發(fā)布時(shí)間：2018-01-09 03:08

  本文關(guān)鍵詞：茶黃素與TGF-β3對(duì)兔骨髓間充質(zhì)干細(xì)胞向軟骨細(xì)胞增殖和誘導(dǎo)分化地影響　出處：《安徽醫(yī)科大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 軟骨細(xì)胞 骨髓間充質(zhì)干細(xì)胞 茶黃素 轉(zhuǎn)化生長(zhǎng)因子β3

【摘要】：目的:軟骨損傷在臨床上非常多見,但修復(fù)非常困難,組織工程的發(fā)展使軟骨修復(fù)的研究得到長(zhǎng)足的發(fā)展,間充質(zhì)干細(xì)胞被認(rèn)為是一種理想的種子細(xì)胞。轉(zhuǎn)化生長(zhǎng)因子β3可以誘導(dǎo)軟骨的代謝和增殖,而茶黃素也有促進(jìn)軟骨代謝和增殖的作用,本實(shí)驗(yàn)探討在茶黃素和轉(zhuǎn)化生長(zhǎng)因子β3的聯(lián)合作用下對(duì)骨髓間充質(zhì)干細(xì)胞向軟骨細(xì)胞誘導(dǎo)分化和增殖的影響。 方法:本實(shí)驗(yàn)于2009-09/12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。⑴實(shí)驗(yàn)動(dòng)物:健康6~7周齡清潔級(jí)新西蘭大白兔15只,雌雄不拘,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)家兔處置符合動(dòng)物倫理學(xué)標(biāo)準(zhǔn)。⑵實(shí)驗(yàn)方法:全身肝素麻醉狀態(tài)下處死動(dòng)物,取雙側(cè)肱骨和股骨,剔去附麗軟組織,切除兩側(cè)骺端(包括骺板),全骨髓法分離及培養(yǎng)骨髓間充質(zhì)干細(xì)胞,按6×10~4 /L的細(xì)胞密度接種,當(dāng)細(xì)胞生長(zhǎng)至90%左右融合時(shí)消化傳代。取第3代細(xì)胞按1×10~4 /ml地密度接種,置入含地塞米松10~(-8)mmol/L、轉(zhuǎn)化生長(zhǎng)因子β3 10μg/L和重組人胰島素15mg/L的DMBM成軟骨誘導(dǎo)液培養(yǎng)二周,取第三代骨髓間充質(zhì)干細(xì)胞分為3組:對(duì)照組:完全培養(yǎng)基加地塞米松10~(-8) mmol/L、維生素C 10mmol/L。轉(zhuǎn)化生長(zhǎng)因子組:完全培養(yǎng)基加地塞米松10~(-8) mmol/L、維生素C 10mmol/L、轉(zhuǎn)化生長(zhǎng)因子β3 10μg/L。茶黃素組:完全培養(yǎng)基加地塞米松10~(-8) mmol/L、維生素C 10mmol/L、轉(zhuǎn)化生長(zhǎng)因子β3 10ng/mL、茶黃素30mg/L。⑶評(píng)估:在倒置顯微鏡下觀察細(xì)胞的生長(zhǎng)狀況和形態(tài)變化,用甲苯胺藍(lán)染色和阿利新藍(lán)比色法測(cè)定各板每孔中葡萄糖氨基聚糖含量,免疫檢測(cè)儀測(cè)定3組的吸光度值鑒別II型膠原。 結(jié)果:骨髓基質(zhì)干細(xì)胞增值能力旺盛,培養(yǎng)24小時(shí)為細(xì)胞適應(yīng)期,72小時(shí)為對(duì)數(shù)增長(zhǎng)期,一周后進(jìn)入平臺(tái)期,以后速度減慢,細(xì)胞數(shù)減少;骨髓中分離獲得地骨髓間充質(zhì)干細(xì)胞在體外增殖旺盛,細(xì)胞經(jīng)茶黃素和轉(zhuǎn)化生長(zhǎng)因子β3誘導(dǎo)后生長(zhǎng)明顯減緩。加入誘導(dǎo)液誘導(dǎo)二周后,誘導(dǎo)組與對(duì)照組均有顯著的差異性(P0.05),誘導(dǎo)組之間同樣具有差異性(P0.05)。經(jīng)甲苯胺藍(lán)染色和免疫組化測(cè)定結(jié)果,經(jīng)誘導(dǎo)后透明軟骨特征性標(biāo)志II型膠原化學(xué)染色陽(yáng)性,甲苯胺藍(lán)異染明顯。 結(jié)論:茶黃素與轉(zhuǎn)化生長(zhǎng)因子β3聯(lián)合作用能有效促進(jìn)骨髓間充質(zhì)干細(xì)胞在體外向軟骨細(xì)胞分化。
[Abstract]:Objective: cartilage injury is very common in clinic, but it is very difficult to repair. The development of tissue engineering has made great progress in the research of cartilage repair. Mesenchymal stem cells are considered to be ideal seed cells. Transforming growth factor 尾 3 can induce the metabolism and proliferation of cartilage, while theaflavin can promote the metabolism and proliferation of cartilage. The effects of theaflavine and transforming growth factor 尾 3 on the differentiation and proliferation of bone marrow mesenchymal stem cells into chondrocytes were investigated. Methods: this experiment was completed in the Central Laboratory of Provincial Hospital affiliated to Anhui Medical University on 2009-09 / 12. 