人羊膜间充质干细胞移植对兔角膜碱烧伤的疗效及磁标记示踪分析

发布时间：2018-07-23 14:22

【摘要】：背景和目的角膜碱烧伤后,碱性物质能够迅速渗透眼表面,通过细胞渗透、蛋白水解酶和细胞因子的分泌导致严重的特征性的炎症反应。为保护角膜上皮的完整性,阻止基质层溃疡发生,国内外学者们已经应用了多种治疗方法,均取得了一定的疗效,但是何为最佳疗法,至今仍有争议,并未确定。本研究分别通过四个阶段探讨磁标记人羊膜间充质干细胞(human amniotic membrane-derived mesenchymal stem cells, hAM-dMSCs)移植对兔角膜碱烧伤治疗的影响,以及碱烧伤后泪液乳铁蛋白(lactoferrin, LF)的表达,研究泪液LF与碱烧伤的关系及其意义,以期获得对于角膜急性碱烧伤较为简易且适于临床使用的最佳治疗方法。1.通过观察人羊膜组织来源的细胞形态学、流式细胞仪检测和MTT法活性细胞检测等鉴定原代获取的间充质干细胞,为后续的实验提供干细胞。2.不同方法标记原代培养的羊膜间充质干细胞,探索最合适的细胞标记方法。3.超顺磁性氧化铁纳米颗粒(Superparamagnetic iron oxide nanoparticles, SPIONs)标记的hAM-dMSCs局部移植入角膜碱烧伤眼结膜下,观察细胞在宿主存活和迁徙情况；联合传统的羊膜移植,通过临床检查及检测IL-1p和TNF-α评估治疗效果,探讨干细胞在角膜碱烧伤后组织再生中扮演的角色。4.采集正常和碱烧伤治疗后的兔泪液,采用放射免疫分析方法,分别测定其中LF的含量,研究泪液LF与碱烧伤的关系及其对碱烧伤愈合的意义。材料和方法 1羊膜间充质干细胞的原代培养和鉴定使用酶消化和贴壁筛选法获取人羊膜组织中的间充质干细胞,反复传代对其扩增和纯化细胞。通过观察细胞学形态、MTT法活性检测和流式细胞仪检测等鉴定人羊膜来源的间充质干细胞。通过显微镜观察细胞的形态变化；采用流式细胞术检测人羊膜来源的细胞膜表面特异性蛋白分子CD31、CD44、CD45、 CD90、CD29、CD34的表达。 2.SPIONs标记的羊膜间充质干细胞在体外的活性监测本研究应用SPIONs标记的细胞生长培养基(Cytokine Growth Medium, CGM),标记浓度分别为3.5、7、14和28μg/ml,细胞标记采用单次SPIONs标记法。培养的细胞分别于标记后第1、2、4和7d收获,各时间点获得的细胞分为五组,分别行MTT法、普鲁氏蓝染色和流式细胞仪检测；无SPIONs标记的细胞生长培养基组作为对照组。 3.羊膜间充质干细胞的标记分别采用不同浓度的Brdu、DAPI和SPIONs标记原代培养的人羊膜间充质干细胞,确定较好的羊膜间充质干细胞标记途径和方法,为羊膜间充质干细胞移植治疗角膜碱烧伤的活体示踪进行前期准备。 4.羊膜间充质干细胞移植对重度角膜碱烧伤的疗效待移植细胞的制备：应用浓度为14μg/ml SPIONs标记原代培养获取的羊膜间充质干细胞,标记时间24h,收集细胞制成1×106细胞混悬液备用。实验动物分组和细胞移植的方法：动物被随机分成四组,对照组(n=10)；人羊膜间充质干细胞注射组(干细胞注射组,n=12)：结膜下注射含标记后的hAM-dMSCs注射液；单纯羊膜移植组(羊膜组,n=12)：行单纯羊膜移植；人羊膜间充质干细胞联合羊膜移植组(联合组,n=12)：结膜下注射含标记后的羊膜间充质干细胞注射液联合羊膜移植。