IL-10-hAMSCs的体外鉴定及对小鼠创面愈合中血管相关因子表达的影响

发布时间：2018-06-19 19:34

本文选题：人羊膜间充质干细胞 + 细胞因子　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:对IL-10修饰后的h AMSCs(IL-10-h AMSCs)是否改变h AMSCs的表型、生长特性和多向分化能力的生物学特性进行评价,并就IL-10-h AMSCs局部移植对小鼠皮肤全层缺损创面血管相关因子VEGF、b FGF表达的影响进行检测分析,为IL-10以及IL-10修饰hAMSCs的临床应用提供实验依据。方法:1.采用流式细胞术(FCM)分别对培养的h AMSCs、IL-10修饰的h AMSCs和空质粒转染的h AMSCs进行CD73、CD90、CDl05及CD44表型分子鉴定,MTT法绘制各组h AMSCs生长曲线,并用成骨、成脂诱导培养检测各组h AMSCs的多向分化能力。2.采用细胞培养划痕实验,检测h AMSCs、IL-10-h AMSCs以及空质粒-h AMSCs的迁移能力。3.选择7周龄健康雄性C57BL/6野生小鼠80只,随机分成PBS移植组、h AMSCs移植组、IL-10-h AMSCs移植组、空质粒转染h AMSCs移植组,每组各20只小鼠。麻醉后在小鼠背部两侧各切取1cm×1cm大小的全层皮肤缺损创面模型。根据分组不同,在建模即刻于每个创面周围皮下多点(创缘各边中点距创面1mm处)注射100μl PBS悬浮的不同组别的h AMSCs(细胞总数为1×106个),PBS移植组注射等量PBS。4.建模后1d、3d、7d、14d,分别处死各组损伤小鼠5只,沿创缘切取创面全层组织。HE染色观察创面组织中炎性细胞浸润及新生血管情况。ELISA检测小鼠创面组织匀浆中VEGF、b FGF的表达。免疫荧光染色观察各组小鼠背部全层皮肤缺损创面组织中VEGF、b FGF的表达情况。结果:1.经流式细胞仪检测结果表明,h AMSCs高表达CD73(99.85%)、CD90(90.55%)、CDl05(97.95%)及CD44(99.57%),IL-10-h AMSCs高表达CD73(98.6%)、CD90(93.8%)、CD105(99.8%)及CD44(97.7%),空质粒-h AMSCs高表达CD73(94.1%)、CD90(98.1%)、CD105(96.6%)及CD44(94.8%),三者均不表达CD34、CD45、CDl1b、CDl9、HLA-DR。表型符合国际细胞治疗协会就间充质干细胞表面标志的规定。各组细胞生长曲线呈“S”形。IL-10转染后的h AMSCs增殖能力较未转染的h AMSCs以及空质粒转染的h AMSCs有所下降。生长曲线显示第3代IL-10-h AMSCs细胞生长曲线呈“S”形。细胞培养到21d时,行成骨诱导的茜素红S染色,可见红色钙化结节,行成脂诱导的油红O染色,红色显示在脂滴处,IL-10-h AMSCs与h AMSCs、空质粒-h AMSCs诱导能力无明显差别。2.划痕实验结果显示,IL-10-h AMSCs细胞在0h与h AMSCs组、空质粒组迁移能力基本一致,6h、12h、18h的IL-10-h AMSCs组细胞向空白区域迁移能力稍大于h AMSCs组、空质粒组,表明IL-10可以增强hAMSc的迁移能力。3.HE结果显示:在显微镜下观察,各组小鼠创面组织第1d均有大量炎性细胞浸润,可见坏死组织;第3d时各组创面组织炎性细胞浸润较第1d减少,出现大量新生毛细血管,成纤维细胞逐渐出现,其中h AMSCs组、IL-10-h AMSCs组及空质粒组小鼠创面组织炎性细胞浸润较PBS组少,成纤维细胞数量以及新生毛细血管生成数量较PBS组多,其中以IL-10-h AMSCs组最优,典型新鲜肉芽组织形成;第7d时各组创面组织炎性细胞浸润较第3d明显减少,成纤维细胞成熟,数量较第3d增加,毛细血管数量较第3d减少,其中IL-10-h AMSCs组成纤维细胞数量较其余三组少,以PBS组最多;第14d时各组小鼠创面未见明显炎性细胞,毛细血管数量较第7d减少,成纤维细胞数量较第7d多,其中IL-10-h AMSCs组成纤维细胞数量较其余三组少,PBS组最多。4.ELISA法检测各组标本全层皮肤缺损模型组织中VEGF及b FGF含量,在建模后第1d、3d、7d、14d,PBS组VEGF含量分别为:96.78±4.87、100.84±4.69、103.08±4.21、99.70±5.05ng/L;h AMSCs组VEGF含量分别为:101.77±4.05、115.2±4.03、117.48±6.1、112.61±2.23ng/L;IL-10-h AMSCs组VEGF含量分别为:132.71±3.09、136.86±4.75、142.17±7.39、138.93±5.84ng/L;空质粒组VEGF含量分别为:105.31±4.70、111.89±4.41、115.02±6.96、110.79±3.97ng/L。PBS组b FGF含量分别为:8.78±0.70、11.61±0.22、13.11±0.36、11.20±0.49ng/L;h AMSCs组b FGF含量分别为:9.90±0.44、15.73±1.43、19.50±1.11、16.20±1.96ng/L;IL-10-h AMSCs组bFGF含量分别为:16.70±1.78、21.73±1.85、23.13±1.67、22.19±1.55ng/L;空质粒组b FGF含量分别为:9.10±0.64、15.93±1.51、18.90±1.49、16.40±1.66ng/L。各组VEGF、b FGF含量第1d、第3d、第7d逐渐增加,第14d含量较第7d有所下降,h AMSCs组、IL-10-h AMSCs组和空质粒组较PBS组在第1d、第3d、第7d的VEGF、b FGF含量明显增高,且IL-10-h AMSCs组升高更明显,差异具有统计学意义(P0.05),hAMSCs组和空质粒组未见明显差异。5.免疫荧光染色观察各组标本创面组织中VEGF与b FGF表达情况,在建模后第1d、3d、7d、14d,PBS组VEGF阳性率分别为:33.24±0.85、35.65±1.75、38.57±1.32、34.94±1.25;h AMSCs组VEGF阳性率分别为:37.25±1.05、42.87±0.79、48.92±1.46、43.93±1.68;IL-10-h AMSCs组VEGF阳性率分别为:54.57±1.38、67.10±0.94、71.11±1.53、68.35±1.09;空质粒组VEGF阳性率分别为:35.29±0.57、41.34±0.95、48.22±1.44、43.61±1.56。PBS组b FGF阳性率分别为:16.99±1.04、22.00±0.46、25.97±0.70、21.85±1.15;h AMSCs组b FGF阳性率分别为:20.13±1.06、26.20±1.15、31.17±1.21、28.18±0.76;IL-10-h AMSCs组b FGF阳性率分别为:26.41±0.86、34.42±1.85、38.57±1.33、33.97±0.38;空质粒组b FGF阳性率分别为:19.23±1.02、25.16±1.11、30.27±0.93、27.22±1.17。结果见自第1d、第3d、第7d,VEGF及b FGF阳性率逐渐增加,第14d阳性表达较第7d稍降低,IL-10-h AMSCs组、h AMSCs组及空质粒组阳性率在各时间点均高于PBS组,其中IL-10-h AMSCs组阳性表达最高,差异具有统计学意义(P0.05)。结论:(1)采用慢病毒载体搭载IL-10基因修饰后的h AMSCs除细胞增殖能力受到一定程度的抑制外,不影响h AMSCs的表型特征和多向分化能力的基本生物学特性;(2)IL-10可增强h AMSCs的迁移能力,移植IL-10-h AMSCs能够上调创面组织VEGF以及b FGF的表达,促进血管再生,并促进创面愈合。
