IGF-1对成兔关节软骨细胞体外增殖的影响
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摘要
目的
在临床上,由外伤或疾病引起的关节软骨损伤很常见,多为成年人和老年人,尤其是骨关节炎病,在老年人中更为常见。由于软骨组织中不具有血管与神经,其损伤后难以自我恢复。区别以往的实验研究对象大多为胎兔或幼兔关节软骨细胞,我们以成兔的关节软骨细胞为研究对象,在IGF-1的单独作用下,通过细胞形态学,细胞计数,细胞计量等检测手段,了解单独应用IGF-1对其增殖的促进作用,为成人关节软骨的体外培养扩增提供理论基础和临床依据。
材料与方法
1.成年兔关节软骨分离、培养:
取22月龄(属于老龄兔)的日本大耳白兔,处死,片状削取关节软骨,手术刀将软骨切成1.0mm~3大小碎块,消化、过滤、离心,搜集关节软骨细胞,加入DMEM培养液(含20%胎牛血清,青霉素100U/ml,链霉素100μg/ml),血细胞计数仪计数,调整细胞数量4.0×10~5每毫升,置于37℃,5%CO_2孵育箱内培养。3天后第一次换液,以后隔日换液,至细胞85%融合后,胰酶消化、传代。
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比色实验测定软骨细胞存活数。
5.细胞计数绘制生长曲线观察在IGF-1作用下软骨细胞的增殖情况。
6.采用t检验,进行统计学分析。
ObjectiveArticular cartilage injury caused by trauma or disease is common clinically. Most of patients are adult and old people, especially in orthopaedis' sufferers. Then there are not blood vessels and nerves in the cartilage tissues, so self -repaired of the injury is not easy. Discriminating with those studies' animals are always the fetal rabbits or immature rabbits'articular cartigate in previous, whose prolifeation are more fast than the adult rabbits'. we used the adult rabbits articular chondrocytes as the object of study. Under the effect of IGF - 1 alone to promote chondrocytes proliferation by drawing cell growth curve and detecting the survival number of the chondrocytes by MTT test, We can provide theortical-ly basis of culture and proliferation in vitro of adult people articular chondrocytes.Materials and Methods1. Isolation and Culture of adult rabbits chondrocytes: under the sterile condition , slice the articular cartigate of one Japanese big ear rabbit with the age of 22 months, the cartigate slices was cut into small cubes(1.0mm × 1.0mm × 1. 0mm or so) , digested, centrifuged. The collected cells were then resupended in DMEM culture medium ( containing 20% fetal bovine serum, penicillin 100u/ ml, streptomycin 100μg/ml) , adjust the number of cells to 4 × 10~5 and cultivated in culture flask at 37℃ ,containing 5% of CO_2. First change the liquid at the day after tomorrow, later, change the liquid every 2 days, till cartilage cell together with 85%.
2. Observe the form 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 chondro-cytes .5. Cell counting and draw growth curve to observe the proliferation state of the chondrocytes under the effect of IGF - 16. Statistics with analysis of t testDiscussion1. Using the invert microscope:Chondrocytes began to sticking to the wall of the dish at 6 hours after inoculated and after 72 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 because to be unanimous. The chondrocytes of the test group which be added with IGF - 1 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. MTT 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 directra-tio. According to the histogram with various concentrations of IGF -1, analyzed statistically, the cell nonmember is more super than the control group remarka-blely. So we determined that IGF -1 has the strongest effect of stimulating chon-
drocyte proliferation and has the best stimulating concentration.4. Cell counting and draw growth curve:Chondrocytes appeared incubation after cell seeding firstly. 4 days later, Proliferation of cells of the test group were enhanced and the multiplication time in the logarithm period is 120 hours. Meanwhile, the control group appeared cell proliferation slowly and has no obvious logarithm period. At 16 days, the cell number of test group was 3 times that of control group and has outstanding pronounced. This was in accordanced with the MTT test result. Which showed that IGF -1 can advanced cell proliferation of the adult rabbits articular chondrocytes.Results;Under tissue engineering techniques get large number autogenous chondrocytes to repair articular cartilage defect has made significant progress, Even though early hyaline cartilage has been observed in some of these studies, complete and consistent, long - term reheneration of articular cartilage in an adult animal has not yet been achieved. In previous studies, the donors of chondrocytes almost were immature articular cartilage, it is not apply to adult or order patients who want to repair articular defect with autogenous chondrocytes. At present study, we cultured adult rabbits articular chondrocytes in vitro which similar with the adult or older people. We found that the adult rabbits articular chondrocytes were survived in no growth factor condition, but they growed slowly, keep phenotypic bad and excretion weaken. This illustrate the most chondrocytes of adult articular cartilage is in tranquillization state that their dirrerentia-tion and proliferation is ceased, but part of them still has the capability to proliferation in order to complement the reduce of cells because of the damaging and cell ageing. Yet, want to get large nonmember functional cells in tissue engineering various complicated factors must be regulated and controlled.With the developing of cell biology and the study is deeping on expression of special gene of chondrocytes cultured in vitro. By drawing cell growth curve and detecting the survival number of the chondrocytes by MTT test, it proved
