBD-PTHrP and NAT-PTHrPprotein expression, and the protein was purified by Ni-column.0,0.5,1,2,4,8,12,16μmfusion protein were added to the96well plate coated with collagen, collagen binding capacity of the protein was detected by ELISAs.
2. Human bone marrow mesenchymal stem cells were isolated, cultured andindentified.
Human bone marrow mesenchymal stem cell was isolated from the human bonemarrow by gradient centrifugation. The cells were cultured with DMEM/F12+10%FBSmedium, and the flow cytometry was used to detect the molecular markers of theBM-MSCs: CD34, CD44, CD45, CD73, CD90, CD105, when the cells transmitted to thethird generation.
3. CBD-PTHrP promotes chondrocyte differentiation and inhibits chondrocytedifferentiation toward hypertrophy from BM-MSCs.
The pellet was made from BM-MSCs, and cultured with cartilage inducing medium.The medium was changed every2-3days. Two weeks later,100ng/ml NAT-PTHrP andCBD-PTHrP protein was added to the medium separately, and a blank control group isnecessary. Two weeks after that, total RNA and protein was extracted and Real-time PCRand Western blot was used to detect the expression of COL1A1, COL2A1, COL10A1andSox-9at mRNA and protein level. The pellet was also embedded by paraffin. Safranin-Ostaining was used to detect the cartilage formation, and the COL1A1, COL2A1, COL10A1and Sox-9expression in the pellet was measured by immunohistochemical staining.
Results:
1. pET-32a-NAT-PTHrP and pET-32a-CBD-PTHrP recombinant plasmid areconstructed, and the protein of CBD-PTHrP and NAT-PTHrP are induced and purified.Compared with NAT-PTHrP protein, CBD-PTHrP protein has better collagen bindingcapacity.
2. Human BM-MSCs is isolated and indentified, the results show that the immunemarkers for CD34and CD45are negative, excluding the divided cells are hematopoieticcells or leukocytes; while CD44, CD73, CD90and CD105surface antigen are positive, toindentify the cells is the BM-MSCs.
3. Real-time PCR and Western blot results indicate that, compared with the controlmedium (CM) group, CBD-PTHrP and NAT-PTHrP protein can significantly induced COL2A1and Sox-9gene expression, and inhibit COL1A1and COL10A1gene expressionat the mRNA and protein levels. Safranin-O staining demonstrate that compared with theCM group, CBD-PTHrP and NAT-PTHrP induce cartilage cell formation in the BM-MSCscell pellet. The immunohistochemical detection results also indicate that the CBD-PTHrPand NAT-PTHrP can induce the COL2A1and Sox-9protein expression and inhibitCOL1A1and COL10A1protein expression, which is consistent with the experimentalresults of the Real-time PCR and Western blot.
Conclusion:
Compared with the NAT-PTHrP protein, CBD-PTHrP protein has bettercollagen-binding capacity by the use of CBD peptide. It indicates that CBD-PTHrP couldbind collagen I scoffolds more tightly in vitro and in vivo and continue promoting seed celldifferentiation. At the same time, CBD-PTHrP gets the same biological activity ofNAT-PTHrP. It could induce chondrocytes from BM-MSCs and inhibit chondrocytedifferentiation toward hypertrophy. These results indicate that adding CBD peptide at theN-terminus of PTHrP has no effect on the biological activity. Moreover, CBD peptidepromotes PTHrP binding collagen I scoffolds for a long time. Therefore it could continueinducing chondrocytes from BM-MSCs and inhibiting chondrocyte differentiation towardhypertrophy. In conclusion, our study suggests CBD-PTHrP could be an efficient system fortargeting cartilage tissue engineering in clinical application.
