稳定分化软骨前体细胞株的建立与鉴定及其NMR波谱和材料相容性研究
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摘要
本文旨在建立分离培养和鉴定大鼠骨骺生长板软骨细胞亚群的实验方法,探讨其静止区软骨细胞的生物学特性;并通过基因工程技术,诱导静止区骺软骨细胞稳定分化,筛选稳定分化株,研究其基因和蛋白质表达,监测细胞表面的代谢变化和细胞内离子浓度,全面评估其遗传特征保存情况;分析稳定分化软骨前体细胞与纳米组织工程材料的复合黏附特性及其增殖分化的变化。本课题深入研究骨骺软骨细胞增殖、分化规律,为人工调控骨与软骨的生长和通过现代组织工程技术修复软骨的研究建立实验基础。
通过显微外科技术解剖大鼠长骨骨骺生长板软骨,酶消化法获得分散的单细胞悬液;采用percoll不连续密度梯度离心法分离生长板细胞亚群,分层培养、观察不同层细胞形态,并进行细胞生长动力学分析,电子显微镜观察细胞超微结构,组织化学和免疫细胞化学方法检测不同细胞FGFR-3、X和II型胶原的表达。利用获得的骨骺生长板增殖区软骨细胞亚群,提取总RNA,RT-PCR方法获得PTHrp基因的全长cDNA,扩增后经纯化、双酶切连接到真核表达载体pEGFP-IRES2;转化扩增后提取重组质粒pEGFP-IRES2-PTHrp,进行酶切鉴定和序列测定。利用脂质体介导的基因转染技术将含有PTHrp基因的重组质粒转染至原代培养的新生大鼠软骨前体细胞,经G418筛选,抗性克隆扩大培养传代。应用FGFR-3、II型胶原、X型胶原和PTHrp抗体进行免疫细胞化学鉴定,检测其表型及分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern blot和western blot从不同层次鉴定PTHrp基因在转染细胞中的表达。以重水(D2O)缓冲液重悬传代培养的稳定分化软骨前体细胞,在灌流条件下,利用inova 600型液态核磁共振谱仪测定细胞的31P-NMR波谱,和细胞内游离Mg2+浓度,观察波谱组成及各波峰高度,评价细胞代谢情况。尝试细胞与nanoHA/PDLLA多孔材料复合培养7天后,进行形态学观察和扫描电镜观察及MTT测定,了解细胞增殖及黏附情况,获得了稳定分化骨骺干细胞与组织工程材料的相容性、黏附性的初步资料。
我们获得了四层生长板软骨细胞亚群,细胞活性达到了95%以上;原代培养细胞呈多角形,第8代细胞仍保持多角的形态;前4代细胞的每日倍增指数随着传代次数的增加而增加,第8代后显著降低。依照细胞成熟程度,体积逐渐增大,密度逐层减小;软骨前体细胞表面有FGFR-3的表达,其他层细胞均为阴性;随着细胞的逐渐成熟,II型胶原表达逐渐减弱,而X型胶原的表达逐渐增强;随着传代次数增加阳性细胞的比例下降。从增殖区细胞提取的总RNA纯度、含量均符合要求,RT-PCR和PCR产物电泳可在预期大小处得到清晰条带,酶切、连接、转化后获得2株阳性转化大肠杆菌,大提产物经限制性内切酶酶切图谱分析可得到目的基因条带和载体条带,DNA序列测定证实目的基因已插入重组质粒。将重组质粒转染入软骨前体细胞后获得1个阳性细胞克隆,免疫细胞化学证实为具有较强增值能力和多分化潜能。经Southern印迹杂交和Western证实,PTHrp已稳定转染入软骨前体细胞,表达其mRNA及其蛋白。转染细胞经扩大培养,命名为稳定分化软骨前体细胞(immortalized precartilaginous stem cell, IPSC)。贴壁培养的IPSC,群体倍增时间为22.92h,传代、冻存和复苏对细胞形态及生长无明显影响。本实验成功获得稳定分化软骨前体细胞株31P-NMR波谱,发现其由PCr、ATP、Pi等五个共振峰组成,细胞内游离Mg2+浓度为0.326mmol/L。稳定分化软骨前体细胞与nanoHA/PDLLA多孔材料具有良好的相容性,细胞在材料上生长良好,材料对细胞增殖无不良影响,为进一步应用研究研究打下基础。
通过上述实验,本研究发现能够通过密度梯度离心法分离骨骺生长板细胞亚群,并可以获得四个高纯度、高活性的软骨细胞亚群。第4代以前细胞表型稳定,可以作为研究软骨增殖分化调控的细胞平台。从骨骺生长板增殖区软骨细胞亚群中克隆PTHrp基因构建其真核表达载体,可诱导软骨前体细胞稳定分化。稳定分化软骨前体细胞的31P-NMR波谱形态正常,代谢正常,内环境稳定;细胞与nanoHA/PDLLA多孔材料相容性良好,黏附能力强。本实验所获得的稳定分化软骨前体细胞株为骺软骨细胞的实验研究及其介导的细胞移植治疗提供了稳定的细胞来源。为进一步揭示PTHrp的生物学功能及其在在软骨细胞的分化和骨骼形态发生中的作用奠定了良好的基础,可望成为一种新的治疗手段。
This paper is to establish the methods for separation and culture the subpopulations chondrocyte of rat tibiofibula epiphysis growth plate, and to investigate their biological features. Then this ressarch cloned the parathroid hormone-related peptide gene from proliferating zone cell and constructed its eukaryotic expression vector by gene engineering