脂肪来源干细胞在整形外科的应用研究
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摘要
研究目的：建立兔脂肪间充质干细胞的体外分离培养方法,鉴定并分析其生物学特性,并探索脂肪组织来源干细胞体外培养的最佳条件。观察大鼠自体脂肪来源干细胞的皮下移植成活的情况。尝试用兔脂肪来源干细胞来解决扩张皮瓣淤血问题。探索脂肪来源干细胞在人体体表软组织缺损复合脂肪组织移植的临床应用。观察人脂肪来源干细胞复合脂肪移植人体表软组织缺损临床效果。
研究方法：①选用新西兰大白兔,无菌取出腹股沟脂肪,胶原酶法分离出兔脂肪干细胞,并进行体外培养,取2代以后的兔脂肪干细胞观察其形态特征,并绘制细胞生长曲线及倍增时间。比较了不同胶原酶浓度、胶原酶消化时间和血清浓度对于脂肪来源干细胞体外培养的不同效果。②60只Vistar大鼠随机分为3组,每组20只,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后用脂质体法对细胞进行eGFP(增强型绿色荧光蛋白)转染标记。实验对照组的细胞转染标记后,利用G418将细胞其灭活。空白对照组为生理盐水。分别将其注时移植至大鼠自体耳廓皮下层次,分别于注射后第2、4、6周时进行冰冻切片观察并定量分析所形成组织量的转归情况。③20只新西兰大耳白,左耳、右耳随机排列,10ml扩张器在耳背皮下植入,定期注水,于第20天取出扩张器,并形成皮瓣,制做淤血模型。、同时,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后对细胞Dil染色标记。空白对照组为生理盐水。分别将其注射移植至兔形成皮瓣的皮下层次,在注射后于第2周时观察皮瓣的成活情况,并进行病理切片观察和CD34兔疫组织化学染色,定量分析所形成组织量的成活情况和血管发生情况。2008年4月至2010年4月共收治体表软组织缺损患者共12例,病因有放疗遗留软组织萎缩、手术遗留缺损、年龄老化致鼻唇沟加深、眉间纹加深等。与患者交流后,都愿意尝试脂肪来源干细胞复合脂肪移植填充体表软组织缺损。局部肿胀麻醉下,取自体腹部或双侧大腿皮下脂肪组织,用I型胶原酶消化法提取脂肪间充质干细胞,浓集(2×106／ml)后即时与自体脂肪混合静置半小时后注射移植,采用多点、多层次、少量多次的注射方法,术后适度加压包扎。
结果：①兔脂肪干细胞原代及传代细胞形态：原代及传代的兔脂肪干细胞均呈长梭形或多边形贴壁生长,生长分化活跃。②兔脂肪干细胞的生长曲线及倍增时间：传代的兔脂肪干细胞生长曲线呈“s”形,倍增时间为55 h。③兔脂肪干细胞的成脂分化诱导,经油红染色的办法,其成脂分化的阳性率为65%④采用0.5mg/ml、1mg/ml、1.5mg/ml、2mg/ml四种不同胶原酶浓度进行消化后发现,在消化时间为1h时,2mg/ml组细胞总数最多,活细胞比率与其他组有统计学差异。在消化时间为2h时,细胞总数较1h各组均增加,同时,1mg/ml组、1.5mg/ml组和2mg/ml组消化细胞总数无统计学差异,活细胞比率1mg/ml组和1.5mg/ml组最高。采用5%、10%、15%、20%五种不同血清浓度培养后发现,15%和20%组生长特性最佳,明显优于另三组。④实验组切片内可见明显绿色荧光组织层存留,其体积在各个时间点没有明显的变化。(P<0.01)⑤实验组皮瓣成活的长度明显长于对照组,病理切片显示CD34染色密度明显高于对照组。(P<0.01)。术后半年随访填充脂肪维持注射量,无明显吸收。
结论：①本次实验所分离出来的兔脂肪干细胞在体外具有生长稳定,增殖较快的特点。脂肪组织来源干细胞,胶原酶浓度lmg/ml、消化时间2h、血清浓度15%是最佳的培养条件。②单纯自体脂肪来源干细胞的皮下移植可以存活,并且形成均一稳定的组织。③脂肪来源干细胞可以增加血管的发生,改善皮瓣淤血情况,从而提高扩张皮瓣的成活长度。④脂肪来源干细胞可以增加脂肪移植的成活率。
Objective:To investigate the methods of isolating and culturing rabbit adipose—derived stem cells (ASCs), and to identify and analyze the biological characteristics of rabbit ADSCs and To explore the optimized condition for adipose-derived stem cells(ADSC) culture in vitro. To observe the result of mouse's ASCs autotransplantation subcutaneously. To solve the problem of congestion in the expanded flap by use of the ASCs. To observe the therapeutic effect of ASCs assisted adipose transfer for soft tissue defect
Methods:①The ASCs was derived from the inguinal fat tissue of Vistar mouse and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with eGFP transfection at second or third passage. The ASCs of experimental control group was killed by G418 after labled by eGFP. The blank control group normal saline.They were injected into the auricle of mouse subcutaneously seperately and the results was observed at 2nd,4th,6th week with frozen section.②Adipose was collected from the inguinal region of News Zealand rabbits and the ASCs were isolated with adherent method and collagenase method and cultured in vitro. The morphology of ADSCs after passage 2 was analyzed. The growth curve and doubling time were drawn. The collagenase with different concentration and duration and FCS with different concentration were used.