本文關(guān)鍵詞：嗅鞘細(xì)胞的培養(yǎng)及其生物學(xué)特性的實驗研究　出處：《第四軍醫(yī)大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 脊髓損傷 嗅鞘細(xì)胞 嗅粘膜 細(xì)胞培養(yǎng) 脊髓背根神經(jīng)節(jié)神經(jīng)元 共培養(yǎng) 凋亡 基因 細(xì)胞移植

【摘要】： 近年來,脊髓損傷(Spinal cord injury ,SCI)的再生與修復(fù)研究取得了很大進(jìn)展,細(xì)胞移植被認(rèn)為是最有前景的治療方法之一。嗅鞘細(xì)胞(Olfactory ensheathing cells,OECs)因其獨特的生物學(xué)特性越來越受到人們的注意。OECs移植入受損的脊髓部位后,可以排列成細(xì)胞鏈,引導(dǎo)促進(jìn)神經(jīng)元軸突再生通過損傷部位,并可促進(jìn)多種神經(jīng)細(xì)胞的生長及軸突的延長,研究發(fā)現(xiàn),OECs可成功促進(jìn)下行傳導(dǎo)通路的再生。因此,OECs被認(rèn)為是細(xì)胞移植修復(fù)SCI最有希望的種子細(xì)胞之一。 一、OECs對培養(yǎng)神經(jīng)元生長狀態(tài)的影響 培養(yǎng)新生SD大鼠脊髓背根神經(jīng)節(jié)神經(jīng)元(Dorsal root ganglion neurons, DRGn)與成年SD大鼠OECs,將DRGn與不同濃度(8×10~5/ml、4×10~5/ml、2×10~5/ml、10~5/ml、10~4/ml)OECs共培養(yǎng)。共培養(yǎng)3天后在倒置相差顯微鏡下觀察神經(jīng)元生長發(fā)育情況;行抗NSE SABC免疫組織化學(xué)染色并進(jìn)行細(xì)胞計數(shù);行抗GAP-43免疫熒光染色;同時采用MTT法測定神經(jīng)元活性。對各組結(jié)果進(jìn)行統(tǒng)計學(xué)分析。結(jié)果表明:各共培養(yǎng)組神經(jīng)元生長密度明顯高于對照組,神經(jīng)元胞體大而飽滿,突起較長,細(xì)胞活性也高于對照組。且在一定范圍內(nèi)，神經(jīng)元生長密度隨共培養(yǎng)的OECs接種密度的增加而增加。但當(dāng)OECs達(dá)到一定密度后(2×10~5／ml)，再增加其接種密度，測量神經(jīng)元生長密度并不繼續(xù)隨之增高。結(jié)論：OECs可明顯促進(jìn)體外培養(yǎng)DRGn的生長，提高細(xì)胞活性，且在一定范圍內(nèi)存在密度依賴效應(yīng)。 二、OECs對H_2O_2誘導(dǎo)神經(jīng)元凋亡的影響 培養(yǎng)新生SD大鼠DRGn與成年SD大鼠OECs。向DRGn培養(yǎng)基內(nèi)加入終濃度為1mmol／L的H_2O_2誘導(dǎo)其凋亡，然后立刻與不同密度(10~4／ml、10~5／ml、2×10~5／ml、8×10~5／ml)的OECs共培養(yǎng)；以及在加入H_2O_2后的不同時間(0h、4h、8h、12h、24h)與OECs共培養(yǎng)。各組DRGn分別于共培養(yǎng)24h后進(jìn)行Tunel凋亡染色、流式細(xì)胞技術(shù)測定細(xì)胞凋亡率及MTT法檢測細(xì)胞活性。結(jié)果表明：DRGn培養(yǎng)基中加入H_2O_2誘導(dǎo)凋亡后，與不同密度OECs共培養(yǎng)24h，，檢測細(xì)胞凋亡率均明顯低于對照組，細(xì)胞活性明顯高于對照組，且隨著共培養(yǎng)的OECs接種密度的增加，細(xì)胞凋亡率隨之降低、細(xì)胞活性升高。但當(dāng)OECs接種密度升高至2×10~5／ml后，再增加OECs的接種密度，上述指標(biāo)變化并不明顯；DRGn培養(yǎng)基中加入H_2O_2誘導(dǎo)凋亡，于加入H_2O_2后不同時間與OECs共培養(yǎng)24h。檢測DRGn凋亡率在0h、4h、8h、12h組均明顯低于對照組，且隨著與OECs共培養(yǎng)時間的推遲，凋亡率隨之升高。