technology. After established the immortalized precartilaginous stem cell strain, this project could provide stable cell resource for cell-transplantation and gene therapies. The 31P-NMR and the ion concentration of the IPSCs were investigated by using MR spectroscopy. The compatibility between IPSCs and nanoHA/PDLLA was evaluated through combination culture. Thereby this paper could provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation and for repairing of cartilage using tissue-engineering materials.
The chondrocyte of rat tibiofibula epiphysis growth plate was obtained by microdissection and digestion. Subpopulations of these chondrocytes were separated by discontinues percoll gradient and were cultured in monolayer. Morphological changes of the serial passage of subpopulations and the cell growth kinectics were observed. The ultramicro structures were observed by electron microscope. The cellular FGFR-3, collagen type II and type X expression were detected by histochemistry and immunocytochemistry. The total RNA was extracted from the proliferating zone cells and the full length cDNA encoding PTHrp gene was obtained by RT-PCR method. Product of PCR, amplified by transformed into E-coli DH5α, was inserted into the eukaryotic expression vector pEGFP-IRES2 after digestion and ligation by using restriction endonucleases and ligase. Recombinated plasmid pEGFP-IRES2-PTHrp containing the PTHrp gene was transfected into the primarily cultured precartilaginous stem cell(PSC) of newborn rat by using lipofectin transfection method. Colonies were isolated by G418 selection and expanded to immortalized cell strains. FGFR-3, collagen type II and type X antibodies were used to identify the cultured cells and to investigate the capability of differentiation of the transfected cells. The expression of PTHrp in expanded cells was identified by RT-PCR, Southern blot, western blot and immunocytochemistry method. IPSCs were resuspension by tris-buffer prepared in D2O. 31P-NMR spectroscopy and Mg2+ concentration were carried by using high magnetic field spectrometer inova 600. At last, cells were seeded on the tissue-engineering material nanoHA/PDLLA, and were observed under inverted microscope and environmental scanning electron microscope and recorded. The number of the cell adhered on the materials was calculated 7days after the culture.