③The ASCs was derived from the inguinal fat tissue of rabbit and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with Dil at second or third passage. The blank control group normal saline.They were injected into the expanded flap with congestion after transplantation. The result was observed at 2nd week with pathologic section and CD34 immunohistochemistry.④12 patients diagnosed as soft tissue defect received the ASCs assisted adipose transfer for soft tissue atrophy after radiotherapy, surgical treatment and aging. The patients want to received the treatment after consultation. The ASCs was isolated with collagenase I from the adipose in patients abdomen or thigh under local anesthesia Then the ASCs(2×106/ml) was mixed with the adipse for half an hour before being injected to the soft tissue defect region. Moderate pressure should be given after the mixture transplantation delicately.
Results:①Formation of primary and passage of ADSCs:ADSCs had spindle and polygon shape adherent growth, and their growth and differentiation were active.②Growth curve and doubling time of ADSCs:The growth curve was like "S" shape. The doubling time of ADSCs was about 55 hours.δThe ADSCs adipogenic differentiation was induced and the rate of positive rate was 65% through the Oil Red O stain and positive cell counting.4 Based on the collagenase concentration, cells were divided into four groups, i.e.,0.5mg/ml, 1mg/ml,1.5mg/ml and 2mg/ml groups. When the duration was 1 hour,2mg/ml group had the most total cell number(TCN) and the living cell rate(LCR) was statistically different from other groups. When the duration was 2 hours, TCN increased in every group and there were no statistical differences among 1mg/ml,1.5mg/ml and 2mg/ml groups. LCR was the highest in 1mg/ml and 1.5mg/ml. 2.Based on FBS concentration, the cells were divided into five groups, i.e.,0,5%,10%, 15%and 20%groups. The growth character was the best in 15%and 20%groups.⑤The new formed tissue labled by GFP was found in the subcutaneous tissue.⑥The survived length of flap with ASCs injection was longer than the flap in the control group. The CD34 immunohistochemistry showed more vessel formation in the flap section with ASCs injection than the flalp in the control group.⑦No obvious absorbtion was found after follow-up for half year.
Conclusion①The ASCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro. The optimized conditions for human adipose-derived stem cells culture are collagenase concentration of 1mg/ml, digestion duration of 2 hour and FBS concentration of 15%.②Autotransplanted ASCs can survive without scaffold.③.ASCs injection could improved the survived length by enhance the vessel formation.④The ASCs could improve the survive rate of adipose transfer.