[Abstract]:Objective: To evaluate whether the H AMSCs (IL-10-h AMSCs) modified by IL-10 changes the phenotype of H AMSCs, the growth characteristics and the biological characteristics of multidirectional differentiation, and the effect of IL-10-h AMSCs local transplantation on the expression of vascular related factor VEGF and B FGF in the full-thickness skin defect wound of mice. Methods: 1. using flow cytometry (FCM), H AMSCs, IL-10 modified h AMSCs and empty plasmid transfected h AMSCs were used to identify CD73, CD90, CDl05 and CD44 phenotypic molecules. Force.2. used cell culture scratch test, and detected the migration ability of H AMSCs, IL-10-h AMSCs and empty plasmid -h AMSCs to select 80 healthy male C57BL/6 mice of 7 weeks old, randomly divided into PBS transplantation group, H AMSCs group, transplantation group, and empty plasmid transfected 20 mice. A full-thickness skin defect wound model of 1cm * 1cm size was cut on both sides. According to the different groups, H AMSCs (the total number of 1 x 106 cells) of 100 mu L PBS suspension was injected subcutaneously around each surface of each wound near each wound. PBS transplantation group was injected with equal amount of PBS.4. modeling 1D, 3D, 7d, respectively, respectively. 5 mice were killed and 5 mice were injured. The infiltration of inflammatory cells and the neovascularization in the wound tissue were observed along the wound edge. The expression of VEGF, B FGF in the tissue homogenate of the wound was detected by.ELISA. The expression of VEGF and B FGF in the whole layer skin defect wound tissue of the mice was observed by immunofluorescence staining. Results: 1 The results of flow cytometry showed that h AMSCs expressed CD73 (99.85%), CD90 (90.55%), CDl05 (97.95%) and CD44 (99.57%), IL-10-h AMSCs high expression CD73 (98.6%), CD90 (93.8%), CD105 (99.8%) and CD44 (97.7%), 98.1%, 96.6% and 94.8%. The LA-DR. phenotype accords with the regulation of the international cell therapy association on the surface markers of mesenchymal stem cells. The proliferation ability of H AMSCs after transfection of "S" form.IL-10 in each group is lower than that of H AMSCs without transfection and H AMSCs transfected by empty plasmid. The growth curve shows that the growth curve of third generation IL-10-h AMSCs cells is "S". When the cells were cultured to 21d, the osteogenesis induced alizarin red S staining, the red calcified nodule, the fat induced oil red O staining, the red display at the lipid droplets, the IL-10-h AMSCs and H AMSCs, and the AMSCs induction ability of the empty plasmid -h, the.2. scratch test results showed that IL-10-h AMSCs cells were in the group and the space plasmid group migration ability base. The migration ability of 6h, 12h and 18h IL-10-h AMSCs cells to the blank area was slightly greater than that of the H AMSCs group. The empty plasmid group showed that IL-10 could enhance the migration ability of hAMSc.3.HE. The results of.3.HE showed that there was a large number of inflammatory cells infiltration and necrotic tissue in the wound tissue in each group. Cell infiltration was less than 1D, a large number of new capillaries appeared, and fibroblasts gradually appeared. The infiltration of inflammatory cells in H AMSCs group, IL-10-h