本文選題：脂肪移植　切入點(diǎn)：自體骨髓　出處：《北京協(xié)和醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的：目前,多種細(xì)胞輔助脂肪移植已被證實(shí)可有效提高移植脂肪的成活率,然而細(xì)胞培養(yǎng)、擴(kuò)增過程繁瑣、再次移植風(fēng)險(xiǎn)較高。一種能夠在一次手術(shù)中完成、感染及免疫排斥風(fēng)險(xiǎn)小的細(xì)胞輔助脂肪移植手段仍然缺乏。本實(shí)驗(yàn)研究致力于探討新鮮自體骨髓及自體富集骨髓改善自體脂肪移植效果及相關(guān)機(jī)制,為解決自體脂肪移植術(shù)后吸收率較高的問題提供思路。方法：本實(shí)驗(yàn)建立了自體脂肪移植兔耳模型。由實(shí)驗(yàn)兔髂部抽取新鮮自體骨髓；通過梯度密度離心法離心富集得到富集骨髓,計(jì)數(shù)其中有核細(xì)胞數(shù)目并使用熒光染料PKH26進(jìn)行標(biāo)記。將24只實(shí)驗(yàn)兔隨機(jī)分為三組：實(shí)驗(yàn)組A,實(shí)驗(yàn)組B及空白對照組。實(shí)驗(yàn)組A將新鮮自體骨髓(Bone marrow aspirate, BMA) 1 ml與自體脂肪顆粒1.5 ml混合后注射于家兔耳部；實(shí)驗(yàn)組B將1 m1富集骨髓(Bone marrow concentrate, BMC)與自體脂肪顆粒1.5 m1混合后注射于家兔耳部；對照組將PBS緩沖液1 ml與自體脂肪顆粒1.5 ml混合后注射于家兔耳部。術(shù)后1天、2周、4周及12周進(jìn)行了小動(dòng)物活體熒光檢測。選取4周及12周兩個(gè)時(shí)間點(diǎn)將脂肪移植物切取觀察,通過移植脂肪大體觀察、移植脂肪存活質(zhì)量、移植物內(nèi)脂肪細(xì)胞成活情況及移植脂肪血管新生情況四個(gè)方面評估移植脂肪的成活及移植物內(nèi)血運(yùn)重建情況,并觀察骨髓細(xì)胞在移植物內(nèi)的分化情況,初步探討其作用機(jī)制。檢測手段包括移植物體積、濕重的測量,移植物活組織染色、組織學(xué)切片HE染色及評分、CD31免疫組化染色及微血管密度計(jì)數(shù)。使用SPSS 19.0進(jìn)行方差分析檢驗(yàn),設(shè)定P值為0.05。結(jié)果：術(shù)后活體熒光檢測結(jié)果顯示B組實(shí)驗(yàn)兔耳部在移植術(shù)后的第1天、2周、4周及12周均可檢測到熒光信號,移植部位以外的其他區(qū)域未檢測到熒光信號,說明骨髓細(xì)胞可在移植物局部存活至術(shù)后12周。術(shù)后4周及12周,A、B兩組脂肪移植物的濕重及體積均高于對照組(P0.05)；HE染色及評分結(jié)果顯示：術(shù)后4周,A、B兩組在脂肪組織完整性方面強(qiáng)于對照組,炎性細(xì)胞浸潤及纖維結(jié)締組織增生程度低于對照組(p0.05),術(shù)后12周,A、B兩組與對照組在脂肪組織完整性、囊腫及空泡出現(xiàn)程度、炎細(xì)胞浸潤程度、纖維結(jié)締組織增生情況四個(gè)方面均有顯著差異(p0.05)；活組織染色、CD31免疫組化染色結(jié)果顯示：術(shù)后4周及12周,A、B兩組活體脂肪細(xì)胞及新生微血管數(shù)目高于對照組(p0.05)同時(shí),通過移植物活組織染色可觀察到骨髓細(xì)胞可向脂肪細(xì)胞分化并參與血管形成。結(jié)論：自體骨髓及自體富集骨髓輔助脂肪移植是一種有效的細(xì)胞療法,可有效促進(jìn)移植脂肪的成活及血管新生,應(yīng)用這種方法可以避免細(xì)胞培養(yǎng)及擴(kuò)增的復(fù)雜過程,節(jié)省手術(shù)時(shí)間,降低感染及免疫排斥風(fēng)險(xiǎn),在脂肪移植領(lǐng)域具有一定的應(yīng)用價(jià)值。
[Abstract]:Objective: at present, a variety of cell-assisted fat transplantation has been proved to be effective to improve the survival rate of fat transplantation, but cell culture, amplification process is cumbersome, the risk of re-transplantation is high.A cell-assisted fat transplant that can be performed in a single operation with low risk of infection and immune rejection is still lacking.The purpose of this study was to investigate the effects of fresh autologous bone marrow and autologous enriched bone marrow on the improvement of autogenous fat transplantation and its related mechanisms, and to provide some ideas for solving the problem of high absorption rate after autogenous fat transplantation.Methods: the model of autologous fat transplantation was established.The fresh autologous bone marrow was extracted from the iliac region of the experimental rabbits and the bone marrow was enriched by centrifugation with gradient density centrifugation. The number of nucleated cells was counted and labeled with fluorescent dye PKH26.Twenty-four rabbits were randomly divided into three groups: experimental group A, experimental group B and blank control group.In experiment group A, 1 ml of fresh autologous bone marrow marrow aspirate (BMA) was mixed with 1.5 ml of autologous fat particles and injected into rabbit ear, while in group B, 1 ml of bone marrow was enriched with bone marrow centrate (BMC) and 1. 