脂肪干细胞辅助脂肪移植治疗局限性硬皮病的相关研究

发布时间：2018-04-01 17:22

本文选题：局限性硬皮病　切入点：脂肪移植　出处：《北京协和医学院》2017年博士论文

【摘要】：目的:分析脂肪移植治疗局限性硬皮病的相关影响因素,探讨脂肪干细胞(ADSCs)辅助脂肪移植治疗局限性硬皮病的可行性。方法:(1)21例脂肪标本:局限性硬皮病患者脂肪标本6例(未使用药物治疗),局限性硬皮病患者脂肪标本5例(曾使用药物治疗),正常脂肪患者脂肪标本10例,提取脂肪标本中的干细胞,分离培养,比较脂肪干细胞数量、形态、增殖能力及成脂能力。(2)激光散斑对比成像方法测定8例局限性硬皮病患者病变部位及正常对侧部位的血流情况。(3)取局限性硬皮病病变部位皮肤标本2例及非硬皮病患者皮肤标本2例,行成纤维细胞培养后与脂肪颗粒混匀后移植入裸鼠,观察脂肪的存活情况。(4)博来霉素硬皮病造模裸鼠18例,正常裸鼠18例,各分为三组:对照组,脂肪移植组,脂肪干细胞+脂肪移植组。术后1月取移植脂肪称重,取注射部位裸鼠皮肤免疫组化测定TGF-β 1及Ⅲ型胶原含量。结果:(1)21例患者脂肪干细胞形态学无差异,成脂能力无差异,流式细胞仪检测第3代患者脂肪干细胞的表面标志CD44+都是90%以上,无明显差异。但局限性硬皮病患者(行激素治疗)的干细胞数量较正常人及局限性硬皮病患者(未行激素治疗)明显减少,细胞生长曲线延迟。(2)局限性硬皮病患者病变部位血流灌注为92.69±22.04,正常对侧部位为95.70 ±20.43,局限性硬皮病病变部位与对侧正常部位血流灌注无明显差异。(3)局限性硬皮病患者成纤维细胞+脂肪移植后脂肪重量约68.24±19.20mg,正常人成纤维细胞+脂肪移植后脂肪重量约88.42±20.15mg。(4)正常裸鼠脂肪移植组脂肪重量102.97±17.87mg,脂肪干细胞+脂肪移植组脂肪重量135.05 ±27.28 mg,硬皮病裸鼠移植脂肪重量77.54±12.40mg,脂肪干细胞+脂肪移植组脂肪重量102.48 ±23.16 mg。脂肪干细胞+脂肪移植的存活率均较单纯的脂肪移植存活率高。(5)对于正常裸鼠,TGF-β 1含量对照组为939.34±216.92,脂肪移植组为974.98±470.18,脂肪干细胞+脂肪移植组为967.66±448.74,Ⅲ型胶原含量对照组为623.85±113.84,脂肪移植组为605.73 ±217.17,脂肪干细胞+脂肪移植为599.34±89.39。对于硬皮病裸鼠,TGF-β1含量对照组为1945.95 ±330.77,脂肪移植组为1418.86 ±376.82,脂肪干细胞+脂肪移植为1132.12±190.69,Ⅲ型胶原含量对照组为2629.39±746.62,脂肪移植组为1531.74±836.41,脂肪干细胞+脂肪移植为946.92±448.90。正常裸鼠经脂肪移植或脂肪干细胞+脂肪移植后TGF-β 1含量及Ⅲ型胶原含量无明显改变。但对于硬皮病裸鼠,脂肪移植或者脂肪干细胞+脂肪移植可降低硬皮病组裸鼠的TGF-β 1含量及Ⅲ型胶原含量,尤其是脂肪干细胞移植后效果更加明显。结论:(1)局限性硬皮病患者的脂肪移植不同于正常人群的脂肪移植,长期应用激素治疗病史及病变部位的微环境是影响硬皮病患者脂肪移植存活的关键因素,而局限性硬皮病患者的病变部位血供较正常侧无明显改变。(2)脂肪干细胞辅助脂肪移植不仅可提高脂肪存活率,同时还可减轻硬皮病裸鼠的皮肤胶原沉积。
[Abstract]:Objective: to analyze the effect of fat transplantation for the treatment of localized scleroderma related factors, to explore the feasibility of adipose derived stem cells (ADSCs) transplantation for the treatment of localized scleroderma assisted fat. Methods: (1) 21 cases of fat samples: 6 cases of patients with localized scleroderma fat samples (without drugs use), 5 cases of fat were localized scleroderma patients (used drug treatment), 10 cases of patients with normal fat fat samples, extracted fat samples of stem cells, isolation and culture, compare adipose stem cell number, morphology, proliferation and adipogenic ability. (2) laser speckle contrast imaging method for the determination of 8 cases of patients with localized scleroderma lesions and normal contralateral parts of the blood flow. (3) the limited scleroderma skin lesions were 2 cases and non skin specimens of 2 cases of scleroderma patients, for fibroblast culture and fat particles after mixing transplanted into nude mice, observe the fat deposit Live. (4) bleomycin scleroderma model in nude mice in 18 cases, 18 cases of normal mice, were divided into three groups: control group, fat transplantation group, adipose derived stem cells + fat transplantation group. Postoperative January transplanted fat weight, take the injection site skin of nude mice was detected by immunohistochemistry and TGF- beta 1 type III collagen content. Results: (1) 21 cases of patients with adipose derived stem cells morphological differences, adipogenic ability difference, detection of third generation patients with adipose derived stem cells