研究方法：①选用新西兰大白兔,无菌取出腹股沟脂肪,胶原酶法分离出兔脂肪干细胞,并进行体外培养,取2代以后的兔脂肪干细胞观察其形态特征,并绘制细胞生长曲线及倍增时间。比较了不同胶原酶浓度、胶原酶消化时间和血清浓度对于脂肪来源干细胞体外培养的不同效果。②60只Vistar大鼠随机分为3组,每组20只,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后用脂质体法对细胞进行eGFP(增强型绿色荧光蛋白)转染标记。实验对照组的细胞转染标记后,利用G418将细胞其灭活。空白对照组为生理盐水。分别将其注时移植至大鼠自体耳廓皮下层次,分别于注射后第2、4、6周时进行冰冻切片观察并定量分析所形成组织量的转归情况。③20只新西兰大耳白,左耳、右耳随机排列,10ml扩张器在耳背皮下植入,定期注水,于第20天取出扩张器,并形成皮瓣,制做淤血模型。、同时,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后对细胞Dil染色标记。空白对照组为生理盐水。分别将其注射移植至兔形成皮瓣的皮下层次,在注射后于第2周时观察皮瓣的成活情况,并进行病理切片观察和CD34兔疫组织化学染色,定量分析所形成组织量的成活情况和血管发生情况。2008年4月至2010年4月共收治体表软组织缺损患者共12例,病因有放疗遗留软组织萎缩、手术遗留缺损、年龄老化致鼻唇沟加深、眉间纹加深等。与患者交流后,都愿意尝试脂肪来源干细胞复合脂肪移植填充体表软组织缺损。局部肿胀麻醉下,取自体腹部或双侧大腿皮下脂肪组织,用I型胶原酶消化法提取脂肪间充质干细胞,浓集(2×106／ml)后即时与自体脂肪混合静置半小时后注射移植,采用多点、多层次、少量多次的注射方法,术后适度加压包扎。
结果：①兔脂肪干细胞原代及传代细胞形态：原代及传代的兔脂肪干细胞均呈长梭形或多边形贴壁生长,生长分化活跃。②兔脂肪干细胞的生长曲线及倍增时间：传代的兔脂肪干细胞生长曲线呈“s”形,倍增时间为55 h。③兔脂肪干细胞的成脂分化诱导,经油红染色的办法,其成脂分化的阳性率为65%④采用0.5mg/ml、1mg/ml、1.5mg/ml、2mg/ml四种不同胶原酶浓度进行消化后发现,在消化时间为1h时,2mg/ml组细胞总数最多,活细胞比率与其他组有统计学差异。在消化时间为2h时,细胞总数较1h各组均增加,同时,1mg/ml组、1.5mg/ml组和2mg/ml组消化细胞总数无统计学差异,活细胞比率1mg/ml组和1.5mg/ml组最高。采用5%、10%、15%、20%五种不同血清浓度培养后发现,15%和20%组生长特性最佳,明显优于另三组。④实验组切片内可见明显绿色荧光组织层存留,其体积在各个时间点没有明显的变化。(P<0.01)⑤实验组皮瓣成活的长度明显长于对照组,病理切片显示CD34染色密度明显高于对照组。(P<0.01)。术后半年随访填充脂肪维持注射量,无明显吸收。
结论：①本次实验所分离出来的兔脂肪干细胞在体外具有生长稳定,增殖较快的特点。脂肪组织来源干细胞,胶原酶浓度lmg/ml、消化时间2h、血清浓度15%是最佳的培养条件。②单纯自体脂肪来源干细胞的皮下移植可以存活,并且形成均一稳定的组织。③脂肪来源干细胞可以增加血管的发生,改善皮瓣淤血情况,从而提高扩张皮瓣的成活长度。④脂肪来源干细胞可以增加脂肪移植的成活率。
Objective:To investigate the methods of isolating and culturing rabbit adipose—derived stem cells (ASCs), and to identify and analyze the biological characteristics of rabbit ADSCs and To explore the optimized condition for adipose-derived stem cells(ADSC) culture in vitro. To observe the result of mouse's ASCs autotransplantation subcutaneously. To solve the problem of congestion in the expanded flap by use of the ASCs. To observe the therapeutic effect of ASCs assisted adipose transfer for soft tissue defect
Methods:①The ASCs was derived from the inguinal fat tissue of Vistar mouse and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with eGFP transfection at second or third passage. The ASCs of experimental control group was killed by G418 after labled by eGFP. The blank control group normal saline.They were injected into the auricle of mouse subcutaneously seperately and the results was observed at 2nd,4th,6th week with frozen section.②Adipose was collected from the inguinal region of News Zealand rabbits and the ASCs were isolated with adherent method and collagenase method and cultured in vitro. The morphology of ADSCs after passage 2 was analyzed. The growth curve and doubling time were drawn. The collagenase with different concentration and duration and FCS with different concentration were used.③The ASCs was derived from the inguinal fat tissue of rabbit and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with Dil at second or third passage. The blank control group normal saline.They were injected into the expanded flap with congestion after transplantation. The result was observed at 2nd week with pathologic section and CD34 immunohistochemistry.④12 patients diagnosed as soft tissue defect received the ASCs assisted adipose transfer for soft tissue atrophy after radiotherapy, surgical treatment and aging. The patients want to received the treatment after consultation. The ASCs was isolated with collagenase I from the adipose in patients abdomen or thigh under local anesthesia Then the ASCs(2×106/ml) was mixed with the adipse for half an hour before being injected to the soft tissue defect region. Moderate pressure should be given after the mixture transplantation delicately.
