ZVAD-fmk、L-NAME对体外组织培养法保存异体软骨组织活性的影响

发布时间：2018-06-12 21:36

本文选题：关节软骨 + 异体移植　；参考：《泰山医学院》2013年硕士论文

【摘要】：目的在体外组织培养液中加入ZVAD-fmk、L-NAME用于保存同种异体骨软骨组织一定时间，观察其是否具有提高软骨细胞成活率、保持软骨组织活性的作用，进而探索具有提高软骨组织保存效果的培养液成分。材料与方法以羊膝关节骨软骨为研究标本，无菌条件下，用自制取软骨器获取同样大小的骨软骨块270枚，包括软骨下骨全长约5mm，直径约为4.5mm，PBS液冲洗3遍，随机分配到A组、B组、C组三个不同培养液成分的无菌培养瓶中进行保存，分别为A组(对照组）：基础保存液，B组：基础保存液中添加50uMol/L的ZVAD-fmk，C组：基础保存液中添加15mg/L的L-NAME。基础培养液成份为99%EMEM培养液和1%的青链霉素（各100UI/ml）。在4℃、每隔两天换液的条件下，分别于保存第1、21、35天每瓶取出30块进行检测，包括EB/FDA双荧光法测软骨细胞存活率，Annexin V-FITC与PI双染色法测定软骨细胞凋亡率（流式细胞仪定量检测），软骨组织番红-O染色。结果第1天时,三组细胞成活率均保持在90%以上，各组之间差异无统计学意义（P＞0.05）。三组细胞凋亡率凋亡率均在5%左右，差异不具有统计学意义（P＞0.05）。软骨基质番红-O染色IOD值均在190左右，三组之间比较差异无统计学意义（P＞0.05）。第21天时,三组软骨细胞成活率均出现下降，其中A组(基础保存液)下降明显，C组（基础保存液中+L-NAME）次之，B组（基础保存液+ZVAD-fmk）仍能保持在85%，三组间差异有统计学意义(P0.05)。各组细胞凋亡率均有上升，以其中C组最为明显，三组之间比较差异有统计学意义(P0.05)。各组软骨IOD值均有下降，B组较高，C组次之，，A组最低。各组之间差异比较具有统计学意义（P0.05）。第35天时，各组成活率均出现下降，其中A组(基础保存液)、C组（基础保存液中+L-NAME）下降明显，已经降至50%以下。B组（基础保存液+ZVAD-fmk）仍能保持在70%以上，且三组间差异比较有统计学意义(P0.05)。各组凋亡率均出有上升，其中A组(基础保存液)、C组（基础保存液中+L-NAME）上升明显，已经升至70%。B组（基础保存液+ZVAD-fmk）仍能保持在25%左右，且各组间差异有统计学意义(P0.05)。三组软骨IOD值均继续下降，其中B组软骨IOD值仍能保持在60以上，而其他两组则下降明显，三组间差异比较有统计学意义(P0.05)。结论在基础保存液中添加50uMol/L的ZVAD-fmk，能够有效提高体外液体保存的异体软骨组织活性以及软骨细胞存活率；在基础保存液中添加15mg/L的L-NAME，没有提高体外保存的异体软骨组织活性以及软骨细胞存活率。意义关节软骨全层缺损的修复方法与效果不佳一直是困扰临床运动医学医生与骨科医生的一大难题。虽然，国外临床上异体骨软新鲜骨移植治疗全层软骨损伤取得了肯定的效果，其优点为取材相对容易、无需二次手术等，但由于没有理想的体外液体保存软骨移植物的方法，影响了临床应用与推广。因此，延长同种异体软骨移植物的体外保存时间并保持其活性成为学者研究的焦点。本课题研究发现组织体外培养液中加入ZVAD-fmk具有保持软骨细胞成活率以及组织活性的作用，为下一步研究理想的组织培养保存液奠定了基础。
[Abstract]:objective
ZVAD-fmk was added to the tissue culture medium in vitro, and L-NAME was used to preserve the allogenic osteochondral tissue for a certain time, to observe whether it could improve the survival rate of cartilage cells and maintain the activity of cartilage tissue, and then explore the differentiation of culture fluid that could improve the preservation of cartilage tissue.
Materials and methods
Under the aseptic condition, 270 bone cartilage blocks of the same size were obtained with the homemade cartilage apparatus, including the total length of 5mm, the diameter of about 4.5mm, and 3 times of PBS solution, which were randomly assigned to the A, B and C group of three sterile culture bottles, A group (control group), respectively. Base preservation solution, group B: ZVAD-fmk of 50uMol/L in base preservation solution, group C: L-NAME. base medium containing 15mg/L in basic preservation solution is 99%EMEM culture solution and 1% penicillin (100UI/ml). At 4 C, every two days of liquid exchange, 30 blocks per bottle are detected, including EB/FD, respectively. The survival rate of chondrocytes was measured by A double fluorescence method. The apoptosis rate of chondrocytes was measured by Annexin V-FITC and PI double staining method (quantitative detection of flow cytometer), and the cartilaginous tissue red -O staining.
Result
