【摘要】：研究背景 骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是來(lái)源于中胚層的具有多向分化能力的干細(xì)胞,具有向軟骨細(xì)胞、骨細(xì)胞、肌細(xì)胞、肌腱細(xì)胞、脂肪細(xì)胞、干細(xì)胞和造血細(xì)胞等多向分化及自我更新的功能,并且于一定條件下在體內(nèi)及體外可橫向分化為神經(jīng)細(xì)胞和膠質(zhì)細(xì)胞。近來(lái),已經(jīng)有很多動(dòng)物實(shí)驗(yàn)研究報(bào)道,可以用移植骨髓間充質(zhì)干細(xì)胞的方法來(lái)治療各種神經(jīng)系統(tǒng)的變性疾病、腦卒中及中樞損傷等,并取得了一定的效果。 Rho/Rho激酶(ROCK)信號(hào)通路包括有三種成分：Rho蛋白、Rho激酶及Rho激酶的效應(yīng)分子(ROCK),是體內(nèi)一條重要的信號(hào)通路,主要參與了調(diào)控細(xì)胞骨架形成、細(xì)胞增殖、細(xì)胞遷移、基因轉(zhuǎn)錄和凋亡等生物行為及功能,主要通過(guò)小G蛋白GDP-GTP之間的轉(zhuǎn)換,來(lái)調(diào)節(jié)細(xì)胞內(nèi)肌動(dòng)蛋白骨架的聚合狀態(tài),從而扮演著“分子開(kāi)關(guān)”的角色,并參與調(diào)節(jié)細(xì)胞骨架蛋白的合成、降解、移動(dòng)和收縮等,因此對(duì)細(xì)胞的分裂、黏附、收縮、遷移和分泌等活動(dòng)具有非常重要的調(diào)節(jié)作用。我們觀(guān)察了ROCK抑制劑法舒地爾在體外誘導(dǎo)大鼠MSCs向神經(jīng)細(xì)胞分化中的作用。 TRP (transient receptor potential)通道是一類(lèi)六次跨膜的非選擇性陽(yáng)離子通道。它們?cè)谶M(jìn)化中高度保守,在哺乳動(dòng)物體內(nèi)廣泛表達(dá),參與了許多重要的生理學(xué)功能,如對(duì)溫度、痛覺(jué)、聽(tīng)覺(jué)的感知。TRPM8通道是TRP的一個(gè)亞家族,在人的中樞神經(jīng)系統(tǒng)中表達(dá)豐富,其主要生理功能是感知低溫。令人驚奇的是,本研究在誘導(dǎo)MSCs分化為神經(jīng)細(xì)胞時(shí),首次發(fā)現(xiàn)TRPM8出現(xiàn)表達(dá)。 microRNA-124a是腦組織中表達(dá)最為豐富的一類(lèi)microRNA,約占腦組織microRNA總量的25%-48%。Lim等人的研究表明,將microRNA-124a感染到HeLa細(xì)胞中導(dǎo)致一系列非神經(jīng)細(xì)胞轉(zhuǎn)錄物的表達(dá)受到抑制,HeLa細(xì)胞的基因組表達(dá)模式向神經(jīng)方向轉(zhuǎn)化。在小鼠腦組織,microRNA-124只限于在已經(jīng)分化的和成熟的神經(jīng)細(xì)胞中表達(dá),而在神經(jīng)前體細(xì)胞中表達(dá)很少。本研究通過(guò)構(gòu)建攜帶含有綠色熒光蛋白(green fluorescence protein, GFP)報(bào)告基因的大鼠microRNA-124a慢病毒載體,將其感染至MSCs,并傳代,觀(guān)察其對(duì)骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化的影響。 第一部分法舒地爾誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞向神經(jīng)細(xì)胞分化 目的 探討Rho/Rho激酶(ROCK)抑制劑法舒地爾在體外誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)向神經(jīng)細(xì)胞分化中的可行性。 方法 全骨髓培養(yǎng)法分離培養(yǎng)大鼠MSCs；采用法舒地爾誘導(dǎo)MSCs;倒置顯微鏡觀(guān)察誘導(dǎo)后各組的細(xì)胞形態(tài)學(xué)變化；AO-EB染色法鑒定誘導(dǎo)后各組細(xì)胞存活率；免疫熒光法鑒定誘導(dǎo)后NSE、NF200、GFAP的表達(dá),判斷其分化情況。 結(jié)果 AO-EB染色法鑒定誘導(dǎo)后細(xì)胞存活率 誘導(dǎo)后存活細(xì)胞胞質(zhì)呈綠色,核為亮綠色,核形態(tài)規(guī)則,為圓形或橢圓形,死亡細(xì)胞胞質(zhì)呈紅色,核呈亮紅色,核皺縮或碎裂。隨著誘導(dǎo)時(shí)間延長(zhǎng),死亡細(xì)胞數(shù)量增加。通過(guò)AO-EB染色法鑒定法舒地爾誘導(dǎo)30 min、90min、120min及180min,細(xì)胞的存活率分別為96.7±2.2%、95.3±1.9%、93.8±1.8%、92.5±2.1%及90.1±1.3%。 誘導(dǎo)后免疫熒光鑒定 法舒地爾誘導(dǎo)組,隨著誘導(dǎo)時(shí)間延長(zhǎng), NSE、NF200表達(dá)顯著增加,GFAP表達(dá)較少。誘導(dǎo)后60、90、120及180 min,通過(guò)免疫熒光鑒定,NSE陽(yáng)性率(分別為66.5±1.9%、88.1±3.2%、93.6±1.9%、93.5±5.4%),NF200陽(yáng)性率(分別為70.1±2.9%、89.5±1.3%、98.1±1.6%、98.3±1.9%)并呈逐漸增加的趨勢(shì),而各組GFAP表達(dá)率均小于5%。 