海馬放射狀膠質(zhì)細(xì)胞的體外誘導(dǎo)激活及向膽堿能神經(jīng)元的分化
發(fā)布時(shí)間：2019-05-24 17:27
【摘要】：第一部分：海馬放射狀膠質(zhì)細(xì)胞的體外誘導(dǎo)激活及其神經(jīng)干細(xì)胞樣特性 目的探討海馬放射狀膠質(zhì)細(xì)胞在切割穹窿海馬傘去神經(jīng)支配海馬提取液誘導(dǎo)下的形態(tài)學(xué)變化以及細(xì)胞增殖、胚胎源性和向神經(jīng)元及膠質(zhì)細(xì)胞分化的神經(jīng)干細(xì)胞樣特性。 方法取生后1 d的SD大鼠海馬組織,應(yīng)用差速貼壁及搖床振蕩法分離純化海馬放射狀膠質(zhì)細(xì)胞。將純化的海馬放射狀膠質(zhì)細(xì)胞接種于24孔培養(yǎng)板中,分成切割組和正常組,切割組加入含5%(v/v)切割穹窿海馬傘去神經(jīng)支配海馬提取液的DMEM/F12培養(yǎng)液,正常組加入含5%(v/v)正常側(cè)海馬提取液的DMEM/F12培養(yǎng)液,同時(shí)在兩組中加入5-溴-2-脫氧尿嘧啶(BrdU)標(biāo)記增殖的細(xì)胞,并分別于培養(yǎng)后1、3、7和14 d時(shí)行BLBP免疫熒光檢測(cè)和Hoechst標(biāo)記；同時(shí)用BLBP/BrdU、BLBP/nestin、BLBP/MAP-2、BLBP/GFAP、BLBP/CNP免疫熒光雙標(biāo)技術(shù)觀察其增殖特性和胚胎源性以及向神經(jīng)元、星型膠質(zhì)細(xì)胞、少突膠質(zhì)細(xì)胞分化的情況,檢測(cè)BrdU陽(yáng)性細(xì)胞占BLBP陽(yáng)性細(xì)胞的百分比、BLBP陽(yáng)性細(xì)胞的周長(zhǎng)和面積,以及兩組中BLBP/nestin、BLBP/MAP-2、BLBP/GFAP、BLBP/CNP雙標(biāo)細(xì)胞占BLBP陽(yáng)性細(xì)胞的百分比。應(yīng)用Stata10.0統(tǒng)計(jì)軟件行組間比較。 結(jié)果BLBP免疫熒光檢測(cè)結(jié)果顯示,通過(guò)上述的純化培養(yǎng)方法,我們獲得了幾近100%的海馬放射狀膠質(zhì)細(xì)胞,BLBP在細(xì)胞漿、細(xì)胞核及突起中均有表達(dá)。切割組BrdU陽(yáng)性細(xì)胞占BLBP陽(yáng)性細(xì)胞的百分比明顯高于正常組(切割組：56.86±8.52%；正常組：31.11±4.28%,P0.01)。接種1 d后,兩組細(xì)胞的胞體均較小,突起較短較細(xì)；3 d時(shí),與正常組相比,切割組BLBP陽(yáng)性細(xì)胞的胞體稍變大,突起稍變得粗而長(zhǎng)；7 d時(shí),與正常組相比,切割組BLBP陽(yáng)性細(xì)胞的胞體明顯變大,突起明顯變粗、變長(zhǎng)且交織成網(wǎng)狀；14 d時(shí)兩組細(xì)胞的胞體和突起均開始變細(xì)變短,正常組較為明顯。兩組各d BLBP陽(yáng)性細(xì)胞的周長(zhǎng)和面積的統(tǒng)計(jì)分析結(jié)果表明除1 d時(shí)兩組BLBP陽(yáng)性細(xì)胞的周長(zhǎng)和面積無(wú)明顯差異(P0.05)外,其余各d切割組BLBP陽(yáng)性細(xì)胞的周長(zhǎng)和面積均高于正常組(P0.01)。切割組nestin陽(yáng)性細(xì)胞占BLBP陽(yáng)性細(xì)胞的百分比也明顯高于正常組(切割組：57.92±17.93%；正常組：23.26±9.85%,P0.01)。并可見切割組中較多的BLBP陽(yáng)性細(xì)胞向MAP-2陽(yáng)性的神經(jīng)元分化(切割組：46.13±14.92%；正常組：29.13±10.07%,P0.05),雖然兩組向GFAP陽(yáng)性的星型膠質(zhì)細(xì)胞和CNP陽(yáng)性的少突膠質(zhì)細(xì)胞分化在數(shù)量上無(wú)明顯差異,但切割組中兩種膠質(zhì)細(xì)胞的胞體明顯增大、突起明顯豐富。 結(jié)論切割穹窿海馬傘去神經(jīng)支配海馬提取液不僅可以明顯誘導(dǎo)BLBP陽(yáng)性放射狀膠質(zhì)細(xì)胞增殖、胞體明顯增大、突起變粗變長(zhǎng)呈“激活”狀態(tài),具胚胎源性,而且還可使其向神經(jīng)元、星型膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞分化,表現(xiàn)為神經(jīng)干細(xì)胞樣特性。 第二部分：去神經(jīng)支配海馬提取液誘導(dǎo)海馬放射狀膠質(zhì)細(xì)胞向膽堿能神經(jīng)元的分化 目的在體外細(xì)胞培養(yǎng)中加入切割穹窿海馬傘去神經(jīng)支配海馬提取液,模擬在體海馬神經(jīng)再生微環(huán)境,觀察海馬放射狀膠質(zhì)細(xì)胞向膽堿能神經(jīng)元分化的情況。 方法將純化的海馬放射狀膠質(zhì)細(xì)胞分成切割組和正常組,切割組加入含5%(v/v)切割穹窿海馬傘去神經(jīng)支配海馬提取液的DMEM/F12培養(yǎng)液,正常組加入含5%(v/v)正常側(cè)海馬提取液的DMEM/F12培養(yǎng)液,分別在培養(yǎng)7d后應(yīng)用BLBP/ChAT免疫熒光雙標(biāo)、Real-time PCR及Western blot等技術(shù)觀察兩組放射狀膠質(zhì)細(xì)胞向膽堿能神經(jīng)元的分化,以及表達(dá)ChAT mRNA和蛋白的情況。 結(jié)果ChAT免疫熒光結(jié)果顯示,加入切割穹窿海馬傘側(cè)海馬提取液后,與正常組相比,切割組中ChAT陽(yáng)性的膽堿能神經(jīng)元占BLBP陽(yáng)性細(xì)胞的比例(41.62±9.97%)明顯多于正常組(16.08±7.31%) (P0.01),且切割組中ChAT陽(yáng)性的膽堿能神經(jīng)元的胞體較正常組明顯增大,突起明顯增粗增長(zhǎng)。切割組ChAT mRNA水平明顯高于正常組,約是正常組的5倍(P0.01)。ChAT蛋白的表達(dá)量雖較低,但切割組(0.1141±0.0380)仍高于正常組(0.0423±0.0106)(P0.05)。 結(jié)論切割穹窿海馬傘側(cè)海馬提取液可明顯誘導(dǎo)BLBP陽(yáng)性放射狀膠質(zhì)細(xì)胞向膽堿能神經(jīng)元分化。
[Abstract]:The first part: the in vitro induction activation of the radial glial cells in the hippocampus and its neural stem cell-like characteristics Objective To study the morphological changes and the increase of the cells in the hippocampus of the hippocampus of the hippocampus from the hippocampus of the hippocampus. Neural stem cell samples for the differentiation of colonizing, embryonic-derived and neuron-and glial cells Sex. The rat hippocampal tissue was isolated and purified by differential malapposition and shaking method in the