局部应用血管内皮生长因子及间充质干细胞培养物提高皮瓣成活率的实验性研究
摘要
针对各种原因造成的组织缺损进行的皮瓣移植术中,如何提高皮瓣的成活率一直是损伤与重建外科的研究热点问题。血管内皮生长因子(VEGF)是目前所知道的唯一作用于血管内皮细胞表面,直接刺激血管形成的细胞因子。它可通过细胞间隙的形成增加内皮通透性,还可以通过自分泌和旁分泌作用,刺激血管内皮细胞有丝分裂和增加血管内皮细胞渗透性促进血管生成,进而起到促进组织愈合的作用。骨髓间充质干细胞(MSCs)是存在于骨髓中的具有多种分化潜能的祖细胞,能增殖迅速,并且有低免疫原性的特点。它能够在特定的条件下向所有起源于中胚层的细胞分化。
本实验采用密度梯度离心法和贴壁筛选法分离培养人骨髓间充质干细胞(hMSCs),经免疫组化、流式细胞分析方法及根据MSCs具有多向分化潜能的特点采用的反证法证明所分离的细胞是hMSCs,而不是成熟细胞。传代培养至三代以上的细胞用于动物实验。本研究根据VEGF和MSCs的生物学特性,将实验动物随机分成四组,即空白对照组、VEGF组、hMSCs组,VEGF+hMSCs组。通过对不同分组实验动物的术后临床观察、皮瓣成活率检测、组织病理检测、免疫组化检测及微血管密度计数等实验结果进行统计学分析,得出采用密度梯度离心法和贴壁筛选法可以得到纯度较高、生物学性能稳定的骨髓间充质干细胞;hMSCs具有低免疫原性或无免疫反应性的特性;单独将VEGF或hMSCs应用于超比例皮瓣均可提皮瓣的成活率以及VEGF和hMSCs的联合应用对提高超比例皮瓣成活率的效果明显优于单独应用VEGF或hMSCs的效果。针对提高皮瓣成活率的研究很多,但将VEGF和hMSCs联合应用考察其有效性的研究尚少。本实验在证明了骨髓间充质干细胞具有低免疫原性或无免疫反应性的特性的同时也得出了VEGF和hMSCs的联合应用在短期内可重建缺血区域的血液循环,解决超比例皮瓣的远蒂端缺血问题,提高皮瓣成活率和创面组织的修复质量的结论。
Objective
The biological characteristics of VEGF and hMSC will be involved in this experiment. Utilizing the Wistar rat to establish ultra-ratio random flap, observe the reestablishment of blood circulation in ischemic region of different division of animal skin flap in short term, by trying the local application of the VEGF, hMSCs, and the culture of VEGF and hMSC. Investigate the effect that the combination of VEGF and hMSCs make to the solution of the distal pedicle ischemia of ultra-ratio random flap, the enhancement of the skin flap survival rate, and the quality restoration of wound tissues.
Methods
1. Isolation, culturing and identification of human bone marrow mesenchymal stem cells:
Isolate and culture bone marrow mesenchymal stem cells by both adherence separation and density gradient centrifugation. The cells which are sub-cultured for more than three generations can be used in the follow-up animal experiment. Immunohistochemistry and flow cytometry (FACS) united identification will be adopted. Besides, according to MSCs has the characteristic of multiplex differentiation potential, use reduction to absurdity.
2. Establishment of animal model:
Use rat back as the operation area, designing the skin flap approach after routine disinfection. Mark out the 2×5cm rectangular flap by gentian violet. Strengthen infiltration anesthesia in local area by 0.5% lidocaine. Incise full-thickness skin along the designed line down to the superficial layer of muscular fasciae by the 11th scissors slice. Use the dissector peel off the clearance on the muscular fasciae, make the distal pedicle of the skin flap completely free, and then establish the random flap model. Use lukewarm physiological saline gauze to stop bleeding by compression in local bleeding point. Stitch up the wound by the technique of interrupted apposition sutures along number 0 line. Besmear with aureomycin eye ointment, inject Penicillin 200000RU, and inject RECOVERY-4 in a ratio of 1.5:1 till the end of suturing. Put the rat into the isolated cages after it can move about freely.
