【摘要】：背景和目的 角膜堿燒傷后,堿性物質(zhì)能夠迅速滲透眼表面,通過細(xì)胞滲透、蛋白水解酶和細(xì)胞因子的分泌導(dǎo)致嚴(yán)重的特征性的炎癥反應(yīng)。為保護角膜上皮的完整性,阻止基質(zhì)層潰瘍發(fā)生,國內(nèi)外學(xué)者們已經(jīng)應(yīng)用了多種治療方法,均取得了一定的療效,但是何為最佳療法,至今仍有爭議,并未確定。本研究分別通過四個階段探討磁標(biāo)記人羊膜間充質(zhì)干細(xì)胞(human amniotic membrane-derived mesenchymal stem cells, hAM-dMSCs)移植對兔角膜堿燒傷治療的影響,以及堿燒傷后淚液乳鐵蛋白(lactoferrin, LF)的表達,研究淚液LF與堿燒傷的關(guān)系及其意義,以期獲得對于角膜急性堿燒傷較為簡易且適于臨床使用的最佳治療方法。1.通過觀察人羊膜組織來源的細(xì)胞形態(tài)學(xué)、流式細(xì)胞儀檢測和MTT法活性細(xì)胞檢測等鑒定原代獲取的間充質(zhì)干細(xì)胞,為后續(xù)的實驗提供干細(xì)胞。2.不同方法標(biāo)記原代培養(yǎng)的羊膜間充質(zhì)干細(xì)胞,探索最合適的細(xì)胞標(biāo)記方法。3.超順磁性氧化鐵納米顆粒(Superparamagnetic iron oxide nanoparticles, SPIONs)標(biāo)記的hAM-dMSCs局部移植入角膜堿燒傷眼結(jié)膜下,觀察細(xì)胞在宿主存活和遷徙情況；聯(lián)合傳統(tǒng)的羊膜移植,通過臨床檢查及檢測IL-1p和TNF-α評估治療效果,探討干細(xì)胞在角膜堿燒傷后組織再生中扮演的角色。4.采集正常和堿燒傷治療后的兔淚液,采用放射免疫分析方法,分別測定其中LF的含量,研究淚液LF與堿燒傷的關(guān)系及其對堿燒傷愈合的意義。 材料和方法 1羊膜間充質(zhì)干細(xì)胞的原代培養(yǎng)和鑒定 使用酶消化和貼壁篩選法獲取人羊膜組織中的間充質(zhì)干細(xì)胞,反復(fù)傳代對其擴增和純化細(xì)胞。通過觀察細(xì)胞學(xué)形態(tài)、MTT法活性檢測和流式細(xì)胞儀檢測等鑒定人羊膜來源的間充質(zhì)干細(xì)胞。通過顯微鏡觀察細(xì)胞的形態(tài)變化；采用流式細(xì)胞術(shù)檢測人羊膜來源的細(xì)胞膜表面特異性蛋白分子CD31、CD44、CD45、 CD90、CD29、CD34的表達。 2.SPIONs標(biāo)記的羊膜間充質(zhì)干細(xì)胞在體外的活性監(jiān)測 本研究應(yīng)用SPIONs標(biāo)記的細(xì)胞生長培養(yǎng)基(Cytokine Growth Medium, CGM),標(biāo)記濃度分別為3.5、7、14和28μg/ml,細(xì)胞標(biāo)記采用單次SPIONs標(biāo)記法。培養(yǎng)的細(xì)胞分別于標(biāo)記后第1、2、4和7d收獲,各時間點獲得的細(xì)胞分為五組,分別行MTT法、普魯氏藍(lán)染色和流式細(xì)胞儀檢測；無SPIONs標(biāo)記的細(xì)胞生長培養(yǎng)基組作為對照組。 3.羊膜間充質(zhì)干細(xì)胞的標(biāo)記 分別采用不同濃度的Brdu、DAPI和SPIONs標(biāo)記原代培養(yǎng)的人羊膜間充質(zhì)干細(xì)胞,確定較好的羊膜間充質(zhì)干細(xì)胞標(biāo)記途徑和方法,為羊膜間充質(zhì)干細(xì)胞移植治療角膜堿燒傷的活體示蹤進行前期準(zhǔn)備。 4.羊膜間充質(zhì)干細(xì)胞移植對重度角膜堿燒傷的療效 待移植細(xì)胞的制備：應(yīng)用濃度為14μg/ml SPIONs標(biāo)記原代培養(yǎng)獲取的羊膜間充質(zhì)干細(xì)胞,標(biāo)記時間24h,收集細(xì)胞制成1×106細(xì)胞混懸液備用。 實驗動物分組和細(xì)胞移植的方法：動物被隨機分成四組,對照組(n=10)；人羊膜間充質(zhì)干細(xì)胞注射組(干細(xì)胞注射組,n=12)：結(jié)膜下注射含標(biāo)記后的hAM-dMSCs注射液；單純羊膜移植組(羊膜組,n=12)：行單純羊膜移植；人羊膜間充質(zhì)干細(xì)胞聯(lián)合羊膜移植組(聯(lián)合組,n=12)：結(jié)膜下注射含標(biāo)記后的羊膜間充質(zhì)干細(xì)胞注射液聯(lián)合羊膜移植。結(jié)膜下注射一律選擇眼球結(jié)膜顳上部位,注射量100微升,約含1×106個羊膜間充質(zhì)干細(xì)胞,干細(xì)胞注射組和聯(lián)合組對側(cè)眼分別注射同等量含14μg/ml SPIONs的生理鹽水作為對照。 羊膜移植方法：堿燒傷后,實驗側(cè)眼開瞼器開瞼,1：1000慶大霉素液沖洗結(jié)膜囊,沿角膜緣環(huán)形剪開球結(jié)膜,分離結(jié)膜下組織,刮除角膜表面壞死組織,將羊膜上皮面朝上平鋪使覆蓋整個角膜和角膜緣,用10.0尼龍線于角膜緣附近間斷縫合,并固定在淺層鞏膜上,將球結(jié)膜游離緣固定在羊膜上,剪去多余羊膜組織。術(shù)畢結(jié)膜下注射慶大霉素2萬u+地塞米松2mg,結(jié)膜囊內(nèi)涂紅霉素眼膏,縫合瞼緣防止兔損傷手術(shù)創(chuàng)面。術(shù)后局部常規(guī)點抗生素,激素。 5.兔角膜堿燒傷后淚液乳鐵蛋白的表達 淚液乳鐵蛋白(lactoferrin, LF)檢測選擇在角膜堿燒傷治愈后,停藥2周進行。治療方法采用本實驗得出的最佳治療方法：人羊膜間充質(zhì)干細(xì)胞聯(lián)合羊膜移植治療堿燒傷。將30只大白兔隨機分為三組：空白組(n=10)：不進行任何處理；對照組(n=10)：制作兔眼角膜堿燒傷模型,不進行間充質(zhì)干細(xì)胞或羊膜移植；實驗組(n=10)：造模后,局部結(jié)膜下行人羊膜間充質(zhì)干細(xì)胞移植聯(lián)合羊膜移植。造模動物均局部常規(guī)用藥,停藥2周開始實驗檢測。用微量吸管吸取結(jié)膜囊內(nèi)的淚液80-100ul,置于0.5m1無菌靜脈輸液管中,管端加熱封閉管口,置于深低溫(-30℃)冰箱內(nèi)保存待檢。放射免疫分析方法,測定正常角膜和堿燒傷治愈后的兔淚液LF的含量。 結(jié)果 1.用胰酶和膠原酶消化羊膜組織,將獲得的細(xì)胞懸液接種在細(xì)胞培養(yǎng)瓶內(nèi)進行原代培養(yǎng)。