hippocampus of SD rats. culturing the purified hippocampal radial glial cells in a 24-well culture plate, dividing into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole hippocampal umbrella to the neurodominant hippocampus extract, and adding DMEM/ F12 containing 5% (v/ v) normal-side hippocampal extract in the normal group; The cells were labeled with 5-bromo-2-deoxy-1-detrusor (BrdU) in both groups. The BLBP/ BrdU, BLBP/ nestin, BLBP/ MAP-2, BLBP/ G were also used. The proliferation and embryo-origin of the BLBP-positive cells, the percentage of BLBP-positive cells, the perimeter and the area of the BLBP-positive cells and the BLBP/ nestin, BLBP/ MAP-2 and BLBP/ G in the two groups were examined by means of the two-standard technique, such as FAP, BLBP/ CNP. FAP, BLBP/ CNP double-labeled cells account for BLBP positive cells %. Applying the Staa10.0 Statistics Software Line Group The results of the results of the BLBP immunofluorescence test show that, by the above-mentioned purification and culture method, we obtained nearly 100% of the hippocampal radial glial cells, and the BLBP is in the cytoplasm, nucleus and protrusion of the cells. The percentage of BrdU positive cells in the cut group was significantly higher than that in the normal group (56.86-8.52%, normal group: 31.11-4.28%, P 0.01) After 1 day of inoculation, the cell bodies of the two groups were small and the protrusions were shorter and thinner; when compared with the normal group, the cells of the BLBP positive cells of the cut group were slightly larger and the protrusions were slightly thicker and longer; at the time of 7 d, the cells of the BLBP positive cells of the cut group were compared with the normal group. The body of the two groups of cells began to become thinner and shorter, and the body and the protrusion of the two groups of cells started to become shorter and normal at the time of 14 d. The results of the statistical analysis of the perimeter and area of each d BLBP positive cells in the two groups showed no significant difference in the circumference and the area of the BLBP positive cells in the two groups (P <0.05), and the perimeter and area of the BLBP positive cells in the remaining d groups were higher than that in the normal group (P The percentage of nestin positive cells in the cut group was significantly higher than that in the normal group (group: 57.92-17.93%, normal group: 23.26-9.85%, P 0.01). It was found that more of the BLBP positive cells in the cut group were differentiated into the MAP-2-positive neurons (group: 46.13-14.92%, normal group: 29.13-10.07%, P0.05). There was no significant difference, but the cell bodies of the two kinds of glial cells in the cutting group were obviously increased, and the process of the process Conclusion The removal of the hippocampal extract from the hippocampus of the rat hippocampus can not only obviously induce the proliferation of the BLBP-positive radial glial cells, the cell body is obviously increased, the growth of the protrusion becomes the "activate" state, the embryo-derived, but also can differentiate into neurons, astrocytes, and oligodendrocytes and act as a god Stem cell-like properties. Part 2: The removal of the hippocampal extract from the hippocampus in the innervation of the hippocampal extract. The purpose of the differentiation of cholinergic neurons in the in vitro cell culture is to add a cut hole in the hippocampus