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背景:软组织充填是临床的难题,已经证实超声波作用后的成熟脂肪细胞不能用于细胞移植,脂肪组织工程有希望解决这一问题。目的:观察超声波对前脂肪细胞体外培养与增殖的影响,验证超声辅助吸脂术后前脂肪细胞作为脂肪组织工程种子细胞的可行性。设计:对照观察实验。单位:武汉大学人民医院整形外科。材料:实验于2003-07/2004-09在武汉大学医学院完成。3月龄本地杂交猪1头(武汉大学医字院实验动物中心提供)氯胺酮麻醉后,于背部两侧各标记10cm×20cm的矩形取材区,右侧为实验组,左侧为对照组。方法:实验组用体外超声波预处理8min,能量3W/cm2。无菌条件下掀开皮肤层,分别切取两侧皮下脂肪组织各100g,放入预先已备好的盛有冷D-hanks液容器中待用。将经过超声波作用后的实验组脂肪组织猪前脂肪细胞进行分离和体外培养,并每隔2d测定其细胞数量及脂质含量,结果以3孔的细胞均数表示。对照组除了不进行超声波预处理处,前脂肪细胞的培养方法同实验组。绘制两组生长曲线。采用油红O染色提取法测定细胞内的脂肪含量,于HITACHIG2000型分光光度计510nm波长处测定吸光度。主要观察指标:①猪前脂肪细胞培养的形态学观察。②实验组及对照组的生长曲线。③实验组与对照组脂质含量的变化。结果:①对照组6～24h细胞完成贴附,实验组一两天才基本贴壁,且未贴壁细胞较多;4d后对照组细胞内见脂肪颗粒积聚,实验组6d后才见到;12d时对照组基本分化成单脂滴或多脂滴的细胞,并有少量漂浮的成熟脂肪细胞,实验组产生脂滴的细胞较少。②在细胞数量相同的情况下,实验组细胞倍增时间约为72h,对照组为36h,培养9d后实验组与对照组的细胞总数分别为13×104个/孔、18×104个/孔,实验组细胞增殖速率缓慢。③实验组脂质出现时间约为培养后6d,对照组为4d,吸光度峰值分别为0.32、0.68。结论:超声波对前脂肪细胞的增殖分化有明显的抑制作用,经超声波处理的前脂肪细胞不宜用作种子细胞。
BACKGROUND: Soft tissue filling is a clinical problem. It has been demonstrated that mature adipocytes after ultrasonic treatment can not be used for cell transplantation. Adipose tissue engineering has the potential to solve this problem. OBJECTIVE: To observe the effect of ultrasound on the culture and proliferation of preadipocytes in vitro and to verify the feasibility of using preadipocytes as adipose tissue-engineered seed cells after ultrasound-assisted liposuction. Design: Control observation experiment. Unit: People 's Hospital of Wuhan University, Plastic Surgery. Materials: The experiment was performed at Wuhan University Medical College from July 2003 to September 2004. A local hybrid pig of 3 months old (provided by Laboratory Animal Center of Medical College of Wuhan University) was anesthetized with ketamine. Rectangular drawing area of 10cm × 20cm was marked on both sides of the back, the experimental group on the right and the control group on the left. Methods: The experimental group was pretreated with ultrasound in vitro for 8 min and the energy was 3 W / cm2. Open the skin layer under aseptic conditions, were cut on both sides of the subcutaneous fat tissue 100g, into the pre-prepared filled with cold D-hanks liquid container stand. The experimental group adipose preadipocytes were isolated and cultured in vitro. The number of cells and the lipid content were determined every 2 days. The results were expressed as the number of 3-well cells. In addition to the control group without ultrasonic pretreatment, preadipocyte culture method with the experimental group. Draw two sets of growth curves. The fat content in the cells was determined by oil red O staining extraction and the absorbance was measured at 510 nm wavelength of a HITACHIG 2000 spectrophotometer. MAIN OUTCOME MEASURES: ① Morphological observation of porcine preadipocyte culture. ② experimental group and control group growth curve. ③ experimental group and control group changes in lipid content. Results: ① The cells in control group were attached after 6 ~ 24h, and the experimental group adhered to the wall almost once or two days without adherent cells. After 4 days, the accumulation of fat particles in the control group was observed, When the control group basically differentiated into single lipid droplets or multiple lipid droplets of cells, and a small amount of floating mature adipocytes, experimental group lipid droplets less cells. In the same cell number, the cell doubling time was about 72 hours in the experimental group and 36 hours in the control group. The total number of cells in the experimental group and the control group was 13 × 104 cells / well and 18 × 104 cells / well, Experimental group of cells proliferation rate is slow. ③ The experimental group lipid appeared about 6d after culture, the control group was 4d, the peak absorbance was 0.32,0.68. Conclusion: Ultrasound can significantly inhibit the proliferation and differentiation of preadipocytes. Ultrasound-treated preadipocytes should not be used as seed cells.