研究背景：

脂肪移植是整形外科中修复软组织缺损的重要治疗手段，脂肪移植后出现的囊肿、结节等并发症以及较低的体积保持率等是限制该技术应用的关键问题，这些问题都与脂肪移植物坏死有密不可分的关系。

    脂肪移植物的坏死是一个十分复杂的过程，伴随着脂肪细胞代谢功能与形态的变化，其演化过程与机制至今尚未清楚。弄清脂肪移植物坏死的演化规律，对临床干预并减少脂肪移植物坏死的发生，提高脂肪移植的成活率具有十分重大的意义。研究发现，白色脂肪细胞在受到一定外界刺激（寒冷，运动，肿瘤等）后可形成与棕色脂肪细胞形态及生理均类似的米色脂肪细胞，这一过程被称为脂肪细胞的棕色化。

在对临床脂肪移植坏死组织的病理观察中，我们偶然发现：在移植后坏死的脂肪组织中有大量脂肪细胞棕色化现象的存在。正常情况下，棕色脂肪仅存在于普通成年人的特定部位，含量极少，棕色脂肪细胞能够特异性地表达解耦连蛋白（UCP-1），具有高代谢、易坏死的特点。因此我们推测脂肪移植后脂肪细胞的棕色化可能与脂肪坏死高度相关，可能是脂肪坏死普遍存在的伴随现象，提示有脂肪坏死发生。为了证实这一现象，我们进行了脂肪移植后坏死组织中脂肪细胞棕色化现象的临床研究。

近年来，有研究发现在动物实验的脂肪移植过程可诱导移植物中白色脂肪细胞发生棕色化，形成与棕色脂肪细胞类似的米色脂肪细胞，但其发生机制及影响因素尚不明了，对米色脂肪细胞的来源和生物学性质尚不清楚，基于既往对棕色脂肪细胞生物学特性的研究，棕色脂肪细胞具有基础代谢率高、更容易坏死的特点；在创伤、慢性创面组织中出现的米脂肪细胞被证实为活力不强、更易于坏死的细胞；结合我们在临床研究中发现的脂肪移植后脂肪坏死与脂肪细胞棕色化现象的伴随关系，我们推测：动物实验中出现的米色脂肪细胞与我们在临床上发现的脂肪移植后脂肪细胞棕色化具有相同的起源与生物学特性，是脂肪移植后脂肪细胞成活不良的表现。为验证这一假设，我们进行了脂肪移植后脂肪细胞棕色化与脂肪坏死关系的实验研究。在进一步证实临床发现的基础上，揭示米色脂肪细胞的来源和生物学本质。

目前尚没有研究揭示脂肪移植过程中脂肪细胞棕色化的发生机制与调控机制。现有研究证实：炎症微环境对于移植脂肪细胞的存活具有十分重要的影响。其中，作用最重要的是巨噬细胞，其两个亚型，即M1和M2巨噬细胞，在炎症反应中扮演重要的角色。既然脂肪移植后的棕色化与脂肪坏死密切相关，其调控也必然受到炎症微环境中巨噬细胞的作用和影响。因此，我们认为，也对脂肪细胞棕色化的调控机制与巨噬细胞作用密不可分，且M1和M2巨噬细胞的作用有所不同。为而进一步明确脂肪棕色化的机制，我们进行了M1/M2巨噬细胞对脂肪细胞棕色化影响的研究