Results:①Formation of primary and passage of ADSCs:ADSCs had spindle and polygon shape adherent growth, and their growth and differentiation were active.②Growth curve and doubling time of ADSCs:The growth curve was like "S" shape. The doubling time of ADSCs was about 55 hours.δThe ADSCs adipogenic differentiation was induced and the rate of positive rate was 65% through the Oil Red O stain and positive cell counting.4 Based on the collagenase concentration, cells were divided into four groups, i.e.,0.5mg/ml, 1mg/ml,1.5mg/ml and 2mg/ml groups. When the duration was 1 hour,2mg/ml group had the most total cell number(TCN) and the living cell rate(LCR) was statistically different from other groups. When the duration was 2 hours, TCN increased in every group and there were no statistical differences among 1mg/ml,1.5mg/ml and 2mg/ml groups. LCR was the highest in 1mg/ml and 1.5mg/ml. 2.Based on FBS concentration, the cells were divided into five groups, i.e.,0,5%,10%, 15%and 20%groups. The growth character was the best in 15%and 20%groups.⑤The new formed tissue labled by GFP was found in the subcutaneous tissue.⑥The survived length of flap with ASCs injection was longer than the flap in the control group. The CD34 immunohistochemistry showed more vessel formation in the flap section with ASCs injection than the flalp in the control group.⑦No obvious absorbtion was found after follow-up for half year.
Conclusion①The ASCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro. The optimized conditions for human adipose-derived stem cells culture are collagenase concentration of 1mg/ml, digestion duration of 2 hour and FBS concentration of 15%.②Autotransplanted ASCs can survive without scaffold.③.ASCs injection could improved the survived length by enhance the vessel formation.④The ASCs could improve the survive rate of adipose transfer.