近年来，由于软骨组织退行性改变和缺损而引起的诸如腰椎间盘突出症、退行性骨关节炎等在内的退行性骨关节疾病越来越多。关节软骨相关的退化性关节疾病严重影响超过三分之一的世界人口的关节健康。虽然治疗软骨组织缺损的外科技术已得到广泛的研究和发展，但是现存的治疗手段中没有任何一种能从根本上治疗软骨组织缺损。因此，优化关节软骨病变的治疗策略具有很高的社会和经济重要性。随着组织工程技术在骨科领域相关研究的不断深入和发展，为骨科退行性相关疾病的治疗开辟出一条崭新的治疗途径。
目前采用组织工程学技术构建的椎间盘替代物研究尚处于起步阶段，研究主要集中于支架材料、种子细胞和生物活性因子三个方面。骨髓间充质干细胞(Bone marrowmesenchymal stem cells，BM-MSCs)作为一种常用的种子细胞，已广泛应用于组织工程修复中。为克服组织工程中种子细胞不断衰减，传代后种子细胞老化退变，退变的种子细胞生物学功能下降等问题，大量的外源性生长因子加入到支架材料中，以维持种子细胞的生物学功能。但所添加的生长因子由于不具有靶向结合支架材料的能力，不能很好滞留在支架材料中发挥生物学功能，从而不能对种子细胞起到持续性的促进作用。胶原蛋白，尤其是I型胶原蛋白是目前最常用的组织工程支架材料，因此I型胶原是组织工程材料靶向性的重要靶点。
甲状旁腺激素相关蛋白(Parathyroid hormone related protein，PTHrP)是1987年从恶性高血钙肿瘤患者的肿瘤中提取纯化的。它是由141个氯基酸组成，其N-端PTHrP(1-34)与N-端甲状旁腺激素(N-PTH)氨基酸序列具有很高的相同性。PTHrP的生理活性除了类似N-PTH外，还具有其他的生物功能，如促进钙的传递，抑制骨的重吸收，促进骨髓间充质干细胞成软骨分化，同时抑制软骨细胞老化。同时，胶原结合域(collagen-binding domain, CBD)是由七个氨基酸构成的寡肽序列(TKKTLRT)组成，具有与胶原紧密结合的功能，被广泛用于胶原结合相关活性分子的重组表达中。
目的：
在本实验中，我们利用基因重组技术构建了新型生物活性重组蛋白CBD-PTHrP，该重组蛋白具有双重的生物学功能：一方面可以通过其中的PTHrP成分发挥诱导骨髓间充质干细胞成软骨分化和抑制软骨细胞肥大的生物学功能，另一方面该融合蛋白又能够通过CBD序列的偶联，将PTHrP锚定在胶原蛋白支架材料上，从而使PTHrP能对种子细胞起到持续靶向性的作用。因此，利用基因重组技术构建新型生物活性因子解决种子细胞在实验中易老化退变的问题能为构建新型软骨组织工程替代材料奠定基础。
方法：
1、CBD-PTHrP重组融合蛋白的表达、纯化和胶原靶向性结合能力鉴定
将CBD的编码序列构建到引物两端，以cDNA为模板利用PCR的方法获得编码CBD-PTHrP和NAT-PTHrP两种融合蛋白的基因序列，然后连接到pET-32a原核表达载体中。转化TOP10感受态细胞后筛选鉴定出阳性克隆，然后提取质粒转化Rosetta(DE3)感受态细胞，利用IPTG诱导CBD-PTHrP和NAT-PTHrP两种融合蛋白表达，经Ni柱纯化得到目的蛋白。将浓度为0,0.5,1,2,4,8,12,16μm的融合蛋白添加到铺有胶原的96孔板中，利用类ELISA的方法检测两种融合蛋白对胶原的靶向结合能力。
2、人源骨髓间充质干细胞的分离培养和传代鉴定
抽取人骨髓，利用密度梯度离心法分离得到骨髓间充质干细胞，然后利用DMEM/F12+10%FBS培养基进行培养，在细胞传至三代时，收取细胞利用流式细胞术对细胞表面抗原：CD34、CD44、CD45、CD73、CD90、CD105进行检测，对所分BM-MSCs的分子标记进行鉴定。
3、CBD-PTHrP促BM-MSCs向软骨细胞分化和抑制软骨细胞老化的实验研究
收集培养的BM-MSCs细胞，制成细胞小球后利用成软骨细胞诱导培养基进行培养，每2-3天换液一次，两周后用100ng/ml的NAT-PTHrP和CBD-PTHrP蛋白分别处理，并设空白对照组，再诱导培养两周后，收集细胞小球，分别提取RNA和总蛋白，利用Real-time PCR和Western blot检测COL1A1、COL2A1、COL10A1和Sox-9在mRNA和蛋白水平上的表达情况，细胞小球石蜡包埋切片后，利用番红-O染色检测软骨形成情况，免疫组织化学染色检测COL1A1、COL2A1、COL10A1和Sox-9在细胞小球组织中的表达情况。
结果：
1、构建了pET-32a-NAT-PTHrP和pET-32a-CBD-PTHrP重组质粒，并诱导表达和纯化得到了CBD-PTHrP和NAT-PTHrP两种融合蛋白。ELISA检测405nm吸光度结果提示：相比于NAT-PTHrP蛋白，CBD-PTHrP蛋白在胶原材料上的残留量更多，具有更强的胶原结合能力。
2、分离得到了原代人BM-MSCs，免疫标记的鉴定结果为CD34和CD45阴性，排除了所分细胞为造血细胞和内皮细胞的可能性；同时CD44、CD73、CD90和CD105表面抗原为阳性，明确了所分细胞为BM-MSCs。
3、 Real-time PCR和Western blot的结果显示，相比于空白对照组，CBD-PTHrP和NAT-PTHrP蛋白都能显著的诱导COL2A1和Sox-9基因在mRNA和蛋白水平的表达，有效的抑制COL1A1和COL10A1基因在mRNA和蛋白水平的表达。番红-O染色表明相比于空白对照组，添加CBD-PTHrP和NAT-PTHrP两种融合蛋白进行诱导的细胞小球中有更多的软骨细胞形成。免疫组织化学的检测结果也表明CBD-PTHrP和NAT-PTHrP都能诱导COL2A1和Sox-9蛋白的表达和抑制COL1A1和COL10A1蛋白的表达，这与Real-time PCR和Western blot的实验结果是一致的。
结论：
相比于NAT-PTHrP蛋白，在CBD短肽的作用下，CBD-PTHrP蛋白能更好的锚定在I型胶原上，提示CBD-PTHrP蛋白在动物和体外水平上都能更好的结合到胶原支架材料上，对其中的种子细胞起到持续的诱导作用。同时，CBD-PTHrP蛋白具有与NAT-PTHrP蛋白一样的促进BM-MSCs向软骨细胞分化的生物学作用，并能够有效的抑制软骨细胞的肥大，提示在PTHrP的N端加上CBD短肽对其生物学活性没有任何影响，使得PTHrP能通过CBD短肽持续的锚定在胶原支架上，进而持续的诱导胶原支架中的BM-MSCs向软骨细胞分化和抑制软骨细胞肥大。以上实验结果为CBD-PTHrP应用到基于BM-MSCs的软骨组织工程中奠定了坚实的实验基础。