第二部分TRPM8在體外骨髓間充質(zhì)干細(xì)胞分化為神經(jīng)細(xì)胞中的表達(dá)變化 瞬時(shí)受體電位(transient receptor potential, TRP)是一類(lèi)廣泛存在于細(xì)胞膜上的跨膜離子通道,可以辨別味覺(jué)、溫度覺(jué)等特殊感覺(jué)。TRP通道亞型TRPM8主要存在于特定神經(jīng)細(xì)胞的細(xì)胞膜上,當(dāng)溫度低于27℃或者薄荷醇存在的條件下,TRPM8通道開(kāi)放,使Ca2+等帶正電的粒子進(jìn)入細(xì)胞,具有重要的生理意義。 目的 本研究探討TRPM8在大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)分化為神經(jīng)細(xì)胞中的表達(dá)變化和基本作用。 方法 在建立體外法舒地爾誘導(dǎo)大鼠MSCs分化為神經(jīng)細(xì)胞的基礎(chǔ)上,采用免疫細(xì)胞化學(xué)法、Western Blot法檢測(cè)TRPM8的表達(dá)變化。 結(jié)果： 誘導(dǎo)前大鼠MSCs不表達(dá)TRPM8;誘導(dǎo)30min; MSCs開(kāi)始表達(dá)TRPM8(45.3%±1.58%)；誘導(dǎo)60main,表達(dá)較前增加(57.50%±2.45%)；誘導(dǎo)后90min,TRPM8表達(dá)最高(89.56%±12.24%)；誘導(dǎo)120 min,TRPM8表達(dá)逐漸下降(59.25%±9.15%), Western Blot也有類(lèi)似的趨勢(shì)。 第三部分microRNA-124a慢病毒載體的構(gòu)建及感染大鼠骨髓間充質(zhì)干細(xì)胞的研究 目的 探討:microRNA-124a在法舒地爾誘導(dǎo)大鼠骨髓間充質(zhì)干細(xì)胞(MSCs)向神經(jīng)細(xì)胞分化中的作用。 方法 本研究構(gòu)建攜帶含有綠色熒光蛋白(green fluorescence protein, GFP)報(bào)告基因的大鼠microRNA-124a慢病毒載體,將其感染至大鼠骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells,MSCs),并傳代,采用法舒地爾誘導(dǎo)大鼠MSCs分化為神經(jīng)細(xì)胞。并于倒置熒光顯微鏡下觀(guān)察MSCs感染后的熒光表達(dá)情況；采用免疫細(xì)胞化學(xué)染色和western印跡檢測(cè)神經(jīng)元烯醇化酶(NSE)、神經(jīng)微絲蛋白(NF200)及膠質(zhì)纖維酸性蛋白(GFAP)的表達(dá)變化,MTT方法檢測(cè)細(xì)胞存活率。 結(jié)果 免疫細(xì)胞化學(xué)染色法 感染組誘導(dǎo)1h后,NSE、NF200的表達(dá)率分別為83.2±2.0%,79.6±0.4%,顯著高于其它兩組(P0.05)。各組GFAP的表達(dá)率都小于5%,無(wú)顯著性差異。 Western Blot結(jié)果 三組分別誘導(dǎo)分化1h后均表達(dá)NSE、NF200,未感染組和陰性對(duì)照組無(wú)明顯的統(tǒng)計(jì)學(xué)差異,而感染組表達(dá)的NSE、NF200較其它兩組明顯增高,有顯著差異。 結(jié)論 1本研究發(fā)現(xiàn)Rho/Rho激酶(ROCK)抑制劑法舒地爾可以在體外快速,高效的誘導(dǎo)大鼠MSCs向神經(jīng)細(xì)胞分化。 2本研究首次報(bào)道TRPM8這種神經(jīng)細(xì)胞特殊蛋白MSCs分化為神經(jīng)細(xì)胞中出現(xiàn)表達(dá),而且可能在其分化過(guò)程中起到一定的作用。 3 microRNA-124a可促進(jìn)大鼠骨髓間充質(zhì)干細(xì)胞在向神經(jīng)細(xì)胞分化
[Abstract]:Research background
Bone marrow mesenchymal stem cells (MSCs) are mesenchymal stem cells derived from the mesoderm and have the ability to differentiate into chondrocytes, osteocytes, muscle cells, tendon cells, adipocytes, stem cells and hematopoietic cells, and self-renewal. Under certain conditions, MSCs can be transverse in vivo and in vitro. Recently, many animal experiments have reported that bone marrow mesenchymal stem cells can be transplanted to treat various degenerative diseases of the nervous system, stroke and central injury, and some results have been achieved.
