【作者】 毋巨龙；

【导师】 李世荣；

【作者基本信息】 第三军医大学 ， 外科学， 2007， 博士

【摘要】 背景软组织在维持面部和躯体轮廓中起着重要作用,软组织缺损的修复重建目前仍然是整形外科面临的挑战之一,严重的深度烧伤,肿瘤切除术后,半侧颜面萎缩等先天性缺损疾病等都存在软组织缺损的问题。其中脂肪组织缺损是其主要表现之一。自Neuber在1893年完成了第1例用多个自体游离的小脂肪块填充软组织缺损的脂肪移植手术取得满意的疗效后,到20世纪初脂肪移植手术甚为流行。因此项技术操作简单,不需附加切口,不留瘢痕,组织相容性好,所以在整形及美容外科的许多方面得到广泛的应用.但是因为存在着脂肪细胞成活率低,较高的吸收率和纤维化问题,临床效果并不理想.另外脂肪移植后的吸收率的检查缺乏一个客观的标准。对前脂肪细胞性质的认识为将脂肪视为可移植的组织材料奠定了坚实的基础。前脂肪细胞不含脂滴,体积小,有促血管生成特性,在细胞的收集与移植过程中比成熟的脂肪细胞更能耐受缺血与创伤,在脂肪移植后的缺血期,使单个细胞比块状脂肪更易通过细胞融合而成活.如果我们利用现有的理论,首先得到患者的前体脂肪细胞,然后在体外促进其增殖分化成成熟的脂肪细胞,就可以随时用它来填充自身的软组织缺损。这就等于建立了一个无限量的脂肪组织库,我们可以根据需要随时取货。目前对人前脂肪细胞的生物学特性还处于研究阶段。目的1、通过临床研究建立一个客观脂肪移植成活率的影象学判断标准,并借此观察临床bFGF对注射脂肪成活率的作用;2、观察bFGF对体外培养的前脂肪细胞增殖和分化的影响。3、观察bFGF对移植于裸鼠体内前脂肪细胞成脂的影响。为脂肪细胞移植的临床应用开辟一条新的思路。方法（一）1、无菌条件下抽吸的脂肪组织消化、用酶消化法,体外分离培养Pra,并进行传代扩增;观察细胞形态。2、将第二代细胞按10⁴个/孔（含100μl培养液）接种于96孔板（每板分2组,每组3×8孔,其中1组为对照）,实验组加入生长因子（bFGF10 ng/ml）,每天以MTT法观测细胞增殖情况,绘制生长曲线。3、将第二代细胞按104个/孔（含100μl培养液）接种于96孔板（每板分2组,每组3×8孔,其中1组为对照）。培养2天待细胞融合后后加入分化培养基,实验组加入生长因子（bFGF10 ng/ml）,对照组为仅含10%小牛血清的人前脂肪细胞分化培养液。测定脂肪细胞甘油三酯的合成量。4、统计学方法处理。（二）1、制备前脂肪细胞悬液:第二代前脂肪细胞以3×10⁴/ml密度接种培养瓶中,在37℃,5%CO₂培养,第2天,换分化培养基培养2天后,胰酶消化,以普通培养液制成3×10⁴/ml细胞悬液,分成两组,A组内含10 ng/ml重组人b FGF;B组不含重组人b FGF。2、ADM用15%胎牛血清DMEM/F12培养液浸泡24h待用。3、细胞生物支架复合体的制备:将经15%胎牛血清DMEM/F12培养液浸泡过的片状生物支架置于培养板内,每孔1片,共12孔。实验分为三组,每组4孔,a组加入A组细胞悬液100ul;b组加入B组细胞悬液100ul;c组加入不含细胞的普通培养液100ul.各组液体由移液枪均匀滴在ADM表面,静置10分钟。4、细胞支架复合体植入裸鼠皮下,8周后取材。5、组织学检查:将上述标本材料经10%中性甲醛固定后,石蜡包埋并切片,HE染色,观察其组织学变化。6、在200倍光镜下,每张切片选5个视野,记数生成脂肪细胞数目,取平均植（x±s表示）代表脂肪密度,比较三组间差异。7、免疫组织化学检查鉴定脂肪组织来源。（三）1、2006年1月-2007年1月,选取女性双侧颞部凹陷要求手术者20例行颗粒脂肪注射填充术。2、自体脂肪颗粒的制备:采用注射器针式吸脂法（16号针头）,于皮下脂肪层放射状吸取足量脂肪混悬液,过滤、冲洗,每20ml脂肪颗粒中注入碱性成纤维细胞生长因子2000U及庆大霉素8000U,混匀备用。3、脂肪颗粒的注射:注射总量约为预测脂肪量×150%+局部浸润麻药量。4、注射手术结束后第二天、三月分别照相,CT成像。5、3月后技算脂肪吸收率,判断疗效。结果（一）1、重组人bFGF对前脂肪细胞有明显的促增殖作用,与对照组比较第1,2天差异不明显,随时间的推移,第6-7天到达高峰,与对照组比较差异显著;bFGF刺激前脂肪细胞倍增时间提前12小时。2、加入bFGF（10 ng/ml）的前脂肪细胞分化提前,细胞内合成的脂肪小滴更多;诱导分化第3天时,前脂肪细胞中甘油三酯总量轻度升高,到分化6天和9天时,甘油三酯总量明显升高,与对照组相比差异均有显著性（二）1、8周后取材的细胞支架复合体HE染色显示（1）c组,在裸鼠皮下,脱细胞异体真皮支架表面和真皮内都没有观察到脂肪细胞:（2）b组,在裸鼠皮下,脱细胞异体真皮支架表面和真皮内有脂肪细胞存活,但脂肪细胞散在,密度较a组低:（3）a组,成活的脂肪细胞较多,在真皮表面有成团的脂肪细胞,脂肪细胞可深入真皮内。在放大200倍镜下a组脂肪细胞的数目（77±56/视野）较b组（66±44/视野）增加明显。支架内有新生血管形成。2、免疫组化显示a、b组组都有阳性细胞染色,证明存活的脂肪细胞是人源性的。a组阳性细胞较b组阳性细胞多,c组无阳性细胞染色。（三）1、本组20例40个凹陷部位,每部位每次脂肪颗粒注射量10ml～16ml,平均11ml术,后无感染、血肿、脂肪液化、脂肪栓塞发生,1-3月内均可见移植物部分吸收体积变小,3个月后外形基本稳定。一次注射后优16个部位（40%）,良20个部位（50%）,差4个部位（10%）。2、CT检测3月后注射脂肪吸收率27%±5.6%结论1、b FGF对体外培养的前脂肪细胞的有明显的促增殖分化作用。2、b FGF对移植于裸鼠体内的人前脂肪细胞有明显的促成脂作用。其可能原因为bFGF促进了脂肪细胞的在体内的增殖和分化;bFGF缩短了移植组织内形成血管的时间,使移植的脂肪细胞缩短了体内缺血缺氧的时间,有更多的脂肪细胞存活。3、脱细胞异体真皮是一种良好的脂肪组织化工程的支架材料4、bFGF能够提高临床脂肪移植的成活率。5、计算机辅助CT成像技术检测注射脂肪后吸收率这一无创方法客观、准确,值得在临床研究中推广。更多还原