研究目的：

1.       明确临床脂肪移植后脂肪细胞棕色化现象与脂肪坏死的关系；

2.       明确动物实验中脂肪细胞棕色化与脂肪坏死的关系，揭示米色脂肪细胞的来源及生物学性质；

3.       探讨脂肪移植后脂肪细胞棕色化的调控机制，明确M1和M2巨噬细胞在脂肪细胞棕色化中的调控作用。

研究方法：

（一）临床实验

1. 临床样本收集与分组：

1)      实验组（坏死结节组）：脂肪坏死结节6例，取自自体脂肪颗粒隆胸手术后坏死结节；

2)      对照组1(坏死皮瓣组)：移植后坏死脂肪组织5例，取自DIEP皮瓣乳房再造术后切除的皮瓣坏死组织；

3)      对照组2(成活脂肪组)：移植后成活脂肪组织5例，取自脂肪移植乳房再造术后修整术。

2. 样本米色脂肪细胞的检测与鉴定：

1)      免疫组化特异性棕色化脂肪标志物UCP-1抗体染色；

2)      电镜观察脂肪细胞形态及线粒体含量；

3)      实时荧光定量PCR检测UCP-1基因表达水平；

4)      Western Blot检测UCP-1蛋白表达水平。

（二）动物实验

1. 动物模型及分组：

1)      于Balb/c裸鼠背部，对称注射定量人体颗粒脂肪抽吸物，建立人来源脂肪移植坏死组织动物模型；

2)      实验组（坏死组）：颗粒脂肪注射量为每个位点100μl；

3)      对照组（成活组）：颗粒脂肪注射量为每个位点500μl；

4)      空白组（新鲜组）：未移植的新鲜脂肪组织。

2. 取样时间：移植后2周、4周，8周和12周。

3. 米色脂肪细胞的检测与鉴定：

1)          解耦连蛋白（UCP-1）特异性免疫组化染色；

2)          透射电镜观察米色脂肪细胞形态以及脂肪细胞内线粒体含量；

3)          实时荧光定量PCR和Western blot检测UCP-1基因及蛋白表达水平；

4)          Western blot检测cleaved caspase-3坏死因子水平；

5)          Western blot和ELISA检测CD206等M1/M2细胞表面标志物；

6)          Western blot和ELISA检测IL-6, TNFα，TGFβ，IL-10, IL1β，MCP-1等炎性因子。

研究结果：

1.       免疫组化UCP-1抗体特异性染色显示临床出现坏死的脂肪移植物组织样本中染色阳性脂肪细胞，细胞内含多房脂滴，形态较小。透射电镜观察结果进一步明确该细胞中含有丰富线粒体，符合米色脂肪细胞特征。而在正常脂肪移植物中并未观察到该类型脂肪细胞。

2.       实时荧光定量PCR结果显示，临床出现坏死的脂肪移植物组织样本中UCP-1基因表达水平较正常移植脂肪组明显升高（>5倍, **p<0.01)。

3.       Western Blot半定量检测提示动物实验坏死组较成活组和空白组表达更高的细胞坏死标志物cleaved caspase-3蛋白水平，提示其组织坏死水平较高，

4.       免疫组化UCP-1抗体特异性染色显示动物实验坏死组样本中含有染色阳性脂肪细胞，细胞内含多房脂滴，形态较小。透射电镜观察结果进一步明确该细胞中含有丰富线粒体，符合米色脂肪细胞特征。而在成活组和新鲜组样本中并未观察到该类型脂肪细胞。

5.       Western Blot半定量检测进一步明确动物实验坏死组较成活组和空白组表达更高的米色脂肪细胞标志物UCP-1蛋白水平，提示其组织棕色化水平较高，

6.       ELISA检测提示在坏死组脂肪移植物中存在M2型巨噬细胞的持续激活和局部浸润（> 2倍），伴随移植物中的棕色化标志物UCP1水平升高。同时还伴有促脂肪细胞棕色化因子IL-6的显著升高（749.0±134.1 pg / ml）。

研究结论：

1. 在脂肪移植后的坏死组织中有脂肪棕色化现象，而在存活良好的脂肪移植物中未发现该现象，说明脂肪细胞的棕色化并非脂肪移植所伴随的必然结果，也非脂肪移植本身“诱导”的反应，而是与脂肪移植物坏死密切相关的产物，脂肪细胞棕色化高度提示有脂肪坏死发生。

2. 移植后坏死脂肪组织所出现的米色细胞体现为细胞形态较小，含多房脂滴，富含线粒体，在基因和蛋白层面表达高水平UCP-1。该米色脂肪细胞与棕色脂肪细胞以及在创伤组织中发现的米色脂肪细胞具有相同的生物学特征，且发生棕色化的脂肪细胞来源于脂肪移植物而非宿主，是脂肪移植后脂肪细胞成活不良的表现。

3. 脂肪移植后脂肪细胞的棕色化过程受到巨噬细胞的调控，巨噬细胞类型及浸润程度可影响脂肪棕色化水平，M2型巨噬细胞水平与脂肪棕色化呈正相关。另外，由M1型巨噬细胞分泌的IL-6，具有促M2型巨噬细胞极化进而促进脂肪棕色化的作用，其在脂肪组织中含量与脂肪移植后脂肪细胞棕色化呈正相关。

Background:

Fat transfer is an important treatment method for repairing soft tissue defects in plastic and reconstructive surgeries. However, complications such as necrotic cysts and nodules after fat grafting and low volume retention are the key issues that limit the application of this technology.

