嗅鞘細(xì)胞對(duì)螺旋神經(jīng)節(jié)細(xì)胞離體培養(yǎng)的影響及其機(jī)制的研究
發(fā)布時(shí)間：2018-08-03 13:13
【摘要】： 第一部分嗅鞘細(xì)胞的體外培養(yǎng)、純化及鑒定 目的：純化培養(yǎng)和鑒定成年大鼠嗅球嗅鞘細(xì)胞(olfactory ensheathing cells OECs),為后續(xù)實(shí)驗(yàn)做好準(zhǔn)備。方法：取成年大鼠嗅球神經(jīng)層,經(jīng)酶消化的方法獲得含有OECs的混合細(xì)胞懸液,采用多次長(zhǎng)時(shí)差速貼壁的方法純化OECs,用含有10%FBS的DF12培養(yǎng)基培養(yǎng),2d后培養(yǎng)基中加入bFGF (20ng/ml)和forskolin(2uM)促進(jìn)OECs的增殖,每隔3d半量更換培養(yǎng)基。倒置相差顯微鏡下觀察OECs的生長(zhǎng)狀態(tài),采用P75NTR和GFAP細(xì)胞免疫組化的方法鑒定OECs,熒光顯微鏡下觀察OECs的形態(tài)并統(tǒng)計(jì)其純度。結(jié)果：采用多次長(zhǎng)時(shí)差速貼壁的方法能夠去除絕大部分的污染細(xì)胞,包括成纖維細(xì)胞和星形膠質(zhì)細(xì)胞,接種的OECs約24小時(shí)后貼壁,倒置相差顯微鏡下觀察見大部分細(xì)胞胞體呈梭形、部分細(xì)胞胞體為多角形,伸出突起；細(xì)胞在接種培養(yǎng)的前2d生長(zhǎng)緩慢,2d后培養(yǎng)基中加入bFGF (20ng/ml)和forskolin(2uM)可見細(xì)胞增殖加快,細(xì)胞突起細(xì)長(zhǎng),培養(yǎng)7d后見OECs覆蓋皿底的90%,形成—細(xì)胞單層。P75NTR和GFAP染色見約92%的細(xì)胞為P75NTR(+),分布在突起、胞體,其中胞核被深染；約12%的細(xì)胞為GFAP (+)。結(jié)論：采用多次長(zhǎng)時(shí)差速貼壁的方法能夠獲得實(shí)驗(yàn)所需純度的OECs,培養(yǎng)基(DF12+10%FBS)中加入bFGF(20ng/ml)和forskolin(2uM)能夠顯著促進(jìn)OECs的增殖。 第二部分嗅鞘細(xì)胞對(duì)耳蝸螺旋神經(jīng)節(jié)細(xì)胞離體培養(yǎng)的影響 目的：探索在離體實(shí)驗(yàn)中OECs有無(wú)促進(jìn)耳蝸聽覺傳入神經(jīng)元—螺旋神經(jīng)節(jié)細(xì)胞(spiral ganglion cells SGCs)存活的作用。方法：取成年大鼠嗅球和新生大鼠蝸軸組織塊進(jìn)行OECs與SGCs的培養(yǎng),采用多次長(zhǎng)時(shí)差速貼壁的方法純化培養(yǎng)OECs。實(shí)驗(yàn)分OECs與SGCs共培養(yǎng)組、SGCs在OECs條件培養(yǎng)液中(OEC-CM)培養(yǎng)組和SGCs單獨(dú)培養(yǎng)組。倒置相差顯微鏡下觀察OECs和SGCs生長(zhǎng)狀態(tài),P75NTR免疫組化法鑒定OECs,神經(jīng)元特異性標(biāo)志物βⅢ-tubulin標(biāo)記SGCs。結(jié)果：OECs貼壁培養(yǎng)7天后形成—細(xì)胞單層,在OECs與SGCs共培養(yǎng)體系中,SGCs在OECs形成的細(xì)胞單層的表面生長(zhǎng),并伸出長(zhǎng)突起,呈現(xiàn)典型的雙極神經(jīng)元形態(tài)；在培養(yǎng)的前6d,隨著培養(yǎng)時(shí)間的延長(zhǎng),SGCs較接種前減少,但共培養(yǎng)組中SGCs存活數(shù)量明顯高于SGCs單獨(dú)培養(yǎng)組(P0.01)；與SGCs在OEC-CM中培養(yǎng)比較,共培養(yǎng)組更能促進(jìn)SGCs存活,并且單獨(dú)培養(yǎng)組的SGCs數(shù)量在培養(yǎng)的第6d出現(xiàn)大幅度減少；在培養(yǎng)的第9d,單獨(dú)培養(yǎng)組中幾乎沒有SGCs生長(zhǎng)；而在共培養(yǎng)組,SGCs的數(shù)量未見明顯變化(P0.05)；統(tǒng)計(jì)發(fā)現(xiàn)單獨(dú)培養(yǎng)組中存活的SGCs由第3d的14.6±1.1SGCs/視野(×200)減少到第14d的3.3±0.3SGCs/視野；OEC-CM組中的SGCs由35.3±2.1SGCs/視野下降到12.9±0.3SGCs/視野；共培養(yǎng)組中存活的SGCs較其它組明顯增多,由第3d的66.9±1.2SGCs/視野(×200)減少到第14d的38.9×1.0 SGCs/視野。結(jié)論：OECs與SGCs共培養(yǎng)能夠促進(jìn)新生大鼠SGCs存活。 第三部分嗅鞘細(xì)胞促進(jìn)螺旋神經(jīng)節(jié)細(xì)胞存活的分子機(jī)制 目的：初步研究OECs促進(jìn)SGCs體外存活的分子機(jī)制。方法：建立OECs與SGCs共培養(yǎng)體系,實(shí)驗(yàn)分三組：共培養(yǎng)+腦源性神經(jīng)營(yíng)養(yǎng)因子(BDNF,500 pg/ml)組；共培養(yǎng)+BDNF抗體(IgY型,50 ug/ml)組；對(duì)照組為OECs與SGCs共培養(yǎng)。共培養(yǎng)3d后固定,βⅢ-tubulin免疫組化染色鑒定SGCs,每個(gè)培養(yǎng)皿隨機(jī)取4個(gè)視野,熒光顯微鏡下統(tǒng)計(jì)βⅢ-tubulin標(biāo)記的SGCs,統(tǒng)計(jì)各培養(yǎng)組中的SGCs。