There were four subpopulations in the tibiofibula epiphysis growth plate, were more than 96% viable cells in the obtained subpopulations. The morphology of primary cultured subpopulations was fusiform shape or polygon. In this experiment, the eighth passage cell was still maintained and showed polygonal morphology. The index of duplicating day increased in the preceding fourth passage cell and decreased afterwards. There was more than 95 % cells expressed collagen type II in C4 but type X in C1. As the passagenumber increasing, the ratio of these collagens expression and dropped abruptly. The size was smaller in C4, but the density smaller in C1. FGFR-3 was expressed only in the cell membrane of C4. Both the content and the purity quotient of total RNA were qualified. The correct PCR production was obtained and it was confirmed that PTPrp gene was inserted into the eukaryotic expression vector correctly by using digestion identification and sequencing. One anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive PSC with the capability of proliferation after transfection. mRNA and protein of PTHrp were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains maintained for more than 50 passages, named as immortalized precartilaginous stem cell (IPSCs). IPSCs were elliptic or triangular cells with two or three short axons. The population doubling time of IPSC was 23.52 h. Subculture, freezing and recovering had no effect on cellular shape and proliferation. There were five resonance peaks in the 31P-NMR Spectra of IPSCs, as PCr, ATP and Pi, et al. Intracellular free Mg2+ was 0.326mmol/L. IPSCs were attached to nanoHA/PDLLA and grew favorably on surface of it.
The separation and culture methods adopted in this study can obtain high pure and viable subpopulations of epiphysis growth plate. The preceding four passage cells maintains their in vivo phenotype, they are ideal experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering. The PThrp gene was cloned accurately from proliferating zone cell. The recombinant eukaryotic expression vector pEGFP-IRES2-PTHrp is successfully constructed, which may be a promising for studying the biological function of the PTHrp gene and its role in chondrocyte differentiation and bone formation. Transfection PTHrp gene could immortalize precartilaginous stem cells. The establishment of FGFR-3 positive IPSC line may provide stable cell resource for the basic researches and cell-transplantation therapies with PSC. The metabolism and internal environment of IPSCs were normal and stable. There was a good compatibility between IPSCs and bone tissue-engineering material nanoHA/PDLLA. The proliferation and differentiation of IPSCs adhered on materials were normal. This material is a safe tissue-engineering material, IPSCs and this material complex could be substitute for bone graft.
