丹参注射液对自体脂肪移植存活率影响的实验与临床研究

发布时间：2019-03-07 17:06

【摘要】：先天性和后天性的组织缺损的修复是整形外科的难点和重点。缺损的组织类型各种各样,但是其中重要的组成部分是皮下脂肪。整复外科通常应用各种组织的移植、充填方法,来修复软组织缺损患者的畸形缺损。临床上常用的充填软组织的材料有自体组织和人工合成材料两种。由于合成人工材料有组织相容性差,轮廓感明显、非永久性植入等缺陷,而限制了其更广泛的应用。脂肪组织是一种常用的自体软组织修复材料。其游离组织移植亦是整形外科最常用的组织修复方法之一。它具有来源丰富,取材容易,疤痕隐蔽,操作简单,组织相容性好而无异物排斥反应等优点,然而,自体脂肪移植其较高且难以预测的吸收率是其固有的缺点,虽然一系列涉及脂肪移植、提高脂肪成活率的新技术和方法获得较为广泛的认可。但是脂肪移植存活率仍不尽如人意且治疗效果不可确定仍存在。极大地限制了其在临床上的广泛应用。脂肪细胞自身耗能高而对缺血、缺氧耐受力较差,游离移植后其需迅速从受区获得足够的营养供应。在移植物与宿主建立充分的血供之前,脂肪组织只能靠周围组织液的浸润和渗透来维持营养供应,而这种供应的距离是极为有限的。超过该距离就需要生成新的血管来提供营养,然而自发的血管生成速度每天只生长十几微米,一般要在移植后5天逐渐长入,并且新生血管只能侵入移植物的周边部位,这时中央部分的脂肪组织已经发生了细胞坏死、脂肪液化。由此可见,、尽早建立充分血供,以促进游离移植脂肪组织的再血管化是问题的关键。为加快移植体内血供的重建,从而增加移植体血供,缩短脂肪细胞缺血周期,学者们做了许多深入研究。其中较有代表性的研究方法大致包括：ASC, SVF, BRAVA的应用等等。虽然他们获得了令人鼓舞的效果,但存在的体外培养,操作繁琐,易污染,消耗大量可充填脂肪组织,患者依从性差等局限性。同时即使应用了这些辅助手段,大容量脂肪移植的成活率仍不足以令人满意。本研究通过动物实验和临床研究,观察在使用丹参注射液的情况下,自体游离脂肪移植的大体和组织学等方面的变化,游离移植脂肪中毛细血管数量和密度的变化趋势,从而评估中药制剂对脂肪颗粒移植物成活率的影响,为临床通过利用该技术手段,提高自体脂肪移植成活率提供实验及临床研究依据。探讨提高移植脂肪成活率的新途径。目的：1.探讨丹参注射液对自体脂肪移植存活率的影响2.筛选提高自体脂肪移植存活率的中药制剂3.研究丹参注射液在自体脂肪移植隆乳中应用的有效性方法1.以大白兔自体脂肪游离移植动物为模型,分为实验组和对照组,分别予以腹腔注射丹参、黄芪注射液,通过以下方法探讨对自体游离移植脂肪的作用。(1) Elisa法,在不同时间点测定血清VEGF表达水平(2)术后第2、4、12周取材,精密天平测量移植体残余质量,计算脂肪移植存活南(3)HE染色,光镜下观察组织细胞形态,并作半定量分析。(4)免疫组化染色内皮细胞因子CD3l观察毛细血管生长情况。(5)周脂素免疫组化染色观察移植脂肪细胞活性。2.自体脂肪移植隆乳术中分实验组和对照组,实验组应用丹参注射液每天一次,共4周。随访3个月,通过以下方法探讨丹参注射液在自体脂肪移植隆乳中应用的有效性。(1)三维扫描(Konica Minota, Vivid 910)测量脂肪移植术前后体积,并计算乳房体积增大率、脂肪存活率和乳房体积增加率。(2) Hochest33342和周脂素免疫荧光染色观察移植脂肪细胞活性。(3)vWF免疫荧光染色观察移植脂肪中血管分布情况。(4) BZ-X Analyzer软件抽提vWF免疫荧光染色血管计数,计算每高倍镜视野下微血管密度。结果1.动物模型研究：(1)自体脂肪移植术后第12周,对照组与各实验组在移植脂肪重量上的差别均具有统计学意义；(2) ELISA结果表明中药各组在各时间点的血清VEGF表达水平均明显高于对照组；(3)HE染色示中药组在脂肪细胞数量、形态均一性等方面均明显优于对照组；(4)内皮细胞CD31免疫组化染色示实验组的新生毛细血管多于对照组。实验组间比较,以黄芪组血管数量较多。(5)周脂素免疫组化染色显示实验组的移植脂肪细胞活性高于对照组。中药两实验组比较,又以丹参组较多于黄芪组和丹参黄芪组。2.自体脂肪移植隆乳的临床研究：(1)乳房体积增大量、移植脂肪存活率和乳房体积增加率等方面,丹参组高于对照组。(2) Hochest33342和周脂素免疫荧光染色观察移植脂肪细胞形态和饱满程度,丹参组高于对照组。(3)vWF免疫荧光染色的微血管分布表明实验组高于对照组。(4) BZ-X Analyzer软件抽提的vWF免疫荧光染色微血管计数和密度,均是实验组高于对照组。结论l丹参注射液能增加自体脂肪移植中微血管的数量和密度。2.丹参注射液能提高自体脂肪移植的成活率。
[Abstract]:The repair of congenital and acquired tissue defects is the difficulty and focus of plastic surgery. The tissue type of the defect is various, but one of the important components is the subcutaneous fat. The reconstructive surgery is usually applied to the transplantation and filling of various kinds of tissue to repair the defect of the defect of the soft tissue defect. The materials commonly used in the filling of soft tissue include both autograft and synthetic materials. The synthetic artificial material has the defects of poor tissue compatibility, obvious contour sense, non-permanent implantation and the like, and the wider application of the synthetic artificial material is limited. Adipose tissue is a common soft tissue repair material. The free tissue transplantation is one of the most commonly used tissue repair methods in plastic surgery. It has the advantages of abundant source, easy material acquisition, hidden scar, simple operation, good tissue compatibility and no foreign body rejection, And the new technology and the method for improving the survival rate of the fat are widely accepted. However, that survival rate of the fat transplantation is still unsatisfactory and the therapeutic effect is not determine to be present. And can greatly limit the wide application of the medicine in clinic. The fat cell has high self-energy