TGF-β_1作用下成年兔关节软骨细胞的体外培养
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摘要
前言
外伤或疾病引起的关节软骨损伤在临床上很常见，但软骨组织中不具有血管与神经，其损伤后不易自我修复。对于关节软骨损伤的修复在目前的骨科临床、基础研究中仍是一个重要的课题。如何体外大量扩增得到数量足够的自体软骨细胞以进行关节软骨缺损的组织工程方法修复是目前实验研究的重点。但以往的实验研究对象大多为胎兔或幼兔关节软骨细胞，其增殖能力明显好于成年兔。临床上关节软骨损伤或严重骨关节炎的患者多为老年或成年人，其自我修复能力极差，以往研究对此是不适用的。许多研究表明，生物活性因子在关节软骨的损伤、修复机制中，起着重要的调节作用。许多生长因子促进软骨细胞增殖和基质表达已被证实。其中，TGF-β_1就是一个重要的因子。我们以成兔的关节软骨细胞为对象，研究了TGF-β_1对其增殖和特异性基质合成的促进作用，为成人关节软骨细胞的体外培养扩增提供理论和临床依据。
材料与方法
1．成年兔关节软骨细胞分离、培养：
取18月龄的成年日本大耳白兔1只，处死，片状切取关节软骨，解剖刀将软骨切成1.0mm~3大小碎块，消化，过滤，离心，收集关节软骨细胞，加入DMEM培养液(含20％胎牛血清、青霉素100U／ml，链霉素100U／ml)，调整细胞密度为2.5×10~5／ml，置于37℃，5％CO_2孵育箱内培养。
2．倒置显微镜下观察软骨细胞生长状况。
3．细胞爬片HE染色光镜下观察软骨细胞形态。
4．MTT比色试验测定软骨细胞存活数及TGF-β_1促进其增殖的最佳浓度。
5．细胞计数绘制生长曲线观察在TGF-β_1作用下软骨细胞的增殖情况。
6．Ⅱ型胶原免疫组织化学检测证实在TGF-β_1作用下软骨细胞Ⅱ胶原
的表达。
7.采用方差分析法，进行统计分析。
实验结果
1.倒置显微镜下观察:
软骨细胞接种4小时后开始贴壁，培养48小时后大部分细胞贴壁，形
态由球形逐渐向外伸展突起而转变为多边形，少数呈梭形。随着细胞数量
增加，体积增大，细胞形态趋于一致。加人因子TGF一日1实验组软骨细胞以
多角形为主，分裂相多见，接种后16一18天后逐渐融合成单层，呈“铺路
石”样外观。对照组细胞生长较为缓慢，梭形细胞比例逐渐增高，分裂相少
见。
2.细胞爬片HE染色光镜观察:
通过组织学染色，可以更清楚观察软骨细胞形态:软骨细胞多数为小细
胞，形态有多边形、圆形及梭形，可以见到核分裂。
3.M竹比色试验:
以酶联免疫检测仪(MTT)测定所得的光密度(OD)值，此值与细胞存
活数成正比，即作为细胞相对数，绘出加人不同浓度TGF一pl后的OD值直
方图，其结果经过统计学处理分析，表明加入TGF一日，实验组的细胞数显著
高于对照组，且最佳浓度为10n扩血。所以我们认为，TGF一p，对软骨细胞的
具有促增殖作用，有最佳刺激浓度。
4.细胞计数绘制生长曲线:
细胞接种后出现潜伏期，实验组自第5天起细胞增殖能力增强，对数期
细胞数倍增时间为72小时。对照组自第5天起细胞缓慢增殖，没有明显的
对数增殖期。第巧天时实验组细胞为对照组的4倍以上，明显高于对照
组。与M竹试验结果一致，说明TGF~日1对成年兔关节软骨细胞增殖的促
进作用。
5 .n型胶原免疫组织化学检测:
细胞爬片n型胶原免疫组化染色光镜观察，可见软骨细胞的胞浆被染
成棕黄色，细胞相互接触少的地方棕黄色较淡，细胞密集生长的地方棕黄色
较深，并且细胞外也可见到棕黄色。实验组着色强于对照组，说明TGRpl
在促进软骨细胞增殖的同时能保持分泌n型胶原，能够维持经过较长时间
体外培养的软骨细胞的功能。
讨论
采用组织工程学方法体外扩增获得大量的自体细胞进行修复关节软骨
缺损的研究取得了重大进展，在许多试验中可早期发现透明软骨修复组织
的形成，但在成年动物中尚未发现完全的、不断进行再生和重建的关节软骨
组织。以往实验中，软骨细胞供体大多是处于幼稚状态的关节软骨，对于成
年或老年患者自体软骨细胞进行软骨缺损修复明显是不适用的。本实验选
择了与成年或老年人相近的成年兔关节软骨细胞进行体外培养研究，在实
验中发现，成年兔关节软骨细胞在体外无因子培养条件下可以成活，但表现
出了细胞生长缓慢、细胞表型维持不佳，细胞分泌功能减弱的特性。说明了
成熟的关节软骨组织中的大部分软骨细胞是处于一种停止分化和增殖的静
息状态，但仍有部分细胞保持一定的增殖能力，以便可以补充由于损伤或细
胞老化造成的细胞减少。但要获得组织工程所需的大量的有功能的细胞则
需进行多种复杂因素的调控。
随着细胞生物学的发展，软骨细胞体外培养特异基因表达的研究日益
深人，生物活性因子调节软骨细胞的增殖、分化过程逐渐被人们所揭示。这
对成熟阶段的关节软骨细胞的体外培养研究又指出了一个方向。转化生长
因子p(TGF一p)最初是由Robert等学者在1 978年作为一种可诱导大鼠成
纤维细胞增殖因子而描述。TGF一日可以诱导间充质细胞转化为软骨细胞。
现已实验证明TGF一pl具有促进软骨细胞增殖，调节其分化和细胞外基质合
