促进脂肪移植的最佳SVFs浓度的实验研究

目的 探讨应用自人脂肪组织新鲜分离的不同浓度的血管基质层细胞( stromal vasvular fraction cells,SVFs)辅助脂肪移植,从而提高移植物的存活率的方法.方法 脂肪组织来自临床行吸脂术者,提取SVFs,将0.3 ml待移植的脂肪颗粒分别与浓度为①5×105个/ml(A组);②1 × 106个/ml(B组);③2×106个/ml(C组)的SVFs混合,另设对照组为完全培养基+单纯脂肪颗粒(D组),随机注射移植于6只裸鼠背部皮下.术后3个月观察移植物情况:①湿重.②切片HE 染色计数血管密度.③方网测试系统“点计数”法检测存活脂肪细胞计数以及纤维坏死组织计数.结果 ①湿重:A组(60.000±6.325) mg,B组(81.670±7.528) mg,C组(68.330±7.528) mg,D 组(48.330±7.528) mg,B组脂肪存活率均高于A、C、D组(P<0.05),A、C2组之间比较差异无统计学意义(P＞0.05).②血管密度:A、B、C组血管密度均高于D组,且B组明显高于其他3组(P＜0.05),A、C2组之间比较差异无统计学意义(P＞0.05).③点计数:B组存活脂肪细胞计数均高于A、C、D组(P＜0.05),纤维组织计数均低于D组(P ＜0.05),A、C2组之间比较差异均无统计学意义(P＞　0.05).结论 来源于人自体SVFs复合脂肪颗粒能够显著提高移植脂肪组织的成活率,其中1×106个/ml数量级的SVFs移植脂肪的存活率最高,更适合用于脂肪移植,具有广阔的临床应用前景.     

自体基质血管成分改善脂肪组织移植效果的实验研究

目的：分析自体脂肪基质血管成分（stromal vascular fraction cells,SVF）对脂肪颗粒（adipose granule,AG）移植的作用。方法：从6只健康新西兰家兔背部肩胛区获取脂肪组织,实验组将自体SVF与自体AG复合,植入家兔耳部皮下,对照为单纯脂肪移植。在术后1、3、6个月,用B超和游标卡尺测量移植脂肪体积;术后6个月取材常规组织学观察。结果：术后1、3、6个月对照组脂肪组织存活率分别为：（64.35±8.36）%、（58.22±2.88）%、（50.61±9.47）%;实验组脂肪组织存活率分别为：（77.42±5.1）%、（67.95±6.09）%、（72.75±4.37）%。两组比较均存在显著性差异（P〈0.05）。术后6个月组织学观察两组呈正常脂肪组织形态,未见明显差异。结论：自体SVF复合脂肪颗粒能够显著提高移植脂肪组织的成活率,为临床脂肪移植提供实验依据。     

细胞移植在自体脂肪填充中的应用进展

脂肪移植广泛应用于软组织的充填,但是其局部存活率不稳定的问题一直未能得到有效解决。越来越多的证据表明,各类间充质干细胞、血小板能够通过多种机制促进缺血组织新血管的生成,对促进移植脂肪的存活具有一定的作用。在脂肪移植中应用细胞辅助移植可以提高远期疗效,降低移植后脂肪组织液化吸收,减少局部并发症的发生。     

颗粒状脂肪组织游离移植后的体积变化

实验选用４８只大鼠进行颗粒状脂肪组织游离移植的研究。应用外科手术的方法切取腹股沟区的脂肪垫，修剪成４－６ｍｍ直径的脂肪颗粒，分别移植到腹部皮下，腹直肌内和大腿内侧皮下，实验组将移植的脂肪颗粒经过胰岛溶液处理，移植前定量，移植后１－６个月切取标测量体积，经过统计分析，同时标本进行光镜和电镜观察。     

脂肪瓣延迟术后瓣内脂肪细胞移植增加成活的实验研究

目的探讨脂肪瓣延迟术后瓣内脂肪细胞移植成活率和成活时间。方法以兔为模型，在腹股沟处形成脂肪瓣。12h时，用Elisa法检测瓣内血管内皮生长因子（VEGF）的表达，21d后把瓣内脂肪剪成颗粒状移植至兔的背部，移植后1、3、6、9和12个月取出移植脂肪组织，进行肉眼观察，称重和组织切片检查，用CD34染色检测血管增生情况。结果在脂肪瓣形成12h后VEGF显著增高（P〈0．05），移植后1和3个月，实验组移植脂肪血管数显著高于对照组（P〈0．01），成活率未见差异（P〉0．05），但存活时间明显延长，瓣内脂肪细胞在移植后12个月仍然存活，而对照组6个月时已基本吸收。结论脂肪瓣延迟术后移植瓣内脂肪细胞能增加成活率及成活时间，可为临床脂肪移植提供新的思路和方法。     