結(jié)果：共培養(yǎng)+BDNF組中有大量SGCs在OECs形成的單層細(xì)胞毯表面生長(zhǎng),但與對(duì)照組比較,SCG的數(shù)目無(wú)統(tǒng)計(jì)學(xué)差異；加入BDNF抗體(IgY)的共培養(yǎng)組中的SGCs數(shù)量明顯減少(P0.01)。結(jié)論：OECs與SGCs共培養(yǎng)中加入BDNF對(duì)SGCs存活無(wú)明顯影響,而加入BDNF抗體(IgY)后存活的SGCs減少。OECs分泌BDNF可能是其促進(jìn)SGCs體外培養(yǎng)的存活,發(fā)揮對(duì)SGCs的營(yíng)養(yǎng)保護(hù)作用的分子機(jī)制之一。
[Abstract]:Part one: culture, purification and identification of olfactory ensheathing cells in vitro
Objective: to purify and cultivate the olfactory olfactory ensheathing cells (olfactory ensheathing cells OECs) of adult rats, and to prepare for the follow-up experiment. Methods: the olfactory bulb nerve layer of adult rats was taken and the mixed cell suspension containing OECs was obtained by enzyme digestion. OECs was purified by the method of multiple long time differential adherence, and DF12 culture containing 10%FBS was used. In the culture medium, bFGF (20ng/ml) and forskolin (2uM) were added to the medium of 2D to promote the proliferation of OECs. The culture medium was replaced every 3D and a half quantity. The growth state of OECs was observed under the inverted phase contrast microscope. P75NTR and GFAP cell immunohistochemistry were used to identify the OECs. The morphology of OECs was observed under the fluorescence microscope and the purity of the OECs was observed. Most of the contaminated cells, including fibroblasts and astrocytes, were removed, including fibroblasts and astrocytes. The OECs inoculated for about 24 hours was attached to the wall. The cell bodies of most cells were spindle shaped and some cells were polygons and protuberances. The cells grew slowly in the 2D before inoculation. After 2D, bFGF (20ng/ml) and forskolin (2uM) were added in the culture medium to accelerate the proliferation of cell proliferation, and the cell protruded long. After 7d, 90% of OECs covered dish bottom, and the cell monolayer.P75NTR and GFAP staining showed that about 92% of the cells were P75NTR (+), distributed in the protuberance, the nucleus was deeply dyed; about 12% of the cells were GFAP (+). Conclusion: the use of more (+). Conclusion: adopt more (+). "Conclusion: adopt more (+)." conclusion: adopt more (+). "Conclusion: adopt many (+). It is possible to obtain the purity of OECs for the experiment, and the addition of bFGF (20ng/ml) and forskolin (2uM) in the medium (DF12+10%FBS) can significantly promote the proliferation of OECs.