Rho/Rho kinase (ROCK) signaling pathway consists of three components: Rho protein, Rho kinase and Rho kinase effector molecule (ROCK), which is an important signaling pathway in vivo, mainly involved in the regulation of cytoskeleton formation, cell proliferation, cell migration, gene transcription and apoptosis, and other biological behaviors and functions, mainly through the transduction of small G protein GDP-GTP. In other words, it regulates the aggregation of actin cytoskeleton, thus acting as a "molecular switch" and regulating the synthesis, degradation, movement and contraction of cytoskeleton proteins. Therefore, it plays an important role in regulating cell division, adhesion, contraction, migration and secretion. In vitro, the effects of MSCs on the differentiation of rat neurons into neurons were observed.
TRP (transient receptor potential) channels are a class of six-time transmembrane nonselective cationic channels. They are highly conserved in evolution, widely expressed in mammals, and participate in many important physiological functions, such as temperature, pain, and auditory perception. TRPM8 channels are a subfamily of TRP, which are found in the human central nervous system. Surprisingly, TRPM8 was found for the first time in this study when MSCs were induced to differentiate into neural cells.
MicroRNA-124a is one of the most abundant microRNAs in brain tissues, accounting for about 25-48% of the total microRNA in brain tissues. Lim et al. showed that infection of microRNA-124a into HeLa cells resulted in a series of non-neuronal transcripts being inhibited, and the genomic expression pattern of HeLa cells was transformed to neural direction. Tissue, microRNA-124 is only expressed in differentiated and mature neural cells, but rarely in neural precursor cells. In this study, rat microRNA-124a lentiviral vector containing green fluorescence protein (GFP) reporter gene was constructed to infect MSCs and pass it on to observe its expression in bone marrow. The effect of mesenchymal stem cells on differentiation of neurons.
The first part is fasudil induced differentiation of rat bone marrow mesenchymal stem cells into neurons.
objective
Objective To investigate the feasibility of fasudil, a Rho/Rho kinase inhibitor, in inducing rat bone marrow mesenchymal stem cells (MSCs) to differentiate into neural cells in vitro.
