大鼠骨髓間充質(zhì)干細胞體外誘導(dǎo)分化為血管平滑肌樣細胞的實驗研究
發(fā)布時間：2019-04-23 20:41
【摘要】：目的探討骨髓間充質(zhì)干細胞(BMSCs)體外分離培養(yǎng)以及擴增的方法并鑒定細胞性質(zhì)。 方法取100 g左右雄性SD大鼠后肢股骨、脛骨骨髓,原代全骨髓培養(yǎng)法,多次傳代純化,體外擴增后,觀察細胞形態(tài),繪制細胞生長曲線,并利用免疫組化及流式細胞學(xué)檢測培養(yǎng)細胞中CD34、CD90、CD105細胞標(biāo)志性因子,鑒定原代培養(yǎng)獲取的細胞是否為BMSCs。 結(jié)果原代培養(yǎng)所獲取的細胞形態(tài)呈長梭形,生長呈現(xiàn)特征性的漩渦狀,生長曲線為典型的S型,免疫組化結(jié)果顯示CD34陰性,CD44陽性,CD54陽性,流式細胞學(xué)檢測結(jié)果顯示CD34陰性,CD90、CD105陽性。 結(jié)論利用全骨髓培養(yǎng)法可成功分離骨髓間充質(zhì)干細胞,全骨髓培養(yǎng)較為簡便、易行。經(jīng)傳代后細胞純度增高,10代以內(nèi)的細胞純度高,活性好。 目的探討骨髓間充質(zhì)干細胞(BMSCs)作為構(gòu)建小口徑血管種子細胞的可行性,及其誘導(dǎo)機制。 方法取100g左右雄性SD大鼠后肢股骨、脛骨骨髓,原代全骨髓培養(yǎng)法,多次傳代純化,體外擴增后,觀察細胞形態(tài),并免疫熒光及流式細胞儀檢測CD34、CD90、CD105細胞因子,鑒定是否為BMSCs。將BMSCs分為實驗組及對照組:實驗組使用含全反式維甲酸及二丁酰環(huán)磷酸腺苷(db-cAMP)的低糖基本培養(yǎng)基(DMEM-LG)培養(yǎng)基培養(yǎng);對照組采用普通的DMEM-LG培養(yǎng)基培養(yǎng)。觀察誘導(dǎo)后的細胞形態(tài)并利用免疫熒光技術(shù)及流式細胞儀檢測誘導(dǎo)后第5代細胞平滑肌α-肌動蛋白(SM-α-actin)、鈣結(jié)合蛋白(calponin)、肌球蛋白重鏈(SMMHC)的表達情況。 結(jié)果原代培養(yǎng)所獲取的細胞經(jīng)過多次傳代培養(yǎng)后呈長梭形,呈現(xiàn)特征性的漩渦狀生長,檢測CD34陰性,CD90、CD105陽性。經(jīng)誘導(dǎo)后的BMSCs生長較緩慢,略呈橢圓形,檢測SM-α-actin、calponin、SMMHC顯著表達;對照組細胞的形態(tài)及生長速度和BMSCs相似,不表達SM-α-actin、calponin、SMMHC。 結(jié)論BMSCs在全反式維甲酸的誘導(dǎo)下可向血管平滑肌樣細胞表型分化,為組織工程構(gòu)建小口徑血管提供平滑肌種子細胞。 目的探討血管平滑肌樣細胞在體外體外靜態(tài)培養(yǎng)的可能性以及觀察細胞生長狀態(tài)。 方法取成年雄性比格犬,處死后,取胸主動脈,長約6cm,并對所取血管行脫細胞實驗,獲取脫細胞基質(zhì)支架,并行快速冰凍染色檢查,檢測脫細胞效果。將獲取的脫細胞基質(zhì)支架材料剪成2x2cm大小的薄片,將誘導(dǎo)完成的血管平滑肌樣細胞種植在薄片上,隔日換液,種植15d后將支架薄片行快速冰凍檢查、電子掃描電鏡檢查,觀察細胞生長狀態(tài)。 結(jié)果快速冰凍染色檢查可見誘導(dǎo)完成的血管平滑肌樣細胞可種植在血管脫細胞基質(zhì)支架表面生長,電子掃描電鏡可見細胞復(fù)雜在支架材料上。 結(jié)論誘導(dǎo)完成的血管平滑肌樣細胞可靜態(tài)種植在血管脫細胞基質(zhì)材料上。
[Abstract]:Objective to investigate the methods of isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in vitro and to identify the cell properties. Methods the femoral and tibial bone marrow of the male SD rats with 100g or so were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed and the growth curve of the cells was drawn. Immunohistochemistry and flow cytometry were used to detect the marker factors of CD34,CD90,CD105 cells in order to identify whether the cells obtained in primary culture were BMSCs. or not. Results the morphology of the cells was spindle-shaped and the growth curve was typical S-type. The results of immunohistochemistry showed that CD34 was negative, CD44 was positive, CD54 was positive, and CD34 was negative by flow cytometry. CD90,CD105 positive. Conclusion Bone marrow mesenchymal stem cells can be successfully isolated by whole bone marrow culture, and the whole bone marrow culture is simple and easy. After passage, the cell purity increased, the cell purity within 10 generations was high, and the cell activity was good. Objective to investigate the feasibility and induction mechanism of bone marrow mesenchymal stem cells (BMSCs) as seed cells of small diameter blood vessels. Methods the femoral and tibial bone marrow of 100 g male SD rats were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed, and the CD34,CD90,CD105 cytokines were detected by immunofluorescence and flow cytometry. Identify BMSCs. BMSCs was divided into experimental group and control group: the experimental group was cultured in low glucose basic medium (DMEM-LG) containing all-trans retinoic acid and dibutyryl cyclic adenosine monophosphate (db-cAMP), and the control group was cultured in common DMEM-LG medium. The morphology of induced cells was observed and the expression of SM- 偽-actin), calcium binding