Necrosis of fat grafts is a very complicated process, accompanied by changes in the metabolic function and morphology of adipocytes. The mechanism behind it has not yet been clear. Understanding the development of fat graft necrosis is of great significance for clinical intervention and reducing the occurrence of fat grafts necrosis, and improving the survival of fat grafts.

In the pathological observation of the necrotic tissue of fat grafts, we accidentally discovered that there is a large number of browning adipocytes in the area of necrotic adipose tissue. Under normal circumstances, brown adipocytes only exist in specific parts of humans in a small number. Brown adipocytes can specifically express uncoupling protein (UCP-1), which is responsible for its high metabolism. Therefore, we speculate that browning of adipocytes after fat grafting may be highly related to fat necrosis, suggesting the occurrence of fat necrosis. To confirm this hypothesis, we conducted a clinical study on the browning of adipocytes in necrotic tissue after fat transfer.

In recent years, studies have found that in animal models fat transfer can induce the browning of white adipocytes in the graft, forming beige adipocytes which are similar to brown adipocytes, but the mechanism and influencing factors behind this phenomenon are still unclear. The origin and biological properties of the cells are unclear. Based on previous studies on the biological characteristics of brown adipocytes, brown adipocytes are characteristic of high metablism level and they are more prone to necrosis; beige adipocytes appearing in wounds and chronic wound tissues have been confirmed to be less viable and more prone to necrosis than white adipocytes. Combined with the concomitant relationship between fat necrosis and browning of adipocytes after fat grafting that we found in clinical studies, we speculate that beige adipocytes that appeared in animal experiments and browning of adipocytes we found in humans after fat grafting share same origins and biological characteristics. It is a sign of poor adipocytes survival after fat grafting. To verify this hypothesis, we conducted in vivo experiments on the relationship between browning of adipocytes and fat grafts necrosis after fat grafting. Confirming the clinical findings, the source and biological nature of beige adipocytes were to be revealed.

At present, there is no research reporting the mechanism and regulation mechanism of browning of adipocytes during fat grafting. Existing studies have confirmed that the inflammatory micro-environment has a very important influence on the survival of fat grafts. Among them, the most important factor is macrophages, and its two subtypes, M1 and M2 macrophages, play important roles in the inflammatory response. Since browning of adipocytes after fat grafting is closely related to fat necrosis, its regulation is regulated by macrophages in the inflammatory microenvironment. Therefore, we believe that the regulation mechanism of the browning of adipocytes is inseparable from the functioning of macrophages, and the effects of M1 and M2 macrophages are different. In order to further clarify the mechanism of fat browning, we conducted a study on the effect of M1/M2 macrophages on browning of adipocytes.

Objectives:

1. To clarify the relationship between browning of adipocytes and fat necrosis after fat grafting in humans;

2. To clarify the relationship between browning of adipocytes and fat necrosis in animal experiments, and to reveal the origin and biological properties of browning adipocytes;

3. To explore the regulatory mechanism of browning of adipocytes after fat grafting, and to clarify the regulatory role of M1 and M2 macrophages in browning of adipocytes.

Methods:

(1) Clinical trials

1. Clinical tissue samples:

1) Experimental group (necrotic nodules group): 6 cases of fat necrotic nodules, obtained from necrosis remove surgeries after autologous fat transfer for breast augmentation;

2) Control group 1 (necrotic flaps group): 5 cases of necrotic adipose tissue after grafting were obtained from the necrotic area of the flap after breast reconstruction with abdominal flaps;

3) Control group 2 (survived adipose group): 5 cases of normal adipose tissue from the repair operation after breast reconstruction with abdominal flaps.