不同移植部位对可注射组织工程化脂肪生存率的影响

目的：研究裸鼠腹股沟及背部两个不同部位注射移植组织工程化脂肪的存活率。方法：体外分离培养的兔脂肪来源的脂肪基质干细胞（ADSCs）和富含血小板血浆（PRP）与自体兔脂肪颗粒混合注射于裸鼠皮下，按照移植部位分为背部组和腹股沟组，分别于移植后1周、2周、4周、6周，通过B超检测方法评价移植物体积变化情况。结果：脂肪移植后第1、2、4、6周，背部组脂肪移植物存活率为（84.39±7.41）%、（70.89±4.57）%、（52.49±4.42）%、（42.44±5.42）%；腹股沟组存活率为（82.00±6.93）%、（73.81±5.59）%、（61.82±5.94）%、（51.23±4.37）%；两组存活率随时间的推移逐渐降低，通过重复测量的统计分析显示两组存活率差异具有统计学意义（P<0.05），腹股沟组脂肪移植物存活率高于背部组。结论：裸鼠腹股沟的组织工程化脂肪移植存活率更高。     

影响移植自体脂肪颗粒存活率因素的研究进展

应用自体脂肪游离移植治疗组织缺损和发育不良，已有较长的历史。1893年，Neuber最早完成了用多个自体游离的小脂肪块填充软组织缺损的手术；随后Brunings于1911年报道用注射器注射少量脂肪纠正软组织凹陷。移植脂肪颗粒存在高吸收率和低存活率的问题。Peer的研究发现移植的脂肪颗粒，在体积和质量上减少50％以上，坏死的脂肪颗粒会引起纤维囊性化和假性囊肿。此外，在治疗中还存在液化、坏死、感染等并发症，因此，自体脂肪移植半个多世纪以来没有突破性进展。但是，自体脂肪组织作为理想的软组织填充材料，其生物相容性优于人工组织代用品、异体及异种材料，没有免疫排斥现象，并随着20世纪80年代脂肪抽吸技术的发展，脂肪移植再次得到广泛应用，并体现出相当大的优势。但自体脂肪移植依然存在着术后脂肪吸收、存活率低的问题。对于影响提高存活率的因素，很多学者已经做了相当多的研究，现就国内外有关文献综述如下。     

自体脂肪颗粒移植术后超声观察的应用价值

目的总结自体脂肪颗粒移植术后超声观察的应用价值。方法对2010年10月-2011年4月采用自体脂肪颗粒移植的8例(15个部位)女性患者行超声检查。注射部位:鼻唇沟2例(4个部位),颞部2例(4个部位),眶周2例(4个部位),颏部1例(1个部位),乳房1例(2个部位)。患者均接受1次移植,每个部位注射脂肪颗粒3～150 mL。术前超声观察植入部位皮下各层组织情况,颏部测量皮下脂肪厚度。术后1周、1个月、3个月各进行1次超声检查,测量植入脂肪的体积或厚度,观察其内部回声。结果术前超声检查各层次显示清晰,分界明显。术后1周及1个月15个部位植入脂肪轮廓均清晰,术后1个月脂肪存活率为70.21%～76.63%。3个月时12个部位植入脂肪边界可分辨,3个部位植入脂肪与周围组织分界欠清,脂肪存活率为47.83%～56.93%。1例移植脂肪中出现不均匀低回声区,触诊有硬结;余14个部位未发现并发症。结论通过超声可了解植入脂肪存活情况,评价手术效果。     

