發(fā)布時(shí)間：2018-03-09 20:39

  本文選題：嗅鞘細(xì)胞　切入點(diǎn)：細(xì)胞培養(yǎng)　出處：《復(fù)旦大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分嗅鞘細(xì)胞的體外培養(yǎng)、純化、標(biāo)記及鑒定 目的：純化、培養(yǎng)、標(biāo)記和鑒定成年大鼠嗅球OECs,為后續(xù)實(shí)驗(yàn)做準(zhǔn)備。方法：取新生大鼠嗅球,去除包膜后,經(jīng)胰酶消化,獲得含有OECs的混合細(xì)胞懸液,采用兩次長(zhǎng)時(shí)差速貼壁的方法純化OECs,在含有bFGF、NGF、10%FBS的DF12中培養(yǎng),每隔2d半量更換培養(yǎng)基。倒置顯微鏡下觀察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, Hoechst33342標(biāo)記細(xì)胞核,熒光顯微鏡下觀察OECs的形態(tài)并統(tǒng)計(jì)純度。結(jié)果：嗅球消化后獲得的細(xì)胞懸液經(jīng)過(guò)兩次長(zhǎng)時(shí)差速貼壁后去除了絕大部分的污染細(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,倒置顯微鏡下觀察見大部分細(xì)胞胞體呈現(xiàn)梭形,部分細(xì)胞胞體為多角形,伸出突起,培養(yǎng)7d后見OECs覆蓋皿底的90%,形成一細(xì)胞單層。P75NTR和GFAP染色見約92%的細(xì)胞染色陽(yáng)性,分布在突起、胞體；Hoechst 33342標(biāo)記率幾乎可以達(dá)到100%,可見細(xì)胞核染成藍(lán)色。結(jié)論：采用兩次長(zhǎng)時(shí)差速貼壁的方法能夠獲得實(shí)驗(yàn)所需純度的OECs。 第二部分成年大鼠硫酸卡那霉素耳毒性模型的建立 目的：建立成年大鼠硫酸卡那霉素耳毒性模型,為后續(xù)實(shí)驗(yàn)做準(zhǔn)備。方法：6-7周齡雄性SD大鼠60只,隨機(jī)分為3組：實(shí)驗(yàn)組(1),腹腔注射KM,500 mg/Kg perd (100mg/ml)2周；實(shí)驗(yàn)組(2)腹腔注射KM,400 mg/Kg per d (80mg/ml)2周；對(duì)照組,注射等量生理鹽水2周。采用ABR的方法觀察大鼠聽力變化,基底膜鋪片觀察毛細(xì)胞形態(tài)及數(shù)量變化,耳蝸冰凍切片觀察SGCs的密度及形態(tài)學(xué)變化。結(jié)果：實(shí)驗(yàn)組(1)注射KM 2周后,大鼠在各頻率的聽覺(jué)閾值均有明顯升高,其上升幅度超過(guò)60 dB；實(shí)驗(yàn)組(2)注射KM 2周后,各頻率ABR閾值較注射前上升幅度超過(guò)20dB,特別是在32 KHz時(shí),其上升幅度超過(guò)了30dB。實(shí)驗(yàn)組(1)與實(shí)驗(yàn)組(2)耳蝸細(xì)胞病變類似,只是前者較后者嚴(yán)重。隨著時(shí)間推移,SGCs密度逐漸降低,corti器結(jié)構(gòu)尚存,但OHCs及IHCs均有不同程度的缺失,以O(shè)HCs為甚。結(jié)論：6-7周齡大鼠腹腔注射KM 400 mg/Kg per d 2周比較適合OECs移植。 第三部分OECs對(duì)損傷SGCs保護(hù)和修復(fù)作用的研究 目的：觀察OECs移植對(duì)損傷大鼠SGCs的保護(hù)和修復(fù)作用。方法：將培養(yǎng)7d后的OECs進(jìn)行核標(biāo)記,以1×106個(gè)/ml密度懸浮,移植到腹腔注射KM 400 mg/Kg per d2周的實(shí)驗(yàn)組成年大鼠耳蝸內(nèi),對(duì)照組耳蝸?zhàn)⑸涞攘康腄-Hanks液,以ABR大鼠聽力改變,HE染色和免疫熒光化學(xué)染色觀察SGCs的密度及形態(tài)學(xué)改變。結(jié)果：對(duì)照組與實(shí)驗(yàn)組的ABR閾值在各頻率無(wú)明顯差異；實(shí)驗(yàn)組熒光顯微鏡下見大量Hoechst 33342標(biāo)記的藍(lán)色細(xì)胞核,分布在耳蝸底旋到頂旋；實(shí)驗(yàn)組SGCs密度與對(duì)照組有明顯差異,大部分形態(tài)正常,核膜清晰,核中染色質(zhì)清晰,核仁亦較清晰,排列尚緊密,少部分細(xì)胞核染色質(zhì)邊集,壞死,密度較正常大鼠SGCs明顯降低；對(duì)照組SGCs大量壞死,部分細(xì)胞核結(jié)構(gòu)被吞噬細(xì)胞吞噬,空泡化,細(xì)胞排列疏松、混亂,僅見少量正常的SGCs,免疫熒光見對(duì)照組有大量壞死細(xì)胞的碎片。結(jié)論：OECs可能促進(jìn)SGCs的存活,對(duì)損傷大鼠SGCs有保護(hù)作用。
[Abstract]:In vitro culture, purification, labeling and identification of olfactory ensheathing cells in vitro
Objective: to purify, culture, marking and identification of adult rat olfactory bulb OECs, preparing for the subsequent experiments. Methods: the newborn rat olfactory bulb, removed after coating, obtained by trypsin digestion, mixed cell suspension containing OECs, purification of OECs using the method of two long time differential adherent, in containing bFGF NGF 10%FBS DF 12 2D training, every half amount of medium change. The growth state of OECs was observed under an inverted microscope, identification of OECs using P75NTR and GFAP cells by immunohistochemistry, Hoechst33342 nuclear staining, morphological observation and statistics of purity of OECs by fluorescence microscopy. Results: olfactory bulb digested cell suspension after two long time adherent after the removal of the vast pollution most of the cells, including fibroblasts and astrocytes, inoculated with OECs about 24 hours after the adherent, inverted microscope observed that most cells showed fusiform shape, the Ministry of The part of the cell body is a polygonal protruding, after 7d see OECs cover plate at the end of 90%, the formation of a monolayer of.P75NTR and GFAP staining showed that about 92% of the cells were positive, distribution in neurites, cell body; Hoechst 33342 labeling rate can reach 100%, visible nuclei were stained blue. Conclusion: using the method of two long differential adherent experiment can obtain the required purity of OECs.
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