新鲜自体骨髓及自体富集骨髓促进脂肪移植成活的实验研究

发布时间：2018-03-31 22:36

本文选题：脂肪移植　切入点：自体骨髓　出处：《北京协和医学院》2015年硕士论文

【摘要】：目的：目前,多种细胞辅助脂肪移植已被证实可有效提高移植脂肪的成活率,然而细胞培养、扩增过程繁琐、再次移植风险较高。一种能够在一次手术中完成、感染及免疫排斥风险小的细胞辅助脂肪移植手段仍然缺乏。本实验研究致力于探讨新鲜自体骨髓及自体富集骨髓改善自体脂肪移植效果及相关机制,为解决自体脂肪移植术后吸收率较高的问题提供思路。方法：本实验建立了自体脂肪移植兔耳模型。由实验兔髂部抽取新鲜自体骨髓；通过梯度密度离心法离心富集得到富集骨髓,计数其中有核细胞数目并使用荧光染料PKH26进行标记。将24只实验兔随机分为三组：实验组A,实验组B及空白对照组。实验组A将新鲜自体骨髓(Bone marrow aspirate, BMA) 1 ml与自体脂肪颗粒1.5 ml混合后注射于家兔耳部；实验组B将1 m1富集骨髓(Bone marrow concentrate, BMC)与自体脂肪颗粒1.5 m1混合后注射于家兔耳部；对照组将PBS缓冲液1 ml与自体脂肪颗粒1.5 ml混合后注射于家兔耳部。术后1天、2周、4周及12周进行了小动物活体荧光检测。选取4周及12周两个时间点将脂肪移植物切取观察,通过移植脂肪大体观察、移植脂肪存活质量、移植物内脂肪细胞成活情况及移植脂肪血管新生情况四个方面评估移植脂肪的成活及移植物内血运重建情况,并观察骨髓细胞在移植物内的分化情况,初步探讨其作用机制。检测手段包括移植物体积、湿重的测量,移植物活组织染色、组织学切片HE染色及评分、CD31免疫组化染色及微血管密度计数。使用SPSS 19.0进行方差分析检验,设定P值为0.05。结果：术后活体荧光检测结果显示B组实验兔耳部在移植术后的第1天、2周、4周及12周均可检测到荧光信号,移植部位以外的其他区域未检测到荧光信号,说明骨髓细胞可在移植物局部存活至术后12周。术后4周及12周,A、B两组脂肪移植物的湿重及体积均高于对照组(P0.05)；HE染色及评分结果显示：术后4周,A、B两组在脂肪组织完整性方面强于对照组,炎性细胞浸润及纤维结缔组织增生程度低于对照组(p0.05),术后12周,A、B两组与对照组在脂肪组织完整性、囊肿及空泡出现程度、炎细胞浸润程度、纤维结缔组织增生情况四个方面均有显著差异(p0.05)；活组织染色、CD31免疫组化染色结果显示：术后4周及12周,A、B两组活体脂肪细胞及新生微血管数目高于对照组(p0.05)同时,通过移植物活组织染色可观察到骨髓细胞可向脂肪细胞分化并参与血管形成。结论：自体骨髓及自体富集骨髓辅助脂肪移植是一种有效的细胞疗法,可有效促进移植脂肪的成活及血管新生,应用这种方法可以避免细胞培养及扩增的复杂过程,节省手术时间,降低感染及免疫排斥风险,在脂肪移植领域具有一定的应用价值。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622.9

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