The effect of olfactory ensheathing cells on cochlear spiral ganglion cells in vitro culture in second parts
Objective: To explore the effect of OECs on the survival of the cochlear auditory afferent neurons (spiral ganglion cells SGCs) in vitro. Methods: the cultured rat olfactory bulb and the newborn rat worm axis tissue were cultured for OECs and SGCs, and the OECs. experiment was purified by multiple long time differential adherence methods. S and SGCs co culture group, SGCs in OECs conditioned medium (OEC-CM) culture group and SGCs single culture group. The growth state of OECs and SGCs was observed under the inverted phase contrast microscope. P75NTR immunohistochemical method was used to identify OECs, the neuron specific marker beta III -tubulin labeling SGCs. results: 7 days after the adherence wall culture, the cell monolayer was formed. In the culture system, SGCs grows on the surface of single cell monolayer formed by OECs and protruded long protrusions, showing a typical bipolar neuron form. In the pre culture 6D, with the prolongation of the incubation time, the SGCs is less than before the inoculation, but the survival number of SGCs in the co culture group is significantly higher than that of the SGCs single culture group (P0.01), and compared with SGCs in OEC-CM, The co culture group was more able to promote SGCs survival, and the number of SGCs in the individual culture group decreased significantly in the culture of 6D; in the cultured 9D, there was little SGCs growth in the individual culture group; but in the co culture group, the number of SGCs was not significantly changed (P0.05). The statistics found that the survival SGCs in the individual culture group was 14.6 + 1.1SGCs/ of 3D. The visual field (x 200) was reduced to the 3.3 + 0.3SGCs/ vision of 14d; the SGCs in group OEC-CM decreased from 35.3 + 2.1SGCs/ to 12.9 + 0.3SGCs/, and the surviving SGCs in the co culture group was significantly increased from the 66.9 + 1.2SGCs/ vision of 3D (x 200) to the 38.9 * 1 SGCs/ vision of 14d. Conclusion: OECs and co culture can promote The newborn rat SGCs survived.
The third part of the molecular mechanism of olfactory ensheathing cells promoting the survival of spiral ganglion cells.
Objective: to preliminarily study the molecular mechanism of OECs to promote the survival of SGCs in vitro. Methods: a co culture system of OECs and SGCs was established, and the experiment was divided into three groups: co culture + brain derived neurotrophic factor (BDNF, 500 pg/ml) group; co culture +BDNF antibody (IgY, 50 ug/ml); the control group was co cultured with OECs and SGCs. SGCs was identified by histochemical staining, 4 fields were taken randomly in each culture dish, and the SGCs of beta III -tubulin marked under fluorescence microscope was counted, and the results of SGCs. in each culture group were statistically analyzed. A large number of SGCs in the co culture +BDNF group grew on the surface of monolayer blanket formed by OECs, but the number of SCG was not statistically significant compared with the control group, and BDNF antibody (IgY) was added (IgY). The number of SGCs in the co culture group decreased significantly (P0.01). Conclusion: the addition of BDNF to the co culture of OECs and SGCs has no obvious effect on the survival of SGCs, but the survival of SGCs decreasing.OECs secreted BDNF after BDNF antibody (IgY) may be one of the molecular mechanisms for promoting the survival of SGCs in vitro culture.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329;R764
本文編號(hào)：2161853
[Abstract]:Part one: culture, purification and identification of olfactory ensheathing cells in vitro
Objective: to purify and cultivate the olfactory olfactory ensheathing cells (olfactory ensheathing cells OECs) of adult rats, and to prepare for the follow-up experiment. Methods: the olfactory bulb nerve layer of adult rats was taken and the mixed cell suspension containing OECs was obtained by enzyme digestion. OECs was purified by the method of multiple long time differential adherence, and DF12 culture containing 10%FBS was used. In the culture medium, bFGF (20ng/ml) and forskolin (2uM) were added to the medium of 2D to promote the proliferation of OECs. The culture medium was replaced every 3D and a half quantity. The growth state of OECs was observed under the inverted phase contrast microscope. P75NTR and GFAP cell immunohistochemistry were used to identify the OECs. The morphology of OECs was observed under the fluorescence microscope and the purity of the OECs was observed. Most of the contaminated cells, including fibroblasts and astrocytes, were removed, including fibroblasts and astrocytes. The OECs inoculated for about 24 hours was attached to the wall. The cell bodies of most cells were spindle shaped and some cells were polygons and protuberances. The cells grew slowly in the 2D before inoculation. After 2D, bFGF (20ng/ml) and forskolin (2uM) were added in the culture medium to accelerate the proliferation of cell proliferation, and the cell protruded long. After 7d, 90% of OECs covered dish bottom, and the cell monolayer.P75NTR and GFAP staining showed that about 92% of the cells were P75NTR (+), distributed in the protuberance, the nucleus was deeply dyed; about 12% of the cells were GFAP (+). Conclusion: the use of more (+). Conclusion: adopt more (+). "Conclusion: adopt more (+)." conclusion: adopt more (+). "Conclusion: adopt many (+). It is possible to obtain the purity of OECs for the experiment, and the addition of bFGF (20ng/ml) and forskolin (2uM) in the medium (DF12+10%FBS) can significantly promote the proliferation of OECs.