引文
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[8]Jham BC, Nikitakis NG, Scheper MA, Papadimitriou JC, Levv BA, Rivera H.J Granulomatous foreign-body reaction involving oral and perioral tissues after injection of biomaterials:a series of 7 cases and review of the literature. Oral Mxillofac Surg.2009 Feb;67(2)280
[9]Chalfie M, Tu Y, Euskirchen G,et al. Green fluorescent protein as a marker for gene expression. Science,1994,264(5148):8022805
[10]Nakashima S, Masuyama Y, Nitta A, et al. Highly efficient transfection of human marrow stromal cells by mucleofection. Transplant. Proc 2005:37(5):2290
[11]姜晓丹,徐如祥,张旺明,等。绿色荧光蛋白标记人骨髓源性神经干细胞的体外实验研究[J].中国临床康复,2003,7(25)：3439
[12]Brazelton TR, Blau HM Optimizing techniques for tracking transplanted stem cells in vivo. Stem cells 2005; 23(9):1251
[13]Negasaki T, Zhao J. Uniform distribution of epithelia stem cells in the bulbar conjunciva. Invest Ophthalmol Vis Sci 2005; 46(1):126
[14]李立,应大君,朱楚洪,等。绿色荧光蛋白转基因小鼠骨髓间充质干细胞向内皮细胞定向分化的能力[J]。中国临床康复,2005；9(10)：66-7
[15]Mothe AJ, Kulbatski], Van bendegem RL, et al. Analysisi of gree fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells. J Histochem cytochem cytochem 2005; 53(10):1215
[16]Moore JH Jr, Kolaczynski JW, Morales LM, Considine RV, Pietrzkowski Z, Noto PF, Caro JF. Viability of fat obtained by syringe suction lipectomy:effects of local anesthesia with lidocaine. Aesthetic Plast Surg.1995;19:335
[17]Baglioni S;Francalanci M;Squecco R Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB-J.2009;23(10):3494-505
[18]Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, Fielder MN, Klein-Nulend J, Schouten TE, Ritt MJ, van Milligen FJ. Adipose tissue derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Ytotherapy 2006;8:166
[19]Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E, Sato K, Inoue K, Nagase T, Koshima I, Gonda K. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.J cell physiol.2006;208:64
[1]邱玉金,唐胜建,逢迎春扩张皮肤血液动力学及皮瓣移植实验研究 中华外科杂志2002,40(1)20-23。
[2]You-Bin Wang, Yu-jin Qiu, Sheng-jian,Ke-Ming Qi Microcirculation pattern of expanded skin and time for the formation of stable blood supply.Chinese Journal of Clinical Rhabilitation 2004,8(32)7330-7331
[3]Zuk PA, Zhu M, Ashjian P, De Ugarte Da, Huang.JI, Mizuno H, Alfonso ZCj. Fraser JK. Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002 Dec;13(12):4279-95
[4]Mizuno H Hyakusoku H Mesengernic potential and future clinical perspective of human processed lipoaspirate cells. J Nippon Med Sch.2003 Aug; 70(4):300-6.
[5]Zuk PA. Zhu M, Mizuno H. et al. Multi lineage cells from human adipose:Tissue Eng 2001,7(2):211-228.
[6]Baglioni S; Francalanci M; Squecco R The characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue.FASEB-J 2009;23(10):3494-505
[7]De Ugarte DA, Morizono K, Elbarbary A,et al.Comparison of multi-lineage cells from tissue and bone marrow.Cells Tissue Organs.2003,174(3):101-109.
[8]汪希,崔磊,杨群 骨髓基质干细胞促进轴型皮瓣成活的实验研究 组织工程与重建外科杂志2008,4(1)19-21
[9]雷永红,付小兵,盛志勇 大鼠脂肪干细胞转运VEGF基因促进随意皮瓣成活率的研究中国美容医学2007,16(1)7-10
[10]Jalees Rehman, MD; Dmitry Traktuev, BS;Jingling Li, MS. Secretion of angiogenic and antiapoptotic factors by Human adipose stromal cells. Circulation.2004,109:1292-1298.
[11]S.Hertegard,MD,PhD;J.Cedervall,MSc;B.Svensson,MD. iscoelastic and Histologic Properties in Scarred Rabbit Vocal Folds After Mesenchymal Stem Cell Injection. Laryngoscope 2006,116:1248-1254.
[12]Jessica Cedervall,MSc; Lars Ahrlund-Richter,PhD; Bengt Svensson, MD. Injection of Embryonic Stem Cells Into Scarred Rabbit Vocal Folds Enhances Healing and Improves Viscoelasticity:Short-Term Results. Laryngoscope 2007,117:2075-2081
[13]Feng Lu, M.D.,Ph.D., Hiroshi Mizuno.M.D., Cagri A.Uysal,M.D. Improved viability of random pattern Skin Flaps through the use of adipose-derived stem cells.2008,121:50-58
[14]Chenggang Yi,M.D., Wei Xia,M.D.,M.D., Lingxi Zhang,M.D. Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps. Journal of Surgical Research 2006.135:100-106.
[15]Yan Zheng,M.D., Chenggang,M.D.,Wei Xia,M.D. Mensenchymal stem cells transduced by vascular endothelial growth factors for ischemic random skin flaps. Plastic and Reconstructive Surgery 2008,121:59-69.
