本文選題：軟骨細胞　切入點：水凝膠　出處：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：研究目的構(gòu)建微孔水凝膠,令小鼠胚胎瘤細胞(ATDC5)與原代豬軟骨細胞在微孔水凝膠中直接共培養(yǎng),探索該共培養(yǎng)體系中最適合的細胞環(huán)境,以達到ATDC5最佳的軟骨誘導效果,從而獲得更符合人體的透明軟骨特性的組織工程軟骨,為臨床軟骨修復提供一種理想的方法。方法:一、共培養(yǎng)的細胞采集以及培養(yǎng)共培養(yǎng)所需的原代豬軟骨細胞從鮮豬膝關(guān)節(jié)軟骨中提取,并使用軟骨培養(yǎng)液于175cm2培養(yǎng)瓶進行培養(yǎng)。ATDC5從美國圣地亞哥公司(San Diego,USA)購買,用ATDC5培養(yǎng)液于175cm2培養(yǎng)瓶進行培養(yǎng)。二、明膠微球的制備及微孔水凝膠豬軟骨細胞培養(yǎng)的研究所需的80-120微米大小的明膠微球通過雙乳液法進行制備,然后用羅丹明染色液標記后包埋在水凝膠,觀察明膠微球降解情況。再用此方法獲得的微球制備微孔海藻酸鈉水凝膠培養(yǎng)原代軟骨細胞,通過熒光顯微鏡觀察活/死細胞檢測結(jié)果,并使用CCK8方法測定細胞活力值。三、微孔水凝膠共培養(yǎng)體系的構(gòu)建以1:3、1:1、3:1的軟骨細胞與ATDC5細胞比例(分別為1C3A、AC、3C1A組)混合,對照組分別為單純ATDC5組(A組)和軟骨細胞組(C組)。該5組細胞分別混合到含有適當比例的明膠微球的海藻酸鈉溶液后,制作成細胞/微孔水凝膠復合物,并用軟骨誘導液進行培養(yǎng)。四、共培養(yǎng)細胞的活力以及軟骨特征性表達的檢測整個培養(yǎng)周期中,在對應的時間點收集相應的樣本,通過細胞增殖活力(Cell Counting Kit-8,CCK-8)、軟骨核酸的表達(Quantitative real-time PCR,qRt-PCR)、組織學染色、免疫組織化學染色(Immunohistochemistry,IHC)和細胞代謝相關(guān)蛋白表達(Western blot,WB)的檢測結(jié)果,分析共培養(yǎng)細胞軟骨特征性基因或蛋白的表達水平。結(jié)果:一、成功從新鮮豬軟骨中提取原代軟骨細胞和將P7ATDC5復蘇。成功在體外培養(yǎng)增殖并了解其生長形態(tài)、生長活力和繪制其細胞活力曲線。二、通過不同的攪拌速度,獲得不同直徑的明膠微球。實驗表明,攪拌速度越快,產(chǎn)生的微球粒徑越小。在顯微鏡下觀察到,明膠微球在37℃的培養(yǎng)環(huán)境下能夠自然降解,水凝膠內(nèi)部產(chǎn)生微孔�；�/死細胞染色結(jié)果觀察到原代軟骨細胞能夠在材料中均勻地分布和生長,在微孔邊緣生長的軟骨細胞甚至能夠突破邊緣,將空腔位置占據(jù)生長。細胞活力檢測數(shù)據(jù)表明微孔水凝膠組中的軟骨細胞的增殖能力比對照組中的細胞顯著提高。三、成功構(gòu)建出原代軟骨細胞和ATDC5細胞的微孔水凝膠共培養(yǎng)體系。從共培養(yǎng)細胞的細胞活力,軟骨特異性核酸表達,軟骨相關(guān)蛋白表達以及組織化學染色方面綜合來看,在微孔水凝膠共培養(yǎng)體系中,軟骨細胞和ATDC5以1:3的比例最為理想,并且發(fā)現(xiàn)絲裂原活化蛋白激酶(the mitogen-activated protein kinase,MAPK)中P44/42與該共培養(yǎng)體系中的細胞增殖相關(guān)。結(jié)論:該微孔水凝膠共培養(yǎng)體系能夠為ATDC5提供理想的定向軟骨分化環(huán)境,特別在軟骨與ATDC5以1:3的比例下,ATDC5的定向軟骨分化效果最理想。本研究為軟骨組織工程的進一步應用提供扎實的實驗基礎,為日后臨床治療軟骨損傷提供理想的對策。
[Abstract]:Study objective to construct porous hydrogel, the mouse embryonic cells (ATDC5) and primary porcine chondrocytes in microporous hydrogel in direct co culture, exploring the most suitable environment for cell co culture system, in order to achieve the best effect of ATDC5 induced cartilage, resulting in cartilage tissue engineering with hyaline cartilage properties of the human body, provide an ideal method for clinical cartilage repair. Methods: a cell co culture and co culture collection of primary cultured porcine cartilage cells to extract from fresh porcine articular cartilage, cartilage and use medium in 175cm2 flask and cultured.ATDC5 from the Santiago company (San Diego USA) to buy. Using ATDC5 medium in 175cm2 flask were cultured. Two, gelatin microspheres 80-120 micron size of gelatin microspheres and porous hydrogel cartilage cell culture required by double emulsion method The preparation, then use the Luo Danming dye labeled entrapped in the hydrogel, observe the gelatin microspheres degradation of microspheres obtained by this method. The preparation of microporous sodium alginate hydrogel in primary cultured chondrocytes, live / dead cell results detected by fluorescence microscopy, and the determination of cell viability by CCK8 method. Three, system to construct cartilage cells and ATDC5 cell ratio of 1:3,1:1,3:1 co cultured with