本文選題：骨髓間充質(zhì)干細(xì)胞 + 誘導(dǎo)分化　； 參考：《天津醫(yī)科大學(xué)》2008年博士論文

【摘要】： 目的 隨著眼科臨床和基礎(chǔ)研究的進(jìn)步,白內(nèi)障、角膜病等可得到有效控制和治療,但一些致盲性視網(wǎng)膜疾患如視網(wǎng)膜色素變性、年齡相關(guān)性黃斑變性、青光眼視神經(jīng)視網(wǎng)膜病變等采用目前的方法無法逆轉(zhuǎn)。目前青光眼視神經(jīng)保護(hù)的策略是在有效降低眼壓的基礎(chǔ)上,針對視神經(jīng)損害的不同環(huán)節(jié),利用不同的藥物,保護(hù)患者視功能。此外,通過移植細(xì)胞使視網(wǎng)膜細(xì)胞更新是治療神經(jīng)變性疾病的另一種方法,逐漸受到關(guān)注。用于移植的細(xì)胞有腦源性神經(jīng)干細(xì)胞、視網(wǎng)膜祖細(xì)胞、骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells,BMSCs)等。 本課題旨在探討體外誘導(dǎo)大鼠BMSCs向神經(jīng)細(xì)胞分化的可能性和條件,為青光眼視神經(jīng)視網(wǎng)膜損害及其它視網(wǎng)膜退行性疾病的治療提供種子細(xì)胞奠定實(shí)驗(yàn)基礎(chǔ)。 方法 本課題選用大鼠骨髓間充質(zhì)干細(xì)胞,根據(jù)其取材方便,便于體外擴(kuò)增,可自身取材,來源豐富,免疫源性低等特性,對BMSCs進(jìn)行體外誘導(dǎo)為神經(jīng)元細(xì)胞進(jìn)行研究。本研究包括2部分: 1.大鼠BMSCs的培養(yǎng)和鑒定 采用貼壁篩選法獲得BMSCs,體外增殖純化,獲得純化干細(xì)胞。流式細(xì)胞儀細(xì)胞鑒定。 2.BMSCs經(jīng)bFGF預(yù)誘導(dǎo)后,以β-巰基乙醇(β-BM)和視網(wǎng)膜勻漿上清液為誘導(dǎo)劑進(jìn)行誘導(dǎo)。用倒置相差顯微鏡觀察細(xì)胞形態(tài),細(xì)胞免疫組織化學(xué)法檢測誘導(dǎo)后各組細(xì)胞巢蛋白(Nestin),神經(jīng)絲蛋白(NF),神經(jīng)元特異烯醇化酶(NSE)、膠質(zhì)纖維酸性蛋白(GFAP)等特異性標(biāo)志物的表達(dá)。 結(jié)果 1.大鼠BMSCs的分離、培養(yǎng)和鑒定 (1)細(xì)胞分離培養(yǎng) 通過貼壁法能獲得純度較高的BMSCs,原代培養(yǎng)3天后細(xì)胞呈紡錘形。7天后細(xì)胞呈團(tuán)簇、集落狀生長。細(xì)胞傳代后,24h細(xì)胞大部分貼壁,傳代后細(xì)胞增殖旺盛,仍呈梭形。傳代過多細(xì)胞增殖減慢,可見到一些扁平,寬大的細(xì)胞。 (2)流式細(xì)胞儀檢測細(xì)胞表面標(biāo)志 表達(dá)CD90、CD29、CD44,不表達(dá)CD34、CD45、CD31。 (3)多向分化潛能 在含有誘導(dǎo)劑的培養(yǎng)基中,BMSCs可在體外分化成為成骨細(xì)胞、脂肪細(xì)胞。 (4)10%血清濃度為BMSCs生長最佳濃度。 (5)不同傳代細(xì)胞增殖能力不同,P_1和P_3代細(xì)胞強(qiáng)于P_8代細(xì)胞。 2.體外神經(jīng)分化 (1)β-BM體外誘導(dǎo)BMSCs分化 加入5 mmol/Lβ-BM1h左右開始細(xì)胞質(zhì)向核收縮,胞體變小,變圓,透亮,并出現(xiàn)細(xì)胞突起,隨時(shí)間延長部分細(xì)胞胞體收縮,細(xì)胞突起變細(xì)、變長,呈雙極及多極,但數(shù)量不多。5h后更多細(xì)胞出現(xiàn)類神經(jīng)細(xì)胞改變,細(xì)胞突起相互交織。少數(shù)細(xì)胞懸浮。誘導(dǎo)1h后Nestin陽性率高于NSE和NF,差異有顯著性(P＜0.01);誘導(dǎo)5h后Nestin陽性率低于NSE和NF,差異有顯著性(P＜0.01)。NSE陽性率1h和5h,差異無顯著性(P＞0.05)。Nestin:1h高于5h,差異有顯著性(P＜0.01)。NF:1h低于5h,差異有顯著性(P＜0.01) 加入10 mmol/Lβ-BM誘導(dǎo)30 min左右細(xì)胞開始出現(xiàn)神經(jīng)元細(xì)胞樣形態(tài),但較多細(xì)胞懸浮。誘導(dǎo)1h后陽性率NSE＞NF＞Nestin,差異有顯著性(P＜0.001),誘導(dǎo)5h后Nestin陽性率低于NSE和NF,差異有顯著性(P＜0.01)。