自体脂肪来源的间充质干细胞复合脱细胞真皮基质修复兔关节软骨缺损的实验研究

发布时间：2018-04-27 04:17

本文选题：脂肪来源的间充质干细胞 + 脱细胞真皮基质　；参考：《河北医科大学》2009年硕士论文

【摘要】： 由于负重关节面由透明软骨组成,软骨细胞被细胞外基质包绕,几乎没有迁徙能力,不能聚集到创伤部位,因此,软骨组织一旦发生损伤后自我修复能力十分有限。软骨缺损常常引起关节疼痛及功能障碍甚至残疾,临床上缺乏有效的治疗方法,从而促进了软骨组织工程技术地快速发展。软骨组织工程涉及种子细胞、支架材料及生长因子、信号分子和应力作用等环境方面的内容。其中种子细胞是影响组织工程学的重要因素。脂肪来源的间充质干细胞(adipose-derived mesenchymal stem cells, ADMSCs)由于其强大的增殖和分化能力做为组织工程的种子细胞来源而备受关注。目的: 1探讨脂肪来源的间充质干细胞分离和培养的可行性,为扩大和优化软骨组织工程种子细胞源提供实验依据。 2自体脂肪来源的间充质干细胞与脱细胞真皮基质(acellular derma matrix,ADM)复合修复兔膝关节软骨缺损,对修复组织进行组织学和组织病理学评分,评价修复效果,为临床应用组织工程学方法修复关节软骨缺损提供理论依据和奠定实验基础。方法: 选用健康新西兰兔36只。剪碎的兔脂肪组织使用0.1%Ⅰ型胶原酶消化离心后获得自体ADMSCs后进行体外培养。流式细胞仪检测ADMSCs表面抗原CD44的表达情况。倒置显微镜观察细胞形态。取原代、第3代ADMSCs,采用细胞计数法测定细胞生长曲线。体外特定条件下诱导ADMSCs定向诱导分化为脂肪细胞、成骨细胞及软骨细胞。ADMSCs成脂方向诱导组成为DMEM,10% FBS,异丁基甲基黄嘌呤(IBMX),200μmol/L地塞米松,10μg/ml胰岛素, 1μmol/L吲哚美辛。ADMSCs成骨方向诱导组成为DMEM,10% FBS,0.1μmol/L地塞米松,50μg/mL抗坏血酸,10mmol/Lβ-磷酸甘油。ADMSCs成软骨诱导组成包括10% FBS、高糖DMEM、10μg/L转化生长因子β1、50μg/ml抗坏血酸、6.25 mg/L胰岛素。ADMSCs成脂诱导分化后用油红O染色,成骨方向诱导分化后用钴钙法碱性磷酸酶染色及茜素红钙结节染色,成软骨方向诱导分化后进行Ⅱ型胶原免疫组化染色。使用特殊染色来证明ADMSCs的多向分化潜能。用小牛真皮制备ADM作为细胞载体,ADMSCs体外培养至第3代,制成浓度为5×105/ml的软骨细胞悬液。将自体ADMSCs与ADM体外复合培养2d后,行扫描电镜观察后,移植于兔膝关节软骨缺损处。将36只新西兰兔随机分为ADMSCs /ADM实验组、ADM对照组和空白对照组。实验组髁间窝软骨缺损处植入ADMSCs /ADM,ADM对照组单纯植入脱细胞真皮基质,空白对照组不作任何植入,分别于术后6周、9周和12周每组各处死4只动物,取材对修复组织进行大体、组织学及免疫组化染色观察,根据关节软骨组织学评分及组织病理学评分标准对修复组织进行评分,数据输入SPSS 16.0软件,用随机分组的SNK-q法进行统计分析,比较各组的评分差异是否具有统计学意义。结果: 1接种24h后可见兔ADMSCs呈长梭形或多边形贴壁。细胞增殖迅速,原代培养约10～14d左右可以达到80-90%融合。 2 ADMSCs表面抗原的鉴定:流式细胞仪检测结果显示细胞表面抗原CD44呈阳性表达。 3 ADMSCs生长曲线呈“S”形。贴壁2 d后细胞开始增殖, 7 d进入增殖高峰期, 9 d后进入平台期。 4 ADMSCs成脂诱导分化后经油红染色显示分泌红色脂滴的脂肪细胞;成骨诱导分化后碱性磷酸酶染色呈黑色沉淀,茜素红染色呈钙化结节;成软骨诱导分化后Ⅱ型胶原免疫组化染色阳性。 5电镜下可见ADM呈多孔疏松结构,孔隙率较好,适合ADMSCs生长。 6术后9周ADMSCs /ADM实验组大体观察修复组织整合较好,无支架材料残留。ADM对照组和空白对照组为纤维性修复和无修复。组织学评分ADMSCs /ADM实验组与ADM对照组和空白对照组差别有显著性意义(P0.05), ADM对照组与空白对照组差别无显著性意义(P0.05);组织病理学评分显示ADMSCs /ADM实验组与ADM对照组和空白对照组差别有显著性(P0.05),ADM对照组与空白对照组无显著性差异(P0.05)。 7术后12周ADMSCs /ADM实验组大体观察修复组织表面光滑,与周围软骨整合良好。ADM对照组和空白对照组为纤维性修复和无修复。组织学评分ADMSCs /ADM实验组与ADM对照组和空白对照组差别有显著性(P0.05), ADM对照组与空白对照组差别有显著性(P0.05);组织病理学评分显示ADMSCs /ADM实验组与ADM对照组和空白对照组差别有显著性(P0.05),ADM对照组与空白组差别有显著性(P0.05)。 8 II型胶原免疫组化染色显示ADMSCs /ADM实验组修复区的软骨细胞含II型胶原,与周围软骨结合良好。结论: 1兔脂肪来源的间充质干细胞在体外具有生长稳定,增殖较快的特点,在体外诱导培养体系下可分化为脂肪细胞、成骨细胞和软骨细胞,符合间充质干细胞多向分化潜能的特征。 2脱细胞真皮基质材料适合兔脂肪来源的间充质干细胞的黏附和增殖,是软骨组织工程较为理想的支架,二者复合后植入软骨缺损区能够修复关节软骨。 3组织学观察和Ⅱ型胶原免疫组织化学染色证实脂肪来源的间充质干细胞具有向软骨细胞定向分化的能力。