在临床上,由外伤或疾病引起的关节软骨损伤很常见,多为成年人和老年人,尤其是骨关节炎病,在老年人中更为常见。由于软骨组织中不具有血管与神经,其损伤后难以自我恢复。区别以往的实验研究对象大多为胎兔或幼兔关节软骨细胞,我们以成兔的关节软骨细胞为研究对象,在IGF-1的单独作用下,通过细胞形态学,细胞计数,细胞计量等检测手段,了解单独应用IGF-1对其增殖的促进作用,为成人关节软骨的体外培养扩增提供理论基础和临床依据。
材料与方法
1.成年兔关节软骨分离、培养:
取22月龄(属于老龄兔)的日本大耳白兔,处死,片状削取关节软骨,手术刀将软骨切成1.0mm~3大小碎块,消化、过滤、离心,搜集关节软骨细胞,加入DMEM培养液(含20%胎牛血清,青霉素100U/ml,链霉素100μg/ml),血细胞计数仪计数,调整细胞数量4.0×10~5每毫升,置于37℃,5%CO_2孵育箱内培养。3天后第一次换液,以后隔日换液,至细胞85%融合后,胰酶消化、传代。
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比色实验测定软骨细胞存活数。
5.细胞计数绘制生长曲线观察在IGF-1作用下软骨细胞的增殖情况。
6.采用t检验,进行统计学分析。
ObjectiveArticular cartilage injury caused by trauma or disease is common clinically. Most of patients are adult and old people, especially in orthopaedis' sufferers. Then there are not blood vessels and nerves in the cartilage tissues, so self -repaired of the injury is not easy. Discriminating with those studies' animals are always the fetal rabbits or immature rabbits'articular cartigate in previous, whose prolifeation are more fast than the adult rabbits'. we used the adult rabbits articular chondrocytes as the object of study. Under the effect of IGF - 1 alone to promote chondrocytes proliferation by drawing cell growth curve and detecting the survival number of the chondrocytes by MTT test, We can provide theortical-ly basis of culture and proliferation in vitro of adult people articular chondrocytes.Materials and Methods1. Isolation and Culture of adult rabbits chondrocytes: under the sterile condition , slice the articular cartigate of one Japanese big ear rabbit with the age of 22 months, the cartigate slices was cut into small cubes(1.0mm × 1.0mm × 1. 0mm or so) , digested, centrifuged. The collected cells were then resupended in DMEM culture medium ( containing 20% fetal bovine serum, penicillin 100u/ ml, streptomycin 100μg/ml) , adjust the number of cells to 4 × 10~5 and cultivated in culture flask at 37℃ ,containing 5% of CO_2. First change the liquid at the day after tomorrow, later, change the liquid every 2 days, till cartilage cell together with 85%.
2. Observe the form 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 chondro-cytes .5. Cell counting and draw growth curve to observe the proliferation state of the chondrocytes under the effect of IGF - 16. Statistics with analysis of t testDiscussion1. Using the invert microscope:Chondrocytes began to sticking to the wall of the dish at 6 hours after inoculated and after 72 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 because to be unanimous. The chondrocytes of the test group which be added with IGF - 1 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. MTT 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 directra-tio. According to the histogram with various concentrations of IGF -1, analyzed statistically, the cell nonmember is more super than the control group remarka-blely. So we determined that IGF -1 has the strongest effect of stimulating chon-
drocyte proliferation and has the best stimulating concentration.4. Cell counting and draw growth curve:Chondrocytes appeared incubation after cell seeding firstly. 4 days later, Proliferation of cells of the test group were enhanced and the multiplication time in the logarithm period is 120 hours. Meanwhile, the control group appeared cell proliferation slowly and has no obvious logarithm period. At 16 days, the cell number of test group was 3 times that of control group and has outstanding pronounced. This was in accordanced with the MTT test result. Which showed that IGF -1 can advanced cell proliferation of the adult rabbits articular chondrocytes.Results;Under tissue engineering techniques get large number autogenous chondrocytes to repair articular cartilage defect has made significant progress, Even though early hyaline cartilage has been observed in some of these studies, complete and consistent, long - term reheneration of articular cartilage in an adult animal has not yet been achieved. In previous studies, the donors of chondrocytes almost were immature articular cartilage, it is not apply to adult or order patients who want to repair articular defect with autogenous chondrocytes. At present study, we cultured adult rabbits articular chondrocytes in vitro which similar with the adult or older people. We found that the adult rabbits articular chondrocytes were survived in no growth factor condition, but they growed slowly, keep phenotypic bad and excretion weaken. This illustrate the most chondrocytes of adult articular cartilage is in tranquillization state that their dirrerentia-tion and proliferation is ceased, but part of them still has the capability to proliferation in order to complement the reduce of cells because of the damaging and cell ageing. Yet, want to get large nonmember functional cells in tissue engineering various complicated factors must be regulated and controlled.With the developing of cell biology and the study is deeping on expression of special gene of chondrocytes cultured in vitro. By drawing cell growth curve and detecting the survival number of the chondrocytes by MTT test, it proved
引文
1.陈付国,曹谊林,商庆新.关节软骨缺损修复研究进展.中国修复重建外科杂志,1998;12(5):297
2. Bonavententure J. Kadhon N, Gohen-Solal L, et al, Reexpression of cartilage-specific gene by dedifferentiated human articular chondrocytes cultured in alginate beads [J]. Exp Cell Res, 1994, 212(1): 97.