NSE和NF:陽性率1h低于5h(P＜0.01),Nestin則相反。 各組GFAP和對照組抗體染色陽性率在任何時(shí)間較其它抗體明顯低,差異有顯著性(P＜0.01)。 (2)視網(wǎng)膜勻漿上清液誘導(dǎo)BMSCs向神經(jīng)元細(xì)胞分化 兩組不同濃度視網(wǎng)膜勻漿上清液均能誘導(dǎo)BMSCs向神經(jīng)細(xì)胞分化,與β-BM相比作用比較緩和,3天為分化高峰。誘導(dǎo)細(xì)胞存活時(shí)間1周以上。大鼠視網(wǎng)膜勻漿上清液誘導(dǎo)24h后,不同濃度組Nestin陽性率均明顯高于NSE和NF,差異有顯著性(P＜0.01)。誘導(dǎo)3d后Nestin抗體染色陽性細(xì)胞數(shù)量減少,大部分神經(jīng)元樣細(xì)胞NSE和NF染色陽性,差異有顯著性(P＜0.01)。誘導(dǎo)7d后,Nestin抗體染色陽性細(xì)胞明顯減少,大部分神經(jīng)元樣細(xì)胞NSE和NF染色陽性,差異有顯著性(P＜0.01)。 NSE與NF:誘導(dǎo)24h比3d和7d陽性細(xì)胞率低,差異有顯著性(P＜0.01)。3d和7d差異無顯著性(P＞0.05)。高濃度組陽性細(xì)胞率高,差異有顯著性(P＜0.01)。 Nestin:隨誘導(dǎo)時(shí)間延長陽性率逐漸降低,各組差異均有顯著性(P＜0.01)。高濃度組陽性細(xì)胞率高,差異有顯著性(P＜0.01)。 各組GFAP和對照組抗體染色陽性率在任何時(shí)間較其它抗體明顯低,差異有顯著性(P＜0.01)。 結(jié)論 1.β-BM在體外可誘導(dǎo)BMSCs分化為神經(jīng)元樣細(xì)胞,5mmol/L濃度為最佳濃度。誘導(dǎo)細(xì)胞早期表達(dá)神經(jīng)干細(xì)胞標(biāo)志物Nestin,以后表達(dá)成熟神經(jīng)元標(biāo)志物NF,NSE,不表達(dá)膠質(zhì)細(xì)胞標(biāo)志物GFAP。誘導(dǎo)細(xì)胞存活時(shí)間短。 2.視網(wǎng)膜勻漿上清液可在體外誘導(dǎo)BMSCs分化為神經(jīng)元樣細(xì)胞;誘導(dǎo)細(xì)胞表達(dá)標(biāo)志物與β-BM誘導(dǎo)細(xì)胞相似。勻漿液作用緩和,細(xì)胞存活時(shí)間長,但分化率較低。
[Abstract]:objective
With the progress of clinical and basic research in the ophthalmology, cataracts and keratopathy can be effectively controlled and treated, but some blind retinal diseases such as retinitis pigmentosa, age related macular degeneration, and glaucomatous retinopathy can not be reversed. On the basis of effective reduction of intraocular pressure (IOP), different drugs are used to protect the visual function of the patients in different links of optic nerve damage. In addition, retinal cell renewal is another way to treat neurodegenerative diseases through transplantation of cells. Bone marrow mesenchymal stem cells (BMSCs) and so on.
The purpose of this study is to explore the possibility and conditions for inducing the differentiation of BMSCs into neural cells in vitro, and to provide the experimental basis for the treatment of retinal damage of glaucomatous optic nerve and other retinal degenerative diseases.
