本文選題：間充質(zhì)干細(xì)胞 + IGF-1��； 參考：《華中科技大學(xué)》2009年博士論文

【摘要】： 目的: 1.體外探討分離與培養(yǎng)大鼠骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)的方法,并觀察其體外的多向分化與增殖能力以及其生物學(xué)特性,還對(duì)細(xì)胞表面干細(xì)胞相關(guān)標(biāo)志物進(jìn)行鑒定,為骨髓間充質(zhì)干細(xì)胞的進(jìn)一步研究奠定基礎(chǔ)。 2.探討胰島素樣生長(zhǎng)因子和多種細(xì)胞因子聯(lián)合誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞分化的可行性,實(shí)驗(yàn)條件。并用激光共聚焦顯微鏡觀察分化細(xì)胞的陽(yáng)性率,及形態(tài)學(xué)變化尋找最佳的誘導(dǎo)條件,為獲得大量少突膠質(zhì)細(xì)胞移植修復(fù)脊髓損傷奠定基礎(chǔ)。 3.在實(shí)驗(yàn)二的基礎(chǔ)上,觀察骨髓間充質(zhì)干細(xì)胞的增殖分化過(guò)程,判斷IGF-1在少突膠質(zhì)細(xì)胞分化中的那個(gè)環(huán)節(jié)起作用,尋找胰島素樣生長(zhǎng)因子和多種細(xì)胞因子聯(lián)合誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞分化的作用機(jī)制,并給予一定的干預(yù)措施,進(jìn)一步予以證實(shí)。 方法: 1.取原代細(xì)胞后,用貼壁篩選法和胰蛋白酶消化法分離純化,倒置相差顯微鏡觀察細(xì)胞形態(tài)及生長(zhǎng)特征,研究其增殖過(guò)程,繪制出生長(zhǎng)曲線,采用免疫組化法對(duì)細(xì)胞表面干細(xì)胞標(biāo)志CD44進(jìn)行鑒定,對(duì)第4代細(xì)胞分別用成骨,成軟骨,成脂肪和成神經(jīng)誘導(dǎo)劑培養(yǎng),行堿性磷酸酶染色,Van kossa銀染色法,Ⅱ型膠原免疫組化染色,油紅O染色,NeuN抗體免疫組化染色以及形態(tài)學(xué)觀察,證實(shí)其干細(xì)胞的多向分化潛能。 2.選取生長(zhǎng)良好的第4代骨髓間充質(zhì)干細(xì)胞,胰酶消化后,離心加入誘導(dǎo)液,轉(zhuǎn)移至含有蓋玻片的培養(yǎng)皿中,細(xì)胞數(shù)為1×10~4個(gè)/cm~2,放入體積分?jǐn)?shù)為0.05的CO_2孵箱中培養(yǎng)48h后,誘導(dǎo)組細(xì)胞更換培養(yǎng)液,用含不同濃度IGF-1的分化培養(yǎng)液培養(yǎng)3d后,觀察骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞誘導(dǎo)分化過(guò)程中的形態(tài)學(xué)變化,收取細(xì)胞進(jìn)行特異性標(biāo)志物大鼠磷脂堿性蛋白(MBP)、半乳糖腦苷脂(Glac)、半定量RT-PCR(semi-quatitative reverse transctiption-polymerase chain reaction)檢測(cè),Western-blot測(cè)定大鼠磷脂堿性蛋白(MBP)含量,免疫細(xì)胞化學(xué)染色后激光共聚焦顯微鏡檢測(cè)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞定向分化的陽(yáng)性率。并篩選出最佳IGF-1(Insulin growthfactor-1)濃度劑量的分化培養(yǎng)液。 3.根據(jù)BMP2(bone morphogenetic proteins)在少突膠質(zhì)細(xì)胞的形成中起抑制作用,少突膠質(zhì)細(xì)胞的成熟需要BMP信號(hào)的抑制劑,為了獲得IGF—1誘導(dǎo)少突膠質(zhì)細(xì)胞分化的分子機(jī)制,我們推斷IGF—1的影響是通過(guò)抑制BMP2信號(hào)通路。我將分化培養(yǎng)液中加入不同劑量的BMP2培養(yǎng)3天,收取細(xì)胞進(jìn)行特異性標(biāo)志物大鼠磷脂堿性蛋白(MBP)、半乳糖腦苷脂(Glac)、膠質(zhì)纖維酸性蛋白(GFAP)、微管相關(guān)蛋白—2 (MAP-2)半定量RT-PCR檢測(cè),并進(jìn)行統(tǒng)計(jì)學(xué)處理,免疫細(xì)胞化學(xué)染色后激光共聚焦顯微鏡檢測(cè)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞定向分化的陽(yáng)性細(xì)胞百分比。 結(jié)果: 1.所分離培養(yǎng)的細(xì)胞形態(tài)呈長(zhǎng)梭形或多邊形,細(xì)胞生長(zhǎng)曲線呈S形,群體倍增時(shí)間約為31小時(shí),經(jīng)成骨誘導(dǎo)劑誘導(dǎo)培養(yǎng)2周后,堿性磷酸酶染色成陽(yáng)性,Van kossa銀染色法陽(yáng)性表明該細(xì)胞可向成骨方向分化,經(jīng)成軟骨誘導(dǎo)劑誘導(dǎo)培養(yǎng)2周后,Ⅱ型膠原免疫組化染色陽(yáng)性,表明該細(xì)胞可向成軟骨方向分化,而分離培養(yǎng)的細(xì)胞經(jīng)成脂肪誘導(dǎo)劑誘導(dǎo)培養(yǎng)2周后,油紅O染色可見(jiàn)顯微鏡下大量細(xì)胞內(nèi)充滿紅色脂肪滴,表明該細(xì)胞可向脂肪細(xì)胞方向分化,所培養(yǎng)的細(xì)胞經(jīng)化學(xué)性神經(jīng)誘導(dǎo)劑誘導(dǎo)培養(yǎng)8小時(shí)后,NeuN抗體免疫組化染色陽(yáng)性,細(xì)胞呈神經(jīng)元樣形態(tài)表現(xiàn),表明該細(xì)胞可向神經(jīng)細(xì)胞方向分化。