3. Lee DA, Bentley G, Archer CW. The control of cell division in articular chondrocytes. Osteoarthritis-Cartilage, 1993, 1(2): 137 146.
4. Verschure PJ, Van Noorden CJ, Van Marlej, et al. articular cartilage destruction in experimental inflammatory arthritis: insulin like growth factor-1 regulation of proteoglycan metabolismin chondrocytes. Histochem-J, 1996, 28(12): 835 857
5. Temenoff JS, Mikos AG. Tissue engineering for regeneration of articular cartilage [J]. Biomaterials, 2000, 21(3): 431-440.
6. Nerem RM, Sambanis A. Tissue engineering: from biology to biological substitutes [J]. Tissue Eng, 1995, 1(1): 3-13.
7.付勤,肖逸鹏.生长因子促成兔关节软骨细胞增殖的研究.中国修复重建外科杂志,2002,16(4),219-222.
8. Wei X, Gao J, Messner K. Maturation—dependent repair of untreated osteochondral defects in the rabbit knee joint. Biomed. Mater. Res. 1997,34,63
9. Hunziker, E, B. Articular cartilage repair the intrinsic biological constraints undermining this process insuperable Osteoarthritis Cartilage, 1999,7,15
10. Mankin, H. J. The response of articular cartilage to mechanical injury. [J]. Bone Joint Surg, 1982, 64,460.
11. Robert L, Vacanti JP. Tissue engineering. Science, 1993; 260 (2) :920
12. Buckwalter J, Articular cartilage: injuries and potential for healing. [J]. Orthop Sports Phys Ther, 1998,28,192
13. Mankin H J, Buckwalter J A. Restoration of the osteoarthrotic joint, [J]. Bone Joint Surg Am, 1996,78, 1
14. Luis A. Solchaga, Jizong gao, James E. Dennis at al. Treatment of osteo- chondral defects with Autologous Bone Marrow in a Hyaluronan_Based Delivery Vehicle. Tissue Engineering, 2002; 8 (2), 334
15. Hardingham T, Tews, Murdoch A. Tissue engineering chondrocytes and cartilage. Arthritis Res ,2002,4:65
16.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
17. There_Dimensional Tissue Engineering of Hyaline Cartilage Comparson of Adult Nasal and Articular Chondrocytes. Tissue Engineering, 2002,8 (5), 818
18.解增杰,许建中.IGF-1和TGF-β_1对幼兔和成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
19. Stevens DA and williame CR. Hormone regulation of chondrocyte differentiation and endochondral bone formation [J]. Mol cell Endocrinal, 1999,151:195
20. Chung_Hwan, Jong_chan Lee, Yong_Gi at al. Tissue_Engineered Cartilage on Biodgradable Macroporous Scaffolds: Cell Shape and Phenotypic Expression. The Laryngoscope, 2002, 11, 1050
21. Huckle J, Dootson G, Medcalf N at al. Differentiated chondrocytes for cartilage tissue engineering. Novartis Found Symp, 2003, 24(9), 103
22. Gueme PA, BIcnco F, Kaelin A, at al. Growth factor responsiveness of human articular chondrocytes in aging and development. [J]. Atthritis pheum, 1995,38(7): 960
23. Grande DA, Breitbart AS, Mason J, et al. Cartilage tissue engineering: Current Limitions and sclations. Clin Crthop. 1999, (367, suppl) : S176-S185.