24小時后,倒置相差顯微鏡下觀察見少量細(xì)胞貼壁；培養(yǎng)48小時貼壁細(xì)胞數(shù)量略增多；72小時后,貼壁細(xì)胞數(shù)量顯著增多；原代培養(yǎng)第7天,貼壁細(xì)胞接近50%-60%融合,或基本鋪滿培養(yǎng)瓶底,貼壁細(xì)胞呈成纖維細(xì)胞樣細(xì)胞。 流式細(xì)胞儀檢測細(xì)胞分析顯示：原代培養(yǎng)的第三代細(xì)胞,高表達膜表面蛋白分子CD29、CD44、CD90、CD105;陰性表達CD31、CD34、CD45和CD106。 2.不同濃度的SPIONs進行標(biāo)記羊膜間充質(zhì)干細(xì)胞,普魯氏藍(lán)染色后顯示：胞質(zhì)內(nèi)可見不同數(shù)量的藍(lán)染顆粒,各組SPIONs標(biāo)記細(xì)胞的標(biāo)記率均為100%。隨標(biāo)記濃度增加,藍(lán)染顆粒增加,鐵顆粒呈簇狀聚集分布成堆。MTT結(jié)果明確顯示：SPIONs濃度小于和等于14μg/ml時,標(biāo)記的羊膜間充質(zhì)干細(xì)胞活性大于90%。當(dāng)SPIONs濃度大于14μg/m1時,細(xì)胞胞質(zhì)內(nèi)藍(lán)染顆粒聚集前后無變化。 3.用3種標(biāo)記液標(biāo)記細(xì)胞后細(xì)胞數(shù)目明顯減少,但SPIONs和DAPI標(biāo)記組細(xì)胞數(shù)在3d以后開始增加,其中SPIONs組細(xì)胞生長較好,第6天達到高峰。Brdu標(biāo)記組細(xì)胞數(shù)目始終維持在5×104左右。 4.14μg/ml SPIONs標(biāo)記的羊膜間充質(zhì)干細(xì)胞移植入兔結(jié)膜下,4周后角膜切片行普魯士藍(lán)染色,角膜緣處可見大量的藍(lán)染顆粒,說明hAM-dMSCs存活于角膜緣,生長良好。受體兔全身情況良好,眼局部無排斥反應(yīng)發(fā)生。而注射含147μg/ml SPIONs的生理鹽水的對側(cè)眼,在注射4周后未見藍(lán)染顆粒。 5.角膜堿燒傷的hAM-dMSCs移植結(jié)果：與對照組比較,實驗組愈合率顯著高于對照組(P0.01),而三個實驗組的愈合率差異無顯著性(X2=1.2,P0.05)。4周時各實驗組之間角膜新生血管與對照組相比差異有顯著性(P0.05)；角膜混濁度評分實驗組與對照組相比差異有顯著性(P0.01),；干細(xì)胞注射組和羊膜組與聯(lián)合組相比差異有顯著性(P0.01),干細(xì)胞注射組與羊膜組角膜混濁度評分差異無顯著性(t=0.374,P0.05)；不同治療方法和時間角膜新生血管面積各組與對照組相比差異有顯著性(P0.01或P0.05),實驗組組間比較,聯(lián)合組與其余兩個實驗組相比差異有顯著性(P0.05)。 HE染色顯示角膜上皮層有3-4層細(xì)胞,由少量表皮細(xì)胞、翼狀細(xì)胞和部分基底細(xì)胞組成�；|(zhì)層可見少量新生血管和炎性細(xì)胞。角膜普魯士染色顯示,角膜緣處可見藍(lán)染顆粒,提示hAM-dMSCs仍存活于角膜緣附近,角膜近中央?yún)^(qū)和中央?yún)^(qū)未見遷移的hAM-dMSCs。 6.Western-blot檢測兔角膜堿燒傷不同治療方法后角膜TNF-a和IL-1β的表達：正常對照組及實驗組角膜的蛋白經(jīng)印記實驗檢測可見特異的陽性印跡帶,Western-blot結(jié)果顯示TNF-α和IL-1β在角膜堿燒傷后治療過程中的各個階段都有表達,且隨著治療時間的延長,其表達強度明顯減弱。 ELISA檢測兔角膜堿燒傷后不同治療方法和時間房水TNF-α和IL-1β的變化：實驗眼房水TNF-α含量隨著時間的延長呈下降趨勢,不同時間點之間房水TNF-α含量比較在統(tǒng)計學(xué)上有顯著性差異。對照眼房水TNF-α含量平均值隨著時間的延長亦呈下降趨勢。在同一觀察時間點,實驗眼房水TNF-α含量均低于對照眼,在統(tǒng)計學(xué)上有顯著性差異。實驗組和對照組房水IL-1β含量平均值均隨著時間的延長呈下降趨勢,且實驗組房水IL-1p含量均低于對照眼。在同一觀察時間點,第1天和第3天,實驗組房水IL-1p含量低于對照組,第7天和第14天聯(lián)合組房水IL-1p含量低于對照眼,并且在統(tǒng)計學(xué)上均有顯著性差異。 7.實驗組與對照組比較,各觀察時間點淚液中乳鐵蛋白濃度差異有顯著性(P0.01)；對照組和實驗組各觀察時間點淚液中相對乳鐵蛋白濃度均無統(tǒng)計學(xué)差異(P0.05)；與正常空白組相比,對照組各觀察時間點淚液中乳鐵蛋白濃度差異有顯著性(P0.05),實驗組與空白組相比,其濃度無統(tǒng)計學(xué)意義(P0.05)。經(jīng)Spearman相關(guān)分析,各時間點淚液乳鐵蛋白的含量與Schirmer I試驗呈正相關(guān)(P0.01)。 結(jié)論 1.本實驗從人羊膜中分離間充質(zhì)干細(xì)胞,所得的間充質(zhì)干細(xì)胞純度較高。實驗提示：貼壁篩選和反復(fù)傳代是一種較為理想的提純羊膜間充質(zhì)干細(xì)胞的方法。 2.局部結(jié)膜下注射移植]hAM-dMSCs治療眼表化學(xué)傷安全可行,有助于臨床上新建一種細(xì)胞來源廣泛、無免疫排斥的、微創(chuàng)的治療眼表創(chuàng)傷疾病的新手段。 3.應(yīng)用SPIONs標(biāo)記方法標(biāo)記羊膜間充質(zhì)干細(xì)胞,SPIONs濃度小于和等于14μg/ml,既可使羊膜間充質(zhì)干細(xì)胞得到標(biāo)記,又不影響其活性。 4.SPIONs標(biāo)記hAM-dMSCs移植治療角膜堿燒傷動物模型,可達到示蹤的目的。 5.早期局部結(jié)膜下行hAM-dMSCs移植可顯著抑制傷側(cè)角膜表面新生血管形成,加速重度角膜堿燒傷創(chuàng)傷修復(fù)。hAM-dMSCs在宿主的遷移和分化,與局部微環(huán)境的改變密切相關(guān)。hAM-dMSCs的作用機制可能是通過調(diào)節(jié)細(xì)胞生長因子的分泌,改善殘留的正常細(xì)胞所處的微環(huán)境,促進殘留細(xì)胞的生長來發(fā)揮修復(fù)作用。 6.本研究在角膜堿燒傷早期行hAM-dMSCs移植,與對照組相比,測得淚液LF含量明顯增高。因此,除進行緊急有效的搶救措施外,早期行hAM-dMSCs移植,不僅能夠加快創(chuàng)傷角膜愈合,且能夠提高患者治愈后的視覺質(zhì)量及生活質(zhì)量。
[Abstract]:Background and purpose