The effect of olfactory ensheathing cells on cochlear spiral ganglion cells in vitro culture in second parts
Objective: To explore the effect of OECs on the survival of the cochlear auditory afferent neurons (spiral ganglion cells SGCs) in vitro. Methods: the cultured rat olfactory bulb and the newborn rat worm axis tissue were cultured for OECs and SGCs, and the OECs. experiment was purified by multiple long time differential adherence methods. S and SGCs co culture group, SGCs in OECs conditioned medium (OEC-CM) culture group and SGCs single culture group. The growth state of OECs and SGCs was observed under the inverted phase contrast microscope. P75NTR immunohistochemical method was used to identify OECs, the neuron specific marker beta III -tubulin labeling SGCs. results: 7 days after the adherence wall culture, the cell monolayer was formed. In the culture system, SGCs grows on the surface of single cell monolayer formed by OECs and protruded long protrusions, showing a typical bipolar neuron form. In the pre culture 6D, with the prolongation of the incubation time, the SGCs is less than before the inoculation, but the survival number of SGCs in the co culture group is significantly higher than that of the SGCs single culture group (P0.01), and compared with SGCs in OEC-CM, The co culture group was more able to promote SGCs survival, and the number of SGCs in the individual culture group decreased significantly in the culture of 6D; in the cultured 9D, there was little SGCs growth in the individual culture group; but in the co culture group, the number of SGCs was not significantly changed (P0.05). The statistics found that the survival SGCs in the individual culture group was 14.6 + 1.1SGCs/ of 3D. The visual field (x 200) was reduced to the 3.3 + 0.3SGCs/ vision of 14d; the SGCs in group OEC-CM decreased from 35.3 + 2.1SGCs/ to 12.9 + 0.3SGCs/, and the surviving SGCs in the co culture group was significantly increased from the 66.9 + 1.2SGCs/ vision of 3D (x 200) to the 38.9 * 1 SGCs/ vision of 14d. Conclusion: OECs and co culture can promote The newborn rat SGCs survived.
The third part of the molecular mechanism of olfactory ensheathing cells promoting the survival of spiral ganglion cells.
Objective: to preliminarily study the molecular mechanism of OECs to promote the survival of SGCs in vitro. Methods: a co culture system of OECs and SGCs was established, and the experiment was divided into three groups: co culture + brain derived neurotrophic factor (BDNF, 500 pg/ml) group; co culture +BDNF antibody (IgY, 50 ug/ml); the control group was co cultured with OECs and SGCs. SGCs was identified by histochemical staining, 4 fields were taken randomly in each culture dish, and the SGCs of beta III -tubulin marked under fluorescence microscope was counted, and the results of SGCs. in each culture group were statistically analyzed. A large number of SGCs in the co culture +BDNF group grew on the surface of monolayer blanket formed by OECs, but the number of SCG was not statistically significant compared with the control group, and BDNF antibody (IgY) was added (IgY). The number of SGCs in the co culture group decreased significantly (P0.01). Conclusion: the addition of BDNF to the co culture of OECs and SGCs has no obvious effect on the survival of SGCs, but the survival of SGCs decreasing.OECs secreted BDNF after BDNF antibody (IgY) may be one of the molecular mechanisms for promoting the survival of SGCs in vitro culture.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329;R764
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