of the hippocampus to innervate the hippocampal extract, to simulate In that microenvironment of the neural regeneration in the hippocampus of the body, the radial glial cells of the hippocampus were observe. The method of the invention comprises the following steps of: dividing the purified hippocampal radial glial cells into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole in the hippocampus of the hippocampus, and adding 5% (v/ v) normal-side hippocampal extraction to the normal group; The differentiation of the two groups of radial glial cells to the cholinergic neurons was observed by BLBP/ ChAT immunofluorescence double-label, Real-time PCR and Western blot. Results The results showed that the proportion of ChAT-positive cholinergic neurons in the cut group (41.62% 9.97%) was significantly higher than that of the normal group (16.0%) compared with the normal group (16.0%). 8 (7.31%) (P0.01), and the cells of ChAT-positive cholinergic neurons in the cut group were positive The expression of ChAT mRNA in the cutting group was significantly higher than that in the normal group (P0.01). The expression of ChAT protein was lower, but the cleavage group (0.1141-0.0380) was still higher than that in the normal group (0.0423. Conclusion: It is concluded that the extract of the hippocampus on the side of the hippocampus can induce BLBP-yang obviously.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
本文編號(hào)：2485044
[Abstract]:The first part: the in vitro induction activation of the radial glial cells in the hippocampus and its neural stem cell-like characteristics Objective To study the morphological changes and the increase of the cells in the hippocampus of the hippocampus of the hippocampus from the hippocampus of the hippocampus. Neural stem cell samples for the differentiation of colonizing, embryonic-derived and neuron-and glial cells Sex. The rat hippocampal tissue was isolated and purified by differential malapposition and shaking method in the hippocampus of SD rats. culturing the purified hippocampal radial glial cells in a 24-well culture plate, dividing into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole hippocampal umbrella to the neurodominant hippocampus extract, and adding DMEM/ F12 containing 5% (v/ v) normal-side hippocampal extract in the normal group; The cells were labeled with 5-bromo-2-deoxy-1-detrusor (BrdU) in both groups. The BLBP/ BrdU, BLBP/ nestin, BLBP/ MAP-2, BLBP/ G were also used. The proliferation and embryo-origin of the BLBP-positive cells, the percentage of BLBP-positive cells, the perimeter and the area of the BLBP-positive cells and the BLBP/ nestin, BLBP/ MAP-2 and BLBP/ G in the two groups were examined by means of the two-standard technique, such as FAP, BLBP/ CNP. FAP, BLBP/ CNP double-labeled cells account for BLBP positive cells %. Applying the Staa10.0 Statistics Software Line Group The results of the results of the BLBP immunofluorescence test show that, by the above-mentioned purification and culture method, we obtained nearly 100% of the hippocampal radial glial cells, and the BLBP is in the cytoplasm, nucleus and protrusion of the cells. The percentage of BrdU positive cells in the cut group was significantly higher than that in the normal group (56.86-8.52%, normal group: 31.11-4.28%, P 0.01) After 1 day of inoculation, the cell bodies of the two groups were small and the protrusions were shorter and