According to the need of experiment designing, divide the experimental animals randomly into four groups, 10 each group. Marked with A, B, C, D, which are blank control group, VEGF group, hMSCs group, VEGF+hMSCs group.After blank control group set up, suture the flaps in situ. VEGF group set up in the flaps then inject 300ng VEGF to six different injection sites in the fascia, suture in situ; hMSCs group set up in the flaps then smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells).VEGF + hMSCs group set up in the flaps, inject 300ng VEGF to six different injection sites in the fascia, and smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells),suture in situ.
3. Make continuous clinical observation and record the back flap of the rats during 14 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair, depression and skin ulcers to the experimental animals. Observe the flap color, thickness, elasticity, exudation, hair growth, necrosis area and acupuncture bleeding situation, then record in detail. on the fourteenth day after operation, use sulfuric acid paper describe the area of ischemic necrosis tissue on the distal end of the flaps, then add the number of grids in the grid paper to calculate the survival area and converte to percentage survival, that is the average survival area rate of flap. Data on the results obtained were statistically analyzed.
4. Sacrificing the rats on the fourteenth day after operation, cutting the flap 4cm apart the distal pedicle of each rats, detecting the flaps with the histological examination and immunohistochemical methods. Observing the volume and density of microvessels of the distal pedicle under the microscope. Data on the results obtained were statistically analyzed.
Results
1. The bone marrow-derived mesenchymal stem cells cultured isolated are typical spindle-shaped fibroblast-like. 16th-30th days during isolated culture, the cell confluence is above 80%-90%, which arranges in imbricate or Vortex-like shape. detecting surface marker with Flow Cytometry, we found it didn’t express surface markers of hematopoietic cells, which means CD34、CD45、HLA-DR sustained negative, CD29、CD44、CD73、CD105、CD166 sustained positive. This stem cells can be induced to fat and cartilage differentiation, which proves bone marrow-derived mesenchymal stem cells have multi-directional differentiation potential.
2. There is immunological rejection in each experimental group during 14 days after operation. On the fourteenth day after operation, the survival rate of flap: VEGF+hMSCs group > VEGF group> hMSCs group> blank control group.
3. HE stained histological examination on the fourteenth day after operation, we found in group D, there are the most abundant granulation tissues, cell components and new capillaries, fibroblasts increased; in B group and C group, there are also more abundant granulation tissues, cell components and new capillaries; in A group we could see a large number of inflammatory cell infiltration, necrotic tissues, less cell components and new capillaries.
4. The fourteenth day after operation, the results of the CD34 factor polyclonal antibody immunohistochemical staining showed that the vascular endothelial cells express factor CD34 positive, and the cytoplasm stained brown. in each group under the microscope we could observe the new relationship between the number of microvessels and microvessel density (MVD): VEGF + hMSCs Group> VEGF group> hMSCs group> control group.
Disscution
The separately local application of VEGF and hMSCs could increase the survival rate of the flap. The effects of the united application of VEGF and hMSCs will increase the overwhelming survival rate of the flap is much superior to the separate application of VEGF and hMSCs. The united application of the culture of VEGF and hMSCs may rebuild the ischemic region and blood circulation in the short term, and solve the distal pedicle ischemia of the ultra-ratio flap, increasing the reparation quality of the survival rate of the flap and epidermis organization.
本实验采用密度梯度离心法和贴壁筛选法分离培养人骨髓间充质干细胞(hMSCs),经免疫组化、流式细胞分析方法及根据MSCs具有多向分化潜能的特点采用的反证法证明所分离的细胞是hMSCs,而不是成熟细胞。传代培养至三代以上的细胞用于动物实验。本研究根据VEGF和MSCs的生物学特性,将实验动物随机分成四组,即空白对照组、VEGF组、hMSCs组,VEGF+hMSCs组。通过对不同分组实验动物的术后临床观察、皮瓣成活率检测、组织病理检测、免疫组化检测及微血管密度计数等实验结果进行统计学分析,得出采用密度梯度离心法和贴壁筛选法可以得到纯度较高、生物学性能稳定的骨髓间充质干细胞;hMSCs具有低免疫原性或无免疫反应性的特性;单独将VEGF或hMSCs应用于超比例皮瓣均可提皮瓣的成活率以及VEGF和hMSCs的联合应用对提高超比例皮瓣成活率的效果明显优于单独应用VEGF或hMSCs的效果。针对提高皮瓣成活率的研究很多,但将VEGF和hMSCs联合应用考察其有效性的研究尚少。本实验在证明了骨髓间充质干细胞具有低免疫原性或无免疫反应性的特性的同时也得出了VEGF和hMSCs的联合应用在短期内可重建缺血区域的血液循环,解决超比例皮瓣的远蒂端缺血问题,提高皮瓣成活率和创面组织的修复质量的结论。
Objective
The biological characteristics of VEGF and hMSC will be involved in this experiment. Utilizing the Wistar rat to establish ultra-ratio random flap, observe the reestablishment of blood circulation in ischemic region of different division of animal skin flap in short term, by trying the local application of the VEGF, hMSCs, and the culture of VEGF and hMSC. Investigate the effect that the combination of VEGF and hMSCs make to the solution of the distal pedicle ischemia of ultra-ratio random flap, the enhancement of the skin flap survival rate, and the quality restoration of wound tissues.
