血管基质片段细胞携血管内皮生长因子对移植颗粒脂肪血管化的影响

Experimental study of the effect of adipose stromal vascular fraction cells with VEGF on the neovascularization of free fat transplantation

目的探讨血管内皮生长因子（vascular endothelial growth factor，VEGF）复合血管基质片段细胞（stromal vascular fraction cells，SVFs）细胞疗法对移植颗粒脂肪组织血管化的影响，寻找一种安全、有效、简单的细胞辅助脂肪移植术式。方法取患者植皮术后废弃的皮下脂肪，提取SVFs。用DiI染色观察SVFs染色标记情况，锥虫蓝染色观察SVFs活性。将0．3ml待移植的脂肪颗粒分别与0．2ml下列细胞混合：①复合有VEGF的SVFs（A组）；②SVFs（B组）；③DMEM完全培养基（c组）。按照随机化原则将3组脂肪组织混合物分别注射移植于裸鼠背部皮下，每只裸鼠背上注射3个点（共12只）。观察移植物外形、成活情况、湿重、直径，HE染色及CD31染色镜下观察并计数毛细血管密度。使用SPSS16．0进行方差分析检验。结果新鲜提取的SVFs可被Dil标记染色，加用VEGF仍能保持较好的细胞活性，移植术后2个月3组移植脂肪的湿重为：A组（191．90±9．81）mg、B组（177：01±10．50）mg、C组（92．05±8．30）mg，A组〉B组〉C组（P〈0．01）。移植物直径为：A组（0．49±0．24）cm、B组（0．40±0．26）cm、C组（0．32±-0．28）cm，A组〉B组〉C组（P〈0．01）。CD31染色镜下，每高倍镜视野微血管密度：A组（14．58±2．06）个、B组（11．55±2．18）个、C组（7．87±1．55）个，与B组和C组比较，A组较少见纤维结缔组织增生及坏死凋亡细胞。结论结合VEGF因子的SVFs辅助脂肪移植是一种临床可操作性强、简便可行的较理想的细胞疗法，比单纯SVFs更能有效地促进移植脂肪血管化。

Objective To investigate the effect of adipose stromal vascular fraction cells （SVFs） with VEGF on the neovascularization of free fat transplantation. Methods SVFs were obtained from subcutaneous fat and labelled with DiI. 0. 3 ml autologous fat tissue was mixed with 0. 2 ml cells： （1） autologous SVFs with VEGF（ Group A） ; （2）autologous SVFs （Group B） ;（3）complete DMEM （Group C ） And then the mixture was injected randomly under the back skin of 12 nude mice. The transplanted fat tissue in three groups was harvested at 2 months after implantation. Wet weight and diameter of fat grafts was measured. After HE and CD31 staining,blood vessel density,viable adipocytes and fibrous proliferation were observed. Results Trace of SVFs labeled by DiI in vivo could be detected by fluorescent microscope. The wet weight of fat grafts was（ 191.90 ±9.81 ） mg in group A, （ 177.01± 10.50） mg in group B, and （92.05 ±8.30） mg in group C （ P 〈 0.01 ）. The diameter of fat grafts was （0.49± 0.24） cm in group A, （0.40±0.26） cm in group B, and （0.32±0.28） cm in group C（P〈0. 01）. Histological analysis showed the blood vessel density was （14.58 ± 2.06）/HPL in group A, （11.55 ± 2. 18 ）/HPL in group B, （7.87 ±1.55 ）/HPL in group C. Compared with group B and group C, group A had more adipose tissue with less fat necrosis and fibrosis and had significantly higher capillary density. Conclusions The autologous adipose stromal vascular fraction cells with VEGF could improve the neovascularization of free fat significantly. It indicates a wide clinical application in the future.