consumption and is low in ischemia and hypoxia tolerance, and after free transplantation, it needs to obtain sufficient nutrient supply from the affected area. Prior to the establishment of sufficient blood supply to the graft and the host, the adipose tissue can only maintain the nutritional supply by the infiltration and penetration of the surrounding tissue fluid, and the distance of such supply is extremely limited. more than this distance requires the generation of new blood vessels to provide nutrition, however, the spontaneous blood vessel generation rate is only a few microns per day, typically 5 days after transplantation, and the new blood vessel can only penetrate the peripheral portion of the graft, In this case, the adipose tissue of the central part has undergone cell necrosis, and the fat is liquefied. It can be seen that a sufficient blood supply can be established as soon as possible to promote the re-vascularization of free-transplanted adipose tissue. In order to accelerate the reconstruction of the blood supply in the transplanted body, the blood supply of the transplanted body is increased, the ischemia period of the fat cells is shortened, and the scholars have done a lot of in-depth study. The more representative methods of research generally include the application of ASC, SVF, BRAVA, and so on. Although they have achieved encouraging results, there are limitations such as in-vitro culture, complex operation, easy contamination, a large amount of filling adipose tissue, poor patient compliance, and the like. At the same time, the survival rate of large-capacity fat transplantation is not satisfactory even when these auxiliary means are applied. This study, through animal experiments and clinical studies, observed the changes in the gross and histological aspects of the free fat transplantation in the case of using the Danshen injection, the change of the number and density of the capillaries in the free graft fat, So as to evaluate the effect of the traditional Chinese medicine preparation on the survival rate of the fat granule graft, and to provide an experimental and clinical research basis for clinical application of the survival rate of the autofat transplantation. To explore a new way to improve the survival rate of transplanted fat. Objective:1. To study the effect of Danshen injection on the survival rate of autologous fat transplantation. And the traditional Chinese medicine preparation 3 for improving the survival rate of the autofat transplantation is selected. To study the effectiveness of Danshen injection in the application of autologous fat transplantation. The rabbits were divided into two groups: the experimental group and the control group, and the rabbits were divided into the experimental group and the control group. (1) Elisa method was used to measure the level of VEGF expression in serum at different time points (2). The samples were obtained at the 2nd, 4th and 12th week after operation, and the residual mass of the graft was measured by the precision balance. The survival of fat transplantation was calculated by HE staining. The morphology of the cells was observed under the light microscope, and the semi-quantitative analysis was made. (4) The growth of the capillary was observed by the immunohistochemical staining of the endothelial cell factor CD3l. (5) The activity of transplanted