發(fā)布時間：2018-06-19 19:34

  本文選題：人羊膜間充質(zhì)干細(xì)胞 + 細(xì)胞因子��； 參考：《遵義醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的:對IL-10修飾后的h AMSCs(IL-10-h AMSCs)是否改變h AMSCs的表型、生長特性和多向分化能力的生物學(xué)特性進(jìn)行評價,并就IL-10-h AMSCs局部移植對小鼠皮膚全層缺損創(chuàng)面血管相關(guān)因子VEGF、b FGF表達(dá)的影響進(jìn)行檢測分析,為IL-10以及IL-10修飾hAMSCs的臨床應(yīng)用提供實(shí)驗依據(jù)。方法:1.采用流式細(xì)胞術(shù)(FCM)分別對培養(yǎng)的h AMSCs、IL-10修飾的h AMSCs和空質(zhì)粒轉(zhuǎn)染的h AMSCs進(jìn)行CD73、CD90、CDl05及CD44表型分子鑒定,MTT法繪制各組h AMSCs生長曲線,并用成骨、成脂誘導(dǎo)培養(yǎng)檢測各組h AMSCs的多向分化能力。2.采用細(xì)胞培養(yǎng)劃痕實(shí)驗,檢測h AMSCs、IL-10-h AMSCs以及空質(zhì)粒-h AMSCs的遷移能力。3.選擇7周齡健康雄性C57BL/6野生小鼠80只,隨機(jī)分成PBS移植組、h AMSCs移植組、IL-10-h AMSCs移植組、空質(zhì)粒轉(zhuǎn)染h AMSCs移植組,每組各20只小鼠。麻醉后在小鼠背部兩側(cè)各切取1cm×1cm大小的全層皮膚缺損創(chuàng)面模型。根據(jù)分組不同,在建模即刻于每個創(chuàng)面周圍皮下多點(diǎn)(創(chuàng)緣各邊中點(diǎn)距創(chuàng)面1mm處)注射100μl PBS懸浮的不同組別的h AMSCs(細(xì)胞總數(shù)為1×106個),PBS移植組注射等量PBS。4.建模后1d、3d、7d、14d,分別處死各組損傷小鼠5只,沿創(chuàng)緣切取創(chuàng)面全層組織。HE染色觀察創(chuàng)面組織中炎性細(xì)胞浸潤及新生血管情況。ELISA檢測小鼠創(chuàng)面組織勻漿中VEGF、b FGF的表達(dá)。免疫熒光染色觀察各組小鼠背部全層皮膚缺損創(chuàng)面組織中VEGF、b FGF的表達(dá)情況。結(jié)果:1.經(jīng)流式細(xì)胞儀檢測結(jié)果表明,h AMSCs高表達(dá)CD73(99.85%)、CD90(90.55%)、CDl05(97.95%)及CD44(99.57%),IL-10-h AMSCs高表達(dá)CD73(98.6%)、CD90(93.8%)、CD105(99.8%)及CD44(97.7%),空質(zhì)粒-h AMSCs高表達(dá)CD73(94.1%)、CD90(98.1%)、CD105(96.6%)及CD44(94.8%),三者均不表達(dá)CD34、CD45、CDl1b、CDl9、HLA-DR。表型符合國際細(xì)胞治療協(xié)會就間充質(zhì)干細(xì)胞表面標(biāo)志的規(guī)定。各組細(xì)胞生長曲線呈“S”形。IL-10轉(zhuǎn)染后的h AMSCs增殖能力較未轉(zhuǎn)染的h AMSCs以及空質(zhì)粒轉(zhuǎn)染的h AMSCs有所下降。生長曲線顯示第3代IL-10-h AMSCs細(xì)胞生長曲線呈“S”形。細(xì)胞培養(yǎng)到21d時,行成骨誘導(dǎo)的茜素紅S染色,可見紅色鈣化結(jié)節(jié),行成脂誘導(dǎo)的油紅O染色,紅色顯示在脂滴處,IL-10-h AMSCs與h AMSCs、空質(zhì)粒-h AMSCs誘導(dǎo)能力無明顯差別。2.劃痕實(shí)驗結(jié)果顯示,IL-10-h AMSCs細(xì)胞在0h與h AMSCs組、空質(zhì)粒組遷移能力基本一致,6h、12h、18h的IL-10-h AMSCs組細(xì)胞向空白區(qū)域遷移能力稍大于h AMSCs組、空質(zhì)粒組,表明IL-10可以增強(qiáng)hAMSc的遷移能力。