不同配比冻存液对人脂肪细胞活性影响的实验研究

发布时间：2018-05-07 11:45

本文选题：脂肪颗粒 + 冻存液　；参考：《大连医科大学》2017年硕士论文

【摘要】：研究背景:由于自体脂肪组织的生物相容性优于人工生物制品及异体材料,且无免疫排斥现象,成本相对低廉,目前已成为理想的软组织填充材料,得到了广泛临床应用。然而移植脂肪高吸收率、低存活率的问题一直困扰着整形外科医师,若想达到长期满意效果,常需进行二次手术移植。国内外学者一直在试图寻找更好的方法以提高脂肪移植成活率、尽量减少吸脂手术次数:如一次吸脂后将移植后多余的脂肪颗粒体外冻存,二次移植时复温后再注射;在移植组织中添加PRP、SVF等不同成分;选择合适的纯化方式及抽吸管径、负压等等。各类研究都在进行,关于吸脂尚无一套统一的操作规范,本研究选择脂肪组织移植供区、脂肪组织冻存液配比两个因素对脂肪组织活性的影响进行实验研究。目的:探索不同配比冻存液对不同部位冻存脂肪组织活性的影响,以优化脂肪组织冻存液比例并为临床脂肪移植供区选择提供理论依据。方法:1.常规收集45例吸脂患者大腿内、外侧及腹部的颗粒脂肪。将每例标本平均分为两份分别存入A、B两种比例的冻存液中,于 80°C的深低温冰箱中冻存6个月。其中A组冻存液配比为:60%小牛血清、15%二甲基亚砜(DMSO)、25%高糖培养基(DMEM)。B组为:30%小牛血清、15%二甲基亚砜、55%高糖培养基。2.6个月后将标本复苏,用HE染色观察组织形态,计算完整细胞比率;台盼蓝染色计算死亡细胞比例;肌酸激酶实验测定标本的细胞破坏率;免疫组化CD105检测脂肪干细胞形态及数量。3.不同冻存液储存的脂肪颗粒复温后,每个注射点取0.2ml颗粒脂肪移植于裸鼠背部皮下,术后4周处死裸鼠,取出移植脂肪组织,进行称重、测量体积、HE染色,透射电镜观察细胞形态。结果:HE染色:大腿内侧A组脂肪细胞完整率为78.3±2.03%,B组为66.4±3.23%;大腿外侧A组脂肪细胞完整率为70.5±4.53%,B组为55.4±6.03%;腹部A组脂肪细胞完整率为65.1±4.63%,B组为51.9±5.08%。每个部位的A、B两组之间差异有统计学意义(P0.05),每组冻存液中大腿内侧与其余两个部位间差异有统计学意义。台盼蓝染色示A组冻存液条件下的脂肪细胞拒染率较B组高,同一组内大腿内侧脂肪细胞拒染率较高,肌酸激酶结果与台盼蓝一致,以A组(60%小牛血清、15%DMSO、25%DMEM)长期冻存的脂肪活性较高。来源于大腿外侧、大腿内侧及腹部的脂肪标本中,其中大腿内侧的活性较高,有利于脂肪移植的成活。结论:A组冻存液长期冻存脂肪活性较高。大腿内侧颗粒脂肪活性高于大腿外侧及腹部。
[Abstract]:Background: since the biocompatibility of autogenous adipose tissue is superior to that of artificial biological products and allogeneic materials and there is no immune rejection and the cost is relatively low, autologous adipose tissue has become an ideal soft tissue filling material and has been widely used in clinical practice. However, the problems of high fat absorption rate and low survival rate have been a problem for plastic surgeons. If they want to achieve long-term satisfactory results, it is often necessary to carry out secondary surgery transplantation. Scholars at home and abroad have been trying to find a better way to improve the survival rate of fat transplantation, as far as possible to reduce the number of liposuction operations: such as after the first liposuction transplantation of excess fat particles frozen in vitro, secondary transplantation after rewarming and then injected; Different components such as PRPN SVF were added to the transplanted tissue, and the proper purification methods and suction diameter, negative pressure were selected. All kinds of studies are being carried out, and there is no uniform operation standard for liposuction. In this study, the effects of two factors, adipose tissue transplantation donor area and ratio of frozen fat tissue storage solution, on adipose tissue activity were studied experimentally. Objective: to explore the effect of different proportion of frozen liquid on the activity of frozen adipose tissue in different parts in order to optimize the proportion of frozen liquid of adipose tissue and to provide theoretical basis for the selection of donor area for clinical fat transplantation. Method 1: 1. Granule fat in the inner, lateral and abdominal thighs of 45 patients with liposuction was routinely collected. Each specimen was divided into two parts in two proportions of Agna B, and frozen in 80 掳C deep cryopreservation refrigerator for 6 months. The ratio of cryopreservation solution in group A was: 1. 60% calf serum, 15% DMSOxone, 25% high sugar culture medium, DMEMN. Group B: 30% calf serum, 15% dimethyl sulfoxide, 55% high sugar. After 2.6 months, the specimens were resuscitated, the tissue morphology was observed by HE staining, and the complete cell ratio was calculated. Trypan blue staining was used to calculate the proportion of dead cells, creatine kinase assay was used to determine the cell destruction rate, and immunohistochemical CD105 was used to detect the morphology and number of adipose stem cells. After rewarming the fat particles stored in different frozen solution, the fat particles of 0.2ml were taken from each injection point and transplanted subcutaneously into the back of nude mice. After 4 weeks of operation, the nude mice were killed, the adipose tissue was removed and weighed, and the volume of fat was measured by HE staining. Cell morphology was observed by transmission electron microscope. Results the complete rate of adipocytes in group A was 78.3 卤2.03and 66.4 卤3.23in group B, 70.5 卤4.53 in group B, 55.4 卤6.03in group B, and 65.1 卤4.63in group B, 51.9 卤5.08in group B.Results the complete rate of adipocytes in group A was 78.3 卤2.03g, and that in group B was 61.9 卤5.08.The percentage of adipocytes in group B was 70.5 卤4.53 and 55.4 卤6.03in group B, respectively. There was significant difference between two groups (P 0.05), and there was significant difference between the inner thigh and the other two parts in the frozen liquid of each group. Trypan blue staining showed that the rejection rate of adipocytes in group A was higher than that in group B, and the rejection rate of adipocytes in the inner thigh of the same group was higher than that in group B, and the results of creatine kinase were consistent with those of trypan blue. The fat activity of group A (60% calf serum) was higher than that of group A (DMEM). Among the fat samples from lateral thigh, medial thigh and abdomen, the activity of medial thigh is higher, which is beneficial to the survival of fat transplantation. Conclusion the fat activity of group A was higher than that of group A. The fat activity of medial thigh granules was higher than that of lateral thigh and abdomen.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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