新鲜分离的脂肪SVF细胞促进脂肪移植存活的实验研究

目的 探讨应用自脂肪组织新鲜分离的血管基质层细胞辅助脂肪移植,提高移植物存活率的可行性.方法 将0.5 ml待移植的脂肪颗粒分别与下列细胞混合:①DiI标记的新鲜分离的自体血管基质层细胞(A组);②DiI标记的培养至第4代自体脂肪来源干细胞(B组);③DMEM完全培养基(C组),随机注射移植于14只新西兰兔背部皮下.术后观察:①湿重;②切片HE染色计数血管密度;③方网测试系统"点计数"法检测存活脂肪细胞计数以及纤维组织计数;④荧光显微镜检测DiI标记的细胞在体内的分化转归.结果 ①湿重:A组(291.0±72.1)mg,B组(269.3±67.3)mg,C组(177.8±60.0)mg,A、B两组脂肪存活率均高于C组(P<0.05),两组之间比较差异无统计学意义(P>0.05);②血管密度:A、B两组血管密度均高于C组(P<0.05),两组之间比较差异无统计学意义(P>0.05).③点计数:A、B两组存活脂肪细胞计数均高于C组(P<0.05),纤维组织计数均低于C组(P<0.05),两组之间比较差异均无统计学意义(P>0.05);④荧光显微镜下观察发现自体血管基质层细胞与自体脂肪来源干细胞在体内均可向血管内皮细胞分化.结论 自体血管基质层细胞与培养的自体脂肪来源于细胞均可提高脂肪移植物存活率,但前者操作更方便,安全性更高,具有广阔的临床应用前景.     

自体脂肪移植综合技术矫正面部局限性硬皮病

目的 探讨自体脂肪移植综合技术矫正面部局限性硬皮病的临床效果。方法 2009年1月至2015年8月,对12例面部局限性硬皮病患者应用自体脂肪颗粒注射移植,联合局部皮瓣转移和局部黏连组织松解技术,矫正其组织缺损,自体脂肪颗粒注射3~4次,每次注射间隔6个月。结果 患者术后随访8~18个月,脂肪存活良好,面部形态改善良好,病变区皮肤色泽改善。本组患者术后均无血肿、皮下硬结、感染等并发症发生。结论 自体脂肪移植综合技术矫正面部局限性硬皮病安全有效,值得临床推广应用。     

Pancreatic tissue grafts are reinnervated by neuro-peptidergic and cholinergic nerves within five days of transplantation

The reinnervation process is crucial for the survival and functioning of cell, tissue or organ transplants. This study was designed to examine the exact time of reinnervation of intraocular pancreatic tissue transplants in rats. The rate of survival of neuropeptide-containing cells in pancreatic tissue grafts was also investigated. Calcitonin gene-related peptide (CGRP), galanin (GAL), neuropeptide Y (NPY) were observed in the surviving nerve cell bodies of the grafts. The iridal nerves reinnervating the pancreatic grafts expressed CGRP, GAL, NPY and choline-acetyl-transferase (ChAT) on day 5, and tyrosine hydroxylase (TH) and nitric oxide synthase (bNOS) on day 6 of the transplantation period. The expression of CGRP in the reinnervating nerves was more consistent when compared to GAL, NPY, ChAT, TH and bNOS. Although all of the three neuropeptides (CGRP, GAL, NPY) were present in the surviving nerve cell bodies of the pancreatic tissue graft up to the end (day 9) of the transplantation period, the number of CGRP-immunopositive cells was consistently higher throughout the transplantation period. Hence, the number of CGRP-positive cells in the pancreatic tissue graft was significantly (P < 0.05) higher than that of GAL and NPY. In conclusion, pancreatic fragments were reinnervated by neuropeptidergic (CGRP, NPY) and cholinergic (ChAT) nerves within the first 5 days of transplantation. In addition to the reinnervation of pancreatic tissue grafts, the intrinsic neurones of the grafts also survived after transplantation. The rate of survival of CGRP-containing cells in the pancreatic tissue grafts was more consistent compared to that of NPY and GAL.     

Non-neuronal neuropeptide Y and its receptors during acute rejection of rat pulmonary allografts

This study tested the hypothesis that neuropeptide Y (NPY) and NPY receptors 1 (Y1) and 2 (Y2) participate in lung allograft rejection. Inflammation in grafts may include interaction between blood leukocytes and graft endothelial cells and marked accumulation of intravascular blood leukocytes. Fewer leukocytes accumulate in lung than in kidney allografts. Lung transplantion was performed in the Dark Agouti to Lewis rat strain combination. Intravascular and intraalveolar leukocytes were isolated from the grafts, and we evaluated the mRNA expression of NPY, Y1, and Y2 by real-time RT-PCR as well as the peptide expression of NPY by radioimmunoassay and immunohistochemistry. NPY and Y1 were expressed by pulmonary intravascular and intraalveolar leukocytes. Y1 was up-regulated by pulmonary intravascular and intraalveolar leukocytes during allograft rejection while Y2 could not be detected. Higher NPY expression levels in intravascular leukocytes were observed in lung compared to kidney allografts, which were investigated previously. Our findings suggest that an increased leukocytic expression of NPY in lung compared to kidney allografts results in a reduced accumulation of leukocytes in allograft vessels.     

