本文關(guān)鍵詞： 骨髓間充質(zhì)干細胞 細胞擴增 組織工程　出處：《廣西醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的:通過研究兔骨髓間充質(zhì)干細胞體外擴增培養(yǎng)的各種條件及比較各種影響因素的差異性,探索一種簡單易行、細胞擴增效率高的方法,為干細胞應(yīng)用于創(chuàng)傷修復(fù)及組織器官再造提供必要的前提幫助。 方法:1.從新西蘭白兔(月齡＜3月)股骨轉(zhuǎn)子間區(qū)嚴格無菌條件下負壓抽取骨髓穿刺液5-10ml,經(jīng)密度梯度離心并貼壁法或全骨髓分離法分離提純骨髓間充質(zhì)干細胞,37℃、5%CO_2培養(yǎng)箱中卵育。測定兩種方法培養(yǎng)的原代細胞的細胞克隆集落數(shù)、細胞生長時間和16d細胞消化數(shù)量。2.選取月齡不同的新西蘭白兔分組并在無菌條件下股骨轉(zhuǎn)子間區(qū)負壓抽取骨髓穿刺液5-10ml,經(jīng)密度梯度離心并貼壁法分離提純骨髓間充質(zhì)干細胞,LG-DMEM(10%FBS)培養(yǎng)基中進行原代、傳代培養(yǎng)。分別檢測不同月齡組的骨髓間充質(zhì)干細胞的原代及傳代細胞的生長活性。3.選取月齡＜3月的新西蘭白兔,密度梯度離心并貼壁法體外分離骨髓間充質(zhì)干細胞,LG-DMEM(10%FBS)培養(yǎng)基培養(yǎng)。待細胞傳代后,用LG-DMEM(10%FBS)、LG-DMEM(15%FBS)、LG-DMEM(20%FBS)、HG-DMEM(10%FBS)四種培養(yǎng)基重懸細胞,測定不同培養(yǎng)基培養(yǎng)細胞的擴增倍數(shù)、集落形成率、生長速度和觀察細胞大體形態(tài)。4.選取生長良好的P4代細胞(動物月齡＜3月,密度梯度離心并貼壁法分離,LG-DMEM(15%FBS)培養(yǎng)基體外培養(yǎng))條件培養(yǎng)基成骨誘導(dǎo),顯微鏡下觀察細胞形態(tài)變化和進行茜素紅染色。 結(jié)果:1.通過密度梯度離心并貼壁法分離純化的骨髓間充質(zhì)干細胞在LG-DMEM(10%FBS)培養(yǎng)基中生長良好。對照組中相同培養(yǎng)條件下由全骨髓分離法培養(yǎng)的骨髓間充質(zhì)干細胞的培養(yǎng)成功率明顯比密度梯度離心純化組低。2.不同月齡組中兔骨髓間充質(zhì)干細胞增殖速度不同,月齡＜3月齡的兔骨髓間充質(zhì)干細胞增殖最好,細胞生長活性明顯優(yōu)于其它兩組。3.LG-DMEM(10%FBS)、LG-DMEM(15%FBS)、LG-DMEM(20%FBS)、HG-DMEM(10%FBS)四種培養(yǎng)基中,LG-DMEM(15%FBS)組中細胞擴增倍數(shù)為16.20±1.60倍,第2—3天進入對數(shù)生長期,平均細胞集落形成率為6.11±1.17%。比較LG—DMEM(10%FBS)組和LG—DMEM(20%FBS)組中的細胞擴增倍數(shù)、平均細胞集落形成率和細胞生長速度,差異性不顯著。細胞增殖隨著FBS濃度的增加,形態(tài)發(fā)生了變化。在LG—DMEM(20%FBS)組中的細胞后期增殖過程中出現(xiàn)明顯的老化現(xiàn)象。HG—DMEM(10%FBS)中細胞生長擴增倍數(shù)較其他3組小,平均11.40±2.07倍,平均細胞集落形成率2.67±1.00%,細胞增殖速度低于其他3組。4.P4兔骨髓間充質(zhì)干細胞經(jīng)過成骨誘導(dǎo)18d,硒紅染色陽性,鈣結(jié)節(jié)形成。 結(jié)論:1.相同培養(yǎng)條件下密度梯度離心并貼壁法培養(yǎng)的兔骨髓間充質(zhì)干細胞增殖活性明顯優(yōu)于全骨髓培養(yǎng)法,成功率高。2.月齡＜3月的兔骨髓中含有更多的間充質(zhì)干細胞,且生物活性和增殖特性明顯優(yōu)于老齡組。3.LG—DMEM(15%FBS)培養(yǎng)基較其他三種培養(yǎng)基更適合骨髓間充質(zhì)干細胞體外擴增培養(yǎng),且更好的保持了細胞的正常形態(tài)和生物活性。
[Abstract]:Objective: to study the conditions of rabbit bone marrow mesenchymal stem cells (BMSCs) cultured in vitro and compare the differences of various influencing factors to explore a simple and efficient method for the expansion of rabbit bone marrow mesenchymal stem cells (BMSCs). To provide the necessary prerequisite for the application of stem cells in wound repair and tissue and organ reconstruction. Methods 1. Bone marrow puncture fluid (5-10ml) was extracted from New Zealand white rabbit (age < March) under strict aseptic condition of femoral intertrochanteric region. Bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent method or whole bone marrow separation method. Cell growth time and 16d cell digestibility. 2. New Zealand white rabbits of different ages were divided into groups and bone marrow puncture fluid 5-10ml was extracted under negative pressure of intertrochanteric region of femur under aseptic condition. Bone marrow mesenchymal stem cells (BMSCs) were isolated and purified from LG-DMEM (10S) medium by density gradient centrifugation and adherent method. The growth activity of bone marrow mesenchymal stem cells (BMSCs) in different age groups was detected. 