【摘要】：角膜基質(zhì)細(xì)胞(corneal stroma cells, CSCs)是散在分布于角膜基質(zhì)內(nèi)神經(jīng)嵴來(lái)源的細(xì)胞,對(duì)維持角膜透明性發(fā)揮著重要作用。在體CSCs數(shù)量稀少,所以在體外對(duì)細(xì)胞進(jìn)行培養(yǎng)擴(kuò)增是必經(jīng)的研究路徑。研究表明,培養(yǎng)于含胎牛血清(FBS)的完全培養(yǎng)基內(nèi)的CSCs會(huì)喪失其原有的生物學(xué)特性。然而,當(dāng)培養(yǎng)于無(wú)血清的基礎(chǔ)培養(yǎng)基時(shí),細(xì)胞雖能保持其特性不變,卻無(wú)法進(jìn)行有效增殖。因此,如何在保持細(xì)胞生物學(xué)特性不變的情況下高效擴(kuò)增CSCs,是目前研究難點(diǎn)之一。 研究表明,出生后增殖性CSCs的細(xì)胞數(shù)量會(huì)迅速減少。當(dāng)瞼裂打開后,所有CSCs的細(xì)胞周期進(jìn)入G0期。最近研究證實(shí),CSCs表達(dá)眾多干細(xì)胞標(biāo)記物,并且具有多向分化潛能,與間充質(zhì)干細(xì)胞的生物學(xué)特性十分相似。然而,目前尚缺乏小鼠CSCs是否具有間充質(zhì)干細(xì)胞特性的研究。 樹突狀細(xì)胞(dendritic cells, DCs)是目前已知體內(nèi)功能最強(qiáng)的抗原呈遞細(xì)胞。成熟DCs可引發(fā)機(jī)體免疫反應(yīng),而未成熟DCs則會(huì)誘導(dǎo)機(jī)體免疫耐受。而且,角膜內(nèi)的DCs廣泛的參與了多種角膜相關(guān)疾病以及角膜移植免疫排斥反應(yīng),且以角膜內(nèi)DCs為靶細(xì)胞的治療方法已取得可喜的療效。因此,對(duì)角膜內(nèi)的DCs,尤其對(duì)DCs成熟狀態(tài)的研究具有重要意義。 最近研究表明,位于角膜中央?yún)^(qū)的DCs完全處于未成熟狀態(tài),而位于角膜周邊區(qū)的DCs則大多處于成熟狀態(tài)。局部微環(huán)境對(duì)DCs的成熟狀態(tài)發(fā)揮著重要的調(diào)節(jié)功能。所以,我們推測(cè)CSCs可能具有影響角膜內(nèi)DCs成熟狀態(tài)的功能,然而至今尚未見相關(guān)報(bào)道。 因此,本研究旨在探索如何在體外有效擴(kuò)增小鼠CSCs以及對(duì)CSCs的間充質(zhì)干細(xì)胞特性和抑制DCs成熟的功能進(jìn)行探討。如下: 第一部分小鼠角膜基質(zhì)細(xì)胞的提取、鑒定以及培養(yǎng)擴(kuò)增 目的:研究使用KSFM培養(yǎng)基能否獲取具有增殖能力且保持生物學(xué)特性不變的小鼠CSCs。 方法:將中央?yún)^(qū)角膜置于EDTA液(20mmol/L)內(nèi)孵育45min后,用手術(shù)顯微鑷小心剝離角膜上皮層以及內(nèi)皮層,并將獲取的角膜基質(zhì)置于含300U/mL I型膠原酶的溶液中消化4h。離心后采用DMEM基礎(chǔ)培養(yǎng)基、DMEM完全培養(yǎng)基(含10% FBS)以及KSFM培養(yǎng)基重懸細(xì)胞,接種于培養(yǎng)瓶?jī)?nèi)常規(guī)培養(yǎng),并采用含1U/mL分散酶的EDTA液消化傳代細(xì)胞。同時(shí),觀察細(xì)胞并繪制細(xì)胞生長(zhǎng)曲線;采用逆轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)檢測(cè)細(xì)胞角膜蛋白多糖(keratocan)、乙醛脫氫酶(ALDH)、細(xì)胞角蛋白12(CK12)和神經(jīng)元特異性烯醇化酶(NSE)等基因的表達(dá)情況;采用細(xì)胞免疫熒光染色以及蛋白質(zhì)印跡方法檢測(cè)細(xì)胞keratocan蛋白的表達(dá)情況。 