After alkali burn, alkaline substances can penetrate the surface of the eye quickly, through cell infiltration, protein hydrolase and cytokine secretion, causing serious and characteristic inflammatory reactions. In order to protect the integrity of the corneal epithelium and prevent the occurrence of the stromal ulcer, a variety of treatment methods have been applied by domestic and foreign scholars, and some curative effects have been obtained. But what is the best treatment is still controversial and is not determined. This study investigated the effects of human amniotic membrane-derived mesenchymal stem cells (hAM-dMSCs) transplantation on the treatment of alkali burn in rabbits by four stages, and the lacrimal lactoferrin (lactoferrin, LF) after alkali burn. The relationship and significance of tear LF and alkali burn in order to obtain the best treatment for acute alkali burn of the cornea, which is more simple and suitable for clinical use,.1. by observing the cell morphology of the human amniotic tissue, flow cytometry and MTT assay to identify the primary mesenchymal stem cells obtained. Subsequent experiments provide stem cell.2. with different methods to mark primary cultured amniotic mesenchymal stem cells, and explore the most suitable cell marking method,.3. superparamagnetic iron oxide nanoparticles (Superparamagnetic iron oxide nanoparticles, SPIONs) labeled hAM-dMSCs locally transplanted into the corneal alkali burn eye conjunctiva, and observe the cell in the host Survival and migration, combined with traditional amniotic membrane transplantation, through clinical examination and detection of IL-1p and TNF- alpha, the role of the stem cells in tissue regeneration after alkali burns of the cornea was investigated to collect the tear of rabbits after the treatment of normal and alkali burn. The content of LF was measured by radioimmunoassay, and the content of the.4. was measured by radioimmunoassay, and the tears were studied. Relationship between fluid LF and alkali burn and its significance for alkali burn healing.
Materials and methods
Primary culture and identification of 1 amniotic mesenchymal stem cells
Mesenchymal stem cells in human amniotic tissue were obtained by enzyme digestion and adherent screening, and the cells were amplified and purified repeatedly. The mesenchymal stem cells derived from human amniotic membrane were identified by observation of cytological morphology, MTT activity detection and flow cytometry. Flow cytometry was used to observe the morphological changes of the cells by microscope. The expression of surface specific protein CD31, CD44, CD45, CD90, CD29 and CD34 in human amniotic membrane was detected.
In vitro activity monitoring of 2.SPIONs labeled amniotic mesenchymal stem cells