AMSCs group and empty plasmid group was less than that of the PBS group. The number of fibroblasts and the number of newborn capillaries were more than that of the PBS group, and the group of IL-10-h AMSCs was the best and typical fresh. The infiltration of inflammatory cells in the wound tissue at 7d decreased obviously than that in 3D, the fibroblasts were mature, the number of cells increased and the number of capillaries decreased than that of 3D, and the number of IL-10-h AMSCs components was less than that of the other three groups, which was the most in the PBS group, and no obvious inflammatory cells and capillary blood were found in the wounds of each group at 14d. The number of tube was less than that of 7D, and the number of fibroblasts was more than that of 7D, and the number of IL-10-h AMSCs fibroblasts was less than that of the other three groups. The maximum.4.ELISA method in PBS group was used to detect the FGF content of VEGF and B in the whole layer skin defect model of each group, and the contents of 1D, 3D, 7d, and 7d were 96.78. The contents of VEGF in the group of H AMSCs were 101.77 + 4.05115.2 + 4.03117.48 + 6.1112.61 + 2.23ng/L, and the VEGF content of IL-10-h AMSCs group was 132.71 + and 132.71 +. The contents were 8.78 + 0.70,11.61 + 0.22,13.11 + 0.36,11.20 + 0.49ng/L respectively, and the content of B FGF in H AMSCs group was 9.90 + 0.44,15.73 + 1.43,19.50 + 1.11,16.20 + 1.96ng/L, respectively, and the content of B FGF was respectively: 16.70 + The content of VEGF in each group of 49,16.40 + 1.66ng/L., B FGF content 1D, 3D, and 7d gradually increased, and the content of 14d was lower than that of 7D. There was no significant difference in the expression of VEGF and B FGF in the tissue of the specimens by.5. immunofluorescence staining. The positive rates of VEGF positive in 1D, 3D, 7d, 14d and PBS group after modeling were as follows: 33.24 + 0.85,35.65 + 1.75,38.57 + 1.25, respectively: 37.25 +. The positive rates of VEGF in the group were 54.57 + 1.38,67.10 + 0.94,71.11 + 1.53,68.35 + 1.09, and the positive rates of VEGF positive in the empty plasmid group were: 35.29 + 0.57,41.34 + 1.44,43.61 + 1.56.PBS group B FGF positive rate respectively: 16.99 + 1.04,22.00 + + + 1.15. The positive rate was 20.13 +. The positive rates of B FGF in group IL-10-h AMSCs were: 26.41 + 0.86,34.42 + 1.85,38.57 + 1.33,33.97 + 0.38, and the positive rates of B FGF in the empty plasmid group were: 19.23 + 1.02,25.16 + 1.11,30.27 + + 0.38. In group AMSCs, the positive rate of H AMSCs group and empty plasmid group was higher than that of PBS group at all time points, and the positive expression of IL-10-h AMSCs group was the highest, and the difference was statistically significant (P0.05). Conclusion: (1) the proliferation ability of H AMSCs after IL-10 gene modified by lentivirus carrier is inhibited to a certain extent and does not affect the phenotypic characteristics of H AMSCs. The basic biological characteristics of signs and multidirectional differentiation; (2) IL-10 can enhance the migration ability of H AMSCs. Transplantation of IL-10-h AMSCs can increase the expression of VEGF and B FGF in wound tissue, promote vascular regeneration, and promote wound healing.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R622

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