結(jié)果:通過(guò)膠原酶消化的方法可以從每只小鼠的角膜基質(zhì)獲取約1×104單個(gè)細(xì)胞。RT-PCR結(jié)果顯示:原代細(xì)胞表達(dá)CSCs標(biāo)記物keratocan和ALDH,不表達(dá)角膜上皮細(xì)胞標(biāo)記物CK12以及角膜內(nèi)皮細(xì)胞標(biāo)記物NSE;免疫熒光染色和蛋白質(zhì)印跡結(jié)果顯示:原代細(xì)胞表達(dá)keratocan蛋白。因此,本實(shí)驗(yàn)獲取的原代細(xì)胞為CSCs。培養(yǎng)于DMEM基礎(chǔ)培養(yǎng)基內(nèi)的原代CSCs無(wú)法增殖。培養(yǎng)于DMEM完全培養(yǎng)基內(nèi)的CSCs可增殖,但第3代細(xì)胞不表達(dá)keratocan和ALDH基因以及keratocan蛋白。培養(yǎng)于KSFM培養(yǎng)基內(nèi)的CSCs也可增殖,第3代細(xì)胞仍表達(dá)keratocan和ALDH基因以及keratocan蛋白,且與原代細(xì)胞相比,表達(dá)強(qiáng)度無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。 結(jié)論:KSFM培養(yǎng)基不僅能維持小鼠CSCs的生物學(xué)特性不變,還能有效促進(jìn)細(xì)胞增殖。 第二部分小鼠角膜基質(zhì)細(xì)胞的間充質(zhì)干細(xì)胞樣表型以及多向分化潛能 目的:研究KSFM培養(yǎng)基培養(yǎng)擴(kuò)增后的小鼠CSCs是否具有間充質(zhì)干細(xì)胞樣表型以及多向分化潛能。 方法:在去除角膜上皮層以及內(nèi)皮層后,通過(guò)膠原酶消化的方法獲取小鼠中央?yún)^(qū)角膜來(lái)源的CSCs,并采用KSFM培養(yǎng)基對(duì)其培養(yǎng)擴(kuò)增。收集第2代CSCs,將細(xì)胞與造血干細(xì)胞標(biāo)記物抗體(CD34-FITC、CD45-PE)以及間質(zhì)細(xì)胞標(biāo)記物抗體(CD105-PE、CD90-FITC、CD71-FITC、CD29-APC)共孵育30min后,應(yīng)用流式細(xì)胞技術(shù)進(jìn)行檢測(cè)。當(dāng)培養(yǎng)于KSFM培養(yǎng)基內(nèi)的CSCs達(dá)細(xì)胞融合后,更換成骨細(xì)胞誘導(dǎo)培養(yǎng)基(含10%FBS、100nmol/L地塞米松、10mmol/Lβ-磷酸甘油、50mg/L維生素C的DMEM培養(yǎng)基)、脂肪細(xì)胞誘導(dǎo)培養(yǎng)基(含10% FBS、0.5μmol/L地塞米松、0.5mmol/L 3-異丁基-1-甲基黃嘌呤、10mg/L胰島素的DMEM培養(yǎng)基)以及對(duì)照培養(yǎng)基(含10% FBS的DMEM培養(yǎng)基),進(jìn)行常規(guī)培養(yǎng),每3d更換一次培養(yǎng)基。21d后,對(duì)培養(yǎng)于成骨細(xì)胞誘導(dǎo)培養(yǎng)基以及對(duì)照培養(yǎng)基內(nèi)的細(xì)胞進(jìn)行2%茜素紅S染色,并通過(guò)RT-PCR檢測(cè)細(xì)胞堿性磷酸酶和骨鈣素等基因的表達(dá)情況;對(duì)培養(yǎng)于脂肪細(xì)胞誘導(dǎo)培養(yǎng)基以及對(duì)照培養(yǎng)基內(nèi)的細(xì)胞進(jìn)行0.3%油紅O染色,并通過(guò)RT-PCR檢測(cè)細(xì)胞脂蛋白脂酶和過(guò)氧化物酶增殖物激活受體γ等基因的表達(dá)情況。 