In this study, the cell growth medium (Cytokine Growth Medium, CGM) marked by SPIONs was used in the study. The marker concentration was 3.5,7,14 and 28 mu g/ml respectively. The cell markers were marked by single SPIONs labeling. The cultured cells were harvested at 1,2,4 and 7d after labeling respectively. The cells obtained at each time point were divided into five groups, including MTT method, Prussian blue staining and flow formula, respectively. The cell growth medium without SPIONs labeling was used as control group.
3. markers of amniotic mesenchymal stem cells
Different concentrations of Brdu, DAPI and SPIONs were used to mark the primary cultured human amniotic mesenchymal stem cells, and the better labeling methods and methods of amniotic mesenchymal stem cells were determined to prepare the amniotic mesenchymal stem cells for the treatment of corneal alkali burn.
Effect of 4. amniotic mesenchymal stem cells transplantation on severe corneal alkali burn
The preparation of the transplanted cells: the amniotic mesenchymal stem cells obtained from the primary culture of 14 g/ml SPIONs were used to mark the time 24h, and the collected cells were made up of 1 x 106 cell suspension.
Experimental animal groups and cell transplantation methods: animals were randomly divided into four groups, control group (n=10), human amniotic mesenchymal stem cell injection group (stem cell injection group, n=12): subconjunctival injection of labeled hAM-dMSCs injection; pure amniotic membrane transplantation group (amniotic membrane group, n =12): pure amniotic membrane transplantation; human amniotic mesenchymal stem cells Combined amniotic membrane transplantation group (combined group, n=12): subconjunctival injection of amniotic mesenchymal stem cells and amniotic membrane transplantation with labeled amniotic membrane injection. Subconjunctival injection of the subconjunctival subconjunctival subconjunctiva was selected for 100 microl injections, containing about 1 * 106 amniotic mesenchymal stem cells. The injection group and the combined group were injected with the same amount of 14 in the same amount. The physiological saline of g/ml SPIONs was used as a control.
Amniotic membrane transplantation: after alkali burn, the eyelid opening was opened on the experimental side, the conjunctival sac was washed with 1:1000 gentamicin liquid, the conjunctiva was cut along the rim of the cornea, the tissue under the conjunctiva was separated and the necrotic tissue was removed from the cornea. The upper surface of the amniotic membrane covered the whole cornea and the corner of the keratoid membrane, and the 10 nylon lines were sutured with the limbus cornea. The free amniotic membrane was fixed on the amniotic membrane and the free amniotic membrane was fixed on the shallow sclera. The conjunctival 20 thousand u+ dexamethasone 2mg was injected under the conjunctival conjunctiva. The conjunctival sac was coated with Erythromycin Eye Ointment, and the eyelid margin was sutured to prevent the wound from the wound.
Expression of lacrimal lactoferrin in 5. rabbit cornea after alkali burn
