脂肪来源基质血管成分细胞与脂肪颗粒共移植后的生物学转归
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摘要
研究背景：
自体脂肪组织作为一种理想软组织填充材料,在整形与美容外科领域具有广泛的临床应用前景。但由于脂肪细胞的耐缺血能力较差,失去了原有微血管结构的脂肪组织颗粒移植物,在与受区重新建立血运以前,常因局部血浆营养不足而发生脂肪细胞坏死、脂肪组织吸收及硬结、囊肿形成等并发症。针对这一问题,试图通过促进移植脂肪组织血管化以提高脂肪移植存活率的研究从未中断过,但一直难有重大突破。近年来,脂肪来源基质血管成分(stromal vascular fraction, SVF)细胞的应用为提高脂肪移植存活率提供了一条新的治疗途径。
现已证实,脂肪组织不仅是人体最大的内分泌器官,同时还是间充质干细胞的有效来源,这些具有自我更新和一定分化潜能的原始状态细胞,参与维持脂肪组织的新陈代谢和损伤修复。在脂肪颗粒移植物的获取过程中,大量的脂肪来源干细胞(adipose-derived stem cells, ASCs)存留在供区,只有少数ASCs随脂肪颗粒被移植到受区。这促使研究者们将富含ASCs成分的SVF从脂肪组织中分离出来,并将其重新添加到脂肪移植物内,以恢复脂肪组织中原有的干细胞含量。这一技术由日本学者Yoshimura首先报道,被称为细胞辅助脂肪移植技术(cell-assisted lipotransfer,CAL)。
在自体脂肪注射移植隆乳手术中,Yoshimura等在脂肪移植物内添加了从等量脂肪组织中分离出的SVF细胞,其临床试验结果证实SVF细胞能使脂肪移植物的存活状态得到改善；在超过1年的术后随访中移植的脂肪组织未发生纤维化或粘连；只有2-3%的患者被检测出有囊肿形成或微小钙化灶。一些基础研究结果提示,SVF细胞在脂肪移植中的治疗作用是通过分泌多种生长因子,促进局部组织血管化、抑制细胞凋亡以及动员脂肪祖细胞分化；但对于SVF细胞自身的分化潜能在脂肪组织修复再生中的作用仍不十分清楚。另一个重要问题是,我们对SVF细胞与脂肪颗粒共移植后的存活能力和最终命运还不得而知。SVF细胞能否在脂肪移植物内的局部缺血微环境下长期存活?是否能向某一特定的细胞类型进行分化?目前,国内外尚未有研究报道SVF细胞与脂肪颗粒共移植后的体内存活及分化的动态变化过程。
研究目的：
本课题将重点针对SVF细胞在脂肪移植微环境内的生物学转归展开研究,通过分离绿色荧光蛋白(GFP)转基因小鼠脂肪组织来源的SVF细胞,建立能对SVF细胞进行体内示踪观察的细胞辅助脂肪移植(CAL)模型,探讨SVF细胞与脂肪颗粒共移植后的体内存活和分化情况,为SVF细胞在自体脂肪移植中的应用提供理论依据。
研究方法：
1.分离GFP转基因小鼠腹股沟脂肪组织来源的SVF细胞。用台盼蓝染色法对新鲜分离的GFP阳性SVF细胞(GFP+SVF)进行活细胞计数。切取C57BL/6J小鼠的腹股沟脂肪组织制备成脂肪颗粒。选择BALB/c裸鼠作为脂肪移植受体。将0.5ml(0.360±0.005g)脂肪颗粒与5×105个GFP+SVF细胞混合后,共同注射移植于BALB/c裸鼠头部后方的颅骨表面,建立能对SVF细胞进行体内示踪的细胞辅助脂肪移植(CAL)模型。实验分为2组：(1)以C57BL/6J小鼠脂肪颗粒混合GFP+SVF细胞的注射移植为实验组；(2)以C57BL/6J小鼠脂肪颗粒混合PBS缓冲液的注射移植为对照组。分别于移植术后各个时间点(第1、7、14、28、35、42、56天)切取脂肪移植物标本,称取移植物重量进行组间比较。
2.实验组和对照组分别选取4只实验动物进行应用活体荧光成像技术的动态示踪研究。应用小动物活体荧光成像系统于脂肪移植术后第1天、7天、14天、28天、35天、42天及56天,连续监测GFP+SVF细胞在脂肪移植物局部的存活情况。通过IndiGo操作分析软件对GFP+SVF细胞的荧光信号强度进行定量分析。
3.为明确GFP+SVF细胞在体内向脂肪细胞和内皮细胞自发分化的潜能,我们在移植术后不同时间点(第7、14、28、35、42、56天)切取含有GFP+SVF细胞的脂肪移植物标本,制作成厚度为3μm石蜡切片。分别应用抗Perilipin抗体(脂肪细胞的特异性标志物)和抗CD31抗体(内皮细胞的特异性标志物)对组织切片进行免疫荧光组织化学染色。荧光显微镜下观察染色结果,并用AxioVision软件进行图像分析。
