嗅鞘细胞的培养及其生物学特性的实验研究

发布时间：2017-12-31 00:35

  本文关键词：嗅鞘细胞的培养及其生物学特性的实验研究　出处：《第四军医大学》2010年博士论文　论文类型：学位论文

  更多相关文章： 脊髓损伤 嗅鞘细胞 嗅粘膜 细胞培养 脊髓背根神经节神经元 共培养 凋亡 基因 细胞移植

【摘要】： 近年来,脊髓损伤(Spinal cord injury ,SCI)的再生与修复研究取得了很大进展,细胞移植被认为是最有前景的治疗方法之一。嗅鞘细胞(Olfactory ensheathing cells,OECs)因其独特的生物学特性越来越受到人们的注意。OECs移植入受损的脊髓部位后,可以排列成细胞链,引导促进神经元轴突再生通过损伤部位,并可促进多种神经细胞的生长及轴突的延长,研究发现,OECs可成功促进下行传导通路的再生。因此,OECs被认为是细胞移植修复SCI最有希望的种子细胞之一。 一、OECs对培养神经元生长状态的影响 培养新生SD大鼠脊髓背根神经节神经元(Dorsal root ganglion neurons, DRGn)与成年SD大鼠OECs,将DRGn与不同浓度(8×10~5/ml、4×10~5/ml、2×10~5/ml、10~5/ml、10~4/ml)OECs共培养。共培养3天后在倒置相差显微镜下观察神经元生长发育情况;行抗NSE SABC免疫组织化学染色并进行细胞计数;行抗GAP-43免疫荧光染色;同时采用MTT法测定神经元活性。对各组结果进行统计学分析。结果表明:各共培养组神经元生长密度明显高于对照组,神经元胞体大而饱满,突起较长,细胞活性也高于对照组。且在一定范围内，神经元生长密度随共培养的OECs接种密度的增加而增加。但当OECs达到一定密度后(2×10~5／ml)，再增加其接种密度，测量神经元生长密度并不继续随之增高。结论：OECs可明显促进体外培养DRGn的生长，提高细胞活性，且在一定范围内存在密度依赖效应。 二、OECs对H_2O_2诱导神经元凋亡的影响 培养新生SD大鼠DRGn与成年SD大鼠OECs。向DRGn培养基内加入终浓度为1mmol／L的H_2O_2诱导其凋亡，然后立刻与不同密度(10~4／ml、10~5／ml、2×10~5／ml、8×10~5／ml)的OECs共培养；以及在加入H_2O_2后的不同时间(0h、4h、8h、12h、24h)与OECs共培养。各组DRGn分别于共培养24h后进行Tunel凋亡染色、流式细胞技术测定细胞凋亡率及MTT法检测细胞活性。结果表明：DRGn培养基中加入H_2O_2诱导凋亡后，与不同密度OECs共培养24h，，检测细胞凋亡率均明显低于对照组，细胞活性明显高于对照组，且随着共培养的OECs接种密度的增加，细胞凋亡率随之降低、细胞活性升高。但当OECs接种密度升高至2×10~5／ml后，再增加OECs的接种密度，上述指标变化并不明显；DRGn培养基中加入H_2O_2诱导凋亡，于加入H_2O_2后不同时间与OECs共培养24h。检测DRGn凋亡率在0h、4h、8h、12h组均明显低于对照组，且随着与OECs共培养时间的推迟，凋亡率随之升高。当加入H_2O_2后24h再与OECs共培养组，其细胞凋亡率与对照组相比并无明显区别。结论：OECs可明显抑制H_2O_2诱导的DRGn凋亡，并在一定的范围内存在密度依赖效应与时间依赖效应。 三、OECs对体外诱导神经元凋亡相关基因表达的影响 培养新生SD大鼠DRGn与成年SD大鼠OECs。向DRGn培养基内加入终浓度为1mmol／L的H_2O_2诱导其凋亡，然后与密度为2×10~5／ml的OECs共培养。于共培养24h后采用流式细胞技术测定DRGn细胞凋亡率，RT-PCR及Western blot技术检测DRGn凋亡相关基因FADD、Bcl-2、BAx、Birn、Caspase-3的表达。结果表明：DRGn培养基中加入H_2O_2后与OECs共培养24h，检测细胞凋亡率明显低于对照组，且共培养组BAx、Bim、Caspase-3的表达量明显下调，Bcl-2表达量明显上调。结论：OECs能通过下调BAx、Bitn表达、上调Bcl-2表达的途径抑制DRGn凋亡。 四、OECs移植修复大鼠SCI的试验研究 培养成年SD大鼠OECs，培养14d后将其自培养瓶中消化下来，制成细胞悬液，并用Hoechst33342标记。使用改良的Allen打击法制造大鼠T8／9脊髓损伤模型，于损伤同时用微量注射器向损伤脊髓局部注入5ul密度为2×106／ml的OECs细胞悬液。于移植后2w观察OECs的存活状况及其在脊髓实质内的迁移情况；于移植后24h及2w行Tunel凋亡染色观察原始打击区域临近节段脊髓神经细胞凋亡情况；于移植后4w行NSE及GAP43免疫荧光染色观察打击区域神经再生情况；于移植后4w对试验动物行运动功能评分。结果发现：OECs移植后可在损伤部位存活，并在脊髓实质内迁移；Tunel凋亡染色显示移植组原始打击区域临近节段脊髓神经细胞凋亡数量明显低于于对照组；NSE及GAP43免疫荧光染色显示移植组打击区域神经元轴突再生数量明显高于对照组；但试验组及对照组动物运动功能评分无明显区别。结论：OECs移植可明显减轻脊髓空洞及胶质瘢痕的形成，促进轴突再生，抑制SCI后的神经细胞凋亡。 五、人嗅粘膜来源嗅鞘细胞的分离、培养与纯化 本研究共收集嗅粘膜标本15例，全部来自意外交通或机械事故死亡的成年男性，年龄23-40岁，确认为临床死亡并征得家属同意后，常规碘伏消毒取材区域，无菌条件下，采用硬质鼻内窥镜剥离上鼻甲及中鼻甲内侧的嗅粘膜约5mm~3，采用胰蛋白酶消化、差速贴壁法纯化。在培养的第7、14d，使用倒置相差显微镜进行形态学观察；同时行抗NGFR p75免疫荧光染色，鉴定细胞并计算染色阳性细胞率。结果表明：通过纯化及培养，于14d可获得纯度约为75%的OECs，细胞形态以带有细长突起的双极细胞及三极细胞为主，有少量的扁平细胞。结论：自成人嗅粘膜可成功分离纯化出OECs，来源稳定，纯度达到移植要求，为开展自体嗅粘膜OECs移植修复SCI提供了技术与方法。
[Abstract]:In recent years, spinal cord injury (Spinal cord, injury, SCI) have made great progress in the regeneration and repair of cell transplantation is considered to be one of the most promising treatment methods. Olfactory ensheathing cells (Olfactory ensheathing cells, OECs) for spinal cord and its unique biological characteristics more attention to.OECs transplanted into injured people after the cells can be arranged in a chain guide, promote axon regeneration through the injury site, and can promote the growth and axonal extension of nerve cells in the study found that OECs can successfully promote regeneration of the descending pathway. Therefore, OECs is regarded as one of the seed cells for cell transplantation in the repair of SCI is the most promising.