MSCs抗原CD34免疫細(xì)胞化學(xué)染色呈陰性反應(yīng),CD44免疫細(xì)胞化學(xué)染色見(jiàn)鏡下有棕黃色顆粒沉積。以上結(jié)果提示,用貼壁篩選法所分離培養(yǎng)的細(xì)胞為具有多向分化潛能的大鼠骨髓間充質(zhì)干細(xì)胞。 2.①骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞誘導(dǎo)分化過(guò)程中的形態(tài)學(xué)變化:經(jīng)誘導(dǎo)分化后,大部分骨髓間充質(zhì)干細(xì)胞表現(xiàn)出少突膠質(zhì)細(xì)胞的形態(tài)學(xué)特征,胞質(zhì)向細(xì)胞核回縮,細(xì)胞突起向外延伸,折光性增強(qiáng),隨時(shí)間延長(zhǎng)多個(gè)細(xì)胞突起相互連接形成典型的網(wǎng)狀結(jié)構(gòu)。②少突膠質(zhì)細(xì)胞特異性標(biāo)志物mRNA的表達(dá):細(xì)胞誘導(dǎo)分化后可檢測(cè)到磷脂堿性蛋白mRNA、半乳糖腦苷脂mRNA的特異性條帶。③少突膠質(zhì)細(xì)胞陽(yáng)性率:在誘導(dǎo)分化條件下,半乳糖腦苷脂陽(yáng)性率為65%,磷脂堿性蛋白陽(yáng)性率為45%,微管相關(guān)蛋白2陽(yáng)性率為10%。比較含不同IGF-1濃度的分化培養(yǎng)液,發(fā)現(xiàn)含500μg/L IGF-1濃度的培養(yǎng)液少突膠質(zhì)細(xì)胞標(biāo)志物陽(yáng)性率最高。④Western-blot檢測(cè)磷脂堿性蛋白表達(dá)水平明顯增高,以含500μg/L IGF-1濃度的培養(yǎng)液分化組最為明顯。 3.在分化培養(yǎng)液中加入不同濃度的BMP2,發(fā)現(xiàn)向少突膠質(zhì)細(xì)胞的分化明顯受到抑制,僅部分骨髓間充質(zhì)干細(xì)胞表現(xiàn)出少突膠質(zhì)細(xì)胞的形態(tài)學(xué)特征,與BMP2的濃度成依賴關(guān)系,濃度越高(5μg/L)少突膠質(zhì)細(xì)胞標(biāo)志物(Glac,MBP)的陽(yáng)性細(xì)胞越少,收取細(xì)胞進(jìn)行特異性標(biāo)志物大鼠磷脂堿性蛋白(MBP)、半乳糖腦苷脂(Glac)半定量RT-PCR檢測(cè),未檢查出特異性條帶。在低濃度(0.05μg/L—0.5μg/L)的情況下,可以觀察到部份細(xì)胞向少突膠質(zhì)細(xì)胞分化,這個(gè)結(jié)果與BMPs抑制少突膠質(zhì)分化的特性相一致。 結(jié)論: 1.通過(guò)取大鼠骨髓組織培養(yǎng),用貼壁篩選法和胰蛋白酶消化法分離純化,可以分離出穩(wěn)定的含CD44抗原的有多向分化潛能的骨髓間充質(zhì)干細(xì)胞,該細(xì)胞不斷可以向中胚層成細(xì)胞分化,而且可以向外胚層神經(jīng)細(xì)胞分化,取材方便,容易獲得是體外研究干細(xì)胞定向分化的理想細(xì)胞。 2.合適的培養(yǎng)基和一定濃度的IGF-1有利于骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞定向分化,該結(jié)果通過(guò)探討誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞分化的可行性,實(shí)驗(yàn)條件,為以后的間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞的定向分化移植治療脊髓損傷打下基礎(chǔ)。 3.胰島素樣生長(zhǎng)因子誘導(dǎo)骨髓間充質(zhì)干細(xì)胞向少突膠質(zhì)細(xì)胞定向分化,其主要作用機(jī)理是通過(guò)抑制少突膠質(zhì)細(xì)胞前體在脊髓中特異化的抑制性因子BMPs的信號(hào)通道,促進(jìn)少突膠質(zhì)細(xì)胞的產(chǎn)生。
[Abstract]:Objective:
1. in vitro, the method of separating and cultivating mesenchymal stem cells (MSCs) of rat bone marrow mesenchymal stem cells (MSCs) was studied in vitro, and the ability of multidirectional differentiation and proliferation in vitro and its biological characteristics were observed, and the markers of cell surface stem cells were identified, which lay the foundation for further study of bone marrow mesenchymal stem cells.
2. the feasibility of combining insulin-like growth factors and multiple cytokines to induce differentiation of bone marrow mesenchymal stem cells into oligodendrocytes, and the experimental conditions were investigated. The positive rates of differentiated cells were observed by laser confocal microscopy, and the optimal induction conditions were found by morphological changes, so as to obtain a large number of oligodendrocytes to repair the spinal cord. The pulp injury lays the foundation.
