本文選題：關(guān)節(jié)軟骨 + 異體移植��； 參考：《泰山醫(yī)學(xué)院》2013年碩士論文

【摘要】：目的 在體外組織培養(yǎng)液中加入ZVAD-fmk、L-NAME用于保存同種異體骨軟骨組織一定時間，觀察其是否具有提高軟骨細(xì)胞成活率、保持軟骨組織活性的作用，進(jìn)而探索具有提高軟骨組織保存效果的培養(yǎng)液成分。 材料與方法 以羊膝關(guān)節(jié)骨軟骨為研究標(biāo)本，無菌條件下，用自制取軟骨器獲取同樣大小的骨軟骨塊270枚，包括軟骨下骨全長約5mm，直徑約為4.5mm，PBS液沖洗3遍，隨機(jī)分配到A組、B組、C組三個不同培養(yǎng)液成分的無菌培養(yǎng)瓶中進(jìn)行保存，分別為A組(對照組）：基礎(chǔ)保存液，B組：基礎(chǔ)保存液中添加50uMol/L的ZVAD-fmk，C組：基礎(chǔ)保存液中添加15mg/L的L-NAME�；A(chǔ)培養(yǎng)液成份為99%EMEM培養(yǎng)液和1%的青鏈霉素（各100UI/ml）。在4℃、每隔兩天換液的條件下，分別于保存第1、21、35天每瓶取出30塊進(jìn)行檢測，包括EB/FDA雙熒光法測軟骨細(xì)胞存活率，Annexin V-FITC與PI雙染色法測定軟骨細(xì)胞凋亡率（流式細(xì)胞儀定量檢測），軟骨組織番紅-O染色。 結(jié)果 第1天時,三組細(xì)胞成活率均保持在90%以上，各組之間差異無統(tǒng)計學(xué)意義（P＞0.05）。三組細(xì)胞凋亡率凋亡率均在5%左右，差異不具有統(tǒng)計學(xué)意義（P＞0.05）。軟骨基質(zhì)番紅-O染色I(xiàn)OD值均在190左右，三組之間比較差異無統(tǒng)計學(xué)意義（P＞0.05）。第21天時,三組軟骨細(xì)胞成活率均出現(xiàn)下降，其中A組(基礎(chǔ)保存液)下降明顯，C組（基礎(chǔ)保存液中+L-NAME）次之，B組（基礎(chǔ)保存液+ZVAD-fmk）仍能保持在85%，三組間差異有統(tǒng)計學(xué)意義(P0.05)。各組細(xì)胞凋亡率均有上升，以其中C組最為明顯，三組之間比較差異有統(tǒng)計學(xué)意義(P0.05)。各組軟骨IOD值均有下降，B組較高，C組次之，，A組最低。各組之間差異比較具有統(tǒng)計學(xué)意義（P0.05）。第35天時，各組成活率均出現(xiàn)下降，其中A組(基礎(chǔ)保存液)、C組（基礎(chǔ)保存液中+L-NAME）下降明顯，已經(jīng)降至50%以下。B組（基礎(chǔ)保存液+ZVAD-fmk）仍能保持在70%以上，且三組間差異比較有統(tǒng)計學(xué)意義(P0.05)。各組凋亡率均出有上升，其中A組(基礎(chǔ)保存液)、C組（基礎(chǔ)保存液中+L-NAME）上升明顯，已經(jīng)升至70%。B組（基礎(chǔ)保存液+ZVAD-fmk）仍能保持在25%左右，且各組間差異有統(tǒng)計學(xué)意義(P0.05)。三組軟骨IOD值均繼續(xù)下降，其中B組軟骨IOD值仍能保持在60以上，而其他兩組則下降明顯，三組間差異比較有統(tǒng)計學(xué)意義(P0.05)。 結(jié)論 在基礎(chǔ)保存液中添加50uMol/L的ZVAD-fmk，能夠有效提高體外液體保存的異體軟骨組織活性以及軟骨細(xì)胞存活率；在基礎(chǔ)保存液中添加15mg/L的L-NAME，沒有提高體外保存的異體軟骨組織活性以及軟骨細(xì)胞存活率。 意義 關(guān)節(jié)軟骨全層缺損的修復(fù)方法與效果不佳一直是困擾臨床運動醫(yī)學(xué)醫(yī)生與骨科醫(yī)生的一大難題。雖然，國外臨床上異體骨軟新鮮骨移植治療全層軟骨損傷取得了肯定的效果，其優(yōu)點為取材相對容易、無需二次手術(shù)等，但由于沒有理想的體外液體保存軟骨移植物的方法，影響了臨床應(yīng)用與推廣。因此，延長同種異體軟骨移植物的體外保存時間并保持其活性成為學(xué)者研究的焦點。本課題研究發(fā)現(xiàn)組織體外培養(yǎng)液中加入ZVAD-fmk具有保持軟骨細(xì)胞成活率以及組織活性的作用，為下一步研究理想的組織培養(yǎng)保存液奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：泰山醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R873

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