thinner; when compared with the normal group, the cells of the BLBP positive cells of the cut group were slightly larger and the protrusions were slightly thicker and longer; at the time of 7 d, the cells of the BLBP positive cells of the cut group were compared with the normal group. The body of the two groups of cells began to become thinner and shorter, and the body and the protrusion of the two groups of cells started to become shorter and normal at the time of 14 d. The results of the statistical analysis of the perimeter and area of each d BLBP positive cells in the two groups showed no significant difference in the circumference and the area of the BLBP positive cells in the two groups (P <0.05), and the perimeter and area of the BLBP positive cells in the remaining d groups were higher than that in the normal group (P The percentage of nestin positive cells in the cut group was significantly higher than that in the normal group (group: 57.92-17.93%, normal group: 23.26-9.85%, P 0.01). It was found that more of the BLBP positive cells in the cut group were differentiated into the MAP-2-positive neurons (group: 46.13-14.92%, normal group: 29.13-10.07%, P0.05). There was no significant difference, but the cell bodies of the two kinds of glial cells in the cutting group were obviously increased, and the process of the process Conclusion The removal of the hippocampal extract from the hippocampus of the rat hippocampus can not only obviously induce the proliferation of the BLBP-positive radial glial cells, the cell body is obviously increased, the growth of the protrusion becomes the "activate" state, the embryo-derived, but also can differentiate into neurons, astrocytes, and oligodendrocytes and act as a god Stem cell-like properties. Part 2: The removal of the hippocampal extract from the hippocampus in the innervation of the hippocampal extract. The purpose of the differentiation of cholinergic neurons in the in vitro cell culture is to add a cut hole in the hippocampus of the hippocampus to innervate the hippocampal extract, to simulate In that microenvironment of the neural regeneration in the hippocampus of the body, the radial glial cells of the hippocampus were observe. The method of the invention comprises the following steps of: dividing the purified hippocampal radial glial cells into a cutting group and a normal group, adding a DMEM/ F12 culture solution containing 5% (v/ v) cutting a hole-hole in the hippocampus of the hippocampus, and adding 5% (v/ v) normal-side hippocampal extraction to the normal group; The differentiation of the two groups of radial glial cells to the cholinergic neurons was observed by BLBP/ ChAT immunofluorescence double-label, Real-time PCR and Western blot. Results The results showed that the proportion of ChAT-positive cholinergic neurons in the cut group (41.62% 9.97%) was significantly higher than that of the normal group (16.0%) compared with the normal group (16.0%). 8 (7.31%) (P0.01), and the cells of ChAT-positive cholinergic neurons in the cut group were positive The expression of ChAT mRNA in the cutting group was significantly higher than that in the normal group (P0.01). The expression of ChAT protein was lower, but the cleavage group (0.1141-0.0380) was still higher than that in the normal group (0.0423. Conclusion: It is concluded that the extract of the hippocampus on the side of the hippocampus can induce BLBP-yang obviously.