研究结果：
1.实验组和对照组的脂肪移植物重量均在移植术后第14天出现明显的下降；术后早期两组的脂肪移植物重量差异无统计学意义(p>0.05)；移植术后第28天、35天、42天及56天,实验组的脂肪移植物重量均大于对照组(p<0.05)。
2.活体荧光成像系统的检测结果显示,实验组移植术后不同时间点之间的GFP+SVF细胞荧光强度值差异有统计学意义(p<0.0001)。移植术后第14天GFP+SVF细胞的荧光信号强度出现了显著下降,其平均荧光值下降至术后第1天的47.2%；随后,GFP+SVF细胞的荧光信号强度继续减弱,其术后第56天的平均荧光值仅为术后第1天的17.3%。
3.移植术后第7天的免疫荧光组织化学染色结果显示,一些GFP+SVF细胞能够在脂肪移植物内自发地向脂肪细胞分化。GFP和Perilipin双阳性细胞(GFP+Perilipin+)随时间推移逐渐出现形态改变,在术后第56天形成含有脂滴的成熟脂肪细胞。移植术后第28天,在新生血管管壁内可检测到同时表达GFP和CD31的双阳性细胞(GFP+CD31+),它与GFP阴性的内皮细胞(GFP-CD31+)共同形成微血管结构。
研究结论：
1.应用GFP转基因小鼠作为SVF细胞的有效示踪工具,可通过活体荧光成像技术、免疫荧光组织化学染色技术等多种方法对SVF细胞的体内存活和分化情况进行实时、持续的观察。这一动物模型的建立为研究SVF细胞与脂肪颗粒共移植后的生物学转归及其机制奠定了良好的实验基础。
2. GFP+SVF细胞的活体荧光成像连续监测和定量分析结果显示,SVF细胞与脂肪颗粒共移植后能在脂肪移植物内长期存活,但大部分SVF细胞在脂肪移植早期发生了细胞死亡
3.通过对脂肪移植物的免疫荧光组织化学染色观察,我们验证了SVF细胞自身的分化潜能在脂肪移植中的作用。研究证实,存活的SVF细胞能在脂肪移植物内自发地向脂肪细胞分化,促进脂肪组织再生；同时也能向内皮细胞分化,参与脂肪移植物内新生血管的形成过程。以上研究结果提示SVF细胞可能通过多种机制的共同作用促进移植脂肪组织的存活。
Background:
Autologous fat tissue has been considered as ideal filler for soft tissue augmentation, and has a wide prospect of clinical application in the field of plastic and aesthetic surgery. However, because of the adipocytes have a low tolerance to ischemic stress; they are more susceptible to death before the original microvascular structure of adipose tissue could be re-established. Therefore, complications such as fat necrosis, absorption, indurations and cyst formation, can be frequently seen after fat grafting. Efforts at overcoming this have focused on promoting neovascularization of fat grafts. But there is no breakthrough exists to date on the best fat grafting technique and the longevity of results. Recently, a significant increase in interest in the regenerative medicine using adipose-derived stromal vascular fraction (SVF) cells is emerging as a novel therapeutic option for fat grafting.