surface markers of CD44+ are more than 90% flow cytometry, no significant difference. But the patients with localized scleroderma (hormone treatment) the number of stem cells compared with normal people and patients with localized scleroderma (without hormonal treatment) was significantly reduced, the cell growth curve of delay. (2) patients with localized scleroderma lesions perfusion was 92.69 + 22.04, normal to the contralateral side was 95.70 + 20.43, blood flow of localized scleroderma lesions and contralateral normal area No significant differences in perfusion. (3) patients with localized scleroderma fibroblasts + fat transplantation fat weight about 68.24 + 19.20mg, normal human fibroblasts + fat transplantation fat weight of about 88.42 - 20.15mg. (4) normal nude mice transplantation group fat fat weight 102.97 + 17.87mg, fat stem cells + fat fat transplantation group the weight of 135.05 + 27.28 mg, scleroderma transplanted fat weight 77.54 + 12.40mg, the survival rate of adipose derived stem cells + fat transplantation group fat weight of 102.48 + 23.16 mg. + fat transplantation of adipose derived stem cells were relatively pure fat graft survival rate is high. (5) in normal mice, TGF- beta 1 in the control group was 939.34 + 216.92 that fat transplantation group is 974.98 + 470.18, fat stem cells + fat transplantation group is 967.66 + 448.74, type III collagen content of control group was 623.85 + 113.84, fat transplantation group is 605.73 + 217.17, + fat fat stem cell transplantation was 599.34 For scleroderma + 89.39. nude mice, TGF- beta 1 in the control group was 1945.95 + 330.77, fat transplantation group is 1418.86 + 376.82, + fat transplantation of adipose derived stem cells was 1132.12 + 190.69, type III collagen content of control group was 2629.39 + 746.62, fat transplantation group is 1531.74 + 836.41, fat stem cells + fat transplantation was 946.92. 448.90. normal nude mice by transplantation of adipose derived stem or fat content and collagen content of TGF- beta 1 type III cell transplantation + fat had no obvious change. But for scleroderma mice, fat transplantation or fat content and collagen content of 1 stem cell type III + fat transplantation can reduce the TGF- beta scleroderma mice, especially fat stem cell transplantation the effect is more obvious. Conclusion: (1) patients with localized scleroderma fat transplantation fat transplantation is different from the normal population, the micro environment of long-term use of hormone therapy and the history of the site of the lesion is affected in patients with scleroderma The key point of survival is fat transplantation. However, the blood supply of lesion sites of the patients with localized scleroderma is not significantly different from that of the normal side. (2) adipose tissue derived stem cells assisted fat transplantation can not only increase the fat survival rate, but also reduce the collagen deposition of the scleroderma nude mice.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R593.25

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