Articular cartilage injury caused by trauma or disease is common clinically. But there are not blood vessels and nerves in the cartilage tissues, so self - ple-rosis of the injury is not easy. The plerosis of articular cartilage injury is an important task in the aspects of clinincal and basic research on orthopaedics. How to get large number of autologous chondrocytes is the emphase tesk in the tissue engineering to repair articular cartilage defect. But in previous , the study animals are always the fetal rabbits or immature rabbits'articular cartilage, its'proliferation more fast than the adult rabbits. Many researches show that organism activity gene plays an important role in regulating the mechanism of injury and plerosis of articular cartilage. Many growth factor had been evidenced that their has the effects of promoting proliferation and express the extracellular maxtress. In those, TGF-β1 is a important factor. Clinically,the patients of articular injury or serious osteoarthritis are always the old
and adult people, Their self repair capacity is very poor. In present study, we used the adult rabbits articular chondrocytes as the object of study, we researched the effect of TGF-β1 to promote chondrocytes proliferation and preserve the synthetise of special stroma in order to provide theortically and clinically basis of culture and proliferation in vitro of a-dult people articular chondrocytes.
Materials and Methods
1. Isolation and Culture of adult rabbits chondrocytes: under the sterile
condition, slice the articular cartilage of one Japanese big ear rabbit with the age
of 18 months, the cartilage slices was cut into small cubes (1.0 mm x 1.0 mm
x 1.0mm or so) , digested , filtrated, and centrifuged. The collected cells were
then resuspended in DMED culture mediun(containing20% fetal bovine serum, penicillin l00u/ml,streptomycin l00ug/ml) ,adjust the percentage of cells to 2. 5 x 10Vml and cultivated in culture flask at 37 , containing 5% of C02.
2. Observe the foim and growth condition of the cells under the invert microscope.
3. Observing the cells climbing to the carry sheet glass, which are stained with HE.
4. Detect with MTT test to comprehend the survival numbers of the chon-drocytes and the bestest effective concentration of TGF-β1.
5. Cell counting and draw growth curve to observe the proliferation state of the chondrocytes under the effect of TGF-β1.
6. Detect the expression of n - collagen of the chondrocytes climbing to the carry sheet glass with immunohistochemistry in order to verity the effect of TGF-β1.