At first days, the survival rate of the three groups remained above 90%. There was no significant difference between the groups (P > 0.05). The apoptotic rate of the three groups was around 5%, and the difference was not statistically significant (P > 0.05). The IOD value of the cartilage matrix red -O staining was about 190, there was no statistical difference between the three groups (P > 0.05). The difference between the three groups was not statistically significant. The survival rate of chondrocytes in the three groups decreased, in which group A (base preservation solution) decreased obviously, group C (+L-NAME in basic preservation solution), and group B (basic preservation solution +ZVAD-fmk) remained at 85%, and the difference between the three groups was statistically significant (P0.05). The percentage of cell withering in each group was increased, in which the C group was the most obvious, there was a difference between the three groups. Statistical significance (P0.05). The IOD value of cartilage in each group decreased, B group was higher, C group was the second, group A was the lowest. The difference of each group was statistically significant (P0.05). In the thirty-fifth day, the survival rate of each group decreased, and the A group (Basic preservation solution), C group (the +L-NAME in the basic preservation solution) decreased obviously, and had dropped to the.B group below (base preservation liquid +Z). VAD-fmk) still remained above 70%, and the difference between the three groups was statistically significant (P0.05). The apoptotic rate of each group increased, in which group A (basal preservative) and group C (+L-NAME in the basal preservative) increased obviously, and had been promoted to group 70%.B (basal preservative +ZVAD-fmk) still maintained at about 25%, and there was a significant difference between each group (P0.05). The IOD values of cartilage in the three groups continued to decrease, and the IOD value of cartilage in group B remained above 60, while the other two groups decreased significantly, and the difference between the three groups was statistically significant (P0.05).
conclusion
The addition of 50uMol/L ZVAD-fmk in the basal preservative can effectively improve the tissue activity of cartilage allograft and the survival rate of cartilage cells in vitro. The addition of 15mg/L L-NAME in the base preservative does not improve the activity of cartilage allograft and the survival rate of chondrocytes in vitro.
Significance
The repair method and poor effect of total articular cartilage defect has been a difficult problem in clinical sports medical doctors and Department of orthopedics doctors. Although foreign clinical bone graft and soft fresh bone graft has achieved positive results in the treatment of full-thickness cartilage injury, its advantage is that it is relatively easy to take material, without two operations, but because there is no ideal. The method of preservation of cartilage graft by liquid in vitro affects the clinical application and popularization. Therefore, it is the focus of scholars to prolong the preservation time of allograft cartilage graft in vitro and maintain its activity. It is found that the survival rate and the tissue activity of the cartilage cells with ZVAD-fmk are added to the tissue culture medium in vitro. It will lay a foundation for further study of ideal tissue culture and preservation solution.
【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R873

【参考文献】