As the largest endocrine organ in the body, adipose appears to provide an ideal pool of multipotent, undifferentiated, and nucleated cell populations which are involved in homeostasis and regenerative efforts. During the procedure of liposuction, many ASCs remain in the donor site, only a few ASCs can be transfered with fat grafts. This has led researchers to seek techniques that involve digestion, isolation and concentration of adipose-derived SVF cells, to be added back to the lipoaspirated tissues to restore native cellular quantitative levels. This technique has been reported by Yoshimura, termed cell-assisted lipotransfer (CAL).
In breast augmentation trials, Yoshimura and colleagues have combined SVF cells with lipoaspirates from equal volumes of adipose tissue. The authors note improved fat grafting in the presence of the SVF cells with retention of volume for>1year without evidence of fibrosis or adhesions. Complications such as cyst formation or microcalcifications occur in less than2to3%of patients. Some basic studies implicate that the potential benefits of SVF cells, possibly due to their ability to secrete growth factors that have pro-angiogenic, anti-apoptotic, and pro-adipogenic affects. However, the multipotent differentiation capability of SVF cells in the repair and regeneration process of the grafted adipose tissue is still poorly understood. Of equal importance is to understand the fate of the implanted SVF cells. Can these cells survive for a long time in the ischemic microenvironment of fat grafts? Can these cells develop into specific types of cells? To date, studies that have clearly demonstrated the survival and differentiation of SVF cells as being dynamic phenomena have not been widely reported.
Objective:
In this study, we will focus on the biologic outcomes of SVF cells after co-implantation with fat grafts. We aim to establish a CAL animal model using SVF cells isolated from green fluorescent protein (GFP) transgenic mice, to track and investigate the in vivo survival and differentiation of implanted SVF cells. This study may provide a theoretical basis for the application of SVF cells in autologous fat grafting.
Methods:
1. We isolated the SVF cells from the inguinal adipose tissue of GFP transgenic mice. Cell numbers of freshly isolated GFP positive SVF cells (GFP+SVF) were determined by trypan blue staining. BALB/c nude mice were used as recipients for fat grafting. The inguinal adipose tissue was obtained from C57BL/6J mice and finely minced.0.5ml (0.360±0.005g) of the minced adipose tissue was mixed with5×105GFP+SVF cells and injected subcutaneously onto the skulls of BALB/c nude mice. This CAL model was established for the in vivo tracking of SVF cells after co-implantation with fat grafts. BALB/c nude mice were divided into two groups:(1) C57BL/6J mice-derived adipose tissue mixed with GFP+SVF cells was used in the experimental group;(2) C57BL/6J mice-derived adipose tissue mixed with PBS was used in the control group. The fat grafts were harvested from each mouse at each time point (days1,7,14,28,35,42,56). The graft mass was compared between these two groups.
2. Four nude mice of each group were subjected to fluorescence imaging for the dynamic tracking study. Survival of the implanted GFP+SVF cells in living animals was detected by in vivo fluorescence imaging on days1,7,14.28,35,42and56after CAL. Fluorescence intensity of GFP+SVF cells was quantified using the IndiGo software provided with the in vivo imaging system.
3. To evaluate the in vivo adipogenic and angiogenic potential of the implanted GFP+SVF cells, the grafted adipose tissue containing GFP+SVF cells was dissected from mice at each time point (days7,14,28,35,42,56). The graft samples were embedded in paraffin, and sectioned at3μm. Immunofluorescence staining of sections was performed using anti-perilipin antibody (a specific marker for viable adipocytes) or anti-CD31antibody (a specific marker for endothelial cells). All the sections were imaged on fluorescence microscope. Images were acquired and analyzed by AxioVision software.
Results:
1. In both of the two groups, the graft mass decreased sharply on day14. There was no statistically significance in graft mass between the two groups at the early stage after fat grafting (p>0.05). The graft mass in GFP+SVF cells group was higher than that of control group at28,35,42and56days after fat grafting (p<0.05).