Background:
Recent years, there are more and more degenerative joint diseases such as lumbar discherniation (LDH) and degenerative osteoarthritis caused by articular cartilage degenerativeand defect. These degenerative joint diseases of articular cartilage affect more than a thirdof the world population. Surgical techniques for the treatment of cartilage defects have beenextensive research and development. However, until now, there is no treatment canfundamentally cure the loss of biological function caused by disc degeneration. Thereforeoptimized treatment strategies for articular cartilage lesions are of a high socio-economicimportance. With the research and development of tissue engineering of orthopedics, itoffers a new ideal approach for the treatment of degenerative joint disease.
Intervertebral disc tissue engineering is still in its infancy, and the exploration of disctissue engineering mainly focuses on three parts: seeding cells, scaffolds and growth factors.Bone marrow derived mesenchymal stem cells (Bone marrow mesenchymal stem cells,BM-MSCs) has been widely used as a common seed cells in tissue engineering. Duringtissue engineering, the decay of seed cells, degenerating of seed cells with passaged, theloss of biology function of degeneration seed cells are the major difficulties. In order toovercome these difficulties, a large number of exogenous growth factors have been added tothe scaffolds to maintain the seed cell biology function. However in practice, factor simplydelivered in solution is difficult to be retained in the scoffolds due to its rapid diffusion inextracellular fluids and cannot promote the seed cells persistence. Collagen, especially typeI collagen is the most commonly used tissue engineering scaffolds, so it is an importanttarget of tissue engineering materials targeting.
Parathyroid hormone-related protein (Parathyroid hormone related protein, PTHrP) is identified and purified as a factor involved in human hypocalcaemia of malignancy in1987.It is composed by141amino acid chloride. There is a high homology between itsN-terminal PTHrP (1-34) and the N-terminal parathyroid hormone (N-PTH) amino acidsequence. Apart from similar physiological activity of N-PTH, PTHrP also has otherbiological functions, such as proliferating chondrocytes and inhibits chondrocytedifferentiation toward hypertrophy in the growth plate through the PTHrP-Indian hedgehog(IHH) axis. Meanwhile, the collagen binding domain (collagen-binding domain, CBD),which consists of a sequence of seven amino acids (TKKTLRT),and takes a role of collagenbinding, has been widely used for binding the associated active molecules to collagen.
