SD大鼠螺旋神經(jīng)節(jié)細(xì)胞與骨髓間充質(zhì)干細(xì)胞共培養(yǎng)觀察
發(fā)布時(shí)間：2019-05-17 03:29
【摘要】：目的SD大鼠原代螺旋神經(jīng)節(jié)細(xì)胞(Spiral ganglion cells, SGC)與骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells BMSCs)間接共培養(yǎng),觀察BMSCs是否誘導(dǎo)分化為神經(jīng)細(xì)胞,并觀察其對(duì)SGCs生長(zhǎng)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(jìn)行傳代培養(yǎng),取第3代細(xì)胞進(jìn)行流式細(xì)胞儀檢測(cè)及免疫細(xì)胞化學(xué)染色。2.SGCs的原代培養(yǎng),取新生SD大鼠進(jìn)行離體SGCs離體培養(yǎng),取第3天細(xì)胞用神經(jīng)元特異性烯醇化酶(NSE)進(jìn)行免疫細(xì)胞化學(xué)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(jìn)行共培養(yǎng),每天觀察細(xì)胞形態(tài)。取第7天的BMSCs用NSE一抗進(jìn)行免疫細(xì)胞化學(xué)染色,觀察是否有染色陽(yáng)性的神經(jīng)元樣細(xì)胞。 結(jié)果1.酶消化法SGCs培養(yǎng)24小時(shí)后,大部分細(xì)胞貼壁,并出現(xiàn)較短的軸突,48-72小時(shí)軸突逐漸變長(zhǎng),細(xì)胞胞體呈橢圓形或圓形,突起細(xì)長(zhǎng),常交織成網(wǎng)狀,培養(yǎng)6至14天時(shí),細(xì)胞數(shù)量明顯減少,部分細(xì)胞凋亡。組織塊法接種24小時(shí)后,組織塊貼壁。細(xì)胞形態(tài)與酶消化法相似,但生存時(shí)間比它更長(zhǎng)。2.原代培養(yǎng)的BMSCs24h-48h后貼壁細(xì)胞,呈集落樣生長(zhǎng),細(xì)胞呈短梭型、多角型等。第6d起細(xì)胞迅速增殖,培養(yǎng)11天時(shí),細(xì)胞長(zhǎng)滿瓶底的80-90%左右傳代,第二代細(xì)胞增殖速度較第一代細(xì)胞明顯增快。培養(yǎng)至第3代時(shí),細(xì)胞形態(tài)為均一的“紡錘形”,呈明顯的漩渦樣生長(zhǎng)。流式細(xì)胞儀檢測(cè),造血干細(xì)胞和內(nèi)皮細(xì)胞表面標(biāo)志抗原CD34表達(dá)呈陰性,間充質(zhì)干細(xì)胞表面標(biāo)志抗原CD44表達(dá)陽(yáng)性,免疫細(xì)胞化學(xué)染色呈陽(yáng)性,結(jié)合細(xì)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(kāi)始突起增加,有的呈星狀。共培養(yǎng)組中SGCs與對(duì)照組中SGCs相比,前者SGCs維持時(shí)間相對(duì)更長(zhǎng),第6天神經(jīng)細(xì)胞數(shù)量減少,部分細(xì)胞軸突縮短,而后者第4天數(shù)量就開(kāi)始出現(xiàn)減少,15天時(shí),共培養(yǎng)組中SGCs數(shù)量已明顯減少,而對(duì)照組中SGCs接近凋亡。取第7天的BMSCs進(jìn)行免疫細(xì)胞化學(xué)染色,可見(jiàn)有細(xì)胞著色,染色時(shí)間為5分鐘,陽(yáng)性率約為15%,第一對(duì)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(shí)間更長(zhǎng)。2.單純應(yīng)用貼壁培養(yǎng)法獲得SD大鼠BMSCs簡(jiǎn)單易行,可分離培養(yǎng)出較純化的BMSCs。3.BMSCs與SGCs共培養(yǎng)過(guò)程中,培養(yǎng)第7天,BMSCs部分分化為神經(jīng)元樣細(xì)胞,分化率約為15%；同時(shí),與BMSCs共培養(yǎng),SGCs的生存時(shí)間延長(zhǎng),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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：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(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R329
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