嗅鞘细胞促损伤大鼠耳蜗螺旋神经节细胞保护和修复作用的实验研究

发布时间：2018-03-09 20:39

  本文选题：嗅鞘细胞　切入点：细胞培养　出处：《复旦大学》2011年硕士论文　论文类型：学位论文

【摘要】：第一部分嗅鞘细胞的体外培养、纯化、标记及鉴定 目的：纯化、培养、标记和鉴定成年大鼠嗅球OECs,为后续实验做准备。方法：取新生大鼠嗅球,去除包膜后,经胰酶消化,获得含有OECs的混合细胞悬液,采用两次长时差速贴壁的方法纯化OECs,在含有bFGF、NGF、10%FBS的DF12中培养,每隔2d半量更换培养基。倒置显微镜下观察OECs的生长状态,采用P75NTR和GFAP细胞免疫组化的方法鉴定OECs, Hoechst33342标记细胞核,荧光显微镜下观察OECs的形态并统计纯度。结果：嗅球消化后获得的细胞悬液经过两次长时差速贴壁后去除了绝大部分的污染细胞,包括成纤维细胞和星形胶质细胞,接种的OECs约24小时后贴壁,倒置显微镜下观察见大部分细胞胞体呈现梭形,部分细胞胞体为多角形,伸出突起,培养7d后见OECs覆盖皿底的90%,形成一细胞单层。P75NTR和GFAP染色见约92%的细胞染色阳性,分布在突起、胞体；Hoechst 33342标记率几乎可以达到100%,可见细胞核染成蓝色。结论：采用两次长时差速贴壁的方法能够获得实验所需纯度的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,为后续实验做准备。方法：6-7周龄雄性SD大鼠60只,随机分为3组：实验组(1),腹腔注射KM,500 mg/Kg perd (100mg/ml)2周；实验组(2)腹腔注射KM,400 mg/Kg per d (80mg/ml)2周；对照组,注射等量生理盐水2周。采用ABR的方法观察大鼠听力变化,基底膜铺片观察毛细胞形态及数量变化,耳蜗冰冻切片观察SGCs的密度及形态学变化。结果：实验组(1)注射KM 2周后,大鼠在各频率的听觉阈值均有明显升高,其上升幅度超过60 dB；实验组(2)注射KM 2周后,各频率ABR阈值较注射前上升幅度超过20dB,特别是在32 KHz时,其上升幅度超过了30dB。实验组(1)与实验组(2)耳蜗细胞病变类似,只是前者较后者严重。随着时间推移,SGCs密度逐渐降低,corti器结构尚存,但OHCs及IHCs均有不同程度的缺失,以OHCs为甚。结论：6-7周龄大鼠腹腔注射KM 400 mg/Kg per d 2周比较适合OECs移植。 第三部分OECs对损伤SGCs保护和修复作用的研究 目的：观察OECs移植对损伤大鼠SGCs的保护和修复作用。方法：将培养7d后的OECs进行核标记,以1×106个/ml密度悬浮,移植到腹腔注射KM 400 mg/Kg per d2周的实验组成年大鼠耳蜗内,对照组耳蜗注射等量的D-Hanks液,以ABR大鼠听力改变,HE染色和免疫荧光化学染色观察SGCs的密度及形态学改变。结果：对照组与实验组的ABR阈值在各频率无明显差异；实验组荧光显微镜下见大量Hoechst 33342标记的蓝色细胞核,分布在耳蜗底旋到顶旋；实验组SGCs密度与对照组有明显差异,大部分形态正常,核膜清晰,核中染色质清晰,核仁亦较清晰,排列尚紧密,少部分细胞核染色质边集,坏死,密度较正常大鼠SGCs明显降低；对照组SGCs大量坏死,部分细胞核结构被吞噬细胞吞噬,空泡化,细胞排列疏松、混乱,仅见少量正常的SGCs,免疫荧光见对照组有大量坏死细胞的碎片。结论：OECs可能促进SGCs的存活,对损伤大鼠SGCs有保护作用。
[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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