SD大鼠螺旋神經(jīng)節(jié)細胞與骨髓間充質(zhì)干細胞共培養(yǎng)觀察
發(fā)布時間：2019-05-17 03:29
【摘要】：目的SD大鼠原代螺旋神經(jīng)節(jié)細胞(Spiral ganglion cells, SGC)與骨髓間充質(zhì)干細胞(bone marrow mesenchymal stem cells BMSCs)間接共培養(yǎng),觀察BMSCs是否誘導分化為神經(jīng)細胞,并觀察其對SGCs生長影響,為治療感音神經(jīng)性耳聾治療尋求新的治療方法。 方法1.BMSCs的原代及傳代培養(yǎng)：SD大鼠全骨髓直接貼壁法培養(yǎng)BMSCs并進行傳代培養(yǎng),取第3代細胞進行流式細胞儀檢測及免疫細胞化學染色。2.SGCs的原代培養(yǎng),取新生SD大鼠進行離體SGCs離體培養(yǎng),取第3天細胞用神經(jīng)元特異性烯醇化酶(NSE)進行免疫細胞化學染色。3.采用間接共培養(yǎng)方法,將SGCs與培養(yǎng)第3代BMSCs按1：1比例進行共培養(yǎng),每天觀察細胞形態(tài)。取第7天的BMSCs用NSE一抗進行免疫細胞化學染色,觀察是否有染色陽性的神經(jīng)元樣細胞。 結(jié)果1.酶消化法SGCs培養(yǎng)24小時后,大部分細胞貼壁,并出現(xiàn)較短的軸突,48-72小時軸突逐漸變長,細胞胞體呈橢圓形或圓形,突起細長,常交織成網(wǎng)狀,培養(yǎng)6至14天時,細胞數(shù)量明顯減少,部分細胞凋亡。組織塊法接種24小時后,組織塊貼壁。細胞形態(tài)與酶消化法相似,但生存時間比它更長。2.原代培養(yǎng)的BMSCs24h-48h后貼壁細胞,呈集落樣生長,細胞呈短梭型、多角型等。第6d起細胞迅速增殖,培養(yǎng)11天時,細胞長滿瓶底的80-90%左右傳代,第二代細胞增殖速度較第一代細胞明顯增快。培養(yǎng)至第3代時,細胞形態(tài)為均一的“紡錘形”,呈明顯的漩渦樣生長。流式細胞儀檢測,造血干細胞和內(nèi)皮細胞表面標志抗原CD34表達呈陰性,間充質(zhì)干細胞表面標志抗原CD44表達陽性,免疫細胞化學染色呈陽性,結(jié)合細胞形態(tài)分析符合大鼠BMSCs的特征。3.共培養(yǎng)組中BMSCs第5天開始突起增加,有的呈星狀。共培養(yǎng)組中SGCs與對照組中SGCs相比,前者SGCs維持時間相對更長,第6天神經(jīng)細胞數(shù)量減少,部分細胞軸突縮短,而后者第4天數(shù)量就開始出現(xiàn)減少,15天時,共培養(yǎng)組中SGCs數(shù)量已明顯減少,而對照組中SGCs接近凋亡。取第7天的BMSCs進行免疫細胞化學染色,可見有細胞著色,染色時間為5分鐘,陽性率約為15%,第一對照組BMSCs幾乎呈陰性。 結(jié)論1,體外成功培養(yǎng)了SD大鼠SGCs,組織塊法培養(yǎng)比酶消化法培養(yǎng)SGCs生存時間更長。2.單純應用貼壁培養(yǎng)法獲得SD大鼠BMSCs簡單易行,可分離培養(yǎng)出較純化的BMSCs。3.BMSCs與SGCs共培養(yǎng)過程中,培養(yǎng)第7天,BMSCs部分分化為神經(jīng)元樣細胞,分化率約為15%；同時,與BMSCs共培養(yǎng),SGCs的生存時間延長,凋亡減慢。
[Abstract]:Objective to observe whether BMSCs is induced to differentiate into nerve cells by indirect co-culture of (Spiral ganglion cells, SGC) from primary spiral ganglion cells of SD rats and (bone marrow mesenchymal stem cells BMSCs) of bone marrow mesenchymal stem cells, and to observe its effect on the growth of SGCs. To seek a new treatment for sensorineural deafness. Methods Primary and subculture of 1.BMSCs: BMSCs was cultured by direct adherent method of whole bone marrow of SD rats and subcultured. The third passage cells were detected by flow cytometry and immunochemical staining. 2.SGCs were cultured in primary culture. Neonatal SD rats were cultured in vitro with SGCs in vitro, and the cells were stained with neuron-specific enolase (NSE) on the 3rd day. The SGCs and the third generation BMSCs were co-cultured at 1:1 by indirect co-culture method, and the cell morphology was observed every day. On the 7th day, BMSCs was stained with NSE antibody to observe whether there were positive neuron-like cells. Result 1. After 24 hours of SGCs culture, most of the cells adhered to the wall and appeared short axons. After 48 hours, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. After 6 to 14 days of culture, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. The number of cells decreased significantly and some cells were apoptotic. After 24 hours of vaccination, the tissue block adheres to the wall. The morphology of cells is similar to that of enzyme digestion, but the survival time is longer than that of enzyme digestion. 2. The adherent cells cultured in primary culture showed colony-like growth, short shuttle type, polyangular type and so on. On the 6th day, the cells proliferated rapidly. on the 11th day of culture, the cells grew about 80% of the bottom of the bottle, and the proliferation rate of the second generation cells was significantly faster than that of the first generation cells. When cultured to the third generation, the morphology of the cells was homogeneous "fusiform", showing obvious whirlpool growth. Flow cytometry showed that the expression of surface marker antigen CD34 of hematopoietic stem cells and endothelial cells was negative, the expression of surface marker antigen CD44 of mesenchymal stem cells was positive, and the expression of surface marker antigen CD44 of mesenchymal stem cells was positive. The combined cell morphology analysis was in accordance with the characteristics of rat BMSCs. 3. In the co-culture group, the protrusions of BMSCs began to increase on the 5th day, some of them were starlike. Compared with SGCs in control group, SGCs in co-culture group lasted longer, the number of nerve cells decreased and some axons decreased on the 6th day, while the number of axons in the latter began to decrease on the 4th day, and on the 15th day. The number of SGCs in co-culture group was significantly decreased, while SGCs in control group was close to apoptosis. On the 7th day, BMSCs was stained with cells, the staining time was 5 minutes, the positive rate was about 15%, and the BMSCs in the first control group was almost negative. Conclusion 1. The survival time of SGCs, tissue block culture of SD rats was longer than that of SGCs cultured by enzyme digestion. 2. BMSCs of SD rats was obtained by adherent culture alone. The purified BMSCs.3.BMSCs and SGCs could be isolated and co-cultured. On the 7th day of culture, BMSCs partially differentiated into neuron-like cells, and the differentiation rate was about 15%. At the same time, co-culture with BMSCs prolonged the survival time of SGCs and slowed down apoptosis.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