[16]黄晨昱,沈祖尧 血管内皮细胞生长因子和碱性成纤维细胞生长因子加速预构扩张皮瓣成熟的研究中国修复重建外科杂志2003,17(4)293-297。
1、刘志芳 李华莹 王阳 自体颗粒脂肪注射移植的进展 中国全科医学2004年6月7卷11期
2、易成刚 郭树忠 自体脂肪移植的基础研究与临床应用进展 中国美容医学2003年8月第12卷第4期
3、 Zuk PA,Zhu M,Ashjian P, De Ugarte DA,Huang JI, Mizuno H,Alfonso ZCj,Fraser JK,Benhaim P,Hedrick MH.Human adipose tissue is a source of multipotent stem cells.Mol Biol Cell. 2002 Dec;13(12):4279-95. PMID:12475952 [PubMed-indexed for MEDLINE]
4、崔阳 高旭光 脂肪组织提取细胞—干细胞领域研究的新热点中国航天医药杂志2004年4月第6卷第2期
5、杨立业1 郑佳坤1 惠国桢2 脂肪组织来源的基质细胞研究进展中国修复重建外科杂志2004年第18卷第4期中图分类号：R318 Q813
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10、汪海滨 罗盛康 组织工程支架材料的研究现状中国临床康复2004年7月第8卷第20期
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13、阮建明 邹俭鹏 黄伯云 组织材料学2004年4月 324-338
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15、 Griffith LG, Naughton G:Tissue engineering-current challenges and expanding oppotunities. Science 2002; 295:1009-1014.
16 鞠晓东 娄思权 田华 王卫国 刘延青 脂肪间充质干细胞的基本生物学特性及向成骨细胞诱导分化的实验研究 中华实验外科杂志2004年6月第21卷第6期
17、陈希哲 林云锋 乔鞠 田卫东 闫征斌 李声伟 人体脂肪基质细胞分离培养及其成骨潜能 实用口腔医学杂志2004年2月第20卷第6期
18、友彬 赵敏 戚可名 庄强 瘦素促进移植颗粒脂肪组织血管增生的实验研究中华医学美学美容杂志2004年2月第10卷第1期
19、时安平 罗盛康 罗力生 复脂肪抽吸术相关解剖学基础实用美容外科杂志2001年12、月第12卷第6期
20、易成刚 郭树忠 张琳西 血管生成机制在整形外科中的研究应用 中国实用美容整形外科杂志2004年12月15卷6期
21、李府沈伯均间充质干细胞的来源、特性及临床应用前景国外医学儿科学分册2004年3月延第31卷第2期
22、冯凯 裴雪涛 间充质干细胞——现代组织工程的新资源国外医学生物医学工程分册。中文分类号：R318;Q813。1
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[1]You-Bin Wang. Yu-jin Qiu, Sheng-jian.Ke-Ming Qi Microcirculation pattern of expanded skin and time for the formation of stable blood supply.Chinese Journal of Clinical Rhabilitation 2004,8(32)7330-7331
[2]汪希,崔磊,杨群骨髓基质干细胞促进轴型皮瓣成活的实验研究组织工程与重建外科杂志2008,4(1)19-21
[3]雷永红,付小兵,盛志勇 大鼠脂肪干细胞转运VEGF基因促进随意皮瓣成活率的研究中国美容医学2007,16(1)7-10
[4]Jessica Cedervall,MSc; Lars Ahrlund-Richter.PhD; Bengt Svensson, MD. Injection of Embryonic Stem Cells Into Scarred Rabbit Vocal Folds Enhances Healing and Improves Viscoelasticity:Short-Term Results. Laryngoscope 2007,117:2075-2081
[5]Feng Lu, M.D.,Ph.D., Hiroshi Mizuno,M.D., Cagri A.Uysal,M.D. Improved viability of random pattern Skin Flaps through the use of adipose-derived stem cells.2008,121:50-58
[6]Chenggang Yi,M.D., Wei Xia,M.D.,M.D., Lingxi Zhang, M.D. Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps. Journal of Surgical Research 2006,135:100-106.