1 Experimental Animals: 15 clean New Zealand white rabbits of 6 ~ 7 weeks of age, male and female. The animal test center of Anhui Medical University provided 2 experimental methods for the treatment of rabbits in accordance with animal ethics standard .2: the animals were killed under general heparin anesthesia and bilateral humerus and femur were taken. The bone marrow mesenchymal stem cells were isolated and cultured by removing the soft tissue of epiphysis (including epiphyseal plate) and inoculating with 6 脳 10 ~ (4 / L) / L of cell density. The third passage cells were inoculated at 1 脳 10 ~ (4) / ml density and implanted with dexamethasone 10 ~ 10 ~ (-1) -8 mmol / L. Transforming growth factor 尾 3 10 渭 g / L and recombinant human insulin 15 mg / L DMBM chondrocytes were cultured for 2 weeks. The third generation of bone marrow mesenchymal stem cells were divided into three groups: control group: complete culture medium plus dexamethasone 10 + -8) mmol/L. Vitamin C 10 mmol / L. Transforming growth factor group: complete medium plus dexamethasone 10% -8) mmol / L, vitamin C 10 mmol / L. Transforming growth factor 尾 3 10 渭 g / L. Theafin group: complete medium supplemented with dexamethasone 10% -8) mmol / L, vitamin C 10 mmol / L. Transforming growth factor 尾 3 10 ng / mL, theaflavin 30 mg / L.3 evaluation: cell growth and morphological changes were observed under inverted microscope. The content of glucosaminoglycan in each hole of each plate was determined by toluidine blue staining and alimine blue colorimetry. The absorbance of type II collagen was identified by immunoassay. Results: the proliferation of bone marrow stromal cells (BMSCs) was exuberant. The cells were cultured for 24 hours in adaptation phase and 72 hours in logarithmic growth period. One week later they entered the plateau phase and then the speed slowed down and the number of cells decreased. The proliferation of mesenchymal stem cells isolated from bone marrow was strong in vitro, and the growth of cells induced by theaflavin and transforming growth factor 尾 3 was slowed down obviously. There was significant difference between the induction group and the control group (P 0.05), and there was also difference between the induction group and the control group (P 0.05). The results were determined by toluidine blue staining and immunohistochemistry. The characteristic marker of hyaline cartilage, type II collagen, was positive after induction, and toluidine blue was positive. Conclusion: the combination of theaflavins and transforming growth factor 尾 3 can effectively promote the differentiation of bone marrow mesenchymal stem cells into chondrocytes in vitro.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R329

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本文編號(hào)：1399739