本文選題：耳蝸 + 雪旺細(xì)胞　； 參考：《第四軍醫(yī)大學(xué)》2012年碩士論文

【摘要】：感音神經(jīng)性聾是臨床工作中最常見(jiàn)的疾病，其發(fā)病機(jī)理和治療方法仍在不斷的探索中，目前認(rèn)為主要病變部位在初級(jí)傳入螺旋神經(jīng)元（SGN）和耳蝸毛細(xì)胞。哺乳動(dòng)物螺旋神經(jīng)元和耳蝸毛細(xì)胞損傷后幾乎不能再生，那么如何修復(fù)受損的SGN，如何使SGN增強(qiáng)在復(fù)雜的病理狀態(tài)下的修復(fù)能力，如何使SGN在損傷后的特殊微環(huán)境里提高其存活率是感音神經(jīng)性聾的研究方向之一。雪旺細(xì)胞是從胚胎時(shí)期周圍神經(jīng)系統(tǒng)的神經(jīng)嵴前體細(xì)胞演化而來(lái)的一種膠質(zhì)細(xì)胞。周圍神經(jīng)受損后其遠(yuǎn)端軸突和髓鞘崩解,此時(shí)雪旺細(xì)胞可以大量增殖并且吞噬遠(yuǎn)端軸突和崩解的變性壞死的碎片。雪旺細(xì)胞還可以分泌神經(jīng)生長(zhǎng)因子NGF和神經(jīng)營(yíng)養(yǎng)因子NTF，因此雪旺細(xì)胞具有可以增加神經(jīng)元的存活率、促進(jìn)細(xì)胞軸突生長(zhǎng)和提高神經(jīng)元再生的能力。大量研究顯示，移植雪旺細(xì)胞能夠促進(jìn)脊髓、周圍神經(jīng)損傷的修復(fù)并具有加快中樞神經(jīng)系統(tǒng)脫髓鞘性疾病功能恢復(fù)的作用。然而，目前尚無(wú)關(guān)于耳蝸雪旺細(xì)胞和螺旋神經(jīng)元軸突的相互作用機(jī)制的研究報(bào)道和關(guān)于耳蝸雪旺細(xì)胞與外周神經(jīng)系統(tǒng)的其他雪旺細(xì)胞之間的相似性和差異性的報(bào)道。而弄清楚以上問(wèn)題的關(guān)鍵在于純化和定性耳蝸雪旺細(xì)胞。所以本研究致力于進(jìn)行大鼠仔鼠耳蝸雪旺細(xì)胞的體外培養(yǎng)、純化的試驗(yàn)，并配制一種能使耳蝸雪旺細(xì)胞快速增殖的培養(yǎng)液配方。 實(shí)驗(yàn)一大鼠耳蝸雪旺細(xì)胞體外培養(yǎng)和純化 選用1-3d SD大鼠，無(wú)菌條件下暴露雙側(cè)聽(tīng)泡，在高倍鏡下仔細(xì)剝離蝸殼，開(kāi)放耳蝸，完整取出耳蝸組織，分離并且除去膜蝸管外側(cè)壁的血管紋和基底膜組織，，然后剪碎。用0.25%的胰蛋白酶消化，用胎牛血清中止消化，離心以后加入DMEM/F12培養(yǎng)液培養(yǎng)。3-5天后對(duì)細(xì)胞應(yīng)用免疫磁珠陽(yáng)性分選方法進(jìn)行純化，培養(yǎng)2天后進(jìn)行傳代接種，培養(yǎng)過(guò)程中對(duì)提純后的大鼠耳蝸雪旺細(xì)胞進(jìn)行形態(tài)學(xué)觀察、并繪制其生長(zhǎng)曲線，采用細(xì)胞免疫熒光染色對(duì)細(xì)胞進(jìn)行S-100免疫熒光鑒定并且計(jì)算細(xì)胞純度。結(jié)果：分離培養(yǎng)后所得的細(xì)胞即為雪旺細(xì)胞；利用免疫磁珠陽(yáng)性分選法對(duì)培養(yǎng)所得的細(xì)胞進(jìn)行純化，純化后的大鼠耳蝸雪旺細(xì)胞純度為97％±1.2％。結(jié)論：免疫磁珠法是一種有效的分離純化新生大鼠仔鼠耳蝸雪旺細(xì)胞的方法。所得雪旺細(xì)胞活力強(qiáng)、純度高，可以用于耳蝸雪旺細(xì)胞與螺旋神經(jīng)節(jié)軸突的生長(zhǎng)和再生等相關(guān)研究。 實(shí)驗(yàn)二耳蝸雪旺細(xì)胞增殖的試驗(yàn) 取生長(zhǎng)良好的第三代經(jīng)免疫磁珠分離純化的耳蝸雪旺細(xì)胞，消化后制成細(xì)胞濃度為1×10~4/ml的單細(xì)胞懸液，接種在96孔板上，每塊6×12孔，每孔加入200μl細(xì)胞懸液，共接種10個(gè)樣本。將硫酸乙酰肝素蛋白多糖HSPG和堿性成纖維細(xì)胞生長(zhǎng)因子bFGF配置成不同濃度的的培養(yǎng)液。37℃，5%CO_2培養(yǎng)，每日固定時(shí)間取一塊板，連續(xù)取10天，用激發(fā)波長(zhǎng)為490nm的酶聯(lián)免疫測(cè)試儀測(cè)吸光度值，計(jì)算當(dāng)天各組A值均數(shù)，以光吸收值為縱軸，時(shí)間為橫軸，繪制六組耳蝸雪旺細(xì)胞的生長(zhǎng)曲線。結(jié)果：B、C、D、E、F組優(yōu)于空白對(duì)照組A，有統(tǒng)計(jì)學(xué)意義（P0.05）；B、C、D組之間無(wú)明顯差別，無(wú)統(tǒng)計(jì)學(xué)意義；E、F組優(yōu)于B、C、D組，有統(tǒng)計(jì)學(xué)意義；E、F組之間無(wú)明顯差別，無(wú)統(tǒng)計(jì)學(xué)意義。這說(shuō)明用含硫酸乙酰肝素蛋白多糖HSPG和堿性成纖維細(xì)胞生長(zhǎng)因子bFGF的細(xì)胞培養(yǎng)液能使耳蝸雪旺細(xì)胞快速增殖,其中50ng/mlHSPG+50ng/mlbFGF生長(zhǎng)最旺盛，A、B、C、D四組在第7天達(dá)到分裂頂峰，E、F組在第9天達(dá)到分裂頂峰。
[Abstract]:Sensorineural deafness is the most common disease in clinical work. Its pathogenesis and treatment methods are still in constant exploration. At present, the main lesions are found in primary afferent spiral neurons (SGN) and cochlear hair cells. It is almost impossible to regenerate mammalian spiral neurons and cochlear hair cells, and how to repair damaged SGN, How to enhance the ability to repair SGN in a complex pathological state and how to improve the survival rate of SGN in a special microenvironment after injury is one of the research directions for sensorineural hearing loss. Schwann cells are a glial cell evolved from the neural crest precursor cells from the peripheral nervous system in the embryonic period. With the disintegration of the distal axon and myelin sheath, Schwann cells can proliferate and phagocytic fragments of the distal axons and disintegrating degeneration and necrosis. Schwann cells can also secrete nerve growth factor NGF and neurotrophic factor NTF. Therefore, Schwann cells can increase the survival rate of neurons, promote cell axon growth and increase neurons. A large number of studies have shown that the transplantation of Schwann cells can promote the repair of spinal cord, peripheral nerve damage and accelerate the function recovery of the demyelinating disease of the central nervous system. However, there is no research report on the interaction mechanism of cochlear Schwann cells and spiral neuron axons and the cochlear Schwann cells. The similarity and difference between the other Schwann cells of the peripheral nervous system is reported. The key to the above problem is to purify and determine the cochlear Schwann cells. Therefore, this study is devoted to the culture and purification of the cochlear Schwann cells in rat cochlea, and to make a rapid proliferation of the cochlear Schwann cells. The formula of culture medium.