Searching for the Favorable Donor Site for Fat Injection: In Vivo Study Using the Nude Mice Model

BACKGROUND: The use of suctioned fat grafts for correction of soft tissue defects is a widespread procedure in esthetic and reconstructive surgery. The main disadvantage of this simple and sensible procedure is the unpredictable absorption rate of the fat graft. A lot of research has been performed aiming for enhancement of the take of the fat grafts. OBJECTIVE: Our study was performed to find if there is any favorable donor site for fat harvesting. METHODS: This in vivo experiment using the nude mice model enables the study of the long-term survival of human fat in an animal model. The fat was harvested from three donor areas: the thigh, abdomen, and breast of a 48-year-old woman who came for an elective esthetic procedure. After centrifugation, 1 cc of fat was injected subcutaneously into the scalp of the nude mouse. There were 15 mice in each of the three groups, according to the selected donor sites. The animals were sacrificed 16 weeks after the procedure. The extracted fat was evaluated in terms of weight, volume, and six histologic parameters: integrity, vascularization, cyst formation, fibrosis, necrosis, and inflammation. RESULTS: This study could not find any statistically significant differences between the three investigated donor sites in the evaluated parameters. CONCLUSION: On the basis of this study, there is no favorable area for harvesting fat grafts. The donor site can be chosen according to the preference of the surgeon and the patient.     

Central and peripheral mechanisms of the NPY system in the regulation of bone and adipose tissue

Skeletal research is currently undergoing a period of marked expansion. The boundaries of "bone" research are being re-evaluated and with this, a growing recognition of a more complex and interconnected biology than previously considered. One aspect that has become the focus of particular attention is the relationship between bone and fat homeostasis. Evidence from a number of avenues indicates that bone and adipose regulation are both related and interdependent. This review examines the neuropeptide Y (NPY) system, known to exert powerful control over both bone and fat tissue. The actions of this system are characterized by signaling both within specific nuclei of the hypothalamus and also the target tissues, mediated predominantly through two G-protein coupled receptors (Y1 and Y2). In bone tissue, elevated NPY levels act consistently to repress osteoblast activity. Moreover, both central Y2 receptor and osteoblastic Y1 receptor signaling act similarly to repress bone formation. Conversely, loss of NPY expression or receptor signaling induces increased osteoblast activity and bone mass in both cortical and cancellous envelopes. In fat tissue, NPY action is more complex. Energy homeostasis is powerfully altered by elevations in hypothalamic NPY, resulting in increases in fat accretion and body-wide energy conservation, through the action of locally expressed Y1 receptors, while local Y2 receptors act to inhibit NPY-ergic tone. Loss of central NPY expression has a markedly reduced effect, consistent with a physiological drive to promote fat accretion. In fat tissue, NPY and Y1 receptors act to promote lipogenesis, consistent with their roles in the brain. Y2 receptors expressed in adipocytes also act in this manner, showing an opposing action to their role in the hypothalamus. While direct investigation of these processes has yet to be completed, these responses appear to be interrelated to some degree. The starvation-based signal of elevated central NPY inducing marked inhibition of osteoblast activity, whilst promoting fat accretion, indicating skeletal tissue is a component of the energy conservation system. Moreover, when NPY expression is reduced, consistent with high calorie intake and weight gain, bone formation is stimulated, strengthening the skeleton. In conclusion, NPY acts to regulate both bone and fat tissue in a coordinated manner, and remains a strong candidate for mediating interactions between these two tissues.     

Effect of botulinum toxin A on vasoconstriction and sympathetic neurotransmitters in a murine random pattern skin flap model

Blood supply is the most important factor determining the survival of a skin flap. Botulinum toxin‐A (Botox‐A) is used as pharmacologic agent not only for aesthetic purposes, but also for its vasomotor actions. This study was conducted to establish whether local application of Botox‐A increased survival of random pattern skin flaps in rats by changing the expression of neurotransmitters. Forty adult Sprague–Dawley rats with a caudally‐based random pattern skin flap were divided into two groups: Botox‐A group and saline group. Surviving flap area and cutaneous blood flow in the flap were evaluated on postoperative days 3 and 7. After injection of Botox‐A, changes in vessels were analyzed using immunohistochemical staining. Levels of norepinephrine, neuropeptide‐Y, nitric oxide, and endothelial nitric oxide synthase were analyzed quantitatively by high performance liquid chromatography, Western blot, and colorimetric assay. The survived area in the Botox‐A group was significantly higher than that in the control group on postoperative days 3 and 7. Blood flow in the Botox‐A group was significantly high in the proximal and middle areas immediately after the operation. The number of CD31‐positive vessels in the Botox‐A group was significant greater than that in the control group. Norepinephrine level in the Botox‐A group decreased significantly immediately after flap elevation and at postoperative day 3. There were no significant differences in neuropeptide‐Y level between the two groups. Nitric oxide level did not change significantly in either group despite the increase in endothelial nitric oxide synthase immediately after flap elevation and at 3 days postoperatively. In conclusion, Botox‐A increased vascular blood flow and viable flap area in rats by reducing norepinephrine level. In contrast, neuropeptide‐Y, another vasoconstrictor, was not affected by Botox‐A. Nitric oxide, a vasodilator, was also not affected by Botox‐A, despite the significant increase in endothelial nitric oxide synthase expression in the flaps.     