protein (calponin), myosin heavy chain (SMMHC) was detected by immunofluorescence and flow cytometry. Results the cells obtained from primary culture showed long fusiform shape and characteristic swirl-like growth after repeated subculture. CD34-negative and CD90,CD105-positive cells were detected. After induction, BMSCs grew slowly and showed a slightly oval shape, and the expression of SM- 偽-actin,calponin,SMMHC was detected. In the control group, the morphology and growth rate of the cells were similar to those of BMSCs, but no expression of SM- 偽-actin,calponin,SMMHC. was observed in the cells of the control group. Conclusion BMSCs can differentiate into vascular smooth muscle-like cells induced by all-trans retinoic acid and provide smooth muscle seed cells for tissue engineering. Objective to investigate the possibility of static culture of vascular smooth muscle-like cells (VSMCs) in vitro and to observe the state of cell growth. Methods the adult male Beagle dogs were killed and the thoracic aorta was taken for about 6 cm. The acellular matrix scaffolds were obtained and the acellular effect was detected by rapid frozen staining. The obtained acellular matrix scaffold material was cut into 2x2cm-size thin slices, and the induced vascular smooth muscle-like cells were implanted on the thin slices, then the solution was exchanged every other day. After 15 days of implantation, the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM), and the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM). The state of cell growth was observed. Results Rapid frozen staining showed that the induced vascular smooth muscle-like cells could grow on the surface of the acellular matrix scaffold, and the cells were complicated on the scaffold material by electron scanning electron microscopy (SEM). Conclusion the induced vascular smooth muscle-like cells can be statically implanted on the acellular matrix material.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
本文編號：2463782
[Abstract]:Objective to investigate the methods of isolation, culture and amplification of bone marrow mesenchymal stem cells (BMSCs) in vitro and to identify the cell properties. Methods the femoral and tibial bone marrow of the male SD rats with 100g or so were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed and the growth curve of the cells was drawn. Immunohistochemistry and flow cytometry were used to detect the marker factors of CD34,CD90,CD105 cells in order to identify whether the cells obtained in primary culture were BMSCs. or not. Results the morphology of the cells was spindle-shaped and the growth curve was typical S-type. The results of immunohistochemistry showed that CD34 was negative, CD44 was positive, CD54 was positive, and CD34 was negative by flow cytometry. CD90,CD105 positive. Conclusion Bone marrow mesenchymal stem cells can be successfully isolated by whole bone marrow culture, and the whole bone marrow culture is simple and easy. After passage, the cell purity increased, the cell purity within 10 generations was high, and the cell activity was good. Objective to investigate the feasibility and induction mechanism of bone marrow mesenchymal stem cells (BMSCs) as seed cells of small diameter blood vessels. Methods the femoral and tibial bone marrow of 100 g male SD rats were cultured and purified for many times. After in vitro amplification, the morphology of the cells was observed, and