本文關(guān)鍵詞：OECs無血清上清液誘導UCB-MSCs向神經(jīng)細胞分化　出處：《湖南師范大學》2011年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 嗅鞘細胞 臍血間充質(zhì)干細胞 電生理特性 神經(jīng)細胞

【摘要】：目的：探討大鼠嗅鞘細胞(olfactory ensheathing cells,OECs)無血清上清液體外誘導臍血間充質(zhì)干細胞(human umbilical cord blood mesenchymal stem cells, HUCB-MSCs)向神經(jīng)細胞分化的可行性和可能的機制,以及檢測誘導分化后神經(jīng)元細胞的電生理特性。 方法：從新生大鼠嗅球分離、提取OECs,采用差速貼壁聯(lián)合阿糖胞苷抑制法純化嗅鞘細胞,在細胞最佳狀態(tài)下無血清培養(yǎng)24h后收集上清液。收集正常足月剖腹產(chǎn)胎兒的臍帶血,經(jīng)肝素抗凝,用密度梯度離心法分離獲得臍血的單個核細胞,采用差速貼壁法純化間充質(zhì)干細胞,培養(yǎng)、擴增到第3代,記作臍血MSCsP3。實驗分為實驗組和對照組,DMEM液體中加入OECs無血清上清液培養(yǎng)臍血MSCsP3作為實驗組,用DMEM液體培養(yǎng)臍血MSCsP3作為對照組。在一系列時間點(培養(yǎng)后12小時,24小時,72小時,1周,10天和2周)在倒置顯微鏡下形態(tài)學觀察生長、分化情況,1周后行免疫組化鑒定,神經(jīng)元特異性烯醇化酶(NSE)、神經(jīng)膠質(zhì)纖維酸性蛋白(GFAP)和巢蛋白(nestin)分別鑒定神經(jīng)元、神經(jīng)膠質(zhì)細胞和神經(jīng)干細胞,膜片鉗檢測神經(jīng)元電生理特性。 結(jié)果：用差速貼壁和阿糖胞苷抑制法可獲得純度高達95%的嗅鞘細胞,實驗組細胞在培養(yǎng)誘導后相差顯微鏡下可觀察到典型神經(jīng)元、神經(jīng)膠質(zhì)細胞和神經(jīng)干細胞團,2周后細胞仍維持神經(jīng)細胞樣形態(tài),NSE、GFAP、nestin細胞免疫組化鑒定與形態(tài)學結(jié)果一致。HUCB-MSCsP3誘導分化后的神經(jīng)元樣細胞檢測到快速激活快速失活、能被河豚毒素(TTX)特異性阻斷的鈉離子電流,能被四乙基氯化銨(TEA)特異性阻斷的慢激活慢失活的延遲整流性鉀電流,以及緩慢的內(nèi)向鈣離子電流。對照組臍血MSCs培養(yǎng)后呈多形態(tài)方向分化。 結(jié)論：①OECs無血清上清液能誘導臍血MSCs向神經(jīng)細胞分化;②誘導分化后的神經(jīng)元具備成熟神經(jīng)元的電生理特性。
[Abstract]:Objective: to investigate the olfactory ensheathing cells of rat olfactory ensheathing cells. In vitro induction of umbilical cord blood mesenchymal stem cells (OECs) without serum supernatant. Human umbilical cord blood mesenchymal stem cells. The feasibility and possible mechanism of differentiation of HUCB-MSCs into neurons and the electrophysiological characteristics of neuronal cells induced by differentiation were examined. Methods: OECs were isolated from olfactory bulb of newborn rats. OECs were purified by differential adhesion and cytarabine inhibition. The supernatant was collected after 24 hours of serum-free culture in the best condition of the cells. The umbilical cord blood was collected from the normal full-term caesarean section fetus. The mononuclear cells were isolated by density gradient centrifugation after heparin anticoagulant. Mesenchymal stem cells were purified by differential adherent method, cultured and amplified to the third generation, recorded as cord blood MSCsP3. The experiment was divided into experimental group and control group. OECs serum-free supernatant was added to DMEM fluid to culture umbilical cord blood MSCsP3 as experimental group. Umbilical cord blood MSCsP3 was cultured with DMEM liquid as control group. At a series of time points (12 hours, 24 hours, 72 hours, 1 week). After 10 days and 2 weeks, the growth was observed under inverted microscope, and the differentiation was identified by immunohistochemistry after 1 week. The neuron specific enolase (NSE) was identified. Glial fibrillary acidic protein (GFAP) and nestin (nestin) were used to identify neurons, glial cells and neural stem cells respectively. The electrophysiological characteristics of neurons were detected by patch clamp. Results: olfactory ensheathing cells with purity up to 95% could be obtained by differential adhesion and cytarabine inhibition. Typical neurons could be observed under phase contrast microscope in the experimental group. After 2 weeks, the glial cells and the neural stem cells still maintained the neuron-like morphology of NSE-GFAP. The nestin cells were identified by immunohistochemistry. The neuron-like cells induced by HUCB-MSCsP3 showed rapid activation and rapid inactivation. Sodium ion currents specifically blocked by tetrodotoxin TTX, slow activation of slow inactivated delayed rectifier potassium currents specifically blocked by tetraethyl ammonium chloride. And slow inward calcium current. The cord blood MSCs in the control group differentiated in multiple directions after culture. Conclusion the serum free supernatant of 1: 1 OECs can induce the differentiation of cord blood MSCs into neural cells. The neurons after differentiation can possess the electrophysiological characteristics of mature neurons.
【學位授予單位】：湖南師范大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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