Lactoferrin (LF) was selected for 2 weeks after corneal alkali burn. The best treatment method used in this experiment: amniotic mesenchymal stem cells combined with amniotic membrane transplantation for alkali burn. The 30 rabbits were randomly divided into three groups: blank group (n=10): no treatment; control group (n=10): making rabbit cornea alkali burn model without mesenchymal stem cells or amniotic membrane transplantation; experimental group (n=10): human amniotic mesenchymal stem cell transplantation under local conjunctiva and amniotic membrane transplantation under local conjunctiva. The animal model animals were treated with local conventional medication for 2 weeks, and the tear 80-100ul in the conjunctival sac was sucked with micropipette. In the 0.5m1 aseptic intravenous infusion tube, the tube end was heated to close the pipe mouth and stored in the deep and low temperature (-30) refrigerator to be checked. The radioimmunoassay method was used to determine the content of LF in the tear fluid after the normal cornea and alkali burn were cured.
Result
1. the amniotic membrane tissues were digested with pancreatin and collagenase, and the obtained cell suspension was inoculated in the cell culture bottle for.24 hours. A small amount of cell adherent was observed under the inverted phase contrast microscope, and the number of adherent cells increased slightly for 48 hours. After 72 hours, the number of adherent cells increased significantly; the primary culture was seventh days and adherent cells. Close to the 50%-60% fusion, or basically covered with the bottom of the culture bottle, the adherent cells were fibroblast like cells.
Flow cytometry cell analysis showed that the third generation of primary cultured cells expressed high expression of membrane protein molecules CD29, CD44, CD90, CD105, and negative expression of CD31, CD34, CD45 and CD106..
2. different concentrations of SPIONs were used to mark amniotic mesenchymal stem cells. Prussian blue staining showed that different quantities of blue stained particles were found in the cytoplasm. The labeling rate of SPIONs labeled cells in each group increased with the concentration of 100%., the blue dye particles increased and the iron particles clustered together and distributed into a pile of.MTT. The concentration of SPIONs was clearly showed that the concentration of SPIONs was small. When the sum was equal to 14 g/ml, the marked amniotic mesenchymal stem cells were more active than 90%. when the concentration of SPIONs was greater than 14 g/m1, and the blue stained particles in the cytoplasm were not changed before and after the aggregation.
3. the number of cells labeled with 3 markers decreased significantly, but the number of cells in the SPIONs and DAPI markers began to increase after 3D, of which the cells in the SPIONs group grew better, and the number of cells that reached the peak at the peak of the sixth days was maintained at about 5 * 104.