Expression of donor class I major histocompatibility antigens on the vascular endothelium of mouse skin allografts

The expression of a class I MHC antigen on the vascular endothelium of mouse skin allografts was assessed by in vivo uptake of radiolabeled monoclonal anti-class-I antibody in the grafts after i.v. injection into the recipients. Endothelial localization of the bound antibodies was demonstrated via double-labeling immunofluorescence microscopy using factor-VIII-related antigen as a marker for endothelial cells. Treatment of recipients with cyclosporine was accompanied by low levels of class I antigen expression in the grafts, and similarly low levels were measured in grafts carried by nude recipients in the complete absence of rejection. Withdrawal of immunosuppressive therapy was followed by an increased class I antigen expression in the donor skin. An increase was also observed in skin grafts undergoing first-set rejection. We conclude that the expression of class I antigens on the capillary endothelium of mouse skin allografts in vivo is variable and is under influence of the immune status of the recipient.     

毛囊干细胞表皮膜片修复裸鼠皮肤缺损的实验研究

目的开发新的组织工程表皮替代物,利用毛囊干细胞-壳聚糖明胶膜片(Chitosan-gelatin membrane,CGM)进行修复裸鼠皮肤缺损的实验研究。方法将体外扩增培养的毛囊干细胞接种于CGM,构建组织工程表皮膜片,裸鼠背部作直径1cm的全层皮肤缺损,将毛囊干细胞-CGM复合物覆盖创面。回植后1周、4周和12周分别进行组织学和免疫组织化学检测。结果毛囊干细胞在CGM上生长良好。毛囊干细胞-CGM修复裸鼠后1周切片示创面有新生上皮覆盖,表皮分化良好,而对照组残留较大未愈创面。修复后4周和12周可见对照组收缩较实验组显著。结论体外构建的毛囊干细胞-CGM可以修复裸鼠皮肤缺损。     

复合皮混合移植治疗深Ⅱ度烧伤患者创面疗效观察

目的观察深Ⅱ度烧伤患者创面削痂术后应用复合皮混合移植治疗的效果。方法对23例烧伤患者的30个深Ⅱ度烧伤肢体在伤后3d内分次行削痂术,削至浅筋膜后移植大张异体脱细胞真皮基质,然后切取大张自体刃厚皮(0.10～0.25mm)覆盖于其上。术后10—12d计算移植皮片的存活率,记录创面愈合时间。观察随访3—6个月时患者的肢体外观及功能恢复情况。取1例患者随访3个月时的愈合创面皮肤标本,行病理学观察。结果本组患者复合皮片成活率为93%,7%的皮片因术中固定较差,移植后自体刃厚皮与异体脱细胞真皮基质分离致皮片坏死,或因感染致皮片溶解。随访3—6个月,移植部位皮肤外观、弹性及功能恢复良好。病理学观察显示,成活皮片表皮、真皮结构正常。结论烧伤后早期削痂立即移植复合皮是治疗深Ⅱ度创面的有效方法。     

The Effect of Epidermal Growth Factor on Autogenous Fat Graft

Autogenous fat grafting is widely used for the correction of soft tissue contour deformity. However, the high absorption rate results in the need for overcorrection, and graft longevity is unpredictable. The authors hypothesized that epidermal growth factor (EGF), a potent stimulator of neovascularization, would improve fat graft survival. The experiment used two groups of New Zealand White Rabbit ear. Inguinal fat was harvested and injected with EGF or saline (n = 24, each group). The 48 cases of fat grafting were managed for observation of volume and morphologic change. The fat was harvested 3 months after the autogenous graft. The survival rate and the degree of neovascularization were measured. The grafts in the EGF group had a significantly higher survival rate than those in the control group. Histologic examination of the grafts demonstrated an increase in neovascularization and maintenance of fat cell morphology. These findings show that EGF can enhance fat graft survival and degree of neovascularization. Further well-controlled studies are required before EGF is used for clinical purposes.