7. Statistics with analysis of variance.
Experimental Results
1. Under the invert microscope:
chondrocytes began to sticking to the wall of the Petri dish at 4 hours after inoculated and after 48 hours almost all the cells are sticking to the wall. Their phase changed spherical to polygon and some is fusiformis. With the increase of the quantity and volue,cells phase became to be unanimous. The chondrocytes of the test group which be added with TGF-β1 are polygon mostly and have more division cells and blend to be monolayer as "flagstone", the cells of the control group growth slowly and have a few division cells , the proportion of the fusiformis cells increased.
2. Observing the cells climbing to the carry sheet glass, which are stained with HE:
Under the light microscope, the shapes of chondrocytes may be seen more clearly, being small mostly, in the shapes of fusiform, roundlet and polygon, etc, and karyokinesis may be seen.
3 . MIT colorimetric test;
Determination of the survival numbers of the cells: 0. D. ( optical density ) value determined by MTT and the survival numbers of the cells are in direct ratio. According to the histogram with various concentrations of TGF-β1, analyzed statistically, the cell nonmember is more super than the control group re-markablely. and the best concentration is l0ng/ml. So we determined that TGF-β1 has the strongest effect of stimulating chondrocyte proliferation and has the be
外伤或疾病引起的关节软骨损伤在临床上很常见，但软骨组织中不具有血管与神经，其损伤后不易自我修复。对于关节软骨损伤的修复在目前的骨科临床、基础研究中仍是一个重要的课题。如何体外大量扩增得到数量足够的自体软骨细胞以进行关节软骨缺损的组织工程方法修复是目前实验研究的重点。但以往的实验研究对象大多为胎兔或幼兔关节软骨细胞，其增殖能力明显好于成年兔。临床上关节软骨损伤或严重骨关节炎的患者多为老年或成年人，其自我修复能力极差，以往研究对此是不适用的。许多研究表明，生物活性因子在关节软骨的损伤、修复机制中，起着重要的调节作用。许多生长因子促进软骨细胞增殖和基质表达已被证实。其中，TGF-β_1就是一个重要的因子。我们以成兔的关节软骨细胞为对象，研究了TGF-β_1对其增殖和特异性基质合成的促进作用，为成人关节软骨细胞的体外培养扩增提供理论和临床依据。
材料与方法
1．成年兔关节软骨细胞分离、培养：
取18月龄的成年日本大耳白兔1只，处死，片状切取关节软骨，解剖刀将软骨切成1.0mm~3大小碎块，消化，过滤，离心，收集关节软骨细胞，加入DMEM培养液(含20％胎牛血清、青霉素100U／ml，链霉素100U／ml)，调整细胞密度为2.5×10~5／ml，置于37℃，5％CO_2孵育箱内培养。
2．倒置显微镜下观察软骨细胞生长状况。
3．细胞爬片HE染色光镜下观察软骨细胞形态。
4．MTT比色试验测定软骨细胞存活数及TGF-β_1促进其增殖的最佳浓度。
5．细胞计数绘制生长曲线观察在TGF-β_1作用下软骨细胞的增殖情况。
6．Ⅱ型胶原免疫组织化学检测证实在TGF-β_1作用下软骨细胞Ⅱ胶原
的表达。
7.采用方差分析法，进行统计分析。
实验结果
1.倒置显微镜下观察:
软骨细胞接种4小时后开始贴壁，培养48小时后大部分细胞贴壁，形
态由球形逐渐向外伸展突起而转变为多边形，少数呈梭形。随着细胞数量
增加，体积增大，细胞形态趋于一致。加人因子TGF一日1实验组软骨细胞以
多角形为主，分裂相多见，接种后16一18天后逐渐融合成单层，呈“铺路
石”样外观。对照组细胞生长较为缓慢，梭形细胞比例逐渐增高，分裂相少
见。
2.细胞爬片HE染色光镜观察:
通过组织学染色，可以更清楚观察软骨细胞形态:软骨细胞多数为小细
胞，形态有多边形、圆形及梭形，可以见到核分裂。
3.M竹比色试验:
以酶联免疫检测仪(MTT)测定所得的光密度(OD)值，此值与细胞存
活数成正比，即作为细胞相对数，绘出加人不同浓度TGF一pl后的OD值直
方图，其结果经过统计学处理分析，表明加入TGF一日，实验组的细胞数显著
高于对照组，且最佳浓度为10n扩血。所以我们认为，TGF一p，对软骨细胞的
具有促增殖作用，有最佳刺激浓度。
4.细胞计数绘制生长曲线:
细胞接种后出现潜伏期，实验组自第5天起细胞增殖能力增强，对数期
细胞数倍增时间为72小时。对照组自第5天起细胞缓慢增殖，没有明显的
对数增殖期。第巧天时实验组细胞为对照组的4倍以上，明显高于对照
组。与M竹试验结果一致，说明TGF~日1对成年兔关节软骨细胞增殖的促
进作用。