4.14 mu g/ml SPIONs labeled amniotic mesenchymal stem cells were transplanted into the rabbit conjunctiva. After 4 weeks, the cornea sections were stained with Prussian blue, and a large number of blue stained particles were found in the limbus. It showed that hAM-dMSCs survived the corneal limbus and grew well. The recipient rabbit was in good condition and had no exclusion reaction in the eyes. The physiological salt containing 147 mu g/ml SPIONs was injected. No blue dye granules were found in the contralateral eye after 4 weeks of injection.
5. hAM-dMSCs transplantation results of corneal alkali burn: compared with the control group, the healing rate of the experimental group was significantly higher than that of the control group (P0.01), but there was no significant difference in the healing rate between the three experimental groups (X2=1.2, P0.05), and the corneal neovascularization between the experimental groups was significantly different from the control group at.4 weeks (P0.05); the corneal turbid degree test group and the pair were compared with the control group. There was significant difference between the group and the amniotic membrane group (P0.01), but there was no significant difference (t=0.374, P0.05) between the stem cell injection group and the amniotic group (t=0.374, P0.05), and the different treatment methods and time of corneal neovascularization in different groups were significantly different from those of the control group (P0. 01 or P0.05), the difference between the experimental group and the other two experimental groups was significant (P0.05).
HE staining showed that there were 3-4 layers of cells in the corneal epithelium, consisting of a small amount of epidermal cells, pterygoid cells and some basal cells. A small amount of neovascularization and inflammatory cells were seen in the stroma layer. The cornea Prussian staining showed that blue staining particles were visible in the limbus cornea, suggesting that hAM-dMSCs still survived the corner of the horns, and the corneal near central and central areas were not moved. Moving hAM-dMSCs.
6.Western-blot was used to detect the expression of TNF-a and IL-1 beta in cornea after different treatment of corneal alkali burns in rabbits. The specific positive imprinted bands were detected in the normal control group and the experimental group, and the results of Western-blot showed that TNF- alpha and IL-1 beta were expressed in all stages of the treatment of corneal alkali burns. The expression intensity of the treatment time was obviously weakened.