The effect of OECs on the growth of cultured neurons
Cultured SD rat dorsal root ganglion neurons (Dorsal root, ganglion neurons, DRGn) and adult SD rats OECs, DRGn with different concentration (8 * 10~5/ml, 4 * 10~5/ml, 2 * 10~5/ml, 10~5/ml, 10~4/ml) OECs co culture. The growth and development of neurons were observed under the inverted microscope after 3 days of CO culture; anti NSE SABC immunohistochemical staining and cell counting; anti GAP-43 staining; Determination of neuronal activity by MTT method. Statistical analysis of the results for each group. The results showed that: the co culture group of neurons growth density was significantly higher than the control group, neurons were large and full, long protuberances, cell the activity is higher than that of the control group. And in a certain range, increase the density of OECs was co cultured with the growth density of neurons increased. But when the OECs reaches a certain density (2 * 10~5 / ml), and then increase the inoculum density measurement The growth density of neurons did not continue to increase. Conclusion: OECs can significantly promote the growth of DRGn in vitro, enhance cell viability, and have a density dependent effect in a certain range.
The effect of two, OECs on H_2O_2 induced neuronal apoptosis
Culture of newborn SD rat DRGn and SD adult rat OECs. to DRGn culture medium concentration was added to 1mmol / L H_2O_2 induced apoptosis, and then immediately with different density (10~4 / ml, 10~5 / ml, 10~5 / 2 * ml, 8 * 10~5 / ml) were co cultured with OECs in different time; and after joining the H_2O_2 (0h, 4h, 8h, 12h, 24h) co cultured with OECs. DRGn were co cultured respectively in 24h after Tunel staining, flow cytometry was used to detect the cell apoptosis rate and MTT staining. The results showed that DRGn medium supplemented with H_2O_2 induced apoptosis, and different. OECs co cultured with 24h, detection of cell apoptosis rate was significantly lower than the control group, the cell activity was significantly higher than the control group, and with the increase of OECs inoculum density of co cultured, cell apoptosis rate decreased, cell activity increased. But when the OECs inoculum density increased to 2 x 10~5 / ml after adding OECs inoculation density The above indexes, the change was not obvious; medium added H_2O_2 induced apoptosis in cultured DRGn, joined H_2O_2 in different time after co cultured with OECs DRGn apoptosis detection rate of 24h. in 0h, 4h, 8h, 12h group were significantly lower than the control group, and with OECs co cultured with time delay, the apoptosis rate increase when added. H_2O_2 24h and OECs co culture group, the apoptosis rate compared with the control group no significant difference. Conclusion: OECs can inhibit the apoptosis of DRGn induced by H_2O_2, and in a certain range of density dependent effect and time-dependent effect.
Three, the effect of OECs on the expression of apoptosis related genes induced in vitro
