摘要
种子细胞是组织工程最基本的要素。软骨细胞体外培养极易老化，一般培养至5—6代其特异性标志蛋白Ⅱ型胶原就已经丧失表达。因此，不能通过体外扩增获取大量的软骨细胞来满足构建软骨组织的需要。我们试图通过基因转染的方式建立永生化人关节软骨细胞系并对其生物学特性进行研究，从而为软骨组织工程种子细胞的问题提供新的探索方向。
首先，利用分子克隆技术，构建了含HPV16E7目的基因片段的逆转录病毒载体(pLXSN-HPV16E7)，通过优化各种参数，应用电穿孔的方法将pLXSN-HPV16E7质粒转入PA317包转细胞系中，再利用其分泌的病毒上清转染人关节软骨细胞。结果显示：(1)亚克隆进入逆转录病毒载体的HPV16E7与Genebank的HPV16E7具有高度同源性。(2)电穿孔优化参数为重组质粒用量在20μg左右，电容975μF，电压220V，电击时间15ms时转染效率最高；筛选出了稳定产毒的PA317包转细胞系，病毒滴度在5.0×10~6—6.8×10~6CFU之间。(3)免疫组化、PCR及RT-PCR证明HPV16E7目的基因转染软骨细胞成功，获得了能够长期传代的永生化软骨细胞。
其次，通过比较永生化软骨细胞与未永生化软骨细胞的生物学特性发现：(1)与未永生化软骨细胞典型的三角或多角形相比，永生化软骨细胞的体积变小，形态更显长梭；透射电镜显示未永生化软骨细胞分化成熟而永生化软骨细胞较为幼稚，二者的核浆比都较小；扫描电镜下二者微绒毛均较少。(2)与原代未永生化软骨细胞相比，永生化软骨细胞的贴壁、延展速度较快，冻存复苏后细胞活力及接种率较高。第3代未永生化软骨细胞与永生化软骨细胞的生长曲线相似，但在相同的时相点内，后者的饱和密度高于前者。细胞周期时相比较，永生化软骨细胞处于G0-M以及S期
第三军医大学博士研究生论文
人关节软骨细胞的永生化及其生物学特性
的细胞数目显著高于第3代未永生化软骨细胞(3)从细胞的排列方式、锚
定依赖性、核浆比、微绒毛多少、血清依赖试验、软琼脂克隆形成试验、
染色体核型分析、裸鼠致瘤试验等指标了解永生化软骨细胞的致瘤性，永
生化软骨细胞排列有序，生长具有锚定依赖性，核浆比较小，微绒毛较少，
对血清有依赖性，不能在软琼脂上形成细胞克隆，染色体分析为二倍体核
型，经1年时间观察无裸鼠致瘤性。表明经我们基因转染的永生化软骨细
胞为良性转化。(4)永生化软骨细胞的n型胶原表型不稳，转换为I型胶
原，需要进行n型胶原的诱导。
然后，运用NA无血清诱导培养基、离心管聚集体培养、藻酸盐三维
立体培养诱导永生化软骨细胞的n型胶原。结果表明:(1) NA无血清诱
导培养基使各代转化细胞细胞由长梭形渐变为多角性，并于诱导后第5天
全部细胞n型胶原染色皆为阳性，其阳性染色至少可保持14天已上。(2)
离心管聚集体培养的HE染色发现细胞圆或卵圆形，分布均匀，细胞位于
典型的软骨陷窝内。阿尔新蓝染色强阳性显示有大量的胞外基质堆积。免
疫组化染色显示丰富的n型胶原合成和分泌。(3)藻酸盐三维立体培养可
保持软骨细胞的圆球形态，激光共聚焦显微镜扫描显示圆形的细胞胞浆内
11型胶原荧光染色强阳性。RT一PCR提示上述三种诱导方式均可使n型胶
原的基因表达。(4)3H一脯氨酸掺入胶原定量以及n型胶原SDS一PAGE一
3H一脯氨酸定量检测发现经NA无血清诱导培养的永生化软骨细胞的胶原
及11型胶原诱导后显著增加，ECL一W七Stem一blot检测n型胶原的诱导发
现，藻酸盐三维立体培养效果最佳，其次为离心管聚集体培养和NA无血
清诱导培养。
最后，将永生化软骨细胞经诱导扩增后与藻酸盐载体复合植入裸鼠皮
下，观察其成软骨的能力。结果显示:(1)上述复合体具有良好的成软骨
能力，经1年时间观察，形成的透明软骨无退化倾向。(2)本实验的藻酸
盐载体于12w左右基本降解并且具有较佳的细胞及组织相容性，是软骨细
胞的优秀载体。(3) BrdU标记法证明新形成的透明软骨组织来源于种植的
永生化软骨细胞。
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乙
第三军医大学博士研究生论文
人关节软骨细胞的永生化及其生物学特性
综上所述，我们得出最后结论:1.含HPV 1 6E7的逆转录病毒载体能够
成功永生化人关节软骨细胞。2.永生化人关节软骨细胞(IHAC)经严格致
瘤性检验为良性转化。3.通过3种不同的诱导方法可以快速稳定地诱导并
维持IHAC的11型胶原表型。4.IHAC在体内具有优秀的成软骨能力。
Seeded cells play an important role in tissue engineering. Chondrocytes undergo a limited number of division in culture and enter a non-dividing state called replicative senescence.In vitro mutiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generete neo-cartilage,but is known to induce cell dedifferentiation manifestes losing marker phenotype collagen type II after 5-6 passages,which poses a great difficult problem to cartilage tissue engineering. The attempt to immortalization of human articular chondrocytes shed interesting lights on the seeded cells of cartilage tissue engineering.