The changes of TNF- alpha and IL-1 beta in aqueous humor of rabbit cornea after alkali burn were detected by ELISA. The content of TNF- alpha in experimental aqueous humor decreased with time. The comparison of TNF- alpha content in aqueous humor between different time points was statistically significant. The mean value of TNF- alpha content in the control eye aqueous humor also showed with the prolongation of time. In the same observation time point, the content of TNF- alpha in experimental ocular aqueous humor was lower than that of the control eye. The mean value of IL-1 beta in aqueous humor of experimental and control groups decreased with the prolongation of time, and the content of IL-1p in aqueous humor of experimental group was lower than that of the eye. At the same observation time, first days and third days, The content of IL-1p in the aqueous humor of the experimental group was lower than that of the control group. The IL-1p content in the aqueous humor of the combined group was lower than that of the control group on the seventh day and the fourteenth day, and the difference was statistically significant.
7. compared with the control group, there was a significant difference in the concentration of lactoferrin in the tears in each observation time point (P0.01). There was no significant difference in the relative lactoferrin concentration in the tear liquid between the control group and the experimental group (P0.05). Compared with the normal blank group, the difference of lactoferrin concentration in the tears of the control group was significant. P0.05, the concentration of the experimental group was not statistically significant compared with the blank group (P0.05). After Spearman correlation analysis, the content of lacrimal lactoferrin at all time points was positively correlated with the Schirmer I test (P0.01).
conclusion
1. in this experiment, mesenchymal stem cells were isolated from human amniotic membrane, and the purity of mesenchymal stem cells was high. The experiment suggested that the screening and repeated passages were an ideal method for the purification of amniotic mesenchymal stem cells.
2. local subconjunctival injection of]hAM-dMSCs in the treatment of ocular surface chemical injuries is safe and feasible. It is helpful to establish a new kind of new type of microinvasive and minimally invasive treatment for ocular surface trauma disease.
3. SPIONs labeling method was used to mark amniotic mesenchymal stem cells. The concentration of SPIONs was less than or equal to 14 g/ml, which could not only make amniotic mesenchymal stem cells labeled, but also do not affect their activity.
4.SPIONs labeled hAM-dMSCs transplantation can be used to treat corneal alkali burn animal models and achieve the purpose of tracing.
5. early local conjunctival hAM-dMSCs transplantation could significantly inhibit the formation of neovascularization on the surface of the injured side of the cornea and accelerate the migration and differentiation of.HAM-dMSCs in the host. The mechanism of.HAM-dMSCs is closely related to the change of local microenvironment. The mechanism may be to improve the residual growth factor by regulating the secretion of cell growth factor. normal
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R772.2