3. on the basis of experiment two, the proliferation and differentiation process of bone marrow mesenchymal stem cells (MSCs) was observed and the role of IGF-1 in oligodendrocyte differentiation was judged, and the mechanism of insulin like growth factor and multiple cytokines combined to induce bone marrow mesenchymal stem cells to differentiate into oligodendrocytes was found, and some intervention was given. Measures are further confirmed.
Method:
1. after the primary cells were extracted, the cell morphology and growth characteristics were observed by the adherent screening method and trypsin digestion method. The cell morphology and growth characteristics were observed by inverted phase contrast microscope. The proliferation process was studied, the birth length curve was plotted, and the cell surface stem cell marker CD44 was identified by immunohistochemistry. The fourth generation cells were formed into bone, cartilage, adipose tissue and fat, respectively. Neurogenic inducer culture, alkaline phosphatase staining, Van Kossa silver staining, type II collagen immunohistochemical staining, oil red O staining, NeuN antibody immuno histochemical staining and morphological observation, confirmed the multidirectional differentiation potential of the stem cells.
2. the fourth generation bone marrow mesenchymal stem cells with good growth were selected. After trypsin digestion, the cells were centrifuged and transferred into the culture dish containing the cover glass. The number of cells was 1 x 10~4 /cm~2, and the cells were incubated for 48h in the CO_2 incubator with the volume fraction of 0.05. After the culture, the cells were replaced by the culture solution containing different concentrations of IGF-1, and the culture medium was cultured for 3D, Morphological changes during differentiation of bone marrow mesenchymal stem cells to oligodendrocytes were observed, and cell phospholipid alkaline protein (MBP), galactose brain glycoside (Glac), semi quantitative RT-PCR (semi-quatitative reverse transctiption-polymerase chain reaction) and phosphorus in rats were collected for the determination of phosphorus in rats. Lipid alkaline protein (MBP) content, immunocytochemical staining and laser confocal microscopy were used to detect the positive rate of directional differentiation of bone marrow mesenchymal stem cells to oligodendrocytes, and the optimal IGF-1 (Insulin growthfactor-1) concentration of differentiation culture medium was screened.