結(jié)果:應(yīng)用流式細(xì)胞技術(shù)對(duì)第2代CSCs的表型特征進(jìn)行分析,結(jié)果顯示:細(xì)胞低表達(dá)CD34(3.68%±1.44%)以及CD45(9.56%±1.83%),高表達(dá)CD29(96.85%±1.91%)、CD90(93.62%±1.65%)、CD105(50.91%±2.56%)以及CD71(45.27%±3.56%)。在成骨誘導(dǎo)條件下,3d時(shí),細(xì)胞形態(tài)仍然保持梭形,與對(duì)照組細(xì)胞無(wú)明顯差別。7d時(shí),細(xì)胞形態(tài)逐漸轉(zhuǎn)變?yōu)槎嘟切?胞漿內(nèi)出現(xiàn)黑色顆粒。14d時(shí),開始形成礦化結(jié)節(jié),并逐漸增大,21d時(shí),經(jīng)茜素紅S染色,結(jié)節(jié)呈現(xiàn)鮮紅色。對(duì)照組細(xì)胞未顯現(xiàn)出以上成骨細(xì)胞分化的形態(tài)學(xué)征象,且經(jīng)茜素紅S染色未見陽(yáng)性結(jié)果。通過(guò)RT-PCR檢測(cè)成骨細(xì)胞標(biāo)記物基因的表達(dá)情況,結(jié)果顯示:成骨誘導(dǎo)條件下細(xì)胞高表達(dá)堿性磷酸酶和骨鈣素,而對(duì)照組細(xì)胞低表達(dá)堿性磷酸酶且不表達(dá)骨鈣素。在脂肪誘導(dǎo)條件下,7d時(shí),細(xì)胞形態(tài)逐漸由梭形轉(zhuǎn)變?yōu)轭悎A形,胞漿內(nèi)液滴也逐漸增多。14d時(shí),細(xì)胞胞漿內(nèi)滿布液滴,經(jīng)油紅O染色,液滴被特異性染成橘紅色。RT-PCR結(jié)果顯示:脂肪誘導(dǎo)條件下細(xì)胞表達(dá)脂蛋白脂酶和過(guò)氧化物酶增殖物激活受體γ。而對(duì)照組細(xì)胞未顯現(xiàn)出向脂肪細(xì)胞分化的任何征象。 結(jié)論:經(jīng)KSFM培養(yǎng)基培養(yǎng)擴(kuò)增的小鼠中央?yún)^(qū)角膜來(lái)源的CSCs具有與間充質(zhì)干細(xì)胞相似的表型特征,以及向成骨細(xì)胞和脂肪細(xì)胞分化的能力。 第三部分小鼠角膜基質(zhì)細(xì)胞培養(yǎng)上清液對(duì)樹突狀細(xì)胞成熟的抑制作用 目的:研究小鼠CSCs培養(yǎng)上清液是否具有抑制脂多糖誘導(dǎo)的DCs成熟的作用。 方法:通過(guò)尼龍毛柱法獲取BALB/c小鼠脾臟來(lái)源的T細(xì)胞,并通過(guò)流式細(xì)胞技術(shù)檢測(cè)細(xì)胞表面標(biāo)記物CD3以測(cè)定T細(xì)胞純度。原代小鼠CSCs(105/mL)培養(yǎng)于RPMI 1640基礎(chǔ)培養(yǎng)基內(nèi),3d后半量換液,6d后收集培養(yǎng)上清液以備用。在裂解紅細(xì)胞后,將由C57BL/6小鼠股骨獲取的骨髓單核細(xì)胞培養(yǎng)于含10% FBS以及10ng/mL重組小鼠粒細(xì)胞-巨噬細(xì)胞集落刺激因子的RPMI 1640培養(yǎng)基內(nèi),2d后全量換液,4d后半量換液,6d后收集懸浮和半貼壁細(xì)胞,即為未成熟DCs。通過(guò)流式細(xì)胞技術(shù)檢測(cè)細(xì)胞表面標(biāo)記物CD11c以測(cè)定DCs純度。向DCs培養(yǎng)液內(nèi)加入脂多糖(1μg/mL),48h后未成熟DCs可被誘導(dǎo)成熟。為研究CSCs培養(yǎng)上清液對(duì)DCs成熟的作用,在DCs成熟過(guò)程中,不同濃度的培養(yǎng)上清液(25%、50%)被添加至DCs培養(yǎng)液中。