【參考文獻】

相關(guān)期刊論文 前10條

1 姜廷帥;蔡莉;惠延年;閆峰;;大鼠骨髓間充質(zhì)干細(xì)胞體外可誘導(dǎo)分化為角膜上皮細(xì)胞[J];國際眼科雜志;2007年02期

2 戢維穎;蔡莉;惠延年;姜廷帥;;經(jīng)角膜基質(zhì)細(xì)胞誘導(dǎo)的大鼠骨髓間充質(zhì)干細(xì)胞在人羊膜上構(gòu)建角膜上皮移植片[J];國際眼科雜志;2008年04期

3 姚笠;乳鐵蛋白──一種多功能免疫調(diào)節(jié)蛋白[J];國外醫(yī)學(xué)(免疫學(xué)分冊);1996年06期

4 蔡望;唐仁泓;;準(zhǔn)分子激光原位角膜磨鑲術(shù)后干眼癥的臨床分析[J];醫(yī)學(xué)臨床研究;2007年03期

5 李志杰,C.WayneSMITH;眼表面的防御機制[J];眼科新進展;2002年05期

6 張繼平;賀文鳳;熊華鋒;李潔;吳瓊;戴育成;劉春菊;岳小媚;穆飛飛;;臍帶間充質(zhì)干細(xì)胞對兔眼堿燒傷的治療研究[J];臨床醫(yī)學(xué)工程;2010年11期

7 肖啟國,劉祖國,張梅,張志紅,羅麗輝,姚勇,蔣愛華,林健賢;局部滴用阿托品建立兔干眼模型的評價[J];眼科研究;2005年04期

8 解慧琪,楊志明,魏人前,項舟;ptsA58H質(zhì)粒轉(zhuǎn)化人胚腱細(xì)胞的生物學(xué)特性研究[J];中華手外科雜志;2000年03期

9 郭彤;王薇;張君;陳雪;李炳震;李凌松;;骨髓間充質(zhì)干細(xì)胞移植治療眼表損害的初步實驗研究[J];中華眼科雜志;2006年03期

10 鄭敏文;宦怡;徐健;葛雅麗;趙力;程康;尹濤;孫立軍;趙海濤;;超順磁性氧化鐵標(biāo)記骨髓間充質(zhì)干細(xì)胞的磁共振成像研究[J];中國醫(yī)學(xué)影像技術(shù);2006年08期

相關(guān)博士學(xué)位論文 前1條

1 王旭倩;人脂肪干細(xì)胞移植對兔眼視網(wǎng)膜裂孔修復(fù)作用的研究[D];中國協(xié)和醫(yī)科大學(xué);2008年

相關(guān)碩士學(xué)位論文 前2條

1 李飛;視網(wǎng)膜血管疾病致玻璃體積血術(shù)后視力恢復(fù)相關(guān)因素分析[D];吉林大學(xué);2004年

2 邱婷;利用脂肪源性干細(xì)胞重建角膜上皮的實驗研究[D];復(fù)旦大學(xué);2009年

，

本文編號：2139686