【作者】 梁久龙；

【导师】 刘晓燕；

【作者基本信息】 第二军医大学 ， 整形外科学， 2010， 博士

【摘要】 研究目的：建立兔脂肪间充质干细胞的体外分离培养方法,鉴定并分析其生物学特性,并探索脂肪组织来源干细胞体外培养的最佳条件。观察大鼠自体脂肪来源干细胞的皮下移植成活的情况。尝试用兔脂肪来源干细胞来解决扩张皮瓣淤血问题。探索脂肪来源干细胞在人体体表软组织缺损复合脂肪组织移植的临床应用。观察人脂肪来源干细胞复合脂肪移植人体表软组织缺损临床效果。研究方法：①选用新西兰大白兔,无菌取出腹股沟脂肪,胶原酶法分离出兔脂肪干细胞,并进行体外培养,取2代以后的兔脂肪干细胞观察其形态特征,并绘制细胞生长曲线及倍增时间。比较了不同胶原酶浓度、胶原酶消化时间和血清浓度对于脂肪来源干细胞体外培养的不同效果。②60只Vistar大鼠随机分为3组,每组20只,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后用脂质体法对细胞进行eGFP(增强型绿色荧光蛋白)转染标记。实验对照组的细胞转染标记后,利用G418将细胞其灭活。空白对照组为生理盐水。分别将其注时移植至大鼠自体耳廓皮下层次,分别于注射后第2、4、6周时进行冰冻切片观察并定量分析所形成组织量的转归情况。③20只新西兰大耳白,左耳、右耳随机排列,10ml扩张器在耳背皮下植入,定期注水,于第20天取出扩张器,并形成皮瓣,制做淤血模型。、同时,实验组取自体腹股沟区皮下脂肪组织,用Ⅰ型胶原酶消化法提取脂肪间充质干细胞并进行传代培养,然后对细胞Dil染色标记。空白对照组为生理盐水。分别将其注射移植至兔形成皮瓣的皮下层次,在注射后于第2周时观察皮瓣的成活情况,并进行病理切片观察和CD34兔疫组织化学染色,定量分析所形成组织量的成活情况和血管发生情况。2008年4月至2010年4月共收治体表软组织缺损患者共12例,病因有放疗遗留软组织萎缩、手术遗留缺损、年龄老化致鼻唇沟加深、眉间纹加深等。与患者交流后,都愿意尝试脂肪来源干细胞复合脂肪移植填充体表软组织缺损。局部肿胀麻醉下,取自体腹部或双侧大腿皮下脂肪组织,用I型胶原酶消化法提取脂肪间充质干细胞,浓集(2×106／ml)后即时与自体脂肪混合静置半小时后注射移植,采用多点、多层次、少量多次的注射方法,术后适度加压包扎。结果：①兔脂肪干细胞原代及传代细胞形态：原代及传代的兔脂肪干细胞均呈长梭形或多边形贴壁生长,生长分化活跃。②兔脂肪干细胞的生长曲线及倍增时间：传代的兔脂肪干细胞生长曲线呈“s”形,倍增时间为55 h。③兔脂肪干细胞的成脂分化诱导,经油红染色的办法,其成脂分化的阳性率为65%④采用0.5mg/ml、1mg/ml、1.5mg/ml、2mg/ml四种不同胶原酶浓度进行消化后发现,在消化时间为1h时,2mg/ml组细胞总数最多,活细胞比率与其他组有统计学差异。在消化时间为2h时,细胞总数较1h各组均增加,同时,1mg/ml组、1.5mg/ml组和2mg/ml组消化细胞总数无统计学差异,活细胞比率1mg/ml组和1.5mg/ml组最高。采用5%、10%、15%、20%五种不同血清浓度培养后发现,15%和20%组生长特性最佳,明显优于另三组。④实验组切片内可见明显绿色荧光组织层存留,其体积在各个时间点没有明显的变化。(P<0.01)⑤实验组皮瓣成活的长度明显长于对照组,病理切片显示CD34染色密度明显高于对照组。(P<0.01)。术后半年随访填充脂肪维持注射量,无明显吸收。结论：①本次实验所分离出来的兔脂肪干细胞在体外具有生长稳定,增殖较快的特点。脂肪组织来源干细胞,胶原酶浓度lmg/ml、消化时间2h、血清浓度15%是最佳的培养条件。②单纯自体脂肪来源干细胞的皮下移植可以存活,并且形成均一稳定的组织。③脂肪来源干细胞可以增加血管的发生,改善皮瓣淤血情况,从而提高扩张皮瓣的成活长度。④脂肪来源干细胞可以增加脂肪移植的成活率。更多还原