Culture of newborn SD rat DRGn and SD adult rat OECs. to DRGn culture medium concentration was added to 1mmol / L H_2O_2 induced apoptosis, and then co cultured density was 2 * 10~5 / ml OECs. After 24h co cultured in DRGn were measured by flow cytometry cell apoptosis, apoptosis of DRGn and RT-PCR Western blot Bcl-2, gene FADD, BAx, Birn, Caspase-3 expression. The results showed that DRGn medium supplemented with H_2O_2 after co cultured with OECs 24h, to detect the cell apoptosis rate was significantly lower than the control group, and the co culture group BAx, Bim, Caspase-3 expression was significantly down regulated, Bcl-2 expression was significantly upregulated. Conclusion: OECs can downregulate BAx expression, Bitn, way up regulate Bcl-2 expression and inhibit DRGn apoptosis.
Experimental study on the repair of SCI in rats by four, OECs transplantation
SD in cultured adult rat OECs cultured 14d after the self digestion flask down into cell suspension, and labeled with Hoechst33342. Rats in T8 / 9 spinal cord injury model using modified Allen method to attack, damage at the same time using micro syringe to spinal injection 5ul density was 2 x 106 / ml the OECs cell suspension. After transplantation in 2W OECs to observe the survival and migration in spinal cord parenchyma; after transplantation in 24h and 2W Tunel staining to observe the original combat area near the neuronal apoptosis in the spinal cord segment; after transplantation in 4W NSE and GAP43 was observed by immunofluorescence staining against regional nerve regeneration in the score of 4W on motor function; experimental animal for transplantation. The results showed that OECs transplantation can survive in the injury site, and migration in the spinal cord parenchyma; Tunel staining showed that the apoptosis of transplantation group near the original blow The number of nerve cell apoptosis of spinal cord was significantly lower than that of control group; NSE and GAP43 immunofluorescence staining showed that the transplantation group attack area of axonal regeneration number was significantly higher than the control group; but the experimental group and the control group animal movement function score had no significant difference. Conclusion: OECs transplantation can significantly reduce syringomyelia and glial scar, promote axon regeneration, inhibit the apoptosis of nerve cells after SCI.
Five, isolation, culture and purification of olfactory ensheathing cells from the human olfactory mucous membrane
This study collected olfactory mucosa specimens from 15 patients, all from the deaths of traffic accidents or mechanical adult male, age 23-40, confirmed clinical death and the consent of their families, the routine iodophor disinfection materials, under aseptic condition, using hard endoscopic stripping on the inner side of the middle turbinate and nasal olfactory mucosa of about 5mm~3, using trypsin digestion, differential centrifugation and purification. In the 7,14d medium, using inverted phase contrast microscope was used to observe the morphology; and the anti NGFR p75 immunofluorescence staining, cells were identified and calculated the percentage of positive cells stained. The results show that the purified and cultured in 14d, the purity is about 75% OECs. Cell morphology with elongated protrusion of the bipolar cells and tripolar cells, a small flat cells. Conclusion: the adult olfactory mucosa can be purified OECs, successfully isolated and stable source of purity to transplant The technology and methods are provided for the repair of SCI with autologous olfactory mucosa OECs transplantation.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329

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