而后,通過(guò)流式細(xì)胞技術(shù)檢測(cè)DCs成熟狀態(tài)標(biāo)記物CD80、CD86和主要組織相容性抗原Ⅱ類分子(MHC-Ⅱ),以對(duì)DCs的表型成熟狀態(tài)進(jìn)行鑒定;通過(guò)混合淋巴細(xì)胞反應(yīng)檢測(cè)DCs刺激T細(xì)胞增殖能力以及通過(guò)FITC標(biāo)記葡聚糖內(nèi)吞實(shí)驗(yàn)檢測(cè)抗原吞噬功能,以對(duì)DCs的功能成熟狀態(tài)進(jìn)行鑒定。 結(jié)果:小鼠脾臟細(xì)胞經(jīng)紅細(xì)胞裂解以及尼龍毛柱篩選提取后,可得到大量單個(gè)懸浮的小細(xì)胞。經(jīng)流式細(xì)胞技術(shù)檢測(cè),細(xì)胞高表達(dá)T細(xì)胞標(biāo)記物CD3(93.97%±3.06%)。小鼠骨髓單核細(xì)胞誘導(dǎo)培養(yǎng)6d后,細(xì)胞集落明顯,呈懸浮或半貼壁生長(zhǎng)。細(xì)胞表面可見長(zhǎng)短不一的毛刺狀突起,且高表達(dá)CD11c(78.61%±4.27%),低表達(dá)CD80、CD86和MHC-Ⅱ。細(xì)胞經(jīng)脂多糖刺激48h后,CD80、CD86和MHC-Ⅱ的表達(dá)明顯上調(diào)。在DCs成熟過(guò)程中,將不同濃度的CSCs培養(yǎng)上清液(25%、50%)添加至DCs培養(yǎng)液后,與對(duì)照組相比,DCs CD80、CD86和MHC-Ⅱ的表達(dá)均降低(P0.01),CD11c的表達(dá)無(wú)明顯差異(P0.05);刺激T細(xì)胞增殖能力降低(P0.05);抗原吞噬功能增強(qiáng)(P0.01)。此外,CSCs培養(yǎng)上清液抑制DCs成熟的作用還呈現(xiàn)出劑量依賴性(25% vs. 50%, P0.05)。 結(jié)論:小鼠CSCs培養(yǎng)上清液可以抑制脂多糖誘導(dǎo)的DCs表型以及功能成熟,且呈劑量依賴性。因此,我們推測(cè)CSCs可以通過(guò)分泌可溶性免疫調(diào)節(jié)因子抑制DCs成熟。 第四部分小鼠角膜基質(zhì)細(xì)胞通過(guò)分泌轉(zhuǎn)化生長(zhǎng)因子β2以及前列腺素E2抑制樹突狀細(xì)胞成熟 目的:探索小鼠CSCs是否通過(guò)分泌轉(zhuǎn)化生長(zhǎng)因子β2(TGF-β2)、前列腺素E2(PGE2)、白介素10(IL-10)以及巨噬細(xì)胞集落刺激因子(M-CSF)抑制DCs成熟。 方法:采用RT-PCR檢測(cè)原代小鼠CSCs TGF-β2、IL-10、M-CSF以及前列腺素內(nèi)過(guò)氧化物合酶2(PTGS2)等基因的表達(dá)情況。據(jù)此,通過(guò)酶聯(lián)免疫吸附實(shí)驗(yàn)(ELISA)測(cè)定CSCs培養(yǎng)上清液以及新鮮RPMI 1640培養(yǎng)基內(nèi)PGE2和TGF-β2的含量。而后,通過(guò)應(yīng)用TGF-β2中和抗體(15μg/mL)以及PGE2受體阻滯劑AH6809(100μmol/L),對(duì)CSCs是否通過(guò)分泌TGF-β2以及PGE2抑制DCs成熟作進(jìn)一步鑒定。在DCs成熟過(guò)程中,分別作以下不同處理:1,LPS;2,LPS+50% CSCs培養(yǎng)上清液;3,LPS+50% CSCs培養(yǎng)上清液+AH6809;4,LPS+50% CSCs培養(yǎng)上清液+中和抗體;5,LPS+50% CSCs培養(yǎng)上清液+AH6809+中和抗體。