Objectives:
In this study, we build a new biologically active recombinant protein CBD-PTHrP byrecombinant DNA technology, it has a dual biological function: on the one hand, PTHrPcould induce bone marrow mesenchymal stem cells into cartilage differentiation and inhibitchondrocyte differentiation toward hypertrophy, on the other hand CBD sequence couldbind to the collagen scaffold material, so that the PTHrP play a lasting effect on the seedcells. Therefore, new bioactive factor CBD-PTHrP, building by recombinant DNAtechnology, is used to solve aging of seed cells in the experiment degeneration, and lay thefoundation for building a new type of intervertebral disc.
Methods:
1. The expression and purification of CBD-PTHrP recombinant protein, and theidentification of collagen target capacity.
CBD coding sequence was constructed to the both ends of the primers, and cDNA wasused as a template. The coding gene sequence of CBD-PTHrP and NAT-PTHrP protein wasacquired by PCR, and then inserted into the prokaryotic expression vector pET-32a. Therecombinant plasmid was transformed into TOP10competent cells and the positive cloneswere screened and identified, and then the positive plasmid was transformed into theRosetta (DE3) competent cells. IPTG was used to induce CBD-PTHrP and NAT-PTHrPprotein expression, and the protein was purified by Ni-column.0,0.5,1,2,4,8,12,16μmfusion protein were added to the96well plate coated with collagen, collagen binding capacity of the protein was detected by ELISAs.
2. Human bone marrow mesenchymal stem cells were isolated, cultured andindentified.
Human bone marrow mesenchymal stem cell was isolated from the human bonemarrow by gradient centrifugation. The cells were cultured with DMEM/F12+10%FBSmedium, and the flow cytometry was used to detect the molecular markers of theBM-MSCs: CD34, CD44, CD45, CD73, CD90, CD105, when the cells transmitted to thethird generation.
3. CBD-PTHrP promotes chondrocyte differentiation and inhibits chondrocytedifferentiation toward hypertrophy from BM-MSCs.
The pellet was made from BM-MSCs, and cultured with cartilage inducing medium.The medium was changed every2-3days. Two weeks later,100ng/ml NAT-PTHrP andCBD-PTHrP protein was added to the medium separately, and a blank control group isnecessary. Two weeks after that, total RNA and protein was extracted and Real-time PCRand Western blot was used to detect the expression of COL1A1, COL2A1, COL10A1andSox-9at mRNA and protein level. The pellet was also embedded by paraffin. Safranin-Ostaining was used to detect the cartilage formation, and the COL1A1, COL2A1, COL10A1and Sox-9expression in the pellet was measured by immunohistochemical staining.
Results:
1. pET-32a-NAT-PTHrP and pET-32a-CBD-PTHrP recombinant plasmid areconstructed, and the protein of CBD-PTHrP and NAT-PTHrP are induced and purified.Compared with NAT-PTHrP protein, CBD-PTHrP protein has better collagen bindingcapacity.
2. Human BM-MSCs is isolated and indentified, the results show that the immunemarkers for CD34and CD45are negative, excluding the divided cells are hematopoieticcells or leukocytes; while CD44, CD73, CD90and CD105surface antigen are positive, toindentify the cells is the BM-MSCs.
3. Real-time PCR and Western blot results indicate that, compared with the controlmedium (CM) group, CBD-PTHrP and NAT-PTHrP protein can significantly induced COL2A1and Sox-9gene expression, and inhibit COL1A1and COL10A1gene expressionat the mRNA and protein levels. Safranin-O staining demonstrate that compared with theCM group, CBD-PTHrP and NAT-PTHrP induce cartilage cell formation in the BM-MSCscell pellet. The immunohistochemical detection results also indicate that the CBD-PTHrPand NAT-PTHrP can induce the COL2A1and Sox-9protein expression and inhibitCOL1A1and COL10A1protein expression, which is consistent with the experimentalresults of the Real-time PCR and Western blot.
Conclusion:
Compared with the NAT-PTHrP protein, CBD-PTHrP protein has bettercollagen-binding capacity by the use of CBD peptide. It indicates that CBD-PTHrP couldbind collagen I scoffolds more tightly in vitro and in vivo and continue promoting seed celldifferentiation. At the same time, CBD-PTHrP gets the same biological activity ofNAT-PTHrP. It could induce chondrocytes from BM-MSCs and inhibit chondrocytedifferentiation toward hypertrophy. These results indicate that adding CBD peptide at theN-terminus of PTHrP has no effect on the biological activity. Moreover, CBD peptidepromotes PTHrP binding collagen I scoffolds for a long time. Therefore it could continueinducing chondrocytes from BM-MSCs and inhibiting chondrocyte differentiation towardhypertrophy. In conclusion, our study suggests CBD-PTHrP could be an efficient system fortargeting cartilage tissue engineering in clinical application.
引文
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