microporous hydrogel (1C3A, AC, 3C1A group) mixed control group respectively ATDC5 alone group (A group) and chondrocyte group (C group). The 5 groups of cells were mixed with sodium alginate solution gelatin microspheres containing the appropriate proportion of the, made into cell / porous hydrogel composite, and were cultured in chondrogenic fluid. Four, co cultured cell viability and expression of cartilage characteristic throughout the culture period, at the corresponding time points from the corresponding The sample, through the cell proliferation (Cell Counting, Kit-8, CCK-8), the expression of cartilage (Quantitative real-time nucleic acid PCR, qRt-PCR), histological staining, immunohistochemical staining (Immunohistochemistry, IHC) expression and cell metabolism related protein (Western, blot, WB) of the test results, the expression level of cell specific genes or cartilage protein co culture analysis. Results: a successful, extracted from fresh pig cartilage chondrocytes and P7ATDC5 recovery. Success in vitro proliferation and to understand the growth morphology, growth vigor and the cell viability curve. Two, through different stirring speeds, obtain gelatin microspheres with different diameters. Experiments show that the stirring speed faster, the particle size is smaller. Observed under the microscope, gelatin microspheres can be naturally degraded in the environment under 37 DEG C, hydrogel generated inside the hole. Live / dead cell staining The observed primary chondrocytes can be distributed uniformly and the growth in the material, the chondrocytes in microporous edge growth can even break edge, will occupy the cavity position growth. Cell viability test data showed that the chondrocytes in microporous hydrogel group proliferation significantly increased than in the control group. Cell three, successfully constructed the porous hydrogel primary cartilage cells and ATDC5 cells in co culture system. The co cultured cell viability, expression of cartilage specific expression of cartilage associated protein and nucleic acid staining comprehensive to see, in the microporous hydrogel coculture system, cartilage cells and ATDC5 with the ratio of 1:3 is the most ideal, and found mitogen activated protein kinase (the mitogen-activated protein kinase, MAPK) in P44/42 associated with the co culture system in cell proliferation. Conclusion: the micro pore water gel Co cultured cartilage differentiation system can provide ideal environment for ATDC5, especially in cartilage and ATDC5 with the ratio of 1:3, the most ideal cartilage differentiation effect of ATDC5. This study provides a solid foundation for further experimental application of cartilage tissue engineering, countermeasures provide an ideal clinical treatment of cartilage injury after.

【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R68

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相關(guān)期刊論文 前1條

1 周峰;王英振;張海寧;呂成昱;續(xù)宗耀;;人軟骨細胞培養(yǎng)上清誘導臍血間充質(zhì)干細胞向軟骨細胞分化[J];中國組織工程研究;2013年40期

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本文編號：1672953