[7]Zuk PA, Zhu M, Ashjian P, De Ugarte Da, Huang JI, Mizuno H, Alfonso ZCj, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002 Dec; 13(12):4279-95
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[1]Zuk PA, Zhu M, Ashjian P, De Ugarte Da, Huang JI, Mizuno H, Alfonso ZCj, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002 Dec; 13(12):4279
[2]Mizuno H Hyakusoku H Mesengernic potential and future clinical perspective of human processed lipoaspirate cells. J Nippon Med Sch.2003 Aug; 70(4):300
[3]Zuk PA. Zhu M, Mizuno H. et al. Multi lineage cells from human adipose:Tissue Eng 2001,7(2):211
[4]Gimble Jmand Guilak F Adipose-derived adult stem cells isolation, characterization, and differentiation potential. Cytotherapy.2003;5(5):362
[5]De Ugarte DA, Morizono K, Elbarbary A,et al.Comparison of multi-lineage cells from tissue and bone marrow.Cells Tissue Organs.2003.174(3):101
[6]薛桂松；张英；祁佐良 脂肪来源干细胞研究现状及其在组织工程中的应用组织工程与重建外科2008；4(3)174-6
[7]Aust L, Devlin B, Foster SJ, Halvorsen YD, Hicok K, Du laney T, Sen A, Willingmyre GD, Gimble JM. Yield of human adipose-derived adult stem cells from liposuction aspirates. Cytotherapy.2004;6(1):7
[8]Jham BC, Nikitakis NG, Scheper MA, Papadimitriou JC, Levv BA, Rivera H.J Granulomatous foreign-body reaction involving oral and perioral tissues after injection of biomaterials:a series of 7 cases and review of the literature. Oral Mxillofac Surg.2009 Feb;67(2)280
[9]Chalfie M, Tu Y, Euskirchen G,et al. Green fluorescent protein as a marker for gene expression. Science,1994,264(5148):8022805
[10]Nakashima S, Masuyama Y, Nitta A, et al. Highly efficient transfection of human marrow stromal cells by mucleofection. Transplant. Proc 2005:37(5):2290
[11]姜晓丹,徐如祥,张旺明,等。绿色荧光蛋白标记人骨髓源性神经干细胞的体外实验研究[J].中国临床康复,2003,7(25)：3439
[12]Brazelton TR, Blau HM Optimizing techniques for tracking transplanted stem cells in vivo. Stem cells 2005; 23(9):1251
[13]Negasaki T, Zhao J. Uniform distribution of epithelia stem cells in the bulbar conjunciva. Invest Ophthalmol Vis Sci 2005; 46(1):126
[14]李立,应大君,朱楚洪,等。绿色荧光蛋白转基因小鼠骨髓间充质干细胞向内皮细胞定向分化的能力[J]。中国临床康复,2005；9(10)：66-7
[15]Mothe AJ, Kulbatski], Van bendegem RL, et al. Analysisi of gree fluorescent protein expression in transgenic rats for tracking transplanted neural stem/progenitor cells. J Histochem cytochem cytochem 2005; 53(10):1215
[16]Moore JH Jr, Kolaczynski JW, Morales LM, Considine RV, Pietrzkowski Z, Noto PF, Caro JF. Viability of fat obtained by syringe suction lipectomy:effects of local anesthesia with lidocaine. Aesthetic Plast Surg.1995;19:335
[17]Baglioni S;Francalanci M;Squecco R Characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue. FASEB-J.2009;23(10):3494-505
[18]Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, Fielder MN, Klein-Nulend J, Schouten TE, Ritt MJ, van Milligen FJ. Adipose tissue derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure. Ytotherapy 2006;8:166
[19]Yoshimura K, Shigeura T, Matsumoto D, Sato T, Takaki Y, Aiba-Kojima E, Sato K, Inoue K, Nagase T, Koshima I, Gonda K. Characterization of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.J cell physiol.2006;208:64
[1]邱玉金,唐胜建,逢迎春扩张皮肤血液动力学及皮瓣移植实验研究 中华外科杂志2002,40(1)20-23。
[2]You-Bin Wang, Yu-jin Qiu, Sheng-jian,Ke-Ming Qi Microcirculation pattern of expanded skin and time for the formation of stable blood supply.Chinese Journal of Clinical Rhabilitation 2004,8(32)7330-7331
[3]Zuk PA, Zhu M, Ashjian P, De Ugarte Da, Huang.JI, Mizuno H, Alfonso ZCj. Fraser JK. Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002 Dec;13(12):4279-95
[4]Mizuno H Hyakusoku H Mesengernic potential and future clinical perspective of human processed lipoaspirate cells. J Nippon Med Sch.2003 Aug; 70(4):300-6.