Methods
1. Isolation, culturing and identification of human bone marrow mesenchymal stem cells:
Isolate and culture bone marrow mesenchymal stem cells by both adherence separation and density gradient centrifugation. The cells which are sub-cultured for more than three generations can be used in the follow-up animal experiment. Immunohistochemistry and flow cytometry (FACS) united identification will be adopted. Besides, according to MSCs has the characteristic of multiplex differentiation potential, use reduction to absurdity.
2. Establishment of animal model:
Use rat back as the operation area, designing the skin flap approach after routine disinfection. Mark out the 2×5cm rectangular flap by gentian violet. Strengthen infiltration anesthesia in local area by 0.5% lidocaine. Incise full-thickness skin along the designed line down to the superficial layer of muscular fasciae by the 11th scissors slice. Use the dissector peel off the clearance on the muscular fasciae, make the distal pedicle of the skin flap completely free, and then establish the random flap model. Use lukewarm physiological saline gauze to stop bleeding by compression in local bleeding point. Stitch up the wound by the technique of interrupted apposition sutures along number 0 line. Besmear with aureomycin eye ointment, inject Penicillin 200000RU, and inject RECOVERY-4 in a ratio of 1.5:1 till the end of suturing. Put the rat into the isolated cages after it can move about freely.
According to the need of experiment designing, divide the experimental animals randomly into four groups, 10 each group. Marked with A, B, C, D, which are blank control group, VEGF group, hMSCs group, VEGF+hMSCs group.After blank control group set up, suture the flaps in situ. VEGF group set up in the flaps then inject 300ng VEGF to six different injection sites in the fascia, suture in situ; hMSCs group set up in the flaps then smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells).VEGF + hMSCs group set up in the flaps, inject 300ng VEGF to six different injection sites in the fascia, and smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells),suture in situ.
3. Make continuous clinical observation and record the back flap of the rats during 14 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair, depression and skin ulcers to the experimental animals. Observe the flap color, thickness, elasticity, exudation, hair growth, necrosis area and acupuncture bleeding situation, then record in detail. on the fourteenth day after operation, use sulfuric acid paper describe the area of ischemic necrosis tissue on the distal end of the flaps, then add the number of grids in the grid paper to calculate the survival area and converte to percentage survival, that is the average survival area rate of flap. Data on the results obtained were statistically analyzed.
4. Sacrificing the rats on the fourteenth day after operation, cutting the flap 4cm apart the distal pedicle of each rats, detecting the flaps with the histological examination and immunohistochemical methods. Observing the volume and density of microvessels of the distal pedicle under the microscope. Data on the results obtained were statistically analyzed.
Results
1. The bone marrow-derived mesenchymal stem cells cultured isolated are typical spindle-shaped fibroblast-like. 16th-30th days during isolated culture, the cell confluence is above 80%-90%, which arranges in imbricate or Vortex-like shape. detecting surface marker with Flow Cytometry, we found it didn’t express surface markers of hematopoietic cells, which means CD34、CD45、HLA-DR sustained negative, CD29、CD44、CD73、CD105、CD166 sustained positive. This stem cells can be induced to fat and cartilage differentiation, which proves bone marrow-derived mesenchymal stem cells have multi-directional differentiation potential.
2. There is immunological rejection in each experimental group during 14 days after operation. On the fourteenth day after operation, the survival rate of flap: VEGF+hMSCs group > VEGF group> hMSCs group> blank control group.
3. HE stained histological examination on the fourteenth day after operation, we found in group D, there are the most abundant granulation tissues, cell components and new capillaries, fibroblasts increased; in B group and C group, there are also more abundant granulation tissues, cell components and new capillaries; in A group we could see a large number of inflammatory cell infiltration, necrotic tissues, less cell components and new capillaries.