结膜下注射一律选择眼球结膜颞上部位,注射量100微升,约含1×106个羊膜间充质干细胞,干细胞注射组和联合组对侧眼分别注射同等量含14μg/ml SPIONs的生理盐水作为对照。羊膜移植方法：碱烧伤后,实验侧眼开睑器开睑,1：1000庆大霉素液冲洗结膜囊,沿角膜缘环形剪开球结膜,分离结膜下组织,刮除角膜表面坏死组织,将羊膜上皮面朝上平铺使覆盖整个角膜和角膜缘,用10.0尼龙线于角膜缘附近间断缝合,并固定在浅层巩膜上,将球结膜游离缘固定在羊膜上,剪去多余羊膜组织。术毕结膜下注射庆大霉素2万u+地塞米松2mg,结膜囊内涂红霉素眼膏,缝合睑缘防止兔损伤手术创面。术后局部常规点抗生素,激素。 5.兔角膜碱烧伤后泪液乳铁蛋白的表达泪液乳铁蛋白(lactoferrin, LF)检测选择在角膜碱烧伤治愈后,停药2周进行。治疗方法采用本实验得出的最佳治疗方法：人羊膜间充质干细胞联合羊膜移植治疗碱烧伤。将30只大白兔随机分为三组：空白组(n=10)：不进行任何处理；对照组(n=10)：制作兔眼角膜碱烧伤模型,不进行间充质干细胞或羊膜移植；实验组(n=10)：造模后,局部结膜下行人羊膜间充质干细胞移植联合羊膜移植。造模动物均局部常规用药,停药2周开始实验检测。用微量吸管吸取结膜囊内的泪液80-100ul,置于0.5m1无菌静脉输液管中,管端加热封闭管口,置于深低温(-30℃)冰箱内保存待检。放射免疫分析方法,测定正常角膜和碱烧伤治愈后的兔泪液LF的含量。结果 1.用胰酶和胶原酶消化羊膜组织,将获得的细胞悬液接种在细胞培养瓶内进行原代培养。24小时后,倒置相差显微镜下观察见少量细胞贴壁；培养48小时贴壁细胞数量略增多；72小时后,贴壁细胞数量显著增多；原代培养第7天,贴壁细胞接近50%-60%融合,或基本铺满培养瓶底,贴壁细胞呈成纤维细胞样细胞。流式细胞仪检测细胞分析显示：原代培养的第三代细胞,高表达膜表面蛋白分子CD29、CD44、CD90、CD105;阴性表达CD31、CD34、CD45和CD106。 2.不同浓度的SPIONs进行标记羊膜间充质干细胞,普鲁氏蓝染色后显示：胞质内可见不同数量的蓝染颗粒,各组SPIONs标记细胞的标记率均为100%。随标记浓度增加,蓝染颗粒增加,铁颗粒呈簇状聚集分布成堆。MTT结果明确显示：SPIONs浓度小于和等于14μg/ml时,标记的羊膜间充质干细胞活性大于90%。当SPIONs浓度大于14μg/m1时,细胞胞质内蓝染颗粒聚集前后无变化。 3.用3种标记液标记细胞后细胞数目明显减少,但SPIONs和DAPI标记组细胞数在3d以后开始增加,其中SPIONs组细胞生长较好,第6天达到高峰。Brdu标记组细胞数目始终维持在5×104左右。 4.14μg/ml SPIONs标记的羊膜间充质干细胞移植入兔结膜下,4周后角膜切片行普鲁士蓝染色,角膜缘处可见大量的蓝染颗粒,说明hAM-dMSCs存活于角膜缘,生长良好。受体兔全身情况良好,眼局部无排斥反应发生。而注射含147μg/ml SPIONs的生理盐水的对侧眼,在注射4周后未见蓝染颗粒。 5.角膜碱烧伤的hAM-dMSCs移植结果：与对照组比较,实验组愈合率显著高于对照组(P0.01),而三个实验组的愈合率差异无显著性(X2=1.2,P0.05)。4周时各实验组之间角膜新生血管与对照组相比差异有显著性(P0.05)；角膜混浊度评分实验组与对照组相比差异有显著性(P0.01),；干细胞注射组和羊膜组与联合组相比差异有显著性(P0.01),干细胞注射组与羊膜组角膜混浊度评分差异无显著性(t=0.374,P0.05)；不同治疗方法和时间角膜新生血管面积各组与对照组相比差异有显著性(P0.01或P0.05),实验组组间比较,联合组与其余两个实验组相比差异有显著性(P0.05)。 