[Abstract]:Because the articular surface is composed of transparent cartilage, the chondrocytes are wrapped around the extracellular matrix, and there is little migration ability and can not gather to the site of trauma. Therefore, the self repair ability of cartilage tissue is very limited. Cartilage defects often cause joint pain and dysfunction or even disability. There is no effective treatment in clinic. Methods, thus promoting the rapid development of cartilage tissue engineering technology. Cartilage tissue engineering involves seed cells, scaffold materials and growth factors, signal molecules and stress and other environmental aspects. Seed cells are important factors affecting tissue engineering. Mesenchymal stem cells derived from fat (adipose-derived mesenchymal) Stem cells (ADMSCs) has attracted much attention due to its strong proliferation and differentiation ability as a source of seed cells for tissue engineering.
Objective:
1 to explore the feasibility of isolating and culturing adipose derived mesenchymal stem cells, and to provide experimental evidence for expanding and optimizing the seed cell source of cartilage tissue engineering.
2 autologous fat derived mesenchymal stem cells and acellular derma matrix (ADM) were combined to repair the cartilage defects of the knee joint in rabbits. The histological and histopathological scores of the repaired tissues were evaluated and the effect of repair was evaluated. It provided a theoretical basis for the clinical application of tissue engineering method to repair articular cartilage defects and laid the experimental basis for the repair of articular cartilage defects. Foundation.
Method:
36 healthy New Zealand rabbits were selected. The shears rabbit adipose tissue was digested and centrifuged with 0.1% type collagenase. After the autologous ADMSCs was obtained, the expression of the ADMSCs surface antigen CD44 was detected by flow cytometry. The cell morphology was observed by the inverted microscope. The cell growth curve was measured by the cell count method, and the cell count method was used to determine the cell morphology.
Under specific conditions in vitro, ADMSCs was induced to differentiate into adipocytes, and osteoblasts and chondrocytes were induced by.ADMSCs to become DMEM, 10% FBS, isobutyl methylxanthine (IBMX), 200 mol/L dexamethasone, 10 micron g/ml insulin, and 1 u mol/L indomethacin in.ADMSCs osteogenesis induction group as DMEM, 10% FBS, 0.1 muon mol/L ground plug. Amethasone, 50 g/mL ascorbic acid, 10mmol/L beta phosphoric acid glycerol.ADMSCs formed into cartilage induced composition including 10% FBS, high sugar DMEM, 10 micron g/L transforming growth factor beta 1,50 u g/ml ascorbic acid, 6.25 mg/L insulin.ADMSCs in lipid induced differentiation with oil red O staining, after osteogenesis induced differentiation with cobalt calcium alkaline phosphatase staining and alizarin red calcium nodule Immunohistochemical staining of type II collagen was carried out after induction of differentiation in chondrogenic direction. Special staining was used to demonstrate the multipotency of ADMSCs.