4. The fourteenth day after operation, the results of the CD34 factor polyclonal antibody immunohistochemical staining showed that the vascular endothelial cells express factor CD34 positive, and the cytoplasm stained brown. in each group under the microscope we could observe the new relationship between the number of microvessels and microvessel density (MVD): VEGF + hMSCs Group> VEGF group> hMSCs group> control group.
Disscution
The separately local application of VEGF and hMSCs could increase the survival rate of the flap. The effects of the united application of VEGF and hMSCs will increase the overwhelming survival rate of the flap is much superior to the separate application of VEGF and hMSCs. The united application of the culture of VEGF and hMSCs may rebuild the ischemic region and blood circulation in the short term, and solve the distal pedicle ischemia of the ultra-ratio flap, increasing the reparation quality of the survival rate of the flap and epidermis organization.
引文
[1] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci.2001; 114:853-865.
[2] Stefan Schultze-Mosgau, Improved free vascular graft survival in an irradiated surgical site following topical application of rVEGF. Biol. Phys., 2003,57:803-812.
[3]王炜主编.整形外科学.杭州:浙江科学技术出版社, 1999,9:143
[4] Ferrara N, Henzel W J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific vascular endothelial cells. Biochemeiophys Res Common.1989,161:851-858
[5] Meyer M, Clauss M, Lepple-Wienhues A, et al. A novel vascular endothelial growth factor encoded by orfvirus VEGF-E,mediates an giogenesis viasignaling through VEGFR-2(KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases[J]. EMBO J, 1999,18(2):363-372.
[6] Neufeld G, Cohen T, Gengrinovitch S, et al.Vascular endothelial growth factor(VEGF)and its receptors[J]. FASEB J, 1999,13(1):9-2 2.
[7] Clauss M. Molecular biology of the VEGF and the VEGF receptor family[J]. Semin Thromb Hemost, 2000,26(5):561-569.
[8] Vincenti V, Cassano C, Roschi M, Persico G. Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation. 1996;93:1493-1495.
[9] Lin P, Sankar S, ShanS, etal. Inhibition of tumor growth by targeting tumor endothelium using as oluble vascular endothelial growth factor receptor. Cell Growth Difer. 1998;9(1):49-58.
[10] StepnickD W, PetersonM K, BodganC, etal. Efects of tumor necrosis factor alpha and vascular permeability factor on neovascularization of the rabbit ear flap Arch Otolaryngol Head Neck Surg. 1995;121(6):667-72.
[11] Padubidri A, Browne E J r. Efect of vascular endothelial gr owth factor(VEGF)on survival of random extension of axial patern skin flaps in the rat. Ann Plast Surg.1996;37(6):604-11.
[12]蔚凡,李佛保.血管内皮生长因子影响大鼠皮瓣成活的实验研究.中国修复重建外科杂志, 1997,11(6):376-378.
[13] Kryger Z, Zhang F, Dogan T, etal. The efects of VEGF on survival of a random flap in the rat:examination of various routes of administration. Br J PlastS urg. 2000;53(3):234-9.
[14] Zhang F, Richards L, Angel MF, etal. Accelerating flap maturation by vascular endothelium growth factor in a rat tube flap model.BrJ PlastSurg. 2002;55(1):59-63.
[15] KrygerZ, ZhangF, DoganT, etal. The effects of VEGF on survival of a randomflap in the rat:examination of various routes of administration. BrJ PlastSurg. 2000;53(3):234-9.
[16]刘志辉,刘春丽,张伟.血管内皮生长因子皮下注射对大鼠背部皮瓣成活的实验研究[C].腔领面外科杂志, 2002,12(1):20-23.
[17] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001;114:853-865.
[18] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood.Br J Haematol. 2000,109(1):235-242.
[19] Zvaifler NJ, Mutafchieva LM, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000;2:477-488.
[20] Gronthos S, Zannettino AC, Graves SE, et al. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res, 1999;14(1):47-56.
[21] Zuk PA, Zhu M, Mizuno H,etal. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001, 7(2):211-228.
[22]史春梦,程天民.大鼠真皮多能间充质干细胞的分离培养.第三军医大学学报. 2001,23(9):1068-1070.
[23] Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol, 1976;4:267-274.
[24] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science, 1999,284 (5411):143-147.