In vitro culture and purification of rat Schwann cells from cochlea
1-3D SD rats were exposed to bilateral auditory vesicles under sterile conditions. The cochlea was carefully stripped under high magnification, cochlear was opened and the cochlear tissue was removed completely. The vascular and basal membrane tissues of the outer wall of the cochlear tube were separated and removed, and then shredded. Digestion with 0.25% trypsin, fetal bovine serum digestion and DMEM/F12 culture after centrifugation were added. After culture.3-5 days, the immunomagnetic beads positive separation method was purified and cultured for 2 days. During the culture, the cultured rat cochlear Schwann cells were observed, and the growth curves were plotted. Cell immunofluorescence staining was used to identify the cells by S-100 immunofluorescence and to calculate the cell purity. Results: the cells obtained from the isolation and culture were Schwann cells, and the cultured cells were purified by immunomagnetic beads positive separation. The purity of the purified cochlear Schwann cells after purification was 97% + 1.2%. conclusion: the immunomagnetic bead method was an effective method to separate and purify the cochlear Schwann cells of newborn rat cochlea. The vitality and purity of the cells are strong and can be used for the study of the growth and regeneration of the Schwann cells and the spiral ganglion axons.
Experiment two the proliferation of Schwann cells in the cochlea
A well grown third generation of cochlear Schwann cells were isolated and purified by immunomagnetic beads. After digestion, a single cell suspension with a concentration of 1 x 10~4/ml was prepared, inoculated on 96 orifice plates, each 6 x 12 holes, and 200 mu L cell suspension was added to each hole, and 10 samples were inoculated. The heparan sulfate proteoglycan HSPG and basic fibroblast growth factor bFG were taken. F was formed into a medium of different concentration of.37, 5%CO_2 culture and a plate for 10 days at a fixed time. The absorbance values were measured by an enzyme linked immunosorbent assay with the excitation wavelength of 490nm. The average number of A values in each group was calculated on the same day. The growth curve of six groups of cochlear Schwann cells was drawn with light absorption value as a longitudinal axis and a horizontal axis. Results: B, C, D, E, F group was superior to blank control group A, with statistical significance (P0.05); there was no significant difference between group B, C and D, E, F group was superior to B, C, D group, with statistical significance; E, there was no significant difference between B, C, D group. This shows that the cell culture with the egg white polysaccharide sulfate containing heparin sulfate and basic fibroblast growth factor The liquid can rapidly proliferate the cochlear Schwann cells, in which the 50ng/mlHSPG+50ng/mlbFGF growth is the most vigorous. The four groups of A, B, C, D reach the peak of division at seventh days, E, and the F group reach the split peak at ninth days.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764.43

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相關(guān)期刊論文 前3條

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