Firstly, retrovirus vector pLXSN harbouring HPV16E7 gene segment was constructed through molecular cloning, followed by packaging cell line PA3 17 was electroporated with pLXSN-HPV16E7 after various parameters were optimized,then human articular chondrocytes were transfected with supernatant of viron containing HPV16E7. Results showedr(l) The HFV16E7 held a great homologous with the counterpart in the Genebank. (2)Optimized parameters of electroporation were as follows: recombinant plasmid 20μg, electric capacity 975 μ F, voltage 220v, time 15ms. PA317 packaging cell line of consistant producing viron was isolated with generating viral titer 5.0 × 106-6.8× 106CFU. (3) Immortalized human articular chondrocytes (IHAC) were established with HPV16E7 gene transfection which confirmed by immunohistochemistry, PCR and RT-PCR
Secondly, biological characteristics between HAC and non-immortalized
human articular chondrocytes (NHAC) were compared, we found:(l) While NHAC appear typical trigonal or polygonal form, the size of MAC was slightly decreased and demonstrated spindle morphology. Maturation of NHAC and juvenile state of MAC were revealed by transmission elctronmicroscope. The small neclous plasma ratio as well as sparse microvilli were disclosed by transmission elctronmicroscope and scan elctronmicroscope respectively. (2)Compared with primary NHAC, more quick adhere and elongation rate,more viability and plating efficiency were in MAC. Growth curve of NHAC resembles that of MAC, but more suturation density was in MAC. More cells of MAC were in G0-M and S phase in cell circle than that of NHAC. (3)Judge tumorgenicity of MAC with below cretia: cell arrangement, growth anchorage dependence,neclous plasma ratio,microvilli,serum dependent test,soft agar cell clone forming test,karyotype analysis and nude mice tumor forming test. MAC arrayed orderly and had the characteristics of growth anchorage dependent, small neclous plasma ratio and sparce microvilli. MAC depended on serum to be confluence and had normal diploid karyotype, furthermore, MAC was not form any clones in soft agar and no tumor was observed in nude mice in one year duration. Therefore, IHAC was proved to be benign transformation. (4)Phenotype type II collagen of MAC was instability and convertd to type I collagen, as a result, type II collagen is needed to be inducted.
Third, three different methods including Nutridoma-sp and ascorbate free serum medium(NA), centrifuge tube aggregate culture combinating with BMP-2,insulin,ascorbate and alginate three dimentional culture were performed to induct type II collagen in MAC. Results showed:(l)Fusiform MAC was changed into polygonal form and immunohistochemistry indicted that type II collagen was stained positive after 5d and can maintained as long as 14 d after NA inducted. (2)IHAC inducted with centrifuge tube aggregate culture demonstrated typical hyaline cartilage,round or oval cells inhabited in the
lacuna and distributed evenly stained by HE; Alcian blue stain showed large of great amounts of extracellular matrix accumulation, immunohistochemistry revealed lots of type II collagen secretion.(3)IHAC cultured in three dimentional alginate system maintained a spherical shape,type II collagen was rich in round cells'plasma which evaluated with a confocal laser scanning microscope. Gene of type II collagen was coaxed to express in the above induction
引文
1． Jackson DW, Simon TM,Aberman HM.Symptomatic articular cartilage degeneration. Clin Orthop,2001,391 (suppl):s 14-s25．
2． Langer R, Vacanti JP. Tissue engineering.Science, 1993,260(5110) :920-926．
3． Vacanti CA, Upton J. Tissue-engineered morphogenesis of cartilage and bone by means of cell transplantation using synthetic biodegradable polymer matrices.Clin Plast Surg,1994,21(3) :445-462．
4． Cao Y,Vacanti JP, Paige KT, et al. Transplantation of chondrocytes utilizing a polymer-cell construct to produce tissue-engineered cartilage in the shape of human ear. Plast Reconstr Surg, 1997,100:297-300．
5． 何清义，李起鸿，许建中．软骨细胞的永生化及其生物学特性．中华创伤杂志， 2001,17(1) : 23-25．
6． Bodnar AG, Outellette M, Frolkis, et al. Extension of life-span by introduction of telomerase into normal human ceils. Science, 1998,279(16) :349-352．
7． Jarrard DF, Sarkar S, Shi Y et al. p16/pRB pathway alterations are required for bypassing senescence in human prostate epithelial cells. Cancer Res, 1999, 59 (12) : 2957-2964．