[25] Haynesworth SE, Baber MA, Caplan AI .Cytokine expression by human marrow-derived mesenchymal progenitor cell in vitro:effects of dexamethasone and IL-1 alpha. J Cell Physiol, 1996;166(3):585- 592
[26]刘晓丹,郭子宽,李秀森,等.人骨髓间充质干细胞分离与培养方法的建立.军事医学科学院院刊, 2000;24(4):282-284
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[2] Stefan Schultze-Mosgau, Improved free vascular graft survival in an irradiated surgical site following topical application of rVEGF. Biol. Phys., 2003,57:803-812.
[3]王炜主编.整形外科学.杭州:浙江科学技术出版社, 1999,9:143
[4] Ferrara N, Henzel W J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific vascular endothelial cells. Biochemeiophys Res Common.1989,161:851-858
[5] Meyer M, Clauss M, Lepple-Wienhues A, et al. A novel vascular endothelial growth factor encoded by orfvirus VEGF-E,mediates an giogenesis viasignaling through VEGFR-2(KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases[J]. EMBO J, 1999,18(2):363-372.
[6] Neufeld G, Cohen T, Gengrinovitch S, et al.Vascular endothelial growth factor(VEGF)and its receptors[J]. FASEB J, 1999,13(1):9-2 2.
[7] Clauss M. Molecular biology of the VEGF and the VEGF receptor family[J]. Semin Thromb Hemost, 2000,26(5):561-569.
[8] Vincenti V, Cassano C, Roschi M, Persico G. Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3. Circulation. 1996;93:1493-1495.
[9] Lin P, Sankar S, ShanS, etal. Inhibition of tumor growth by targeting tumor endothelium using as oluble vascular endothelial growth factor receptor. Cell Growth Difer. 1998;9(1):49-58.
[10] StepnickD W, PetersonM K, BodganC, etal. Efects of tumor necrosis factor alpha and vascular permeability factor on neovascularization of the rabbit ear flap Arch Otolaryngol Head Neck Surg. 1995;121(6):667-72.
[11] Padubidri A, Browne E J r. Efect of vascular endothelial gr owth factor(VEGF)on survival of random extension of axial patern skin flaps in the rat. Ann Plast Surg.1996;37(6):604-11.
[12]蔚凡,李佛保.血管内皮生长因子影响大鼠皮瓣成活的实验研究.中国修复重建外科杂志, 1997,11(6):376-378.
[13] Kryger Z, Zhang F, Dogan T, etal. The efects of VEGF on survival of a random flap in the rat:examination of various routes of administration. Br J PlastS urg. 2000;53(3):234-9.
[14] Zhang F, Richards L, Angel MF, etal. Accelerating flap maturation by vascular endothelium growth factor in a rat tube flap model.BrJ PlastSurg. 2002;55(1):59-63.
[15] KrygerZ, ZhangF, DoganT, etal. The effects of VEGF on survival of a randomflap in the rat:examination of various routes of administration. BrJ PlastSurg. 2000;53(3):234-9.
[16]刘志辉,刘春丽,张伟.血管内皮生长因子皮下注射对大鼠背部皮瓣成活的实验研究[C].腔领面外科杂志, 2002,12(1):20-23.
[17] Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001;114:853-865.
[18] Erices A, Conget P, Minguell JJ. Mesenchymal progenitor cells in human umbilical cord blood.Br J Haematol. 2000,109(1):235-242.
[19] Zvaifler NJ, Mutafchieva LM, Adams G, et al. Mesenchymal precursor cells in the blood of normal individuals. Arthritis Res, 2000;2:477-488.
[20] Gronthos S, Zannettino AC, Graves SE, et al. Differential cell surface expression of the STRO-1 and alkaline phosphatase antigens on discrete developmental stages in primary cultures of human bone cells. J Bone Miner Res, 1999;14(1):47-56.
[21] Zuk PA, Zhu M, Mizuno H,etal. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001, 7(2):211-228.
[22]史春梦,程天民.大鼠真皮多能间充质干细胞的分离培养.第三军医大学学报. 2001,23(9):1068-1070.
[23] Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol, 1976;4:267-274.
[24] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells [J]. Science, 1999,284 (5411):143-147.
[25] Haynesworth SE, Baber MA, Caplan AI .Cytokine expression by human marrow-derived mesenchymal progenitor cell in vitro:effects of dexamethasone and IL-1 alpha. J Cell Physiol, 1996;166(3):585- 592
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