5 .n型胶原免疫组织化学检测:
细胞爬片n型胶原免疫组化染色光镜观察，可见软骨细胞的胞浆被染
成棕黄色，细胞相互接触少的地方棕黄色较淡，细胞密集生长的地方棕黄色
较深，并且细胞外也可见到棕黄色。实验组着色强于对照组，说明TGRpl
在促进软骨细胞增殖的同时能保持分泌n型胶原，能够维持经过较长时间
体外培养的软骨细胞的功能。
讨论
采用组织工程学方法体外扩增获得大量的自体细胞进行修复关节软骨
缺损的研究取得了重大进展，在许多试验中可早期发现透明软骨修复组织
的形成，但在成年动物中尚未发现完全的、不断进行再生和重建的关节软骨
组织。以往实验中，软骨细胞供体大多是处于幼稚状态的关节软骨，对于成
年或老年患者自体软骨细胞进行软骨缺损修复明显是不适用的。本实验选
择了与成年或老年人相近的成年兔关节软骨细胞进行体外培养研究，在实
验中发现，成年兔关节软骨细胞在体外无因子培养条件下可以成活，但表现
出了细胞生长缓慢、细胞表型维持不佳，细胞分泌功能减弱的特性。说明了
成熟的关节软骨组织中的大部分软骨细胞是处于一种停止分化和增殖的静
息状态，但仍有部分细胞保持一定的增殖能力，以便可以补充由于损伤或细
胞老化造成的细胞减少。但要获得组织工程所需的大量的有功能的细胞则
需进行多种复杂因素的调控。
随着细胞生物学的发展，软骨细胞体外培养特异基因表达的研究日益
深人，生物活性因子调节软骨细胞的增殖、分化过程逐渐被人们所揭示。这
对成熟阶段的关节软骨细胞的体外培养研究又指出了一个方向。转化生长
因子p(TGF一p)最初是由Robert等学者在1 978年作为一种可诱导大鼠成
纤维细胞增殖因子而描述。TGF一日可以诱导间充质细胞转化为软骨细胞。
现已实验证明TGF一pl具有促进软骨细胞增殖，调节其分化和细胞外基质合
Articular cartilage injury caused by trauma or disease is common clinically. But there are not blood vessels and nerves in the cartilage tissues, so self - ple-rosis of the injury is not easy. The plerosis of articular cartilage injury is an important task in the aspects of clinincal and basic research on orthopaedics. How to get large number of autologous chondrocytes is the emphase tesk in the tissue engineering to repair articular cartilage defect. But in previous , the study animals are always the fetal rabbits or immature rabbits'articular cartilage, its'proliferation more fast than the adult rabbits. Many researches show that organism activity gene plays an important role in regulating the mechanism of injury and plerosis of articular cartilage. Many growth factor had been evidenced that their has the effects of promoting proliferation and express the extracellular maxtress. In those, TGF-β1 is a important factor. Clinically,the patients of articular injury or serious osteoarthritis are always the old
and adult people, Their self repair capacity is very poor. In present study, we used the adult rabbits articular chondrocytes as the object of study, we researched the effect of TGF-β1 to promote chondrocytes proliferation and preserve the synthetise of special stroma in order to provide theortically and clinically basis of culture and proliferation in vitro of a-dult people articular chondrocytes.
Materials and Methods
1. Isolation and Culture of adult rabbits chondrocytes: under the sterile
condition, slice the articular cartilage of one Japanese big ear rabbit with the age
of 18 months, the cartilage slices was cut into small cubes (1.0 mm x 1.0 mm
x 1.0mm or so) , digested , filtrated, and centrifuged. The collected cells were
then resuspended in DMED culture mediun(containing20% fetal bovine serum, penicillin l00u/ml,streptomycin l00ug/ml) ,adjust the percentage of cells to 2. 5 x 10Vml and cultivated in culture flask at 37 , containing 5% of C02.