2. Detection and identification of brown adipocytes in samples:

1) Immunohistochemistry staining with specific browning marker UCP-1 antibody;

2) transmission electron microscope: to observe the morphology of adipocytes and the amount of mitochondria;

3) Real-time PCR: to detect the expression level of UCP-1 gene;

4) Western Blot: to detect the expression level of UCP-1 protein.

(2) Animal study:

1. Animal models:

1) We symmetrically inject different volumes of human adipose tissue on the back of Balb/c nude mice subcutaneously;

2) Experimental group (necrosis group): the injection volume of adipose tissue is 100μl per site;

3) Control group (survival group): the injection volume of adipose tissue is 500μl per site;

4) Control group (Sham): fresh adipose tissue from liposuction without further grafting.

2. Harvest time: 2 weeks, 4 weeks, 8 weeks and 12 weeks after grafting.

3. Detection and identification of browning adipocytes:

1) Immunohistochemistry staining with specific browning marker UCP-1 antibody;

2) transmission electron microscope: to observe the morphology of adipocytes and the amount of mitochondria;

3) Real-time PCR and Western blot: to detect UCP-1 gene and protein expression levels;

4) Western blot: to detect the level of necrosis factor cleaved caspase-3;

5) Western blot and ELISA: to detect CD206 and other M1/M2 cell surface markers;

6) Western blot and ELISA: to detect IL-6, TNFα, TGFβ, IL-10, IL1β, MCP-1 and other M1/M2 inflammatory factors.

Results:

1. UCP-1 antibody-specific staining showed that the fat graft tissue samples with necrosis showed positive staining of adipocytes in humans, which contained multilocular lipid droplets and were small in shape. The results of transmission electron microscopy further confirmed that the cells have abundant mitochondria, which is consistent with the characteristics of browning adipocytes. However, this type of adipocytes is not observed in normal fat grafts.

2. Real-time fluorescent quantitative PCR results showed that the UCP-1 gene expression level in the fat graft tissue with necrosis in humans was significantly higher than that in the normal fat graft group (>5 times, **p<0.01).

3. Western Blot semi-quantitative analysis indicates that in animal studies, necrosis group expresses a higher level of cell necrosis marker cleaved caspase-3 protein than the survival group and the sham group, indicating that the level of tissue necrosis is higher.

4. UCP-1 antibody specific staining showed that necrosis group in mice contained stained-positive adipocytes, which contained multilocular lipid droplets, and were small in shape. The results of transmission electron microscopy further confirmed that the cells are rich in mitochondria, which is consistent with the characteristics of browning adipocytes. However, this type of adipocyte was not observed in the samples of the survival group and the sham group.

5. Western Blot semi-quantitative analysis further clarified that the animal experimental necrosis group expressed a higher level of beige adipocyte marker UCP-1 protein than the survival group and the sham group, indicating that the tissue browning level was higher.

6. ELISA analysis revealed that there was continuous activation and local infiltration of M2 macrophages (> 2 folds) in fat grafts in the necrotic group, accompanied by an increase in the level of browning marker UCP1 in the grafts. At the same time, it was accompanied by a significant increase of IL-6 which could promote browning development (749.0±134.1 pg/ml).

Conclusions:

1. Browning of adipocytes is clearly found in the necrotic tissues after fat grafting in humans. It has been further verified in the necrotic tissues of animal models, but this phenomenon is not found in the fat grafts that survive well, indicating that browning of adipocytes after fat grafting is related to fat necrosis. A large number of browning of adipocytes indicates that fat necrosis has occurred.

2. Brown adipocytes appearing in necrotic adipose tissue after grafting are manifested as small cells, containing multilocular lipid droplets, rich in mitochondria, and expressing high levels of UCP-1 at the gene and protein level. Browning/beige/brite adipocytes share the same biological characteristics as brown adipocytes, which is a sign of poor adipocyte survival after fat grafting.

Browning biogenesis of adipocytes after fat grafting is regulated by macrophages. The type and infiltration of macrophages can affect the level of browning. The recruitment and infiltration of M2 macrophages is positively correlated with browning. Among them, IL-6 is secreted by M1 type macrophages, but it has the effect to promote the polarization of M2 type macrophages and then accelarate browning development. Its level in tissues is positively correlated with the level of browning of adipocytes after fat grafting.