[Abstract]:Objective to observe whether BMSCs is induced to differentiate into nerve cells by indirect co-culture of (Spiral ganglion cells, SGC) from primary spiral ganglion cells of SD rats and (bone marrow mesenchymal stem cells BMSCs) of bone marrow mesenchymal stem cells, and to observe its effect on the growth of SGCs. To seek a new treatment for sensorineural deafness. Methods Primary and subculture of 1.BMSCs: BMSCs was cultured by direct adherent method of whole bone marrow of SD rats and subcultured. The third passage cells were detected by flow cytometry and immunochemical staining. 2.SGCs were cultured in primary culture. Neonatal SD rats were cultured in vitro with SGCs in vitro, and the cells were stained with neuron-specific enolase (NSE) on the 3rd day. The SGCs and the third generation BMSCs were co-cultured at 1:1 by indirect co-culture method, and the cell morphology was observed every day. On the 7th day, BMSCs was stained with NSE antibody to observe whether there were positive neuron-like cells. Result 1. After 24 hours of SGCs culture, most of the cells adhered to the wall and appeared short axons. After 48 hours, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. After 6 to 14 days of culture, the axons gradually became longer, the cell bodies were oval or round, the protrusions were slender, and often intertwined into reticular. The number of cells decreased significantly and some cells were apoptotic. After 24 hours of vaccination, the tissue block adheres to the wall. The morphology of cells is similar to that of enzyme digestion, but the survival time is longer than that of enzyme digestion. 2. The adherent cells cultured in primary culture showed colony-like growth, short shuttle type, polyangular type and so on. On the 6th day, the cells proliferated rapidly. on the 11th day of culture, the cells grew about 80% of the bottom of the bottle, and the proliferation rate of the second generation cells was significantly faster than that of the first generation cells. When cultured to the third generation, the morphology of the cells was homogeneous "fusiform", showing obvious whirlpool growth. Flow cytometry showed that the expression of surface marker antigen CD34 of hematopoietic stem cells and endothelial cells was negative, the expression of surface marker antigen CD44 of mesenchymal stem cells was positive, and the expression of surface marker antigen CD44 of mesenchymal stem cells was positive. The combined cell morphology analysis was in accordance with the characteristics of rat BMSCs. 3. In the co-culture group, the protrusions of BMSCs began to increase on the 5th day, some of them were starlike. Compared with SGCs in control group, SGCs in co-culture group lasted longer, the number of nerve cells decreased and some axons decreased on the 6th day, while the number of axons in the latter began to decrease on the 4th day, and on the 15th day. The number of SGCs in co-culture group was significantly decreased, while SGCs in control group was close to apoptosis. On the 7th day, BMSCs was stained with cells, the staining time was 5 minutes, the positive rate was about 15%, and the BMSCs in the first control group was almost negative. Conclusion 1. The survival time of SGCs, tissue block culture of SD rats was longer than that of SGCs cultured by enzyme digestion. 2. BMSCs of SD rats was obtained by adherent culture alone. The purified BMSCs.3.BMSCs and SGCs could be isolated and co-cultured. On the 7th day of culture, BMSCs partially differentiated into neuron-like cells, and the differentiation rate was about 15%. At the same time, co-culture with BMSCs prolonged the survival time of SGCs and slowed down apoptosis.
【學位授予單位】：中南大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329
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