[5]Zuk PA. Zhu M, Mizuno H. et al. Multi lineage cells from human adipose:Tissue Eng 2001,7(2):211-228.
[6]Baglioni S; Francalanci M; Squecco R The characterization of human adult stem-cell populations isolated from visceral and subcutaneous adipose tissue.FASEB-J 2009;23(10):3494-505
[7]De Ugarte DA, Morizono K, Elbarbary A,et al.Comparison of multi-lineage cells from tissue and bone marrow.Cells Tissue Organs.2003,174(3):101-109.
[8]汪希,崔磊,杨群 骨髓基质干细胞促进轴型皮瓣成活的实验研究 组织工程与重建外科杂志2008,4(1)19-21
[9]雷永红,付小兵,盛志勇 大鼠脂肪干细胞转运VEGF基因促进随意皮瓣成活率的研究中国美容医学2007,16(1)7-10
[10]Jalees Rehman, MD; Dmitry Traktuev, BS;Jingling Li, MS. Secretion of angiogenic and antiapoptotic factors by Human adipose stromal cells. Circulation.2004,109:1292-1298.
[11]S.Hertegard,MD,PhD;J.Cedervall,MSc;B.Svensson,MD. iscoelastic and Histologic Properties in Scarred Rabbit Vocal Folds After Mesenchymal Stem Cell Injection. Laryngoscope 2006,116:1248-1254.
[12]Jessica Cedervall,MSc; Lars Ahrlund-Richter,PhD; Bengt Svensson, MD. Injection of Embryonic Stem Cells Into Scarred Rabbit Vocal Folds Enhances Healing and Improves Viscoelasticity:Short-Term Results. Laryngoscope 2007,117:2075-2081
[13]Feng Lu, M.D.,Ph.D., Hiroshi Mizuno.M.D., Cagri A.Uysal,M.D. Improved viability of random pattern Skin Flaps through the use of adipose-derived stem cells.2008,121:50-58
[14]Chenggang Yi,M.D., Wei Xia,M.D.,M.D., Lingxi Zhang,M.D. Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps. Journal of Surgical Research 2006.135:100-106.
[15]Yan Zheng,M.D., Chenggang,M.D.,Wei Xia,M.D. Mensenchymal stem cells transduced by vascular endothelial growth factors for ischemic random skin flaps. Plastic and Reconstructive Surgery 2008,121:59-69.
[16]黄晨昱,沈祖尧 血管内皮细胞生长因子和碱性成纤维细胞生长因子加速预构扩张皮瓣成熟的研究中国修复重建外科杂志2003,17(4)293-297。
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[4]Jessica Cedervall,MSc; Lars Ahrlund-Richter.PhD; Bengt Svensson, MD. Injection of Embryonic Stem Cells Into Scarred Rabbit Vocal Folds Enhances Healing and Improves Viscoelasticity:Short-Term Results. Laryngoscope 2007,117:2075-2081
[5]Feng Lu, M.D.,Ph.D., Hiroshi Mizuno,M.D., Cagri A.Uysal,M.D. Improved viability of random pattern Skin Flaps through the use of adipose-derived stem cells.2008,121:50-58
[6]Chenggang Yi,M.D., Wei Xia,M.D.,M.D., Lingxi Zhang, M.D. Transplantation of endothelial progenitor cells transferred by vascular endothelial growth factor gene for vascular regeneration of ischemic flaps. Journal of Surgical Research 2006,135:100-106.
[7]Zuk PA, Zhu M, Ashjian P, De Ugarte Da, Huang JI, Mizuno H, Alfonso ZCj, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002 Dec; 13(12):4279-95