然后,應(yīng)用流式細(xì)胞技術(shù)檢測(cè)DCs CD11c、CD80、CD86和MHC-Ⅱ的表達(dá)情況,通過(guò)混合淋巴細(xì)胞反應(yīng)檢測(cè)刺激T細(xì)胞增殖能力,以及通過(guò)FITC標(biāo)記葡聚糖內(nèi)吞實(shí)驗(yàn)檢測(cè)抗原吞噬功能。 結(jié)果:RT-PCR結(jié)果表明:原代小鼠CSCs高表達(dá)TGF-β2和PTGS2,低表達(dá)M-CSF,不表達(dá)IL-10;ELISA數(shù)據(jù)顯示:與新鮮RPMI 1640培養(yǎng)基相比,CSCs培養(yǎng)上清液內(nèi)含有較高濃度的TGF-β2(1.46±0.38 ng/mL)和PGE2(21.27±0.94 ng/mL)。向CSCs培養(yǎng)上清液中加入TGF-β2中和抗體,可以不同程度的逆轉(zhuǎn)CSCs培養(yǎng)上清液對(duì)DCs表型以及功能成熟的抑制作用(P0.05或P0.01)。使用AH6809預(yù)處理未成熟DCs同樣可以不同程度的逆轉(zhuǎn)CSCs培養(yǎng)上清液對(duì)DCs功能成熟的抑制作用(P0.05),以及對(duì)CD86和MHC-Ⅱ表達(dá)的抑制作用(P0.05或P0.01),但不能逆轉(zhuǎn)對(duì)CD80表達(dá)的抑制作用(P0.05)。同時(shí)應(yīng)用TGF-β2中和抗體以及AH6809,可以提高DCs MHC-Ⅱ的表達(dá)和刺激T細(xì)胞增殖能力,且存在交互作用(P0.05);同時(shí)可以提高DCs CD80和CD86的表達(dá)以及降低DCs抗原吞噬功能,但不存在交互作用(P0.05)。此外,同時(shí)應(yīng)用TGF-β2中和抗體以及AH6809未完全逆轉(zhuǎn)CSCs培養(yǎng)上清液對(duì)DCs成熟的抑制作用(P0.05或P0.01)。 結(jié)論:在體外,小鼠CSCs可以通過(guò)分泌TGF-β2以及PGE2抑制DCs成熟,且此兩種細(xì)胞因子可發(fā)揮疊加效應(yīng)。
[Abstract]:Corneal stroma cells (CSCs), which is scattered in the source of the neural crest scattered in the corneal stroma, plays an important role in maintaining corneal transparency. The number of CSCs in the body is scarce, so the culture and expansion of cells in vitro is a required study path. The study shows that the culture of fetal bovine serum (FBS) is completely cultured. CSCs can lose its original biological characteristics. However, when it is cultured on a serum-free basic medium, the cell can not be effectively proliferated, although it can keep its characteristics unchanged. Therefore, it is one of the difficulties at present how to effectively amplify CSCs under the condition of keeping the cell biological characteristics unchanged.