the CD34,CD90,CD105 cytokines were detected by immunofluorescence and flow cytometry. Identify BMSCs. BMSCs was divided into experimental group and control group: the experimental group was cultured in low glucose basic medium (DMEM-LG) containing all-trans retinoic acid and dibutyryl cyclic adenosine monophosphate (db-cAMP), and the control group was cultured in common DMEM-LG medium. The morphology of induced cells was observed and the expression of SM- 偽-actin), calcium binding protein (calponin), myosin heavy chain (SMMHC) was detected by immunofluorescence and flow cytometry. Results the cells obtained from primary culture showed long fusiform shape and characteristic swirl-like growth after repeated subculture. CD34-negative and CD90,CD105-positive cells were detected. After induction, BMSCs grew slowly and showed a slightly oval shape, and the expression of SM- 偽-actin,calponin,SMMHC was detected. In the control group, the morphology and growth rate of the cells were similar to those of BMSCs, but no expression of SM- 偽-actin,calponin,SMMHC. was observed in the cells of the control group. Conclusion BMSCs can differentiate into vascular smooth muscle-like cells induced by all-trans retinoic acid and provide smooth muscle seed cells for tissue engineering. Objective to investigate the possibility of static culture of vascular smooth muscle-like cells (VSMCs) in vitro and to observe the state of cell growth. Methods the adult male Beagle dogs were killed and the thoracic aorta was taken for about 6 cm. The acellular matrix scaffolds were obtained and the acellular effect was detected by rapid frozen staining. The obtained acellular matrix scaffold material was cut into 2x2cm-size thin slices, and the induced vascular smooth muscle-like cells were implanted on the thin slices, then the solution was exchanged every other day. After 15 days of implantation, the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM), and the scaffolds were subjected to rapid freezing examination and electron scanning electron microscopy (SEM). The state of cell growth was observed. Results Rapid frozen staining showed that the induced vascular smooth muscle-like cells could grow on the surface of the acellular matrix scaffold, and the cells were complicated on the scaffold material by electron scanning electron microscopy (SEM). Conclusion the induced vascular smooth muscle-like cells can be statically implanted on the acellular matrix material.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
【參考文獻】
相關(guān)期刊論文 前10條
1 林明山;池一凡;侯文明;孫忠東;牛兆倬;孫勇;生偉;孫龍;;兔主動脈脫細胞血管基質(zhì)的制備[J];青島大學(xué)醫(yī)學(xué)院學(xué)報;2009年02期
2 謝眾上;吳慧穎;趙科研;柳克祥;;犬骨髓間充質(zhì)干細胞體外誘導(dǎo)分化的平滑肌細胞的組織培養(yǎng)及鑒定[J];吉林大學(xué)學(xué)報(醫(yī)學(xué)版);2009年04期
3 董建德;張建;谷涌泉;;脫細胞基質(zhì)在血管組織工程中的應(yīng)用[J];中國肺癌雜志;2009年02期
4 李志勇;骨髓間充質(zhì)干細胞可塑性的研究進展[J];國外醫(yī)學(xué).口腔醫(yī)學(xué)分冊;2004年02期
5 王暉,胡建國,吳忠仕,胡鐵輝,鄧又華,馮耀光,姜志斌,楊彥青;牛頸靜脈經(jīng)不同方法交聯(lián)改性后的免疫原性[J];中南大學(xué)學(xué)報(醫(yī)學(xué)版);2005年02期
6 唐新華;黃玲;曾昭凡;;小口徑血管構(gòu)建的回顧與展望[J];江西醫(yī)學(xué)院學(xué)報;2009年05期
7 馬萍,賈紹斌,劉曉方,沙勇,徐清斌,戴貴東;人骨髓間充質(zhì)干細胞的分離和體外培養(yǎng)[J];寧夏醫(yī)學(xué)雜志;2005年06期
8 趙凌云;錢淑琴;趙廷寶;;人骨髓間充質(zhì)干細胞的貼壁分離與體外培養(yǎng)[J];中國組織工程研究與臨床康復(fù);2007年28期
9 楊麗;張榮華;謝厚杰;蔡宇;黃豐;;建立大鼠骨髓間充質(zhì)干細胞穩(wěn)定分離培養(yǎng)體系與鑒定[J];中國組織工程研究與臨床康復(fù);2009年06期
10 楊瑋;徐道華;周晨慧;劉鈺瑜;戴濱;黃洪新;;小檗堿對骨髓間充質(zhì)干細胞成骨分化的影響[J];中國藥理學(xué)通報;2010年12期
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