本文選題：骨髓基質(zhì)干細胞 + 軟骨細胞�。� 參考：《安徽醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 目的:茶黃素是紅茶中的主要成分,具有調(diào)節(jié)血脂、抗氧化、抗腫瘤、抗心腦血管疾病等多種藥理活性。以往研究茶黃素對骨髓基質(zhì)干細胞轉(zhuǎn)化較少,本實驗探討茶黃素和轉(zhuǎn)化生長因子β1(TGF-β1)對體外培養(yǎng)兔骨髓基質(zhì)干細胞(BMSCs)增殖及向軟骨細胞誘導(dǎo)分化的影響。 方法:實驗于2008-05/8在安徽醫(yī)科大學(xué)附屬省立醫(yī)院中心實驗室完成。①實驗動物:清潔級8周齡家兔12只,由安徽醫(yī)科大學(xué)動物試驗中心提供,實驗過程中對動物的處置符合動物倫理學(xué)標(biāo)準(zhǔn)。②實驗方法:兔全身肝素化后麻醉狀態(tài)下處死,取雙側(cè)股骨和肱骨,去除軟組織,切除包括骺板在內(nèi)的兩側(cè)骺端,采用全骨髓法分離培養(yǎng)骨髓基質(zhì)干細胞,按5×10~4/L的細胞密度接種,當(dāng)細胞生長至80%～90%融合時消化傳代。取傳至第三代的細胞按1×10~4/ml的密度接種,加入含重組人胰島素10mg/L、地塞米松10~(-8)mmol/L、TGF-β_15ng/L的DMBM成軟骨誘導(dǎo)液培養(yǎng)2周。取第3代BMSC分為三組:A對照組,B組:完全培養(yǎng)基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、維生素C(10mmol/L)。C組:完全培養(yǎng)基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、維生素C(10mmol/L)、茶黃素(30 mg/L)。③實驗評估:倒置顯微鏡下觀察細胞生長狀況,甲苯胺藍染色、阿利新藍比色法測定各板每孔中GAG含量、以及Ⅱ型膠原免疫組化鑒別。 結(jié)果:①細胞生長曲線:骨髓基質(zhì)干細胞具有旺盛的增殖能力,培養(yǎng)1d為細胞的適應(yīng)期,3d為對數(shù)增長期,8d時進入平臺期,之后細胞增殖迅速減慢,細胞數(shù)下降。②觀察細胞生長:骨髓中分離獲得的BMSCs在體外增殖旺盛,TGF-β1和茶黃素誘導(dǎo)后細胞生長明顯減緩。與對照組相比,經(jīng)過誘導(dǎo)2周后,誘導(dǎo)組BMSCs均與對照組有顯著的差異性(P＜0.05),誘導(dǎo)組之間也存在差異性(P＜0.05)。③甲苯胺藍染色和免疫組化鑒定結(jié)果:經(jīng)TGF-β1和茶黃素誘導(dǎo)2周后,細胞甲苯胺藍異染明顯,Ⅱ型膠原化學(xué)染色陽性,表現(xiàn)為軟骨細胞生物學(xué)特性。 結(jié)論:茶黃素(30 mg/L)在TGF-β1存在的條件下能有效促進BMSCs在體外向軟骨細胞分化。
[Abstract]:Objective: theaflavin is the main component of black tea. It has many pharmacological activities, such as regulating blood lipid, anti-oxidation, anti-tumor, anti-cardio-cerebrovascular disease and so on. The effect of theaflavine and transforming growth factor 尾 1 (TGF- 尾 1) on the proliferation and differentiation of rabbit bone marrow stromal cells (BMSCs) into chondrocytes in vitro was studied. Methods: the experiment was completed in the Central Laboratory of Provincial Hospital affiliated to Anhui Medical University in 2008-05 / 8. 1 Experimental animals: 12 rabbits of 8 weeks old of clean grade, provided by Animal Test Center of Anhui Medical University. In the course of the experiment, the disposal of animals was in accordance with the standard of animal ethics .2 Experimental methods: rabbits were killed under anesthesia after heparinization, bilateral femur and humerus were removed, soft tissue was removed, epiphysis including epiphyseal plate was removed. Bone marrow stromal cells were isolated and cultured by whole bone marrow method and inoculated with 5 脳 10 ~ (4) / L cell density. When the cells grew to 80% fusion, they were digested and subcultured. The third passage cells were inoculated at the density of 1 脳 10~4/ml, then added 10 mg / L of recombinant human insulin, 10 ~ (-8) mmol / L dexamethasone, TGF- 尾 to form cartilage inducer of 15 ng / L DMBM for 2 weeks. The third generation BMSC was divided into three groups: complete medium plus dexamethasone (10 ~ (-8) mmol / L) TGF- 尾 1 (5 ng / L), vitamin C (10 mmol / L) .C group: complete medium plus dexamethasone (10 ~ (-8) mmol / L) TGF- 尾 1 (5 ng / L), vitamin C (10 mmol / L), tea (30 mg / L) .3. Toluidine blue staining and Alisin blue colorimetry were used to determine the gag content in each hole of each plate, and the type 鈪，

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