Method
In this study, rat bone marrow mesenchymal stem cells (MSCs) were used in vitro. According to their convenience and convenience in extracorporeal expansion, the bone marrow mesenchymal stem cells were easily obtained, rich in source, and low immunogenic characteristics. The BMSCs was induced in vitro to study the neuron cells. This study includes 2 parts:
The culture and identification of BMSCs in 1. rats
BMSCs was obtained by adherence screening method. The cells were purified and purified by flow cytometry.
After bFGF preinduction, 2.BMSCs was induced by beta mercapto ethanol (beta -BM) and the supernatant of retina homogenate. Cell morphology was observed by inverted phase contrast microscope. Cell nestin (Nestin), neurofilament (NSE), glial fibrillary acidic protein (GF), and glial fibrillary acidic protein (GF) were detected by cell immunohistochemistry. AP) the expression of the specific markers.
Result
Isolation, culture and identification of BMSCs in 1. rats
(1) cell isolation and culture
After 3 days of primary culture, the cells displayed a high purity of BMSCs. After 3 days of primary culture, the cells showed a cluster of spindle shaped.7 cells and colonies. After the passage of the cells, most of the cells were adhered to the wall, and the cells proliferated vigorously after the passage, and the cells were still spindle shaped.
(2) detection of cell surface markers by flow cytometry
Express CD90, CD29, CD44, do not express CD34, CD45, CD31.
(3) multidirectional differentiation potential
In the medium containing inducers, BMSCs can differentiate into osteoblasts and adipocytes in vitro.
(4) the concentration of 10% serum was the best concentration of BMSCs growth.
(5) the proliferation ability of cells from different passages is different. The cells of P_1 and P_3 are stronger than those of P_8 cells.
2. in vitro nerve differentiation
(1) induction of BMSCs differentiation in vitro by beta -BM
After adding 5 mmol/L beta -BM1h, the cytoplasm began to shrink to the nucleus, the cell body became smaller, round and bright, and the cell protruded, and the cell bodies contracted with time, the cell protruding became thinner and longer, and the cells were bipolar and multipolar, but more cells appeared to change and the cell protruding interlaced after the number of.5h. A few cells suspended. After 1h, the positive rate of Nestin was higher than that of NSE and NF (P < 0.01), and the positive rate of Nestin was lower than NSE and NF (P < 0.01). There was no significant difference (P < 0.01). There was no significant difference (> 0.05).
After the addition of 10 mmol/L beta -BM, the cells of about 30 min cells began to appear cell like morphology, but more cell suspension. The positive rate of NSE > NF > Nestin after induction of 1H was significant (P < 0.001). The positive rate of Nestin was lower than NSE and NF. The difference was significant (0.01) and the positive rate was lower than that of 0.01.
The positive rates of antibody staining in GFAP and control groups were significantly lower than those in other groups at any time (P < 0.01).
(2) the supernatant of retinal homogenate induces BMSCs to differentiate into neurons.
The two groups of different concentrations of retina homogenate could induce BMSCs to differentiate into nerve cells. Compared with beta -BM, the effect was relatively mild, and the 3 day was the peak of differentiation. The survival time of the cells was more than 1 weeks. After the induction of 24h in the rat retinal homogenate supernatant, the positive rates of Nestin in the different concentration groups were significantly higher than that of NSE and NF (P < 0.01). After 3D, the number of positive cells with Nestin antibody staining decreased, and most of the neuron like cells were positive for NSE and NF staining (P < 0.01). After induced 7d, the positive cells of Nestin antibody staining positive cells were significantly reduced, and most of the neuron like cells were positive for NSE and NF staining, the difference was significant (P < 0.01).
The rate of NSE and NF: induced 24h was lower than that of 3D and 7d positive cells. The difference was significant (P < 0.01) and there was no significant difference between.3d and 7d (P > 0.05). The positive cell rate of high concentration group was high, and the difference was significant (P < 0.01).
The positive rate of Nestin: gradually decreased with the induction time, and there was significant difference in each group (P < 0.01). The rate of positive cells in high concentration group was high, and the difference was significant (P < 0.01).
The positive rates of antibody staining in GFAP and control groups were significantly lower than those in other groups at any time (P < 0.01).
conclusion
1. beta -BM can induce BMSCs to differentiate into neuron like cells in vitro, and the concentration of 5mmol/L is the best concentration. It induces the early expression of neural stem cell marker Nestin, and then expresses the marker of mature neuron NF, NSE, and does not express the glial marker GFAP. to induce cell survival time.
2. the supernatant of the retina homogenate can induce BMSCs to differentiate into neuron like cells in vitro, and the induced cell expression marker is similar to that induced by beta -BM. The effect of homogenate fluid is mild, the cell survival time is long, but the differentiation rate is low.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2008
【分類號】：R329

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