【摘要】：先天性和后天性的組織缺損的修復是整形外科的難點和重點。缺損的組織類型各種各樣,但是其中重要的組成部分是皮下脂肪。整復外科通常應用各種組織的移植、充填方法,來修復軟組織缺損患者的畸形缺損。臨床上常用的充填軟組織的材料有自體組織和人工合成材料兩種。由于合成人工材料有組織相容性差,輪廓感明顯、非永久性植入等缺陷,而限制了其更廣泛的應用。脂肪組織是一種常用的自體軟組織修復材料。其游離組織移植亦是整形外科最常用的組織修復方法之一。它具有來源豐富,取材容易,疤痕隱蔽,操作簡單,組織相容性好而無異物排斥反應等優(yōu)點,然而,自體脂肪移植其較高且難以預測的吸收率是其固有的缺點,雖然一系列涉及脂肪移植、提高脂肪成活率的新技術(shù)和方法獲得較為廣泛的認可。但是脂肪移植存活率仍不盡如人意且治療效果不可確定仍存在。極大地限制了其在臨床上的廣泛應用。脂肪細胞自身耗能高而對缺血、缺氧耐受力較差,游離移植后其需迅速從受區(qū)獲得足夠的營養(yǎng)供應。在移植物與宿主建立充分的血供之前,脂肪組織只能靠周圍組織液的浸潤和滲透來維持營養(yǎng)供應,而這種供應的距離是極為有限的。超過該距離就需要生成新的血管來提供營養(yǎng),然而自發(fā)的血管生成速度每天只生長十幾微米,一般要在移植后5天逐漸長入,并且新生血管只能侵入移植物的周邊部位,這時中央部分的脂肪組織已經(jīng)發(fā)生了細胞壞死、脂肪液化。由此可見,、盡早建立充分血供,以促進游離移植脂肪組織的再血管化是問題的關(guān)鍵。為加快移植體內(nèi)血供的重建,從而增加移植體血供,縮短脂肪細胞缺血周期,學者們做了許多深入研究。其中較有代表性的研究方法大致包括：ASC, SVF, BRAVA的應用等等。雖然他們獲得了令人鼓舞的效果,但存在的體外培養(yǎng),操作繁瑣,易污染,消耗大量可充填脂肪組織,患者依從性差等局限性。同時即使應用了這些輔助手段,大容量脂肪移植的成活率仍不足以令人滿意。本研究通過動物實驗和臨床研究,觀察在使用丹參注射液的情況下,自體游離脂肪移植的大體和組織學等方面的變化,游離移植脂肪中毛細血管數(shù)量和密度的變化趨勢,從而評估中藥制劑對脂肪顆粒移植物成活率的影響,為臨床通過利用該技術(shù)手段,提高自體脂肪移植成活率提供實驗及臨床研究依據(jù)。探討提高移植脂肪成活率的新途徑。目的：1.探討丹參注射液對自體脂肪移植存活率的影響2.篩選提高自體脂肪移植存活率的中藥制劑3.研究丹參注射液在自體脂肪移植隆乳中應用的有效性方法1.以大白兔自體脂肪游離移植動物為模型,分為實驗組和對照組,分別予以腹腔注射丹參、黃芪注射液,通過以下方法探討對自體游離移植脂肪的作用。(1) Elisa法,在不同時間點測定血清VEGF表達水平(2)術(shù)后第2、4、12周取材,精密天平測量移植體殘余質(zhì)量,計算脂肪移植存活南(3)HE染色,光鏡下觀察組織細胞形態(tài),并作半定量分析。(4)免疫組化染色內(nèi)皮細胞因子CD3l觀察毛細血管生長情況。(5)周脂素免疫組化染色觀察移植脂肪細胞活性。2.自體脂肪移植隆乳術(shù)中分實驗組和對照組,實驗組應用丹參注射液每天一次,共4周。隨訪3個月,通過以下方法探討丹參注射液在自體脂肪移植隆乳中應用的有效性。(1)三維掃描(Konica Minota, Vivid 910)測量脂肪移植術(shù)前后體積,并計算乳房體積增大率、脂肪存活率和乳房體積增加率。(2) Hochest33342和周脂素免疫熒光染色觀察移植脂肪細胞活性。(3)vWF免疫熒光染色觀察移植脂肪中血管分布情況。(4) BZ-X Analyzer軟件抽提vWF免疫熒光染色血管計數(shù),計算每高倍鏡視野下微血管密度。結(jié)果1.動物模型研究：(1)自體脂肪移植術(shù)后第12周,對照組與各實驗組在移植脂肪重量上的差別均具有統(tǒng)計學意義；(2) ELISA結(jié)果表明中藥各組在各時間點的血清VEGF表達水平均明顯高于對照組；(3)HE染色示中藥組在脂肪細胞數(shù)量、形態(tài)均一性等方面均明顯優(yōu)于對照組；(4)內(nèi)皮細胞CD31免疫組化染色示實驗組的新生毛細血管多于對照組。實驗組間比較,以黃芪組血管數(shù)量較多。(5)周脂素免疫組化染色顯示實驗組的移植脂肪細胞活性高于對照組。中藥兩實驗組比較,又以丹參組較多于黃芪組和丹參黃芪組。2.自體脂肪移植隆乳的臨床研究：(1)乳房體積增大量、移植脂肪存活率和乳房體積增加率等方面,丹參組高于對照組。(2) Hochest33342和周脂素免疫熒光染色觀察移植脂肪細胞形態(tài)和飽滿程度,丹參組高于對照組。(3)vWF免疫熒光染色的微血管分布表明實驗組高于對照組。(4) BZ-X Analyzer軟件抽提的vWF免疫熒光染色微血管計數(shù)和密度,均是實驗組高于對照組。結(jié)論l丹參注射液能增加自體脂肪移植中微血管的數(shù)量和密度。2.丹參注射液能提高自體脂肪移植的成活率。
[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.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R622.9

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本文編號：2436291