通过显微外科技术解剖大鼠长骨骨骺生长板软骨,酶消化法获得分散的单细胞悬液;采用percoll不连续密度梯度离心法分离生长板细胞亚群,分层培养、观察不同层细胞形态,并进行细胞生长动力学分析,电子显微镜观察细胞超微结构,组织化学和免疫细胞化学方法检测不同细胞FGFR-3、X和II型胶原的表达。利用获得的骨骺生长板增殖区软骨细胞亚群,提取总RNA,RT-PCR方法获得PTHrp基因的全长cDNA,扩增后经纯化、双酶切连接到真核表达载体pEGFP-IRES2;转化扩增后提取重组质粒pEGFP-IRES2-PTHrp,进行酶切鉴定和序列测定。利用脂质体介导的基因转染技术将含有PTHrp基因的重组质粒转染至原代培养的新生大鼠软骨前体细胞,经G418筛选,抗性克隆扩大培养传代。应用FGFR-3、II型胶原、X型胶原和PTHrp抗体进行免疫细胞化学鉴定,检测其表型及分化能力,观察细胞的形态及其生长状况,绘制细胞生长曲线。用RT-PCR、Southern blot和western blot从不同层次鉴定PTHrp基因在转染细胞中的表达。以重水(D2O)缓冲液重悬传代培养的稳定分化软骨前体细胞,在灌流条件下,利用inova 600型液态核磁共振谱仪测定细胞的31P-NMR波谱,和细胞内游离Mg2+浓度,观察波谱组成及各波峰高度,评价细胞代谢情况。尝试细胞与nanoHA/PDLLA多孔材料复合培养7天后,进行形态学观察和扫描电镜观察及MTT测定,了解细胞增殖及黏附情况,获得了稳定分化骨骺干细胞与组织工程材料的相容性、黏附性的初步资料。
我们获得了四层生长板软骨细胞亚群,细胞活性达到了95%以上;原代培养细胞呈多角形,第8代细胞仍保持多角的形态;前4代细胞的每日倍增指数随着传代次数的增加而增加,第8代后显著降低。依照细胞成熟程度,体积逐渐增大,密度逐层减小;软骨前体细胞表面有FGFR-3的表达,其他层细胞均为阴性;随着细胞的逐渐成熟,II型胶原表达逐渐减弱,而X型胶原的表达逐渐增强;随着传代次数增加阳性细胞的比例下降。从增殖区细胞提取的总RNA纯度、含量均符合要求,RT-PCR和PCR产物电泳可在预期大小处得到清晰条带,酶切、连接、转化后获得2株阳性转化大肠杆菌,大提产物经限制性内切酶酶切图谱分析可得到目的基因条带和载体条带,DNA序列测定证实目的基因已插入重组质粒。将重组质粒转染入软骨前体细胞后获得1个阳性细胞克隆,免疫细胞化学证实为具有较强增值能力和多分化潜能。经Southern印迹杂交和Western证实,PTHrp已稳定转染入软骨前体细胞,表达其mRNA及其蛋白。转染细胞经扩大培养,命名为稳定分化软骨前体细胞(immortalized precartilaginous stem cell, IPSC)。贴壁培养的IPSC,群体倍增时间为22.92h,传代、冻存和复苏对细胞形态及生长无明显影响。本实验成功获得稳定分化软骨前体细胞株31P-NMR波谱,发现其由PCr、ATP、Pi等五个共振峰组成,细胞内游离Mg2+浓度为0.326mmol/L。稳定分化软骨前体细胞与nanoHA/PDLLA多孔材料具有良好的相容性,细胞在材料上生长良好,材料对细胞增殖无不良影响,为进一步应用研究研究打下基础。
通过上述实验,本研究发现能够通过密度梯度离心法分离骨骺生长板细胞亚群,并可以获得四个高纯度、高活性的软骨细胞亚群。第4代以前细胞表型稳定,可以作为研究软骨增殖分化调控的细胞平台。从骨骺生长板增殖区软骨细胞亚群中克隆PTHrp基因构建其真核表达载体,可诱导软骨前体细胞稳定分化。稳定分化软骨前体细胞的31P-NMR波谱形态正常,代谢正常,内环境稳定;细胞与nanoHA/PDLLA多孔材料相容性良好,黏附能力强。本实验所获得的稳定分化软骨前体细胞株为骺软骨细胞的实验研究及其介导的细胞移植治疗提供了稳定的细胞来源。为进一步揭示PTHrp的生物学功能及其在在软骨细胞的分化和骨骼形态发生中的作用奠定了良好的基础,可望成为一种新的治疗手段。
This paper is to establish the methods for separation and culture the subpopulations chondrocyte of rat tibiofibula epiphysis growth plate, and to investigate their biological features. Then this ressarch cloned the parathroid hormone-related peptide gene from proliferating zone cell and constructed its eukaryotic expression vector by gene engineering technology. After established the immortalized precartilaginous stem cell strain, this project could provide stable cell resource for cell-transplantation and gene therapies. The 31P-NMR and the ion concentration of the IPSCs were investigated by using MR spectroscopy. The compatibility between IPSCs and nanoHA/PDLLA was evaluated through combination culture. Thereby this paper could provide experimental bases for studying the regulation of chondrocyte proliferation and differentiation and for repairing of cartilage using tissue-engineering materials.