5 ml of autologous fat particles were injected into rabbit ear.In the control group, 1 ml of PBS buffer and 1. 5 ml of autologous fat granules were injected into rabbit ear.The fluorescence of small animals in vivo was measured 1 day, 2 weeks, 4 weeks and 12 weeks after operation.The fat grafts were cut and observed at 4 and 12 weeks, and the quality of fat survival was observed by the gross observation of fat grafts.The survival of adipocytes in grafts and vascularization of grafts were evaluated, and the differentiation of bone marrow cells in grafts was observed and its mechanism was discussed.The detection methods included graft volume, wet weight measurement, grafts living tissue staining, HE staining of histological sections, CD31 immunohistochemical staining and microvessel density count.Using SPSS 19.0 for ANOVA test, set P value as 0.05.Results: the results of in vivo fluorescence test showed that fluorescence signals could be detected in the ears of rabbits in group B on the 1st day after transplantation for 4 weeks and 12 weeks after transplantation, but no fluorescence signals were detected in other areas except the transplant site.The results showed that bone marrow cells could survive locally until 12 weeks after operation.The wet weight and volume of fat grafts in both groups were higher than those in the control group at 4 and 12 weeks after operation. The results of HE staining and scoring showed that the adipose tissue integrity of the two groups was stronger than that of the control group at 4 weeks after operation.The degree of inflammatory cell infiltration and fibrous connective tissue proliferation was lower than that of control group (P 0.05).There were significant differences in the four aspects of fibrous connective tissue proliferation (P 0.05). The results of immunohistochemical staining showed that the number of adipocytes and neovascularization in the two groups were significantly higher than that in the control group at 4 and 12 weeks after operation (p 0.05), meanwhile, the number of adipocytes and neovasculature in the two groups was significantly higher than that in the control group (P 0.05).It was observed that bone marrow cells could differentiate into adipocytes and participate in angiogenesis.Conclusion: autologous bone marrow and autologous enriched bone marrow assisted fat transplantation is an effective cell therapy, which can effectively promote the survival and angiogenesis of transplanted fat. This method can avoid the complex process of cell culture and expansion.It can save the operation time, reduce the risk of infection and immune rejection, and has certain application value in fat transplantation.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R622.9

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