Studies have shown that the number of cells in the proliferative CSCs after birth is rapidly reduced. When the palpebral cleft is opened, the cell cycle of all CSCs enters the G0 phase. Recent studies have confirmed that CSCs expresses numerous stem cell markers and has multiple differentiation potential and is very similar to the biological specificity of mesenchymal stem cells. However, it is still lack of CSCs in mice. Studies of the characteristics of mesenchymal stem cells.
Dendritic cells (DCs) is now known as the most potent antigen presenting cell in the body. Mature DCs can induce immune response, while immature DCs induces immune tolerance. Moreover, the DCs in the cornea is widely involved in a variety of corneal related diseases and corneal transplantation immune rejection, and the DCs in the cornea is in the cornea. Target cell therapy has achieved gratifying results. Therefore, it is important to study DCs in the cornea, especially DCs.
Recent studies have shown that DCs in the central cornea of the cornea is completely in the immature state, while most of the DCs in the peripheral area of the cornea are in mature state. Local microenvironment plays an important role in the maturation of DCs. Therefore, we speculate that CSCs may have the function of affecting the maturity of DCs in the cornea. However, there has been no phase yet. Close the report.
Therefore, the purpose of this study is to explore the effective amplification of mouse CSCs in vitro and the characteristics of CSCs mesenchymal stem cells and the inhibition of DCs maturation.
The first part is the extraction, identification and culture amplification of mouse corneal stromal cells.
Objective: To study whether KSFM medium can obtain CSCs. with the ability to proliferate and maintain biological characteristics.
Methods: after incubating the central region cornea in EDTA liquid (20mmol/L) to incubate 45min, the corneal epithelium and the endothelium were carefully stripped by surgical microtweezers, and the acquired corneal stroma was placed in a solution containing 300U/mL I collagenase, and after 4h. centrifugation, DMEM basal medium was used, DMEM complete medium (containing 10% FBS) and KSFM culture medium suspended fine. The cells were inoculated in the culture bottle for routine culture, and the cells were digested with the EDTA solution containing 1U/mL dispersing enzyme. The cells were observed and the cell growth curves were observed. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the cell corneal proteoglycan (keratocan), acetaldehyde dehydrogenase (ALDH), cytokeratin 12 (CK12) and neuron specific enol The expression of NSE and other genes were detected, and the expression of keratocan protein was detected by cellular immunofluorescence staining and Western blotting.
Results: by collagenase digestion, approximately 1 * 104 single cell.RT-PCR results were obtained from the corneal stroma of each mouse. The CSCs markers keratocan and ALDH were expressed in the primary cells, and the corneal epithelial cell marker CK12 and the corneal endothelial cell marker NSE were not expressed; the immunofluorescence staining and Western blotting results showed that the original cells were original. Therefore, the primary cells obtained in this experiment can not proliferate in the primary cultured CSCs cultured in the basal culture base of DMEM. CSCs can proliferate in the complete culture base of DMEM, but the third generation cells do not express the keratocan and ALDH genes and keratocan protein in the third generation cells. CSCs also can proliferate in the culture base of KSFM, third. The keratocan and ALDH genes and keratocan protein were still expressed in the generation cells, and there was no significant difference in the expression intensity compared with the primary cells (P0.05).
Conclusion: KSFM medium can not only maintain the biological characteristics of CSCs in mice, but also effectively promote cell proliferation.
The second part is the mesenchymal stem cell like phenotype and multipotential differentiation of mouse corneal stromal cells.
Objective: To study whether KSFM CSCs culture medium has mesenchymal stem cell like phenotype and multipotential differentiation potential.