3. according to the inhibition of BMP2 (bone morphogenetic proteins) in the formation of oligodendrocytes, the maturation of oligodendrocytes requires an inhibitor of BMP signal. In order to obtain the molecular mechanism of IGF - 1 induced oligodendrocyte differentiation, we infer that the effect of IGF - 1 is through the inhibition of BMP2 signaling pathway. In different doses of BMP2 culture for 3 days, the cells were collected for specific markers of phospholipid alkaline protein (MBP), galactose brain glycoside (Glac), glial fibrillary acidic protein (GFAP), microtubule related protein 2 (MAP-2) semi quantitative RT-PCR, and the statistics were carried out. After immunocytochemical staining, laser confocal microscopy was used to detect bone marrow The percentage of positive cells differentiated from mesenchymal stem cells to oligodendrocytes.
Result:
The cell morphology of the 1. isolated cultures was long shuttle shaped or polygon, the cell growth curve was S shaped, and the population doubling time was about 31 hours. After 2 weeks of induction of osteogenic inducer, alkaline phosphatase staining was positive. Van Kossa silver staining method showed that the cell could differentiate into osteogenic direction. After induction culture of cartilage inducer for 2 weeks, type II type was found. The collagen immunohistochemical staining was positive, indicating that the cells could differentiate into cartilage. After 2 weeks of induced culture, the isolated cells were filled with red fat droplets in a large number of cells under microscope, indicating that the cells could differentiate into adipocytes, and the cultured cells were induced by chemical nerve inducers. After 8 hours of induction and culture, the immunocytochemical staining of NeuN antibody was positive, and the cells showed neuron like morphology. It showed that the cells could differentiate into.MSCs antigen CD34 by immunocytochemical staining and negative reaction to nerve cells. CD44 immunocytochemical staining showed brown yellow granules under the microscope. The isolated cells are multipotent differentiation rat bone marrow mesenchymal stem cells.
2. (1) morphologic changes during differentiation of bone marrow mesenchymal stem cells into oligodendrocytes: after differentiation, most bone marrow mesenchymal stem cells show morphological characteristics of oligodendrocytes, the cytoplasm retracts to the nucleus, the cell protruding outwards, the refraction is enhanced, and the multiple cell protrusions are connected with time to connect with each other. A typical reticular formation. (2) the expression of oligodendrocyte specific marker mRNA: the specific bands of phospholipid alkaline protein mRNA and galactose brain glycoside mRNA were detected after induction of differentiation. (3) the positive rate of oligodendrocytes: the positive rate of galactose brain glycosides was 65% and the positive rate of phospholipid alkaline protein was 4 under the induced differentiation condition. 5%, the positive rate of microtubule related protein 2 was 10%. compared with the differentiation culture medium containing different IGF-1 concentrations. It was found that the positive rate of oligodendrocyte markers in the medium containing 500 mu g/L IGF-1 was the highest. 4. The expression level of phospholipid alkaline protein was significantly increased by Western-blot, and the most obvious differentiation group with the concentration of 500 mu g/L IGF-1 was the most obvious.
3. with the addition of different concentrations of BMP2 in the differentiated medium, it was found that the differentiation of oligodendrocytes was obviously inhibited. Only some bone marrow mesenchymal stem cells showed morphological characteristics of oligodendrocytes, which were dependent on the concentration of BMP2. The higher the concentration (5 g/L), the less positive cells of oligodendrocyte markers (Glac, MBP) were the less, the less the positive cells of the oligodendrocytes (Glac, MBP) were collected. The specific markers of phospholipid alkaline protein (MBP) and galactose brain glycoside (Glac) were detected by the cells, and the specific bands were not detected. In the case of low concentration (0.05 u g/L - 0.5 mu g/L), some cells could be observed to be divided into oligodendrocytes. This result is in accordance with the characteristics of BMPs inhibition of oligodendrocyte differentiation. Cause.
Conclusion:
1. through the culture of rat bone marrow tissue, we can isolate and purify the bone marrow mesenchymal stem cells with multidirectional differentiation potential with CD44 antigen, which can differentiate into mesoderm cells, and can be differentiated from the mesoderm cells, which can be easily obtained and easily obtained. It is an ideal cell for studying the directional differentiation of stem cells in vitro.
2. a suitable medium and a certain concentration of IGF-1 are beneficial to the differentiation of bone marrow mesenchymal stem cells into oligodendrocytes. The result is to explore the feasibility of inducing bone marrow mesenchymal stem cells to differentiate into oligodendrocytes, and the experimental conditions are for the treatment of spinal cord with oligodendrocyte differentiation and transplantation for the future. Damage lays the foundation.
3. insulin-like growth factors induce bone marrow mesenchymal stem cells to differentiate into oligodendrocytes, the main mechanism of which is to promote the production of oligodendrocytes by inhibiting the signal channel of the inhibitory factor BMPs of the oligodendrocyte precursor in the spinal cord.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329

【共引文獻(xiàn)】

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