24. Solheim SB. Growth factors in bone [J] hat Orthop,1998; 22(6) :410-416.
25. Fortier LA, Last G, Mohammed HO. Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulinlike growth factor-1 [J]. Orthop. Res,1999; 17(4): 467-474.
26. Salmon WD Jr, Daughaday WH. A hormonally controlled serum factor which stimulates-suffate incorporation by cartilage in vitro. J Lab Clin Med, 1990, 116(3): 408-419.
2. Bonavententure J. Kadhon N, Gohen-Solal L, et al, Reexpression of cartilage-specific gene by dedifferentiated human articular chondrocytes cultured in alginate beads [J]. Exp Cell Res, 1994, 212(1): 97.
3. Lee DA, Bentley G, Archer CW. The control of cell division in articular chondrocytes. Osteoarthritis-Cartilage, 1993, 1(2): 137 146.
4. Verschure PJ, Van Noorden CJ, Van Marlej, et al. articular cartilage destruction in experimental inflammatory arthritis: insulin like growth factor-1 regulation of proteoglycan metabolismin chondrocytes. Histochem-J, 1996, 28(12): 835 857
5. Temenoff JS, Mikos AG. Tissue engineering for regeneration of articular cartilage [J]. Biomaterials, 2000, 21(3): 431-440.
6. Nerem RM, Sambanis A. Tissue engineering: from biology to biological substitutes [J]. Tissue Eng, 1995, 1(1): 3-13.
7.付勤,肖逸鹏.生长因子促成兔关节软骨细胞增殖的研究.中国修复重建外科杂志,2002,16(4),219-222.
8. Wei X, Gao J, Messner K. Maturation—dependent repair of untreated osteochondral defects in the rabbit knee joint. Biomed. Mater. Res. 1997,34,63
9. Hunziker, E, B. Articular cartilage repair the intrinsic biological constraints undermining this process insuperable Osteoarthritis Cartilage, 1999,7,15
10. Mankin, H. J. The response of articular cartilage to mechanical injury. [J]. Bone Joint Surg, 1982, 64,460.
11. Robert L, Vacanti JP. Tissue engineering. Science, 1993; 260 (2) :920
12. Buckwalter J, Articular cartilage: injuries and potential for healing. [J]. Orthop Sports Phys Ther, 1998,28,192
13. Mankin H J, Buckwalter J A. Restoration of the osteoarthrotic joint, [J]. Bone Joint Surg Am, 1996,78, 1
14. Luis A. Solchaga, Jizong gao, James E. Dennis at al. Treatment of osteo- chondral defects with Autologous Bone Marrow in a Hyaluronan_Based Delivery Vehicle. Tissue Engineering, 2002; 8 (2), 334
15. Hardingham T, Tews, Murdoch A. Tissue engineering chondrocytes and cartilage. Arthritis Res ,2002,4:65
16.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
17. There_Dimensional Tissue Engineering of Hyaline Cartilage Comparson of Adult Nasal and Articular Chondrocytes. Tissue Engineering, 2002,8 (5), 818
18.解增杰,许建中.IGF-1和TGF-β_1对幼兔和成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
19. Stevens DA and williame CR. Hormone regulation of chondrocyte differentiation and endochondral bone formation [J]. Mol cell Endocrinal, 1999,151:195
20. Chung_Hwan, Jong_chan Lee, Yong_Gi at al. Tissue_Engineered Cartilage on Biodgradable Macroporous Scaffolds: Cell Shape and Phenotypic Expression. The Laryngoscope, 2002, 11, 1050
21. Huckle J, Dootson G, Medcalf N at al. Differentiated chondrocytes for cartilage tissue engineering. Novartis Found Symp, 2003, 24(9), 103
22. Gueme PA, BIcnco F, Kaelin A, at al. Growth factor responsiveness of human articular chondrocytes in aging and development. [J]. Atthritis pheum, 1995,38(7): 960
23. Grande DA, Breitbart AS, Mason J, et al. Cartilage tissue engineering: Current Limitions and sclations. Clin Crthop. 1999, (367, suppl) : S176-S185.
24. Solheim SB. Growth factors in bone [J] hat Orthop,1998; 22(6) :410-416.
25. Fortier LA, Last G, Mohammed HO. Coordinate upregulation of cartilage matrix synthesis in fibrin cultures supplemented with exogenous insulinlike growth factor-1 [J]. Orthop. Res,1999; 17(4): 467-474.
26. Salmon WD Jr, Daughaday WH. A hormonally controlled serum factor which stimulates-suffate incorporation by cartilage in vitro. J Lab Clin Med, 1990, 116(3): 408-419.