【學(xué)位授予單位】：南通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 金國(guó)華,張新化,田美玲,秦建兵,黃鎮(zhèn),徐慧君;穹窿海馬傘切割側(cè)海馬提取液對(duì)神經(jīng)干細(xì)胞分化為神經(jīng)元的促進(jìn)作用[J];解剖學(xué)報(bào);2004年02期
2 朱蕙霞,陳蓉,金國(guó)華,秦建兵,田美玲;非變性聚丙烯酰胺凝膠電泳在大鼠海馬組織蛋白分離中的應(yīng)用[J];南通醫(yī)學(xué)院學(xué)報(bào);2003年04期
3 朱蕙霞;秦建兵;田美玲;陳蓉;金國(guó)華;;切割海馬傘海馬中56kD差異蛋白的質(zhì)譜分析[J];南通大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2006年06期
4 趙善廷,鄧錦波;大鼠海馬結(jié)構(gòu)的組成、細(xì)胞類型及神經(jīng)纖維聯(lián)系 Ⅰ.齒狀回[J];神經(jīng)解剖學(xué)雜志;1999年01期
5 龍大宏,楊丹迪,李佳楣,許孟杰,汪華僑;穹窿海馬傘損傷對(duì)大鼠學(xué)習(xí)記憶和海馬GFAP陽(yáng)性神經(jīng)膠質(zhì)細(xì)胞的影響[J];神經(jīng)解剖學(xué)雜志;2002年01期
6 金國(guó)華,張新化,田美玲,黃鎮(zhèn),秦建兵,徐慧君;大鼠海馬內(nèi)移植神經(jīng)干細(xì)胞的存活和遷移[J];神經(jīng)解剖學(xué)雜志;2003年04期
7 張新化,金國(guó)華,秦建兵,田美玲,黃鎮(zhèn),徐慧君;穹窿海馬傘切割側(cè)海馬對(duì)植入神經(jīng)干細(xì)胞分化為神經(jīng)元的影響[J];神經(jīng)解剖學(xué)雜志;2004年04期
8 陳蓉,金國(guó)華,田美玲,朱蕙霞,秦建兵,譚雪鋒,徐慧君;海馬中56kD蛋白誘導(dǎo)人神經(jīng)干細(xì)胞遷移的作用[J];神經(jīng)解剖學(xué)雜志;2005年04期
9 金國(guó)華;陳蓉;田美玲;秦建兵;譚雪鋒;朱蕙霞;徐慧君;;海馬中56 kD蛋白誘導(dǎo)人神經(jīng)干細(xì)胞向神經(jīng)元分化的作用[J];神經(jīng)解剖學(xué)雜志;2006年04期
10 徐璐璐;王磊;衣昕;秦建兵;田美玲;金國(guó)華;;切割穹窿海馬傘大鼠海馬內(nèi)BLBP的表達(dá)變化[J];神經(jīng)解剖學(xué)雜志;2008年06期
，本文編號(hào)：2485044
本文鏈接：http://sikaile.net/yixuelunwen/shiyanyixue/2485044.html
最近更新
教材專著
