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自体颗粒脂肪移植成活率的实验研究及川芎嗪对提高脂肪移植成活率的影响
Experimental Study on Survival of Fat Particle Auto-transplantation and the Effect of Tetramethylpyrazine Phosphate in Promoting Survival of Fat Particle Auto-transplantation
【作者】 吴慧玲;
【导师】 郑树森;
【作者基本信息】 浙江大学 , 外科学, 2009, 博士
【摘要】 目的:自体脂肪组织移植已有上百年的历史,与其它各种人工合成材料、异种脂肪或异体脂肪相比,由于其具有来源丰富、取材容易、操作简单、充盈外形好、组织相容性好、无排异反应、无过敏反应等优点,倍受整形美容外科医生的重视,可用于填充各种先天性及后天性软组织缺损。但脂肪移植的高吸收率是脂肪移植最大的障碍,也一直是脂肪移植研究的重点。众多学者为了探索脂肪移植的成活机制、提高其成活率、改进其修复效果一直努力开展基础与临床方面的研究。目前认为影响脂肪移植存活的因素有:移植脂肪组织的血运重建、损伤、感染、麻醉方法、供区及受区情况、取材及提纯方法、移植数量、脂肪组织的冷藏术、相应的治疗等。主要归结为:加速移植体的血管重建和促进前脂肪细胞的分化是提高颗粒脂肪移植存活率的关键。目前,国内外对此难题的研究主要集中在生长因子上。bFGF、IGF-1、VEGF等生长因子均被研究有促进植入脂肪成活的作用。但由于生长因子活性受多种因素影响,需要药物载体,价格昂贵,个体差异较大,故未能在临床上广泛应用。而对于促进前脂肪细胞分化从而达到提高脂肪细胞存活率的研究,亦还不成熟。川芎嗪是中药川芎的主要活性成分之一,主要成分为2,3,5,6-四甲基吡嗪,现已可人工合成。药理研究表明,川芎嗪能清除氧自由基,具有抗凝、抑制血小板聚集、抗血栓形成和溶血栓作用、扩张小动脉、改善微循环、保护血管内皮、抗氧化和钙拮抗的作用。对血液系统及肾、心、脑、肺、肝等组织疾病都有治疗保护作用。作为一种成熟的、经济的、方便应用的材料,川芎嗪注射液的药理机制已被较深入地研究。至今,学者对自身脂肪移植后及应用细胞因子等后的局部反应即成活率研究众多,但还未见有对自身脂肪移植后及应用药物等后全身的反应如何进行过较详尽的研究。国内外也未见有学者就中药对脂肪移植存活率的基础和临床应用方面进行研究。本实验希望通过研究,能更详尽地了解自体脂肪移植后局部和全身的反应,了解川芎嗪注射液对脂肪组织移植存活率及全身的影响,是否能早期消除组织水肿,改善组织血流、扩张小动脉、改善微循环,使再血管化能早期进行;能否抑制或廷缓细胞凋亡,促进前脂肪细胞的分化增值以达到提高脂肪组织移植存活率的可能。从而找寻到一种有效的、经济的、方便应用的提高移植脂肪存活率的方法,以便在临床上得到广泛的应用。本实验通过建立SD大鼠自体脂肪移植模型,一方面通过对脂肪移植后各个时间段的全身和局部的动态变化的观察,更详尽地了解脂肪移植后一个连贯的局部和全身的反应以及可能与其它研究所不同的表现;另一方面通过腹腔注射磷酸川芎嗪到脂肪移植的SD大鼠中,探讨磷酸川芎嗪对脂肪移植成活率的影响及全身的反应并分析其可能机制。给以后研究者提供一全面的自体脂肪组织移植研究方法和中药提高脂肪组织移植存活率的研究方向;为临床提高脂肪移植成活率提供理论依据。方法:健康SD大鼠84只,体重205~309(265±5)g,随机分成四组:①空白对照组12只:自体脂肪取出不移植。②实验1组24只:单纯行自体脂肪移植。③实验2组24只:自体脂肪移植+磷酸川芎嗪注射液(50mg·kg^-1·d^-1)。④实验3组24只:自体脂肪移植+磷酸川芎嗪注射液(100mg·kg^-1·d^-1)。剪取大鼠腹腔内脂肪,清洁后修剪成颗粒脂肪,取出1.5 ml按分组要求分别进行自体背部皮下移植。于术后第4、7、14、28、42、70天取材。SD大鼠右侧腹股沟股动脉取血,进行流式全血Annexin V检测,检测血液中的细胞凋亡情况;测定血清VEGF的含量。对于移植局部,取出各组的脂肪移植体,用微刻度试管测量移植体的剩余体积了解各组各个时期的成活率;HE染色后光镜下观察各组移植体组织细胞形态学的动态改变;并用免疫组化法显示微血管,进行微血管密度的定量研究,比较各组脂肪移植体血管生长情况。结果进行统计学处理。结果:1、各组SD大鼠全血检测Annexin V凋亡结果:各组SD大鼠全血Annexin V早期凋亡无显著性差异。晚期凋亡在术后14天,实验1组与空白对照组差别显著,具有统计学意义;实验2、3组与实验1组差别显著,具有统计学意义。实验2组与实验3组基本上无差别。各组SD大鼠血清VEGF的表达水平结果:三实验组的血清VEGF在各个时间段的表达水平与空白对照组相比,均有增高。在术后14天,实验2、3组与实验1组有明显差别。但各组差异无统计学意义。2、移植体存活情况:术后70天实验1组脂肪移植存活率为41.3%,实验2组脂肪移植存活率为58.7%,实验3组脂肪移植存活率为60.7%。实验3组与实验1组在术后14天差别显著,具有统计学意义。实验1组移植体的体积在术后7天就开始下降;实验2、3组体积在术后14天后开始较明显地下降。3、各组移植体组织学动态表现:实验1组的移植组织经历了炎症、坏死吸收、纤维化、组织再生的一系列过程。移植4天后,纤维组织包膜包绕;急性炎性反应明显,未见明显血管新生,脂肪细胞出现坏死,以中央为甚,但细胞壁基本完整。移植7天后,移植体纤维组织增生更加明显,中央区部分脂肪细胞坏死开始融合,形成较大空泡,周边区纤维组织较多,并有分隔倾向,仍无明显血管生成。移植14天后脂肪空泡大量出现,脂肪细胞间纤维组织增厚,微囊增多,移植体边缘血管已较多,但中心区脂肪组织间少见。移植28天后,整个包膜内出现较多的纤维间隔,血管丰富,微囊较多。移植42天后,出现前脂肪细胞。移植70天后,细胞坏死和纤维化稳定。移植体主要由微囊和纤维组织组成,纤维间隔分明,血运丰富,脂肪细胞大小不规则,前脂肪细胞夹杂着大小不一的脂肪细胞大片存在。