本文選題：骨髓間充質干細胞 + 細胞移植　； 參考：《湖北醫(yī)藥學院》2017年碩士論文

【摘要】：目的:(1)探索兔骨髓間充質干細胞(BMSCs)不同提取方法對BMSCs生長及傳代的影響;(2)探索兔耳增生性瘢痕動物模型的可行性,并分析可能的影響因素,以提高造模成功率;(3)研究BMSCs對兔耳增生性瘢痕的預防作用效果。方法:(1)出生25~30天日本大耳白兔2只,獲得股骨、脛骨骨髓后,分別采取全骨髓差速貼壁法、梯度離心法、紅細胞裂解法和梯度離心聯(lián)合差速貼壁法分離BMSCs,比較每種分離方法P0代培養(yǎng)、P3代生長曲線及凍存復蘇率差異,并對所獲BMSCs進行免疫學和多向分化能力鑒定;(2)成年日本大耳白兔12只,每側兔耳腹側面均行4個直徑為1.0 cm全層皮膚缺損創(chuàng)面,觀察造模成功率,分析影響造模成功率的因素;(3)成年日本大耳白兔24只,隨機數(shù)字表法分為兩組:BMSCs組和磷酸鹽緩沖液組(PBS組),每組12只。于手術當天及術后第10天,BMSCs組每個創(chuàng)面周圍注射1ml濃度為1×10~6/ml的BMSCs,PBS組注射1ml PBS溶液。觀察兩組創(chuàng)面愈合時間、再上皮時間、瘢痕高峰時間、瘢痕高峰持續(xù)時間,以及創(chuàng)面愈合后第7、14、21、28天兩組瘢痕顏色、瘢痕面積、瘢痕指數(shù)、肉眼觀及超聲檢查結果差異。結果:(1)梯度離心聯(lián)合差速貼壁法提取BMSCs與全骨髓差速貼壁法、梯度離心法、紅細胞裂解法比較,能提高P0代增殖速度,在傳代、凍存細胞復蘇成活率方面,四種方法無明顯差異(P0.05)。(2)對所獲細胞進行表面抗原及免疫熒光檢測發(fā)現(xiàn),細胞表面CD29、CD71、CD106表達陽性,CD45表達陰性,符合BMSCs表面抗原特點,并且在特定條件誘導下可成骨、成脂分化。(3)兔耳增生性瘢痕動物模型建模成功率為78.13%,皮膚缺損創(chuàng)面位于兔耳腹側面中1/3、盡量剔盡軟骨膜并保持軟骨完整性可提高造模成功率。(4)與PBS比較,BMSCs能改善兔耳增生性瘢痕顏色(P0.05)、減少瘢痕形成;(5)BMSCs不能縮短兔耳全層皮膚缺損愈合時間(P0.05)、再上皮化時間(P0.05)、瘢痕高峰出現(xiàn)時間(P0.05)及高峰持續(xù)時間(P0.05)。(6)肉眼觀及超聲檢測均顯示干細胞治療組兔耳瘢痕組織較對照組小。結論:梯度離心法結合差速貼壁法較常規(guī)的全骨髓差速貼壁法、梯度離心法及紅細胞裂解法分離BMSCs能提加快P0代增殖速度;四種分離方法在BMSCs傳代及復蘇成活率方面無明顯差異;兔耳增生性瘢痕模型制作過程中,皮膚缺損創(chuàng)面位于兔耳腹側面中1/3、盡量剔盡軟骨膜,并保持軟骨完整性可提高造模成功率;BMSCs能改善兔耳增生性瘢痕顏色、減少瘢痕形成;BMSCs不能縮短兔耳全層皮膚缺損愈合時間和再上皮化時間。BMSCs對增生性瘢痕高峰出現(xiàn)時間及高峰持續(xù)時間方面也無明顯影響。
[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.
【學位授予單位】：湖北醫(yī)藥學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R622;R-332

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