2. The results of in vivo fluorescence imaging showed that there was statistically significance in fluorescence intensity of GFP+SVF cells between different time points after fat grafting (p<0.0001). The fluorescence intensity of GFP+SVF cells fell drastically on days14after the co-implantation, with47.2%average signal intensity relative to day1. The fluorescence intensity continued decrease thereafter, with17.3%average signal intensity (relative to day1) at56days.
3. Immunofluorescence staining revealed that some GFP+SVF cells can spontaneously differentiate into adipocytes from day7. The shapes of GFP and Perilipin double positive (GFP+Perilipin+) cells changed over time, as they became lipid droplet-containing adipocytes at56days. At28days, GFP and CD31double positive (GFP+CD31+) vascular endothelial cells were detected in newly formed vessel wall; the microvascular structure was found that was composed of intermingled GFP+CD31+endothelial cells and GFP negative endothelial cells (GFP-CD31+).
Conclusion:
1. The GFP transgenic mice can be used as an effective tool for the in vivo tracking of SVF cells. This method allows us to carry out a real time and continuous observation on the in vivo survival and differentiation of SVF cells by using of fluorescence imaging and immunofluorescence histochemical staining techniques. This animal model was established for investigating the biologic outcomes of SVF cells after co-implantation with fat grafts. It would also provide a good basic for further investigation of the mechanisms of SVF cell in fat grafting.
2. The results of in vivo fluorescence imaging showed that although most implanted SVF cells died shortly at the early stage after fat grafting, some of the SVF cells remained alive in the fat grafts for a long time.
3. The results of immunofluorescence histochemical staining showed convincing evidence of the multipotent differentiation capability of SVF cells in fat grafting. The results prove the principle that a few of the living SVF cells can spontaneously differentiate into adipocytes in fat grafts and contribute to adipose tissue regeneration. Meanwhile, some of the living SVF cells can differentiate into endothelial cells and participate in vascular regeneration of the fat grafts. These findings suggest that the potential benefits of SVF cells in fat grafting may be the results of concurrent effects of multiple mechanisms.
自体脂肪组织作为一种理想软组织填充材料,在整形与美容外科领域具有广泛的临床应用前景。但由于脂肪细胞的耐缺血能力较差,失去了原有微血管结构的脂肪组织颗粒移植物,在与受区重新建立血运以前,常因局部血浆营养不足而发生脂肪细胞坏死、脂肪组织吸收及硬结、囊肿形成等并发症。针对这一问题,试图通过促进移植脂肪组织血管化以提高脂肪移植存活率的研究从未中断过,但一直难有重大突破。近年来,脂肪来源基质血管成分(stromal vascular fraction, SVF)细胞的应用为提高脂肪移植存活率提供了一条新的治疗途径。
