本文選題：骨關(guān)節(jié)炎 + 軟骨單位��； 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:通過Transwell間接共培養(yǎng)技術(shù)研究兔膝關(guān)節(jié)軟骨單位對軟骨細胞生物學(xué)特性的影響,為組織工程技術(shù)修復(fù)損傷軟骨選取良好種子細胞提供依據(jù)。方法:用不同消化酶將2月齡新西蘭兔膝關(guān)節(jié)軟骨分別消化成軟骨細胞和軟骨單位。按2×105個細胞/孔濃度接種于Transwell雙層細胞培養(yǎng)板。實驗組:軟骨單位接種于上室,軟骨細胞接種于下室;對照組:下室接種軟骨細胞,上室未接種。分別在2、4、6、8天提取各組Transwell下室軟骨細胞進行指標檢測。早期凋亡率檢測:Annexin V和7-AAD標記流式細胞儀檢測,TUNEL染色熒光顯微鏡檢測;增殖率檢測:PCNA標記流式細胞儀檢測,Ed U染色熒光顯微鏡檢測;相關(guān)基因相對表達量檢測:PCR技術(shù)檢測AGG、Col-II、MMP-13基因相對表達量。結(jié)果:Annexin V和7-AAD標記流式細胞儀檢測Transwell下室軟骨細胞早期凋亡率發(fā)現(xiàn),在實驗早期(第2、4天)實驗組與對照組Transwell下室軟骨細胞早期凋亡率差異不明顯,差別沒有統(tǒng)計學(xué)意義(p0.05)。在實驗第6天、第8天實驗組軟骨細胞早期凋亡率明顯低于對照組(P0.05)。TUNEL染色熒光顯微鏡檢測Transwell下室軟骨細胞凋亡率發(fā)現(xiàn),實驗組和對照組軟骨細胞凋亡率沒有顯著差異,但有實驗組軟骨細胞凋亡率低于對照組的趨勢。PCNA標記流式細胞儀檢測Transwell下室軟骨細胞增殖率發(fā)現(xiàn),在實驗早期(第2、4天)實驗組與對照組Transwell下室軟骨細胞增值率差異不明顯,差別沒有統(tǒng)計學(xué)意義(p0.05)。在實驗第6天、第8天實驗組軟骨細胞增殖率明顯高于對照組(P0.05)。Ed U染色熒光顯微鏡檢測Transwell下室軟骨細胞增殖率發(fā)現(xiàn),第6天實驗組軟骨細胞增殖率明顯高于對照組(P0.05)。PCR技術(shù)檢測Transwell下室軟骨細胞相應(yīng)基因表達量發(fā)現(xiàn),在實驗早期(第2、4天)實驗組與對照組Transwell下室軟骨細胞AGG、Col-II,MMP-13基因表達較為相似,實驗組與對照組差異不明顯,差別沒有統(tǒng)計學(xué)意義(P0.05)。在實驗后期AGG、Col-II基因相對表達量在第6天、第8天時實驗組明顯高于對照組(P0.05),MMP-13表達量在第8天時對照組明顯高于實驗組(P0.05)。結(jié)論:1.軟骨單位作為關(guān)節(jié)軟骨的基本功能單位,能夠更長時間有效抵抗或延緩軟骨細胞凋亡、維持軟骨細胞增殖及正向調(diào)節(jié)軟骨基質(zhì)相關(guān)物質(zhì)的基因表達。2.軟骨單位有望在組織工程中作為種子細胞用于骨關(guān)節(jié)炎關(guān)節(jié)軟骨缺損的修復(fù)。
[Abstract]:Aim: to study the effect of articular cartilage units on the biological characteristics of chondrocytes in rabbit knee joint by Transwell indirect co-culture technique, and to provide a basis for selecting good seed cells for repairing damaged cartilage by tissue engineering technique. Methods: the articular cartilage of 2 month old New Zealand rabbits was digested with different digestive enzymes to form chondrocytes and chondrocytes respectively. Transwell bilayer cell culture plate was inoculated with 2 脳 105 cells / well concentration. Experimental group: chondrocytes were inoculated in superior chamber and chondrocytes were inoculated in inferior chamber, while in control group, chondrocytes were inoculated in lower chamber without inoculation. The ventricular chondrocytes were extracted from each group of Transwell for 8 days. Early apoptotic rate was detected by flow cytometry and 7-AAD labeled flow cytometry, and proliferation rate was detected by flow cytometry with TUNEL-stained fluorescence microscope, proliferation rate by flow cytometry labeled with 7-AAD and fluorescence microscope with Ed U staining. The relative expression of MMP-13 in AGGN Col-III was detected by PCR. Results the early apoptosis rate of ventricular chondrocytes under Transwell was detected by flow cytometry with 7-AAD and 7% Annexin V. It was found that there was no significant difference in early apoptosis rate between experimental group and control group in the early stage of experiment (2 ~ 4 days), but there was no significant difference between experimental group and control group in early apoptosis rate of ventricular chondrocytes under Transwell (p 0.05). The early apoptotic rate of chondrocytes in the experimental group was significantly lower than that in the control group after 6 days and 8 days. The apoptosis rate of chondrocytes in the experimental group was not significantly different from that in the control group. However, the apoptosis rate of chondrocytes in experimental group was lower than that in control group. The proliferative rate of chondrocytes under Transwell was detected by flow cytometry. It was found that there was no significant difference in the proliferation rate of chondrocytes between experimental group and control group under Transwell at the early stage of experiment (2 ~ 4 days). The difference was not statistically significant (P 0.05). On the 6th and 8th day, the proliferation rate of chondrocytes in the experimental group was significantly higher than that in the control group. On the 6th day, the proliferation rate of chondrocytes in the experimental group was significantly higher than that in the control group (P 0.05). The expression of the corresponding genes in the chondrocytes under Transwell was similar to that in the experimental group and the control group at the early stage of the experiment (on the 2th day), and the expression of AGG- Col-IIMP-13 in the chondrocytes of the experimental group was similar to that of the control group. There was no significant difference between the experimental group and the control group (P 0.05). At the end of the experiment, the relative expression of AGGG Col-II gene was significantly higher in the experimental group than that in the control group on the 6th day and the 8th day. The expression of MMP-13 in the experimental group was significantly higher than that in the control group on the 8th day. The expression of MMP-13 in the experimental group was significantly higher than that in the control group on the 8th day. Conclusion 1. As the basic functional unit of articular cartilage, chondrocytes can effectively resist or delay the apoptosis of chondrocytes for a longer time, maintain the proliferation of chondrocytes and positively regulate the gene expression of cartilage matrix related substances. Cartilage units are expected to be used as seed cells in tissue engineering for the repair of osteoarthritis articular cartilage defects.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R687

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