The chondrocyte of rat tibiofibula epiphysis growth plate was obtained by microdissection and digestion. Subpopulations of these chondrocytes were separated by discontinues percoll gradient and were cultured in monolayer. Morphological changes of the serial passage of subpopulations and the cell growth kinectics were observed. The ultramicro structures were observed by electron microscope. The cellular FGFR-3, collagen type II and type X expression were detected by histochemistry and immunocytochemistry. The total RNA was extracted from the proliferating zone cells and the full length cDNA encoding PTHrp gene was obtained by RT-PCR method. Product of PCR, amplified by transformed into E-coli DH5α, was inserted into the eukaryotic expression vector pEGFP-IRES2 after digestion and ligation by using restriction endonucleases and ligase. Recombinated plasmid pEGFP-IRES2-PTHrp containing the PTHrp gene was transfected into the primarily cultured precartilaginous stem cell(PSC) of newborn rat by using lipofectin transfection method. Colonies were isolated by G418 selection and expanded to immortalized cell strains. FGFR-3, collagen type II and type X antibodies were used to identify the cultured cells and to investigate the capability of differentiation of the transfected cells. The expression of PTHrp in expanded cells was identified by RT-PCR, Southern blot, western blot and immunocytochemistry method. IPSCs were resuspension by tris-buffer prepared in D2O. 31P-NMR spectroscopy and Mg2+ concentration were carried by using high magnetic field spectrometer inova 600. At last, cells were seeded on the tissue-engineering material nanoHA/PDLLA, and were observed under inverted microscope and environmental scanning electron microscope and recorded. The number of the cell adhered on the materials was calculated 7days after the culture.
There were four subpopulations in the tibiofibula epiphysis growth plate, were more than 96% viable cells in the obtained subpopulations. The morphology of primary cultured subpopulations was fusiform shape or polygon. In this experiment, the eighth passage cell was still maintained and showed polygonal morphology. The index of duplicating day increased in the preceding fourth passage cell and decreased afterwards. There was more than 95 % cells expressed collagen type II in C4 but type X in C1. As the passagenumber increasing, the ratio of these collagens expression and dropped abruptly. The size was smaller in C4, but the density smaller in C1. FGFR-3 was expressed only in the cell membrane of C4. Both the content and the purity quotient of total RNA were qualified. The correct PCR production was obtained and it was confirmed that PTPrp gene was inserted into the eukaryotic expression vector correctly by using digestion identification and sequencing. One anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive PSC with the capability of proliferation after transfection. mRNA and protein of PTHrp were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains maintained for more than 50 passages, named as immortalized precartilaginous stem cell (IPSCs). IPSCs were elliptic or triangular cells with two or three short axons. The population doubling time of IPSC was 23.52 h. Subculture, freezing and recovering had no effect on cellular shape and proliferation. There were five resonance peaks in the 31P-NMR Spectra of IPSCs, as PCr, ATP and Pi, et al. Intracellular free Mg2+ was 0.326mmol/L. IPSCs were attached to nanoHA/PDLLA and grew favorably on surface of it.
The separation and culture methods adopted in this study can obtain high pure and viable subpopulations of epiphysis growth plate. The preceding four passage cells maintains their in vivo phenotype, they are ideal experimental materials for studying the regulation of proliferation and differentiation of chondrocyte and tissue engineering. The PThrp gene was cloned accurately from proliferating zone cell. The recombinant eukaryotic expression vector pEGFP-IRES2-PTHrp is successfully constructed, which may be a promising for studying the biological function of the PTHrp gene and its role in chondrocyte differentiation and bone formation. Transfection PTHrp gene could immortalize precartilaginous stem cells. The establishment of FGFR-3 positive IPSC line may provide stable cell resource for the basic researches and cell-transplantation therapies with PSC. The metabolism and internal environment of IPSCs were normal and stable. There was a good compatibility between IPSCs and bone tissue-engineering material nanoHA/PDLLA. The proliferation and differentiation of IPSCs adhered on materials were normal. This material is a safe tissue-engineering material, IPSCs and this material complex could be substitute for bone graft.
引文
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