现已证实,脂肪组织不仅是人体最大的内分泌器官,同时还是间充质干细胞的有效来源,这些具有自我更新和一定分化潜能的原始状态细胞,参与维持脂肪组织的新陈代谢和损伤修复。在脂肪颗粒移植物的获取过程中,大量的脂肪来源干细胞(adipose-derived stem cells, ASCs)存留在供区,只有少数ASCs随脂肪颗粒被移植到受区。这促使研究者们将富含ASCs成分的SVF从脂肪组织中分离出来,并将其重新添加到脂肪移植物内,以恢复脂肪组织中原有的干细胞含量。这一技术由日本学者Yoshimura首先报道,被称为细胞辅助脂肪移植技术(cell-assisted lipotransfer,CAL)。
在自体脂肪注射移植隆乳手术中,Yoshimura等在脂肪移植物内添加了从等量脂肪组织中分离出的SVF细胞,其临床试验结果证实SVF细胞能使脂肪移植物的存活状态得到改善；在超过1年的术后随访中移植的脂肪组织未发生纤维化或粘连；只有2-3%的患者被检测出有囊肿形成或微小钙化灶。一些基础研究结果提示,SVF细胞在脂肪移植中的治疗作用是通过分泌多种生长因子,促进局部组织血管化、抑制细胞凋亡以及动员脂肪祖细胞分化；但对于SVF细胞自身的分化潜能在脂肪组织修复再生中的作用仍不十分清楚。另一个重要问题是,我们对SVF细胞与脂肪颗粒共移植后的存活能力和最终命运还不得而知。SVF细胞能否在脂肪移植物内的局部缺血微环境下长期存活?是否能向某一特定的细胞类型进行分化?目前,国内外尚未有研究报道SVF细胞与脂肪颗粒共移植后的体内存活及分化的动态变化过程。
研究目的：
本课题将重点针对SVF细胞在脂肪移植微环境内的生物学转归展开研究,通过分离绿色荧光蛋白(GFP)转基因小鼠脂肪组织来源的SVF细胞,建立能对SVF细胞进行体内示踪观察的细胞辅助脂肪移植(CAL)模型,探讨SVF细胞与脂肪颗粒共移植后的体内存活和分化情况,为SVF细胞在自体脂肪移植中的应用提供理论依据。
研究方法：
1.分离GFP转基因小鼠腹股沟脂肪组织来源的SVF细胞。用台盼蓝染色法对新鲜分离的GFP阳性SVF细胞(GFP+SVF)进行活细胞计数。切取C57BL/6J小鼠的腹股沟脂肪组织制备成脂肪颗粒。选择BALB/c裸鼠作为脂肪移植受体。将0.5ml(0.360±0.005g)脂肪颗粒与5×105个GFP+SVF细胞混合后,共同注射移植于BALB/c裸鼠头部后方的颅骨表面,建立能对SVF细胞进行体内示踪的细胞辅助脂肪移植(CAL)模型。实验分为2组：(1)以C57BL/6J小鼠脂肪颗粒混合GFP+SVF细胞的注射移植为实验组；(2)以C57BL/6J小鼠脂肪颗粒混合PBS缓冲液的注射移植为对照组。分别于移植术后各个时间点(第1、7、14、28、35、42、56天)切取脂肪移植物标本,称取移植物重量进行组间比较。
2.实验组和对照组分别选取4只实验动物进行应用活体荧光成像技术的动态示踪研究。应用小动物活体荧光成像系统于脂肪移植术后第1天、7天、14天、28天、35天、42天及56天,连续监测GFP+SVF细胞在脂肪移植物局部的存活情况。通过IndiGo操作分析软件对GFP+SVF细胞的荧光信号强度进行定量分析。
3.为明确GFP+SVF细胞在体内向脂肪细胞和内皮细胞自发分化的潜能,我们在移植术后不同时间点(第7、14、28、35、42、56天)切取含有GFP+SVF细胞的脂肪移植物标本,制作成厚度为3μm石蜡切片。分别应用抗Perilipin抗体(脂肪细胞的特异性标志物)和抗CD31抗体(内皮细胞的特异性标志物)对组织切片进行免疫荧光组织化学染色。荧光显微镜下观察染色结果,并用AxioVision软件进行图像分析。
研究结果：
1.实验组和对照组的脂肪移植物重量均在移植术后第14天出现明显的下降；术后早期两组的脂肪移植物重量差异无统计学意义(p>0.05)；移植术后第28天、35天、42天及56天,实验组的脂肪移植物重量均大于对照组(p<0.05)。
2.活体荧光成像系统的检测结果显示,实验组移植术后不同时间点之间的GFP+SVF细胞荧光强度值差异有统计学意义(p<0.0001)。移植术后第14天GFP+SVF细胞的荧光信号强度出现了显著下降,其平均荧光值下降至术后第1天的47.2%；随后,GFP+SVF细胞的荧光信号强度继续减弱,其术后第56天的平均荧光值仅为术后第1天的17.3%。
3.移植术后第7天的免疫荧光组织化学染色结果显示,一些GFP+SVF细胞能够在脂肪移植物内自发地向脂肪细胞分化。GFP和Perilipin双阳性细胞(GFP+Perilipin+)随时间推移逐渐出现形态改变,在术后第56天形成含有脂滴的成熟脂肪细胞。移植术后第28天,在新生血管管壁内可检测到同时表达GFP和CD31的双阳性细胞(GFP+CD31+),它与GFP阴性的内皮细胞(GFP-CD31+)共同形成微血管结构。
研究结论：
1.应用GFP转基因小鼠作为SVF细胞的有效示踪工具,可通过活体荧光成像技术、免疫荧光组织化学染色技术等多种方法对SVF细胞的体内存活和分化情况进行实时、持续的观察。这一动物模型的建立为研究SVF细胞与脂肪颗粒共移植后的生物学转归及其机制奠定了良好的实验基础。
2. GFP+SVF细胞的活体荧光成像连续监测和定量分析结果显示,SVF细胞与脂肪颗粒共移植后能在脂肪移植物内长期存活,但大部分SVF细胞在脂肪移植早期发生了细胞死亡
3.通过对脂肪移植物的免疫荧光组织化学染色观察,我们验证了SVF细胞自身的分化潜能在脂肪移植中的作用。研究证实,存活的SVF细胞能在脂肪移植物内自发地向脂肪细胞分化,促进脂肪组织再生；同时也能向内皮细胞分化,参与脂肪移植物内新生血管的形成过程。以上研究结果提示SVF细胞可能通过多种机制的共同作用促进移植脂肪组织的存活。
Background:
Autologous fat tissue has been considered as ideal filler for soft tissue augmentation, and has a wide prospect of clinical application in the field of plastic and aesthetic surgery. However, because of the adipocytes have a low tolerance to ischemic stress; they are more susceptible to death before the original microvascular structure of adipose tissue could be re-established. Therefore, complications such as fat necrosis, absorption, indurations and cyst formation, can be frequently seen after fat grafting. Efforts at overcoming this have focused on promoting neovascularization of fat grafts. But there is no breakthrough exists to date on the best fat grafting technique and the longevity of results. Recently, a significant increase in interest in the regenerative medicine using adipose-derived stromal vascular fraction (SVF) cells is emerging as a novel therapeutic option for fat grafting.
As the largest endocrine organ in the body, adipose appears to provide an ideal pool of multipotent, undifferentiated, and nucleated cell populations which are involved in homeostasis and regenerative efforts. During the procedure of liposuction, many ASCs remain in the donor site, only a few ASCs can be transfered with fat grafts. This has led researchers to seek techniques that involve digestion, isolation and concentration of adipose-derived SVF cells, to be added back to the lipoaspirated tissues to restore native cellular quantitative levels. This technique has been reported by Yoshimura, termed cell-assisted lipotransfer (CAL).
In breast augmentation trials, Yoshimura and colleagues have combined SVF cells with lipoaspirates from equal volumes of adipose tissue. The authors note improved fat grafting in the presence of the SVF cells with retention of volume for>1year without evidence of fibrosis or adhesions. Complications such as cyst formation or microcalcifications occur in less than2to3%of patients. Some basic studies implicate that the potential benefits of SVF cells, possibly due to their ability to secrete growth factors that have pro-angiogenic, anti-apoptotic, and pro-adipogenic affects. However, the multipotent differentiation capability of SVF cells in the repair and regeneration process of the grafted adipose tissue is still poorly understood. Of equal importance is to understand the fate of the implanted SVF cells. Can these cells survive for a long time in the ischemic microenvironment of fat grafts? Can these cells develop into specific types of cells? To date, studies that have clearly demonstrated the survival and differentiation of SVF cells as being dynamic phenomena have not been widely reported.
Objective:
In this study, we will focus on the biologic outcomes of SVF cells after co-implantation with fat grafts. We aim to establish a CAL animal model using SVF cells isolated from green fluorescent protein (GFP) transgenic mice, to track and investigate the in vivo survival and differentiation of implanted SVF cells. This study may provide a theoretical basis for the application of SVF cells in autologous fat grafting.
