本文選題：骨髓基質(zhì)干細(xì)胞 + 軟骨細(xì)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(duì)骨髓基質(zhì)干細(xì)胞轉(zhuǎn)化較少,本實(shí)驗(yàn)探討茶黃素和轉(zhuǎn)化生長(zhǎng)因子β1(TGF-β1)對(duì)體外培養(yǎng)兔骨髓基質(zhì)干細(xì)胞(BMSCs)增殖及向軟骨細(xì)胞誘導(dǎo)分化的影響。 方法:實(shí)驗(yàn)于2008-05/8在安徽醫(yī)科大學(xué)附屬省立醫(yī)院中心實(shí)驗(yàn)室完成。①實(shí)驗(yàn)動(dòng)物:清潔級(jí)8周齡家兔12只,由安徽醫(yī)科大學(xué)動(dòng)物試驗(yàn)中心提供,實(shí)驗(yàn)過(guò)程中對(duì)動(dòng)物的處置符合動(dòng)物倫理學(xué)標(biāo)準(zhǔn)。②實(shí)驗(yàn)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(xì)胞,按5×10~4/L的細(xì)胞密度接種,當(dāng)細(xì)胞生長(zhǎng)至80%～90%融合時(shí)消化傳代。取傳至第三代的細(xì)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(duì)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(shí)驗(yàn)評(píng)估:倒置顯微鏡下觀察細(xì)胞生長(zhǎng)狀況,甲苯胺藍(lán)染色、阿利新藍(lán)比色法測(cè)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(xì)胞生長(zhǎng)曲線:骨髓基質(zhì)干細(xì)胞具有旺盛的增殖能力,培養(yǎng)1d為細(xì)胞的適應(yīng)期,3d為對(duì)數(shù)增長(zhǎng)期,8d時(shí)進(jìn)入平臺(tái)期,之后細(xì)胞增殖迅速減慢,細(xì)胞數(shù)下降。②觀察細(xì)胞生長(zhǎng):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(xì)胞生長(zhǎng)明顯減緩。與對(duì)照組相比,經(jīng)過(guò)誘導(dǎo)2周后,誘導(dǎo)組BMSCs均與對(duì)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(lán)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(xì)胞甲苯胺藍(lán)異染明顯,Ⅱ型膠原化學(xué)染色陽(yáng)性,表現(xiàn)為軟骨細(xì)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(jìn)BMSCs在體外向軟骨細(xì)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，

本文編號(hào)：2087164