【Abstract】 Background The reconstruction of soft tissue defects remains as a challenge in plastic and reconstructive surgery.Examples for considerable loss of soft connective tissue especially adipose tissue are extensive deep burns,defects after tumor resection, hereditary defects and congenital defects such as Romberg’s disease and Poland syndrome. Soft tissue plays a major role in maintaining contours and also serves as a mechanical cushion for muscles,tendons and bones.Among the various approaches are local and free flaps,dermal fat grafts,collagen injections,the use of synthetic materials and free adipose tissue grafts.Every method shows considerable disadvantages,such as synthetic materials always cause foreign body reactions,biologically derived materials shrink to an unpredictable extend,et.al.Thus,autologous adipose tissue is supposed to be an ideal material,as there usually is sufficient supply for grafting,in spite of the results are generally poor and unpredictable.Studies on free adipose tissue graft showed that most of it was absorbed and replaced by fibrous tissue and oil cysts.These inferior results are thought to be due to the poor rate of revascularization of the graft and the low tolerance of ischemia of fat cells.Even the reviving technique of injecting aspirated fat fragments improved the post-operation results,50%percents of the transplanted tissue shrinked or was totally absorbted.Many studies on the establishment of efficient methods for long survival of transplanted autologous fat-tissues have been performed using animal models.It has been reported that some materials and bioactive peptides can improve the graft maintenance. Researched indicated that the addition of dextran beads with bFGF improves postoperative graft weight maintenance.Preadipocytes,scattering among the mature adipocytes in adipose tissue,are a potential seed cell for soft tissue reconstruction.The proliferative activity is high in preadipocytes,whereas fully differentiated adipocytes have lost their capacity of mitosis.Since it was possible to induce adipogenesis by providing transplanted preadipocytes a suitable microenvironment,we examined the effects of bFGF on preadipocytes ’proliferation and differation in vitro,and on forming adipose tissue in nude mice,and finally on autologous fat grafting on human body.Most published studies agreed that the evaluation of vitality should be mainly based on photographic alteration at different time course,or histopathologic studies,or fatty acid analysis.Only a few studies have attempted to document the persistence of transplanted fat with imaging techniques.The changes of fatty tissue under CT scan, especially of transplanted fat,over the time course post-transplantation has never been reported.For the first time we tried to access the CT signal behavior of transplanted fat over time and compared it with native subcutaneous facial fat in the patients with temporal lipodystrophy.Objectives:1.to Isolate and culture preadipocytes from adult human adipose tissue,2.to study the effects of bFGF on preadipocytes on proliferation and differation in vitro and on forming adipose tissue in nude mice model;3.to study the effects of bFGF on autologous fat grafting;4.to observe the CT signal behavior of transplanted fat over time.Methods1.In vitro tissue culture1.1 Isolation and