【作者】 刘景兰；

【导师】 胡刚；

【作者基本信息】 大连医科大学 ， 外科学， 2012， 硕士

【摘要】 背景：自体脂肪颗粒注射移植因为具备供源充足、取材方便、操作简单、微创无痕、无供区畸形、有良好的组织相容性、手感自然、充盈外形好并能同时获得瘦身塑体的效果等优点，被国内外整形美容外科领域青睐，是人体最理想、最具发展潜力的软组织填充方法，而且脂肪组织最近又被发现是一种内分泌组织，增加了其移植优势。但移植后脂肪细胞吸收率高（30%-70%）、液化、坏死等并发症限制了其在临床的应用。富血小板血浆（platelet-rich plasma, PRP）是将全血通过梯度离心得到的血小板浓缩物，能释放丰富的生长因子。很多研究已经证实PRP中的生长因子对软组织和骨组织的愈合有促进作用，且对脂肪来源干细胞（adipose-derived stem cells, ADSCs）的增殖有促进作用。ADSCs有多向分化潜能，且具有来源广泛、取材方便、组织损伤小、可分泌多种生长因子等优势，使其成为近年来干细胞研究领域的热点。目的：本实验旨在通过建立裸鼠模型，将兔ADSCs、PRP联合加入自体脂肪颗粒中混合移植，来探讨两者联合对移植后血运重建的影响。方法：1取3月龄新西兰大白兔颈背部区脂肪垫，采用Ⅰ型胶原酶消化法体外分离培养兔ADSCs，观察细胞形态特征。2取第3代兔ADSCs进行成脂、成骨诱导分化，油红O、茜素红染色鉴定，确定兔ADSCs具备多向分化潜能。3新西兰大白兔心脏抽血，二次离心法制备PRP，血小板计数。4MTT法检测PRP、全血浆、对照组在不同时段（24h、48h、72h）对体外培养兔ADSCs的增殖的影响。5制备脂肪颗粒、明胶海绵颗粒。28只裸鼠随机编号，按随机表将其分成4组，每组7只。每只鼠后背取5点：左上、右上、左下、右下、正中（每两点间距大于2cm），将5点按照随机表排列。体内实验分为五组：①脂肪颗粒+ADSCs+PRP+明胶海绵；②脂肪颗粒+ADSCs+生理盐水+明胶海绵；③脂肪颗粒+PRP+生理盐水+明胶海绵；④脂肪颗粒+全血浆+生理盐水+明胶海绵；⑤脂肪颗粒+生理盐水+明胶海绵。将各组移植入裸鼠皮下。6分别于7d、14d、30d、90d取材，制作石蜡切片，HE染色法观察对比组织形态，免疫组化法检测血管数。结果：1ADSCs原代培养的细胞接种24h后，可见少量较大的椭圆形、类圆形细胞贴壁，未贴壁的细胞大部分为圆形的红细胞及筋膜杂质。48h后首次换液，大多数细胞贴壁，呈梭形、多角形、类圆形等。3d后可见部分细胞已贴壁伸展，呈长梭形，核居中、可见核分裂相。7d后可见细胞80%-90%融合，细胞呈长梭形成纤维细胞样生长，集体呈旋涡状、鱼群状生长。此时进行胰酶消化传代，4-6h即可贴壁，细胞形态仍呈长梭形，且贴壁速度和生长速度较原代细胞明显增快，连续传代19代细胞形态无明显改变。成脂诱导2W后油红O染色呈阳性结果，成骨诱导3W后茜素红染色呈阳性结果，对照组均为阴性结果。2全血血小板计数结果：(330.2±148.8)×109/L， PRP血小板计数结果：(1398.3±888.5)×109/L，PRP血小板计数是全血的4.23倍。3细胞增殖实验MTT法显示PRP组在24h、48h、72h时对ADSCs的增值较对照组增殖明显，与全血浆组有统计学差异（p≤0.05）。4与其他各组相比，各时间段内脂肪颗粒+ADSCs+PRP+明胶海绵组血管数均明显增多，单独ADSCs或PRP组相对较多，全血浆组、生理盐水组血管数最少。结论：ADSCs与PRP联合可促进脂肪颗粒移植中的血管生成。更多还原

【Abstract】 Objective: To examine whether the rabbit adipose-derived stem cells (ADSCs)either alone or in combination with the platelet-rich plasma (PRP) can improve therevascularization after grainy fat transplantation.Methods:1. Adapted the type Ⅰ collagenase digestion method to isolate andculture the ADSCs on cervicodorsal area fat pads of three-month old New Zealandrabbits.2. Induced the third generation ADSCs into adipose cells or osteoblasts, whichwere identified by the oil red O or alizarin red vital staining method to enable themultiplex differentiation potential of the ADSCs.3. Exsanguinated hearts of the New Zealand rabbits, adapted the secondarycentrifugation on the exsanguinated blood to obtain the PRP, and then recorded thenumber of platelets.4. Detect, used the MTT method, the PRP, plasma and a control group at differentpoints of time (the24th,48th and72th hour).5. Randomly numbered28athymic mice, randomly divided them into four groupseach comprising seven mice. Marked five points on the back of each mouse: upper left,lower left, upper right, lower right, and the center (spacing between any two points isgreater than2cm), and randomly numbered the above five points. Transplanted thefollowing five group grafts into the subcutaneous tissues of the28athymic mice at theabove five points, respectively, where the five group grafts were:①lipochondrias+ADSCs+PRP+spongia gelatinosas;②lipochondrias+ADSCs+saline+spongia gelatinosas;③lipochondrias+PRP+saline+spongia gelatinosas;④lipochondrias+whole plasma+saline+spongia gelatinosas;⑤lipochondrias+saline+spongiagelatinosas, where the lipochondrias and spongia gelatinosas were prepared in advance.6. Procured the above five group grafts from the subcutaneous tissue of theathymic mice on the7th,14th,30th and90th day, respectively, and paraffinized theminto paraffin sections. Observed the paraffin sections and counted the number ofcapillaries using the HE staining method, and the immunohistochemical method.Results:1. The ADSCs grew in the form of the collagenoblasts, there was noobvious variation in the continuous19generations cells. Adipose cells were inductedafter two weeks, the oil red O attaining results were positive. The osteoblasta wereinducted after three weeks, results of the alizarin red staining were positive. The resultsof the control group staining were negative.2. The number of the whole blood platelets was:(330.2±148.8)×109/L, the numberof the PRP platelets was:(1398.3±888.5)×109/L, the number of the PRP platelets was4.23times of that of the whole blood platelet.3. The results of the MTT method showed that the PRP speeded up theproliferation of the ADSCs which was statistically different from that of the plasma andthe control group (p≤0.05).4. The ADSCs in combination with the PRP transplantations therapy producedsignificantly more capillaries than other transplantation therapies, and subjected to lessfat necrosis and fibrosis as revealed by the histological analysis. Also the ADSCs andthe PRP in alone could bring a similar effect which was however not as good as thecombination thereof.Conclusions: The ADSCs in combination with the PRP can boost therevascularization after grainy fat transplantation.更多还原