fat cells was observed by immunohistochemical staining of psoralen. The experimental group and the control group were divided into the experimental group and the control group. The experimental group applied the salvia miltiorrhiza injection once a day for 4 weeks. The effectiveness of the application of Danshen injection in the autologous fat transplantation was discussed by the following method. (1) Three-dimensional scan (Konica Minota, Vivid 910) measures the volume before and after the fat transplantation and calculates the breast volume increase rate, the fat survival rate, and the breast volume increase rate. (2) Hochest33342 and Psoralen immunofluorescence staining were used to observe the activity of transplanted fat cells. (3) The distribution of blood vessels in the transplanted fat was observed by the vWF immunofluorescence staining. (4) BZ-X Analyzer software extracts vWF immunofluorescence staining blood vessel counts, and calculates the microvessel density in the field of each high-power mirror. Results 1. The results of animal model: (1) The difference between the control group and the experimental group on the weight of the transplanted fat was statistically significant at the 12th week and the 12th week after the autograft. (2) The results showed that the expression of VEGF in each group was significantly higher than that of the control group. (3) HE staining showed that the number of the fat cells and the uniformity of the morphology of the traditional Chinese medicine group were superior to those in the control group. (4) The immunohistochemical staining of the CD31 in the endothelial cells showed that the newly-born capillaries in the experimental group were more than the control group. Compared with the experimental group, the number of blood vessels in the yellow blood group was much higher. (5) The immunohistochemical staining of psoralen showed that the activity of the transplanted fat cells in the experimental group was higher than that of the control group. The two experimental groups of the traditional Chinese medicine were compared, and the red-rooted salvia group was more than that of the yellow-rooted salvia group and the radix salviae miltiorrhizae group. The clinical study of the autologous fat-grafted breast augmentation: (1) the volume of the breast is increased, the survival rate of the transplanted fat and the increase rate of the breast volume are increased, and the salvia miltiorrhizae group is higher than that of the control group. (2) Hochest33342 and Psoralen immunofluorescence staining were used to observe the morphology and full extent of the transplanted fat cells, and the group of red-rooted salvia was higher than that of the control group. (3) The microvessel distribution of vWF immunofluorescence staining showed that the experimental group was higher than that of the control group. (4) The microvessel counts and the density of the vWF immunofluorescence staining extracted by the BZ-X Analyzer were both the control group and the control group. Conclusion Salvia miltiorrhiza injection can increase the number and density of microvessels in autologous fat transplantation. Salvia miltiorrhiza injection can improve the survival rate of autologous fat transplantation.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R622.9

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