ADM was prepared by calf dermis as a cell carrier. ADMSCs was cultured to third generations in vitro, and a chondrocyte suspension with a concentration of 5 x 105/ml was prepared. The autologous ADMSCs was cultured with ADM in vitro for 2D and was observed by scanning electron microscope and transplanted into the cartilage defect of the knee joint of rabbit.
The 36 New Zealand rabbits were randomly divided into the ADMSCs /ADM experimental group, the ADM control group and the blank control group. The experimental group was implanted with ADMSCs /ADM in the intercondylar fossa cartilage defect, and the ADM control group was implanted with the acellular dermal matrix alone. The blank control group had no implantation. 4 animals in each group were killed in each group at 6 weeks, 9 and 12 weeks respectively. On the basis of histological and immunohistochemical staining, the repair tissue was scored on the basis of articular cartilage histology score and histopathology score. The data were entered into SPSS 16 software, and the random group SNK-q method was used for statistical analysis to compare the statistical significance of the scores of each group.
Result:
1 after inoculation with 24h, rabbit ADMSCs showed long spindle shape or polygonal adherence. Cell proliferation was rapid and primary culture was about 10 ~ 14d, which could achieve 80-90% fusion.
2 the identification of ADMSCs surface antigen: flow cytometry results showed that CD44 was positive.
3 the growth curve of ADMSCs showed a "S" shape. After adherence to 2 D, the cells began to proliferate, 7 d entered the peak of proliferation, and entered the plateau stage after 9 d.
4 ADMSCs lipid induced adipocytes were stained by oil red staining and red fat droplets were displayed. After osteogenesis, alkaline phosphatase staining showed black precipitation, alizarin red staining showed calcified nodules, and type II collagen immunohistochemical staining was positive after induction of differentiation.
5 under electron microscope, ADM is porous and porous with good porosity. It is suitable for ADMSCs growth.
6, 9 weeks after the operation, ADMSCs /ADM experimental group was better to observe the integration of repair tissue, no scaffold residual.ADM control group and blank control group were fibrous repair and no repair. Histology score of ADMSCs /ADM experimental group was significantly different from that of ADM control group and blank control group (P0.05), ADM control group and blank control group had no significant difference. Sexual significance (P0.05); histopathology score showed that the difference between the ADMSCs /ADM experimental group and the ADM control group and the blank control group was significant (P0.05), and there was no significant difference between the ADM control group and the blank control group (P0.05).
7, 12 weeks after the operation, ADMSCs /ADM experimental group observed the smooth surface of the repaired tissue, the combination with the surrounding cartilage and the.ADM control group and the blank control group were fibrous repair and no repair. The histological score of the ADMSCs /ADM group was significantly different from that of the ADM control group and the blank control group (P0.05), and the difference between the ADM control group and the blank control group was significant. The histopathology score showed that the difference between the ADMSCs /ADM experimental group and the ADM control group and the blank control group was significant (P0.05), and there was a significant difference between the ADM control group and the blank group (P0.05).
8 immunohistochemical staining of type II collagen showed that the chondrocytes in the repair area of ADMSCs /ADM group contained type II collagen, which was well combined with the surrounding cartilage.
Conclusion:
1 rabbit adipose derived mesenchymal stem cells have the characteristics of stable growth and rapid proliferation in vitro, which can differentiate into adipocytes, osteoblasts and chondrocytes under the in vitro culture system, which conforms to the multi differentiation potential of mesenchymal stem cells.
2 acellular dermal matrix material is suitable for the adhesion and proliferation of mesenchymal stem cells derived from rabbit fat source. It is an ideal scaffold for cartilage tissue engineering. The two composite implanted cartilage defect area can repair articular cartilage.
3 histological observation and type II collagen immunohistochemistry confirmed that adipose derived mesenchymal stem cells had the ability to differentiate into chondrocytes.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

【参考文献】