【Abstract】 Objective:To investigate the methods of isolating and culturing rabbit adipose—derived stem cells (ASCs), and to identify and analyze the biological characteristics of rabbit ADSCs and To explore the optimized condition for adipose-derived stem cells(ADSC) culture in vitro. To observe the result of mouse’s ASCs autotransplantation subcutaneously. To solve the problem of congestion in the expanded flap by use of the ASCs. To observe the therapeutic effect of ASCs assisted adipose transfer for soft tissue defectMethods:①The ASCs was derived from the inguinal fat tissue of Vistar mouse and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with eGFP transfection at second or third passage. The ASCs of experimental control group was killed by G418 after labled by eGFP. The blank control group normal saline.They were injected into the auricle of mouse subcutaneously seperately and the results was observed at 2nd,4th,6th week with frozen section.②Adipose was collected from the inguinal region of News Zealand rabbits and the ASCs were isolated with adherent method and collagenase method and cultured in vitro. The morphology of ADSCs after passage 2 was analyzed. The growth curve and doubling time were drawn. The collagenase with different concentration and duration and FCS with different concentration were used.③The ASCs was derived from the inguinal fat tissue of rabbit and was cultured after the fat tissue was degested by collagenase. Then ASCs was labeled with Dil at second or third passage. The blank control group normal saline.They were injected into the expanded flap with congestion after transplantation. The result was observed at 2nd week with pathologic section and CD34 immunohistochemistry.④12 patients diagnosed as soft tissue defect received the ASCs assisted adipose transfer for soft tissue atrophy after radiotherapy, surgical treatment and aging. The patients want to received the treatment after consultation. The ASCs was isolated with collagenase I from the adipose in patients abdomen or thigh under local anesthesia Then the ASCs(2×106/ml) was mixed with the adipse for half an hour before being injected to the soft tissue defect region. Moderate pressure should be given after the mixture transplantation delicately.Results:①Formation of primary and passage of ADSCs:ADSCs had spindle and polygon shape adherent growth, and their growth and differentiation were active.②Growth curve and doubling time of ADSCs:The growth curve was like "S" shape. The doubling time of ADSCs was about 55 hours.δThe ADSCs adipogenic differentiation was induced and the rate of positive rate was 65% through the Oil Red O stain and positive cell counting.4 Based on the collagenase concentration, cells were divided into four groups, i.e.,0.5mg/ml, 1mg/ml,1.5mg/ml and 2mg/ml groups. When the duration was 1 hour,2mg/ml group had the most total cell number(TCN) and the living cell rate(LCR) was statistically different from other groups. When the duration was 2 hours, TCN increased in every group and there were no statistical differences among 1mg/ml,1.5mg/ml and 2mg/ml groups. LCR was the highest in 1mg/ml and 1.5mg/ml. 2.Based on FBS concentration, the cells were divided into five groups, i.e.,0,5%,10%, 15%and 20%groups. The growth character was the best in 15%and 20%groups.⑤The new formed tissue labled by GFP was found in the subcutaneous tissue.⑥The survived length of flap with ASCs injection was longer than the flap in the control group. The CD34 immunohistochemistry showed more vessel formation in the flap section with ASCs injection than the flalp in the control group.⑦No obvious absorbtion was found after follow-up for half year.Conclusion①The ASCs of rabbits isolated in this trial are characterized by stable growth and quick proliferation in vitro. The optimized conditions for human adipose-derived stem cells culture are collagenase concentration of 1mg/ml, digestion duration of 2 hour and FBS concentration of 15%.②Autotransplanted ASCs can survive without scaffold.③.ASCs injection could improved the survived length by enhance the vessel formation.④The ASCs could improve the survive rate of adipose transfer.更多还原