Method
Rat MSCs were isolated and cultured by whole bone marrow culture, induced by fasudil, observed by inverted microscope, identified by AO-EB staining, and identified by immunofluorescence staining the expression of NSE, NF200 and GFAP after induction.
Result
AO-EB staining was used to identify cell viability after induction.
After induction, the cytoplasm of the surviving cells was green, the nucleus was bright green, the nucleus was round or oval, the dead cells were red, the nucleus was bright red, and the number of the dead cells increased with the induction time. It is 96.7 + 2.2%, 95.3 + 1.9%, 93.8 + 1.8%, 92.5 + 2.1% and 90.1 + 1.3%. respectively.
Immunofluorescence identification after induction
The expression of NSE, NF200 and GFAP increased significantly and GFAP decreased with the prolongation of induction time in the fasudil-induced group. After induction, the positive rates of NSE (66.5 -1.9%, 88.1 -3.2%, 93.6 -1.9%, 93.5 -5.4%) and NF200 (89.5 -1.3%, 98.1 -1.6%, 98.3 -1.9% and 98.3 -1.9%) were gradually increased by immunofluorescence assay. The expression rate of GFAP in each group was less than 5%..
The second part is the expression change of TRPM8 in the differentiation of bone marrow mesenchymal stem cells into nerve cells in vitro.
Transient receptor potential (TRP) is a kind of transmembrane ion channel which widely exists on the cell membrane. It can distinguish taste, temperature and other special sensations. TRP channel subtype TRPM8 mainly exists on the cell membrane of specific nerve cells. When the temperature is lower than 27 C or the presence of menthol, TRPM8 channel opens, so that Ca2+ and other positively charged particles enter cells and have important physiological significance.
objective
This study was designed to investigate the expression and role of TRPM8 in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells.
Method
The expression of TRPM8 was detected by immunocytochemistry and Western Blot assay on the basis of inducing rat MSCs to differentiate into neural cells by stereotactic external fasudil.
Result:
Before induction, MSCs did not express TRPM8; 30 minutes after induction; MSCs began to express TRPM8 (45.3% + 1.58%); 60 main cells were induced and the expression increased (57.50% + 2.45%); 90 minutes after induction, the expression of TRPM8 was the highest (89.56% + 12.24%); 120 minutes after induction, the expression of TRPM8 gradually decreased (59.25% + 9.15%) and Western Blot had a similar trend.
The third part is the construction of microRNA-124a lentiviral vector and the infection of rat bone marrow mesenchymal stem cells.
objective
Objective: To investigate the role of microRNA-124a in the differentiation of rat bone marrow mesenchymal stem cells (MSCs) into neural cells induced by fasudil.
Method
In this study, rat microRNA-124a lentiviral vector containing GFP reporter gene was constructed and infected into rat bone marrow mesenchymal stem cells (MSCs) for passage. Rat MSCs were induced to differentiate into nerve cells by Fasudil and then subjected to an inverted fluorescence microscope. The expression of neuron enolase (NSE), neurofilament protein (NF200) and glial fibrillary acidic protein (GFAP) were detected by immunocytochemical staining and Western blot, and the cell viability was detected by MTT assay.
Result
Immunocytochemical staining
One hour after induction, the expression rates of NSE and NF200 were 83.2 (+ 2.0%) and 79.6 (+ 0.4%) respectively, which were significantly higher than those of the other two groups (P 0.05). The expression rates of GFAP in each group were less than 5%, and there was no significant difference.
Western Blot results
The expression of NSE and NF200 was not significantly different between the non-infected group and the negative control group, but the expression of NSE and NF200 was significantly higher in the infected group than in the other two groups.
conclusion
In this study, we found that fasudil, an inhibitor of Rho/Rho kinase (ROCK), could induce rat MSCs to differentiate into neural cells quickly and efficiently in vitro.
This is the first report that TRPM8, a special neuronal protein MSCs, is expressed in differentiated neurons and may play a role in the process of differentiation.
3 microRNA-124a can promote the differentiation of rat bone marrow mesenchymal stem cells into neurons.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R329

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