3. New Zealand white rabbits with the age of less than March were selected. Bone marrow mesenchymal stem cells were isolated from LG-DMEM (10S) medium by density gradient centrifugation and adherent method in vitro. LG-DMEM (15s / LG-DMEM (20S) HG-DMEM (10S) medium) was cultured in four kinds of medium, and the expansion times of the cultured cells in different culture medium were measured. Colony formation rate, growth rate and observed cell morphology. 4. P4 passage cells with good growth (animal age < March, density gradient centrifugation and adherent method) were selected. LG-DMEM (15s) medium was used to induce osteogenesis. Morphological changes of cells and alizarin red staining were observed under microscope. Results 1. Bone marrow mesenchymal stem cells were isolated and purified by density gradient centrifugation and adherent method in LG-DMEM (10S). The success rate of bone marrow mesenchymal stem cells cultured by the whole bone marrow separation method in the control group was significantly lower than that in the density gradient centrifugation group. The proliferation rate of MSCs was different. The proliferation of rabbit bone marrow mesenchymal stem cells (BMSCs) under 3 months old was the best, and the cell growth activity was obviously superior to that of the other two groups. 3. LG-DMEM (10S / LG-DMEM (15S)). The expansion times of LG-DMEM (15S) in LG-DMEM (10S) medium were 16.20 鹵1.60 times. The logarithmic growth period entered into 2-3 days. The average colony-forming rate was 6.11 鹵1.17.The cell expansion times in LG-DMEM10S group and LG-DMEM20S group were compared between LG-DMEM (10S) group and LG-DMEM (20S) group. The average cell colony formation rate and cell growth rate were not significantly different. Cell proliferation increased with the concentration of FBS. In LG-DMEM (20S) group, there were obvious aging phenomena during anaphase proliferation of LG-DMEM (20S). HG-DMEM (10S). Compared with the other three groups, the growth and amplification times of the medium cell were smaller. The average cell colony formation rate was 2.67 鹵1.00 and the average cell colony formation rate was 11.40 鹵2.07 times. The cell proliferation rate was lower than that of the other three groups. 4. P4 rabbit bone marrow mesenchymal stem cells were induced by osteogenesis for 18 days. Selenium red staining was positive and calcium nodules were formed. Conclusion the proliferative activity of rabbit bone marrow mesenchymal stem cells cultured by density gradient centrifugation and adherent method under the same culture conditions is obviously superior to that of whole bone marrow culture. More mesenchymal stem cells were found in bone marrow of rabbits under March. And the biological activity and proliferation characteristics were significantly better than the aging group. 3. LG-DMEM (15S) medium was more suitable for bone marrow mesenchymal stem cell culture in vitro than the other three kinds of medium. And better maintain the normal morphology and biological activity of cells.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R329

【參考文獻】

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本文編號：1491349