相对于实验1组,实验2、3组移植的纤维组织包膜较薄,细胞间新生血管4天后可见,近中央区脂肪细胞坏死融合少,大空泡不多,在移植28天后,周围血管已较丰富,无论周边区还是中央区均可见大量的前脂肪细胞介于坏死的脂肪细胞中。至移植42天后,前脂肪细胞增生明显。移植70天后,整个移植体内都是前脂肪细胞和新生的脂肪细胞,形态规则,血运较丰富。周边区新生脂肪细胞排列整齐,形态一致,4、免疫组化法观察各组移植体各时间段的血管密度:在不同时间段,各脂肪移植组血管密度先逐渐增高,其后稍微下降,实验2、3组均于14天达到最高,实验1组于28天达到最高。在术后4、14、42天,实验2、3组移植体脂肪组织内的新生血管密度均明显高于实验1组,具有统计学意义。但实验2、3组间无显著性差异。结论:1、SD大鼠自体腹腔脂肪移植动物模型优点多。腹腔脂肪集中,质地纯,位置恒定;背部皮下作为移植受区,缺乏皮下脂肪组织,不易与移植体混淆,易于分离。2、自体脂肪移植后全身血液内有相应的改变:血清VEGF表达水平增高,全血Annexin V晚期凋亡增高等。移植早期体积下降明显,移植后70天的体积保持率为41.3%。移植体局部经历了炎症反应、脂肪细胞坏死及增生、纤维化再趋于稳定等过程。3、磷酸川芎嗪注射液能提高SD大鼠自体脂肪移植的成活率。
【Abstract】 Autogenous fat pearl grafting offers a solution to several difficult problems in the reconstruction for soft-tissue and contour defects. The grafting technique is easily learned and patient satisfaction is high. However, the limitations of fat transplantation are well known, because of the high percentage and variable amount of fat resorption reduce the clinical efficacy of this procedure, particularly the long-term unpredictability of volume maintenance and often result in the need for further grafting. Many experts have done a lot of research works to improve the survival rate. The survival of fat grafts depends on many factors, the majors are early revascularization and the differentiation of the adipocytes. Early and adequate revascularization may be important for both the supply of nutrients and vasoactive interactions between vascular endothelial cells and adipocytes in graft. Early studies showed that adipose tissue has a low tolerance to ischemia. Some methods have been described to increase the tolerance of adipose tissue to ischemia. Just like insulin-like growth factor-1; basic-fibroblast growth factor, angiopoietin-1 and leptin can promote transplanted fat survival respectively, but all have localization of clinic application.Tetramethylpyrazine (TMP) is one of major efficient components from Chinese traditional medicine herb Ligusticum Chuanxiong Hort, Which is currently widely used in China for the treatment of coronary atherosclerotic cardiovascular disease and ischemi cerebrocardiac vascular disease. As reported, TMP can inhibit the platelet aggregation, inhibit vasoconstriction in isolated vascular strips and antithrombosis, but until now, there is no report about relationship between Chinese medicine and fat survival.Objective:Through establishing a rat fat-transplantation model, to detect the reaction of the total and the local of fat particle auto- transplantation and to evaluate the effect of the treatment of TMP in promoting survival of fat particle auto- transplantation.Method:Eighty-four SD rats were randomly assigned into four