當(dāng)加入H_2O_2后24h再與OECs共培養(yǎng)組，其細(xì)胞凋亡率與對照組相比并無明顯區(qū)別。結(jié)論：OECs可明顯抑制H_2O_2誘導(dǎo)的DRGn凋亡，并在一定的范圍內(nèi)存在密度依賴效應(yīng)與時間依賴效應(yīng)。 三、OECs對體外誘導(dǎo)神經(jīng)元凋亡相關(guān)基因表達(dá)的影響 培養(yǎng)新生SD大鼠DRGn與成年SD大鼠OECs。向DRGn培養(yǎng)基內(nèi)加入終濃度為1mmol／L的H_2O_2誘導(dǎo)其凋亡，然后與密度為2×10~5／ml的OECs共培養(yǎng)。于共培養(yǎng)24h后采用流式細(xì)胞技術(shù)測定DRGn細(xì)胞凋亡率，RT-PCR及Western blot技術(shù)檢測DRGn凋亡相關(guān)基因FADD、Bcl-2、BAx、Birn、Caspase-3的表達(dá)。結(jié)果表明：DRGn培養(yǎng)基中加入H_2O_2后與OECs共培養(yǎng)24h，檢測細(xì)胞凋亡率明顯低于對照組，且共培養(yǎng)組BAx、Bim、Caspase-3的表達(dá)量明顯下調(diào)，Bcl-2表達(dá)量明顯上調(diào)。結(jié)論：OECs能通過下調(diào)BAx、Bitn表達(dá)、上調(diào)Bcl-2表達(dá)的途徑抑制DRGn凋亡。 四、OECs移植修復(fù)大鼠SCI的試驗研究 培養(yǎng)成年SD大鼠OECs，培養(yǎng)14d后將其自培養(yǎng)瓶中消化下來，制成細(xì)胞懸液，并用Hoechst33342標(biāo)記。使用改良的Allen打擊法制造大鼠T8／9脊髓損傷模型，于損傷同時用微量注射器向損傷脊髓局部注入5ul密度為2×106／ml的OECs細(xì)胞懸液。于移植后2w觀察OECs的存活狀況及其在脊髓實質(zhì)內(nèi)的遷移情況；于移植后24h及2w行Tunel凋亡染色觀察原始打擊區(qū)域臨近節(jié)段脊髓神經(jīng)細(xì)胞凋亡情況；于移植后4w行NSE及GAP43免疫熒光染色觀察打擊區(qū)域神經(jīng)再生情況；于移植后4w對試驗動物行運(yùn)動功能評分。結(jié)果發(fā)現(xiàn)：OECs移植后可在損傷部位存活，并在脊髓實質(zhì)內(nèi)遷移；Tunel凋亡染色顯示移植組原始打擊區(qū)域臨近節(jié)段脊髓神經(jīng)細(xì)胞凋亡數(shù)量明顯低于于對照組；NSE及GAP43免疫熒光染色顯示移植組打擊區(qū)域神經(jīng)元軸突再生數(shù)量明顯高于對照組；但試驗組及對照組動物運(yùn)動功能評分無明顯區(qū)別。結(jié)論：OECs移植可明顯減輕脊髓空洞及膠質(zhì)瘢痕的形成，促進(jìn)軸突再生，抑制SCI后的神經(jīng)細(xì)胞凋亡。 五、人嗅粘膜來源嗅鞘細(xì)胞的分離、培養(yǎng)與純化 本研究共收集嗅粘膜標(biāo)本15例，全部來自意外交通或機(jī)械事故死亡的成年男性，年齡23-40歲，確認(rèn)為臨床死亡并征得家屬同意后，常規(guī)碘伏消毒取材區(qū)域，無菌條件下，采用硬質(zhì)鼻內(nèi)窺鏡剝離上鼻甲及中鼻甲內(nèi)側(cè)的嗅粘膜約5mm~3，采用胰蛋白酶消化、差速貼壁法純化。在培養(yǎng)的第7、14d，使用倒置相差顯微鏡進(jìn)行形態(tài)學(xué)觀察；同時行抗NGFR p75免疫熒光染色，鑒定細(xì)胞并計算染色陽性細(xì)胞率。結(jié)果表明：通過純化及培養(yǎng)，于14d可獲得純度約為75%的OECs，細(xì)胞形態(tài)以帶有細(xì)長突起的雙極細(xì)胞及三極細(xì)胞為主，有少量的扁平細(xì)胞。結(jié)論：自成人嗅粘膜可成功分離純化出OECs，來源穩(wěn)定，純度達(dá)到移植要求，為開展自體嗅粘膜OECs移植修復(fù)SCI提供了技術(shù)與方法。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R329

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