Methods: after removing the corneal epithelium and the endothelium, the CSCs of the cornea of the central region of the mice was obtained by collagenase digestion and was amplified by KSFM medium. Second generations of CSCs were collected, and the cell and hematopoietic stem cell marker antibody (CD34-FITC, CD45-PE) and the antibody of interstitial cell markers (CD105-PE, CD90-FITC, CD71-) were collected. FITC, CD29-APC) were incubated with 30min and detected by flow cytometry. After the fusion of CSCs cells cultured in the KSFM culture base, the culture medium was replaced by osteoblasts (including 10%FBS, 100nmol/L dexamethasone, 10mmol/L beta glycerophosphate, DMEM Pei Yang Ji of 50mg/L vitamin C), and the adipocyte inducible medium (including 10% FBS, 0.5 mu) Siamethasone, 0.5mmol/L 3- isobutyl -1- methyl xanthine, DMEM medium of 10mg/L insulin) and a control medium (10% FBS DMEM medium) were used for routine culture. After each 3D was replaced by a culture medium.21d, 2% alizarin red S staining was performed on the osteoblast induced medium and the cells in the control culture base, and through RT-PCR. The expression of alkaline phosphatase and osteocalcin and other genes were detected, and 0.3% oil red O staining was performed on the cells cultured in the adipocyte induced medium and in the control culture base, and the expression of cell lipoprotein lipase and peroxidase proliferator activated receptor gamma were detected by RT-PCR.
Results: the phenotypic characteristics of the second generation CSCs were analyzed by flow cytometry. The results showed that the cells were low expression of CD34 (3.68% + 1.44%) and CD45 (9.56% + 1.83%), high expression of CD29 (96.85% + 1.91%), CD90 (93.62% + 1.65%), CD105 (50.91% +) and CD71 (45.27% + 3.56%). Under the osteogenic induction condition, the cell morphology remained the shuttle. When there was no obvious difference between the cells of the control group and the control group.7d, the cell morphology gradually changed into polygon, and when the black granule.14d in the cytoplasm appeared, the mineralized nodules began to form and gradually increased. The nodules were bright red when 21d was dyed with alizarin red S. The cells of the control group did not show the morphological signs of the osteoblast differentiation, and were dyed by alizarin red S. No positive results were found. The expression of osteoblast marker gene was detected by RT-PCR. The results showed that the cells expressed high expression of alkaline phosphatase and osteocalcin under the induction of osteogenesis, while the cells in the control group had low expression of alkaline phosphatase and did not express osteocalcin. In the condition of fat induction, the cell morphology gradually changed from spindle shape to round like form when 7d was induced. When the intracellular droplets were gradually increased by.14d, the cells were filled with liquid droplets in cytoplasm, stained with oil red O, and the droplets were stained specifically to orange red.RT-PCR. The results showed that the cells expressed lipoprotein lipase and peroxidase proliferator activated receptor gamma under the condition of fat induction. The cells in the control group did not show any signs of differentiation to adipocytes.
Conclusion: the CSCs of the central region of the mouse cornea derived from KSFM culture medium has similar phenotypic characteristics with mesenchymal stem cells and the ability to differentiate into osteoblasts and adipocytes.
The third part is the inhibitory effect of mouse corneal stromal cell culture supernatant on the maturation of dendritic cells.
Objective: To study whether the supernatant of mouse CSCs can inhibit the maturation of DCs induced by lipopolysaccharide.
Methods: the T cells from the spleen of BALB/c mice were obtained by nylon hairy column method, and the cell surface marker CD3 was detected by flow cytometry to determine the purity of T cells. The primary mouse CSCs (105/mL) was cultured in the basal culture base of RPMI 1640, the second half of the 3D was changed, and the cultured supernatant was collected after 6D. After the lysis of red blood cells, it would be C57BL/6. The bone marrow mononuclear cells obtained from the mouse femur were cultured in the RPMI 1640 culture base containing 10% FBS and 10ng/mL recombinant mouse granulocyte macrophage colony stimulating factor. After 2D, the total amount of fluid was changed, the latter half of the 4D was changed, and the suspension and half adherent cells were collected after 6D, that is, the immature DCs. was used to detect the cell surface marker CD11c by flow cytometry. The purity of DCs was determined. Lipopolysaccharide (1 u g/mL) was added to DCs culture, and immature DCs could be induced to mature after 48h. In order to study the effect of CSCs culture supernatant on DCs maturation, the culture supernatant of different concentrations (25%, 50%) was added to the DCs medium during the maturation of DCs, and then the DCs mature marker CD8 was detected by flow cytometry. 0, CD86 and the main histocompatibility antigen class II molecule (MHC- II), to identify the phenotypic maturation of DCs, and to detect the proliferation of T cells stimulated by DCs by mixed lymphocyte reaction and to detect the phagocytic function of the antigen by the FITC labeled dextran endocytosis test in order to identify the functional maturity of DCs.