Methods:
1. We isolated the SVF cells from the inguinal adipose tissue of GFP transgenic mice. Cell numbers of freshly isolated GFP positive SVF cells (GFP+SVF) were determined by trypan blue staining. BALB/c nude mice were used as recipients for fat grafting. The inguinal adipose tissue was obtained from C57BL/6J mice and finely minced.0.5ml (0.360±0.005g) of the minced adipose tissue was mixed with5×105GFP+SVF cells and injected subcutaneously onto the skulls of BALB/c nude mice. This CAL model was established for the in vivo tracking of SVF cells after co-implantation with fat grafts. BALB/c nude mice were divided into two groups:(1) C57BL/6J mice-derived adipose tissue mixed with GFP+SVF cells was used in the experimental group;(2) C57BL/6J mice-derived adipose tissue mixed with PBS was used in the control group. The fat grafts were harvested from each mouse at each time point (days1,7,14,28,35,42,56). The graft mass was compared between these two groups.
2. Four nude mice of each group were subjected to fluorescence imaging for the dynamic tracking study. Survival of the implanted GFP+SVF cells in living animals was detected by in vivo fluorescence imaging on days1,7,14.28,35,42and56after CAL. Fluorescence intensity of GFP+SVF cells was quantified using the IndiGo software provided with the in vivo imaging system.
3. To evaluate the in vivo adipogenic and angiogenic potential of the implanted GFP+SVF cells, the grafted adipose tissue containing GFP+SVF cells was dissected from mice at each time point (days7,14,28,35,42,56). The graft samples were embedded in paraffin, and sectioned at3μm. Immunofluorescence staining of sections was performed using anti-perilipin antibody (a specific marker for viable adipocytes) or anti-CD31antibody (a specific marker for endothelial cells). All the sections were imaged on fluorescence microscope. Images were acquired and analyzed by AxioVision software.
Results:
1. In both of the two groups, the graft mass decreased sharply on day14. There was no statistically significance in graft mass between the two groups at the early stage after fat grafting (p>0.05). The graft mass in GFP+SVF cells group was higher than that of control group at28,35,42and56days after fat grafting (p<0.05).
2. The results of in vivo fluorescence imaging showed that there was statistically significance in fluorescence intensity of GFP+SVF cells between different time points after fat grafting (p<0.0001). The fluorescence intensity of GFP+SVF cells fell drastically on days14after the co-implantation, with47.2%average signal intensity relative to day1. The fluorescence intensity continued decrease thereafter, with17.3%average signal intensity (relative to day1) at56days.
3. Immunofluorescence staining revealed that some GFP+SVF cells can spontaneously differentiate into adipocytes from day7. The shapes of GFP and Perilipin double positive (GFP+Perilipin+) cells changed over time, as they became lipid droplet-containing adipocytes at56days. At28days, GFP and CD31double positive (GFP+CD31+) vascular endothelial cells were detected in newly formed vessel wall; the microvascular structure was found that was composed of intermingled GFP+CD31+endothelial cells and GFP negative endothelial cells (GFP-CD31+).
Conclusion:
1. The GFP transgenic mice can be used as an effective tool for the in vivo tracking of SVF cells. This method allows us to carry out a real time and continuous observation on the in vivo survival and differentiation of SVF cells by using of fluorescence imaging and immunofluorescence histochemical staining techniques. This animal model was established for investigating the biologic outcomes of SVF cells after co-implantation with fat grafts. It would also provide a good basic for further investigation of the mechanisms of SVF cell in fat grafting.
2. The results of in vivo fluorescence imaging showed that although most implanted SVF cells died shortly at the early stage after fat grafting, some of the SVF cells remained alive in the fat grafts for a long time.
3. The results of immunofluorescence histochemical staining showed convincing evidence of the multipotent differentiation capability of SVF cells in fat grafting. The results prove the principle that a few of the living SVF cells can spontaneously differentiate into adipocytes in fat grafts and contribute to adipose tissue regeneration. Meanwhile, some of the living SVF cells can differentiate into endothelial cells and participate in vascular regeneration of the fat grafts. These findings suggest that the potential benefits of SVF cells in fat grafting may be the results of concurrent effects of multiple mechanisms.
引文
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