HE染色显示角膜上皮层有3-4层细胞,由少量表皮细胞、翼状细胞和部分基底细胞组成。基质层可见少量新生血管和炎性细胞。角膜普鲁士染色显示,角膜缘处可见蓝染颗粒,提示hAM-dMSCs仍存活于角膜缘附近,角膜近中央区和中央区未见迁移的hAM-dMSCs。 6.Western-blot检测兔角膜碱烧伤不同治疗方法后角膜TNF-a和IL-1β的表达：正常对照组及实验组角膜的蛋白经印记实验检测可见特异的阳性印迹带,Western-blot结果显示TNF-α和IL-1β在角膜碱烧伤后治疗过程中的各个阶段都有表达,且随着治疗时间的延长,其表达强度明显减弱。 ELISA检测兔角膜碱烧伤后不同治疗方法和时间房水TNF-α和IL-1β的变化：实验眼房水TNF-α含量随着时间的延长呈下降趋势,不同时间点之间房水TNF-α含量比较在统计学上有显著性差异。对照眼房水TNF-α含量平均值随着时间的延长亦呈下降趋势。在同一观察时间点,实验眼房水TNF-α含量均低于对照眼,在统计学上有显著性差异。实验组和对照组房水IL-1β含量平均值均随着时间的延长呈下降趋势,且实验组房水IL-1p含量均低于对照眼。在同一观察时间点,第1天和第3天,实验组房水IL-1p含量低于对照组,第7天和第14天联合组房水IL-1p含量低于对照眼,并且在统计学上均有显著性差异。 7.实验组与对照组比较,各观察时间点泪液中乳铁蛋白浓度差异有显著性(P0.01)；对照组和实验组各观察时间点泪液中相对乳铁蛋白浓度均无统计学差异(P0.05)；与正常空白组相比,对照组各观察时间点泪液中乳铁蛋白浓度差异有显著性(P0.05),实验组与空白组相比,其浓度无统计学意义(P0.05)。经Spearman相关分析,各时间点泪液乳铁蛋白的含量与Schirmer I试验呈正相关(P0.01)。结论 1.本实验从人羊膜中分离间充质干细胞,所得的间充质干细胞纯度较高。实验提示：贴壁筛选和反复传代是一种较为理想的提纯羊膜间充质干细胞的方法。 2.局部结膜下注射移植]hAM-dMSCs治疗眼表化学伤安全可行,有助于临床上新建一种细胞来源广泛、无免疫排斥的、微创的治疗眼表创伤疾病的新手段。 3.应用SPIONs标记方法标记羊膜间充质干细胞,SPIONs浓度小于和等于14μg/ml,既可使羊膜间充质干细胞得到标记,又不影响其活性。 4.SPIONs标记hAM-dMSCs移植治疗角膜碱烧伤动物模型,可达到示踪的目的。 5.早期局部结膜下行hAM-dMSCs移植可显著抑制伤侧角膜表面新生血管形成,加速重度角膜碱烧伤创伤修复。hAM-dMSCs在宿主的迁移和分化,与局部微环境的改变密切相关。hAM-dMSCs的作用机制可能是通过调节细胞生长因子的分泌,改善残留的正常细胞所处的微环境,促进残留细胞的生长来发挥修复作用。 6.本研究在角膜碱烧伤早期行hAM-dMSCs移植,与对照组相比,测得泪液LF含量明显增高。因此,除进行紧急有效的抢救措施外,早期行hAM-dMSCs移植,不仅能够加快创伤角膜愈合,且能够提高患者治愈后的视觉质量及生活质量。
[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
【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R772.2

【参考文献】