groups and treated as follows: Autogenous fat grafts were excised from abdominal cavity of every rat. Grafts of 1st group were discarded as control group, Grafts of 2nd group were only transplanted, Grafts of 3rd and 4th group were transplanted with which administered TMP in the abdominal cavity at 50mg/kg.d and 100mg/kg.d for 2 weeks after the transplantation.Fat graft harvested from the left abdominal cavity of every rat. The fat grafts of 1st group were discard. For other groups, minimal skin incision and undermining were performed in lateral dorsum of recipient rat, 1.5 ml purified autogenous fat pearl grafts were transplanted in the rats’ dorsum. Four groups were taken samples 4,7,14,28,42,70 days after operation as follows: 1) The harvested blood of the right groin artery of SD rats was processed the measure of Annexin V in order to detect the apoptosis of the cell; and also was to determine the concentration of VEGF in blood serum.2 ) The transplanted fat tissues were extirpated. Skin incision was performed in square to surround on the transplanted region. The half samples of three transplanted groups were taken out only integrality without envelope, and their volumes were measured with their survival rate being calculated.3 ) The samples were stained with HE and VIII factor immunohistochemistry method. Gross and microscopic examinations of autogenous fat grafts were performed. The microvessel density was measured.Results:The VEGF concentration of the blood serum and the apoptosis of the cell were changed in 2nd, 3rd and 4th groups. The values of microvessel density in TMP groups were higher as compared with simplicity graft group in the same observing periods especially in 4,14,42 days. But there was no significant difference between the two TMP groups. Histopathologic evaluation showed degeneration of some adipocytes and inflammatory changes on day 4-14, increased vascularity and fibrosis on day 7-28, as well as fibrosis, microcysties, and the differentiation of the adipocyte on day 42 and later in simplicity graft group. And the two TMP groups had survived better. It was observed that adipose tissue survived in more than 50% of the graft volume in two TMP groups and 41.3% in simplicity graft group. The survival rate of TMP groups were higher than simplicity graft group. There was no significant difference between two TMP groups.Conclusion:A certain dosage Tetramethylpyrazine may be a candidate drug in promoting survival of fat particle auto- transplantation.
【Key words】 fat transplantation; microvessel-density; survival rate; neovascularization; Tetramethylpyrazine; auto- transplantation;