Results: after the splenic cells were lysed and the nylon wool column was screened and extracted, a large number of single suspended small cells were obtained. The cells were detected by flow cytometry, and the high expression of T cell marker CD3 (93.97% + 3.06%). After the mouse bone marrow mononuclear cells were induced and cultured for 6D, the cell colonies were obviously suspended or half adhered to the wall. The surface of the cells was available. The expression of CD11c (78.61% + 4.27%), low expression of CD80, CD86 and MHC- II. The expression of CD80, CD86 and MHC- II was obviously up-regulated after 48h was stimulated by lipopolysaccharide. In the process of DCs maturation, the CSCs culture supernatant (25%, 50%) of different concentrations was added to the DCs culture solution. The expression of - II was decreased (P0.01), the expression of CD11c was not significantly different (P0.05), the proliferation of T cells was reduced (P0.05), and the function of antigen phagocytosis was enhanced (P0.01). In addition, the effect of CSCs culture supernatant on the inhibition of DCs maturation was also dose-dependent (25% vs. 50%, P0.05).
Conclusion: the mouse CSCs culture supernatant can inhibit the DCs phenotype and function maturity induced by lipopolysaccharide, and it is dose-dependent. Therefore, we speculate that CSCs can inhibit the maturation of DCs by secreting soluble immunoregulatory factors.
The fourth part of mouse corneal stromal cells inhibit dendritic cell maturation by secreting transforming growth factor beta 2 and prostaglandin E2.
Objective: To explore whether mouse CSCs inhibits DCs maturation by secreting TGF beta 2 (TGF- beta 2), prostaglandin E2 (PGE2), interleukin 10 (IL-10) and macrophage colony stimulating factor (M-CSF).
Methods: the expression of CSCs TGF- beta 2, IL-10, M-CSF and prostaglandin synthase 2 (PTGS2) were detected by RT-PCR. According to this, the content of CSCs culture supernatant and fresh RPMI 1640 medium PGE2 and TGF- beta 2 were measured by enzyme linked immunosorbent assay (ELISA). Then, the TGF- beta 2 and antibody (1) were used. 5 mu g/mL) and PGE2 receptor blocker AH6809 (100 mu mol/L) for further identification of CSCs through the secretion of TGF- beta 2 and PGE2 to inhibit DCs maturation. In the process of DCs maturation, the following different treatments were made: 1, LPS; 2, LPS+50% CSCs culture supernatant; 3, 4, culture supernatant + neutralization antibody; 5, +50% CSCs culture supernatant +AH6809+ neutralization antibody. Then, flow cytometry was used to detect the expression of DCs CD11c, CD80, CD86 and MHC- II. The proliferation ability of T cells was detected by mixed lymphocyte reaction, and the function of antigen phagocytosis was detected by FITC labelled dextran endocytosis test.
Results: RT-PCR showed that CSCs expressed TGF- beta 2 and PTGS2 with low expression of M-CSF and did not express IL-10. ELISA data showed that the CSCs culture supernatant contained a higher concentration of TGF- beta 2 (1.46 + 0.38 ng/mL) and PGE2 (21.27 + 0.94), compared with the fresh RPMI 1640 medium, and the antibody was added to the culture supernatant. Different levels of CSCs supernatant were reversed to DCs phenotype and functional maturity.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R772.2

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