骨髓间充质干细胞预防兔耳增生性瘢痕的实验研究

发布时间：2018-04-13 09:52

本文选题：骨髓间充质干细胞 + 细胞移植　；参考：《湖北医药学院》2017年硕士论文

【摘要】：目的:(1)探索兔骨髓间充质干细胞(BMSCs)不同提取方法对BMSCs生长及传代的影响;(2)探索兔耳增生性瘢痕动物模型的可行性,并分析可能的影响因素,以提高造模成功率;(3)研究BMSCs对兔耳增生性瘢痕的预防作用效果。方法:(1)出生25~30天日本大耳白兔2只,获得股骨、胫骨骨髓后,分别采取全骨髓差速贴壁法、梯度离心法、红细胞裂解法和梯度离心联合差速贴壁法分离BMSCs,比较每种分离方法P0代培养、P3代生长曲线及冻存复苏率差异,并对所获BMSCs进行免疫学和多向分化能力鉴定;(2)成年日本大耳白兔12只,每侧兔耳腹侧面均行4个直径为1.0 cm全层皮肤缺损创面,观察造模成功率,分析影响造模成功率的因素;(3)成年日本大耳白兔24只,随机数字表法分为两组:BMSCs组和磷酸盐缓冲液组(PBS组),每组12只。于手术当天及术后第10天,BMSCs组每个创面周围注射1ml浓度为1×10~6/ml的BMSCs,PBS组注射1ml PBS溶液。观察两组创面愈合时间、再上皮时间、瘢痕高峰时间、瘢痕高峰持续时间,以及创面愈合后第7、14、21、28天两组瘢痕颜色、瘢痕面积、瘢痕指数、肉眼观及超声检查结果差异。结果:(1)梯度离心联合差速贴壁法提取BMSCs与全骨髓差速贴壁法、梯度离心法、红细胞裂解法比较,能提高P0代增殖速度,在传代、冻存细胞复苏成活率方面,四种方法无明显差异(P0.05)。(2)对所获细胞进行表面抗原及免疫荧光检测发现,细胞表面CD29、CD71、CD106表达阳性,CD45表达阴性,符合BMSCs表面抗原特点,并且在特定条件诱导下可成骨、成脂分化。(3)兔耳增生性瘢痕动物模型建模成功率为78.13%,皮肤缺损创面位于兔耳腹侧面中1/3、尽量剔尽软骨膜并保持软骨完整性可提高造模成功率。(4)与PBS比较,BMSCs能改善兔耳增生性瘢痕颜色(P0.05)、减少瘢痕形成;(5)BMSCs不能缩短兔耳全层皮肤缺损愈合时间(P0.05)、再上皮化时间(P0.05)、瘢痕高峰出现时间(P0.05)及高峰持续时间(P0.05)。(6)肉眼观及超声检测均显示干细胞治疗组兔耳瘢痕组织较对照组小。结论:梯度离心法结合差速贴壁法较常规的全骨髓差速贴壁法、梯度离心法及红细胞裂解法分离BMSCs能提加快P0代增殖速度;四种分离方法在BMSCs传代及复苏成活率方面无明显差异;兔耳增生性瘢痕模型制作过程中,皮肤缺损创面位于兔耳腹侧面中1/3、尽量剔尽软骨膜,并保持软骨完整性可提高造模成功率;BMSCs能改善兔耳增生性瘢痕颜色、减少瘢痕形成;BMSCs不能缩短兔耳全层皮肤缺损愈合时间和再上皮化时间。BMSCs对增生性瘢痕高峰出现时间及高峰持续时间方面也无明显影响。
[Abstract]:Objective to explore the effects of different extraction methods of rabbit bone marrow mesenchymal stem cells (BMSCs) on the growth and passage of BMSCs.To improve the success rate of model making, the preventive effect of BMSCs on hypertrophic scar in rabbit ear was studied.Methods the bone marrow of femur and tibia were obtained from 2 Japanese big ear white rabbits at 2530 days after the birth of 1: 1) the whole bone marrow differential adherent method and gradient centrifugation method were used respectively.BMSCs were separated by RBC lysis and gradient centrifugation combined with differential adherent method. The growth curves and the rate of cryopreservation and resuscitation of P3 generation of P0 culture were compared.The BMSCs was identified by immunology and multidirectional differentiation. 12 adult Japanese white rabbits were treated with 4 full-thickness skin defect wounds with a diameter of 1.0 cm on the ventral side of each ear. The success rate of modeling was observed.24 adult Japanese white rabbits were randomly divided into two groups: BMSCs group (n = 12) and PBS group (n = 12).On the day of operation and on the 10th day after operation, BMSCs PBS solution was injected into each wound area of BMSCs group with 1ml concentration of 1 脳 10~6/ml.The wound healing time, re-epithelium time, scar peak time, scar peak duration and scar color, scar area, scar index, naked eye and ultrasonic examination were observed.Results compared with the whole bone marrow differential adherent method, gradient centrifugation combined with differential adhesion method, gradient centrifugation and erythrocyte lysis method could increase the proliferation rate of P0 generation, and improve the survival rate of passageway and cryopreservation cell recovery, compared with the whole bone marrow differential adherent method, gradient centrifugation method and erythrocyte lysis method.There was no significant difference among the four methods (P0.05P0.05P0.05An. 2) the surface antigen and immunofluorescence assay showed that the positive expression of CD29, CD71, CD106 and CD106 was negative, which was consistent with the characteristics of BMSCs surface antigen, and osteogenesis could be induced under specific conditions.The success rate of animal model of hypertrophic scar in rabbit ear is 78.13, and the wound of skin defect lies in the ventral side of rabbit ear 1 / 3. As far as possible, removing chondrocytes and maintaining the integrity of cartilage can improve the success rate of model making. 4) compared with PBS, BMSCs can be improved.The color of hypertrophic scar in rabbit ear (P0.05A) and the reduction of scar formation (BMSCs) can not shorten the healing time of skin defect in whole layer of rabbit ear (P0.05), the time of re-epithelization (P0.05), the time of peak appearance of scar (P0.05) and the time of peak duration (P0.05).The scar tissue of rabbit ear in cell therapy group was smaller than that in control group.Conclusion: gradient centrifugation combined with differential adhesion method can accelerate the proliferation rate of P0 generation compared with the conventional differential adherent method, gradient centrifugation method and erythrocyte lysis method.There was no significant difference between the four methods in BMSCs passage and survival rate of resuscitation. In the process of making hypertrophic scar model of rabbit ear, the skin defect wound was located in 1 / 3 of the ventral side of rabbit ear, and the chondrocyte membrane was removed as far as possible.Maintaining the integrity of cartilage can improve the success rate of modeling. BMSCs can improve the color of hypertrophic scar in rabbit ear.Reduction of scar formation BMSCs could not shorten the healing time and re-epithelialization time. BMSCs had no significant effect on the peak time and duration of hypertrophic scar.
【学位授予单位】：湖北医药学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622;R-332

【参考文献】