culture:Preadipocytes were isolated out of fragments of freshly obtained human subcutaneous adipose tissue of adults and cultured.1.2 The second passage cells with the density of 10⁴ /cm²were cultured in DMEM containing 10ng/ml bFGF（the control only in DMEM）,counting the number of the cells with MTT and making the growth curve of the cells.1.3 The second passage cells with the density of 10⁴/cm²were cultured in DMEM for 2 days,After cultured with the presence of 50 nm of insulin,100 nM of dexamethasone,10 mg/ml of transferrin,200 pM of triiodet- hyronine,and bFGF（10ng/ml） for 9 days, examining the TG mass synthesized in 3^rd,6^th and 9^th.2.In vivo experiments2.1 Preparation of human preadiocytes/ADM grafts:A suspension of 100 ul, containing 310⁴cells/ml was seeded on the upper surface by gentle dropping one the pre-wetted ADM;group a,the suspension containing 10ng/ml bFGF,group b,the suspension without bFGF,group c having not any cells.2.2.Transplation and explantion:12 fabricated preadipocyte/ADM constructs were transplanted subcutaneously to the nude mice.After 8 weeks,the mice were killed and the grafts were explanted.2.3 Histology/immunohistochemistry:the explanted material was fixed in 4%buffered formaldehyde solution.Embedded,vertically sectioned,and stained.The formalin fixed fragments were dehydrated and embedded in paraffin.Slices were prepared and stained with monoclonal antibodies specific for human vimentin.2.4 Cellularity:Microscopic fields of cuts were examined at 200^* magnification.The overall cellularity was assessed.3.clinical research3.1 20 cases with temporal lipodystrophy underwent fat injection.3.2.the concentrated and purified sample containing 8000u/20ml bFGF was injected into the temporal area of both sides.3.3 CT was performed preoperation and 2days,3monthes post -operation,respectively..3.4 Invested the rate of absorption of the fat.Results1.In vitro1.1 bFGF can improve the proliferation and differentiation of preadipocytes.1.2 bFGFcan increase the synthesis of TG of preadipocytes.2.In vivo2.1 Microscopical examination of the grafts demonstrated viable adipose tissue located on the surfaces of ADM in group a,but group c showed any adipose tissue.2.2 The cellularity was 77±56/field in group a while group b showed 66±44/field.2.3 Staining for human-vimentin indicated that cells within the grafts were significantly positive and human origin in group a.3.clinical application3.1 In 20 cases,16 areas showed excellent results,20areas showed good results,4 areas showed poor results3.2 the rate of absorption of the fat was alleviated with bFGF treatment.Conclusion1.bFGF can improve the proliferation and stimulate differentiation of Pra In vitro2.bFGF can increase the adipogenesis in vivo3.ADM is abetter scaffold for preadipocyte engineer.4.bFGF can increase the survival of injection fat grafts5.CT imaging has the potential to serve as a follow-up method for fat transplantation or even for inter-study comparisons.更多还原