2. Observe the foim and growth condition of the cells under the invert microscope.
3. Observing the cells climbing to the carry sheet glass, which are stained with HE.
4. Detect with MTT test to comprehend the survival numbers of the chon-drocytes and the bestest effective concentration of TGF-β1.
5. Cell counting and draw growth curve to observe the proliferation state of the chondrocytes under the effect of TGF-β1.
6. Detect the expression of n - collagen of the chondrocytes climbing to the carry sheet glass with immunohistochemistry in order to verity the effect of TGF-β1.
7. Statistics with analysis of variance.
Experimental Results
1. Under the invert microscope:
chondrocytes began to sticking to the wall of the Petri dish at 4 hours after inoculated and after 48 hours almost all the cells are sticking to the wall. Their phase changed spherical to polygon and some is fusiformis. With the increase of the quantity and volue,cells phase became to be unanimous. The chondrocytes of the test group which be added with TGF-β1 are polygon mostly and have more division cells and blend to be monolayer as "flagstone", the cells of the control group growth slowly and have a few division cells , the proportion of the fusiformis cells increased.
2. Observing the cells climbing to the carry sheet glass, which are stained with HE:
Under the light microscope, the shapes of chondrocytes may be seen more clearly, being small mostly, in the shapes of fusiform, roundlet and polygon, etc, and karyokinesis may be seen.
3 . MIT colorimetric test;
Determination of the survival numbers of the cells: 0. D. ( optical density ) value determined by MTT and the survival numbers of the cells are in direct ratio. According to the histogram with various concentrations of TGF-β1, analyzed statistically, the cell nonmember is more super than the control group re-markablely. and the best concentration is l0ng/ml. So we determined that TGF-β1 has the strongest effect of stimulating chondrocyte proliferation and has the be
引文
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11．解增杰，许建中．IGF -1和TGF-β_1对幼兔和成年兔关节软骨细胞增殖和代谢作用．第三军医大学学报，2001，23(6)，670
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18．解慧琪．组织工程种子细胞研究．现代高新技术与创伤修复，2002，5，180
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9．王建，杨志明，解慧琪．培养软骨细胞移植修复兔生长板缺损．中国修复重建外科杂志，2001；15(1)：53
10. Three-Dimensional Tissue Engineering of Hyaline Cartilage Comparison of Adult Nasal and Articular Chondrocytes. Tissue Engineering, 2002, 8(5), 818
11．解增杰，许建中．IGF -1和TGF-β_1对幼兔和成年兔关节软骨细胞增殖和代谢作用．第三军医大学学报，2001，23(6)，670
12. Stevens DA and williame CR. Hormone regulation of chondrocyte differentiation and endochondral bone formation[J]. Mol cell Endocrinal, 1999, 151: 195
13. Chung-Hwan, Jong-chan Lee, Yong-Gi at al. Tissue-Engineered Cartilage on Biodegradable Macroporous Scaffolds: Cell Shape and Phenotypic
Expression. The Laryngoscope, 2002, 11, 1050
14. Huckle J, Dootson G, Medcalf N at al. Differentiated chondrocytes for cartilage tissue engineering. Novartis Found Syrup, 2003, 24(9), 103
15. Gueme PA, Blcnco F, Kaelin A, at al. Growth factor responsiveness of human articular chondrocytes in aging and devdopment.[J]. Arthritis pheum, 1995, 38(7): 960
16. Peter C, Yaeger, Tersdl. Synthesis action of transforming growth factor-β and insuline-like growth factor -1 induces espression of type collagen and aggrecan gene in adult human articular chondrocytes[J]. Exp-cellres, 1997, 237: 318
17. Firedini, Matriel. Transforming growth factor beta exert opposite effects from interleukin-β on cultured rabbit articular chondrocytes through reduction of interleukin receptor expression[J]. Arthrits and pheumatism, 1993, 36: 44
18．解慧琪．组织工程种子细胞研究．现代高新技术与创伤修复，2002，5，180