3.HE結(jié)果顯示:在顯微鏡下觀察,各組小鼠創(chuàng)面組織第1d均有大量炎性細(xì)胞浸潤,可見壞死組織;第3d時各組創(chuàng)面組織炎性細(xì)胞浸潤較第1d減少,出現(xiàn)大量新生毛細(xì)血管,成纖維細(xì)胞逐漸出現(xiàn),其中h AMSCs組、IL-10-h AMSCs組及空質(zhì)粒組小鼠創(chuàng)面組織炎性細(xì)胞浸潤較PBS組少,成纖維細(xì)胞數(shù)量以及新生毛細(xì)血管生成數(shù)量較PBS組多,其中以IL-10-h AMSCs組最優(yōu),典型新鮮肉芽組織形成;第7d時各組創(chuàng)面組織炎性細(xì)胞浸潤較第3d明顯減少,成纖維細(xì)胞成熟,數(shù)量較第3d增加,毛細(xì)血管數(shù)量較第3d減少,其中IL-10-h AMSCs組成纖維細(xì)胞數(shù)量較其余三組少,以PBS組最多;第14d時各組小鼠創(chuàng)面未見明顯炎性細(xì)胞,毛細(xì)血管數(shù)量較第7d減少,成纖維細(xì)胞數(shù)量較第7d多,其中IL-10-h AMSCs組成纖維細(xì)胞數(shù)量較其余三組少,PBS組最多。4.ELISA法檢測各組標(biāo)本全層皮膚缺損模型組織中VEGF及b FGF含量,在建模后第1d、3d、7d、14d,PBS組VEGF含量分別為:96.78±4.87、100.84±4.69、103.08±4.21、99.70±5.05ng/L;h AMSCs組VEGF含量分別為:101.77±4.05、115.2±4.03、117.48±6.1、112.61±2.23ng/L;IL-10-h AMSCs組VEGF含量分別為:132.71±3.09、136.86±4.75、142.17±7.39、138.93±5.84ng/L;空質(zhì)粒組VEGF含量分別為:105.31±4.70、111.89±4.41、115.02±6.96、110.79±3.97ng/L。PBS組b FGF含量分別為:8.78±0.70、11.61±0.22、13.11±0.36、11.20±0.49ng/L;h AMSCs組b FGF含量分別為:9.90±0.44、15.73±1.43、19.50±1.11、16.20±1.96ng/L;IL-10-h AMSCs組bFGF含量分別為:16.70±1.78、21.73±1.85、23.13±1.67、22.19±1.55ng/L;空質(zhì)粒組b FGF含量分別為:9.10±0.64、15.93±1.51、18.90±1.49、16.40±1.66ng/L。各組VEGF、b FGF含量第1d、第3d、第7d逐漸增加,第14d含量較第7d有所下降,h AMSCs組、IL-10-h AMSCs組和空質(zhì)粒組較PBS組在第1d、第3d、第7d的VEGF、b FGF含量明顯增高,且IL-10-h AMSCs組升高更明顯,差異具有統(tǒng)計學(xué)意義(P0.05),hAMSCs組和空質(zhì)粒組未見明顯差異。5.免疫熒光染色觀察各組標(biāo)本創(chuàng)面組織中VEGF與b FGF表達(dá)情況,在建模后第1d、3d、7d、14d,PBS組VEGF陽性率分別為:33.24±0.85、35.65±1.75、38.57±1.32、34.94±1.25;h AMSCs組VEGF陽性率分別為:37.25±1.05、42.87±0.79、48.92±1.46、43.93±1.68;IL-10-h AMSCs組VEGF陽性率分別為:54.57±1.38、67.10±0.94、71.11±1.53、68.35±1.09;空質(zhì)粒組VEGF陽性率分別為:35.29±0.57、41.34±0.95、48.22±1.44、43.61±1.56。PBS組b FGF陽性率分別為:16.99±1.04、22.00±0.46、25.97±0.70、21.85±1.15;h AMSCs組b FGF陽性率分別為:20.13±1.06、26.20±1.15、31.17±1.21、28.18±0.76;IL-10-h AMSCs組b FGF陽性率分別為:26.41±0.86、34.42±1.85、38.57±1.33、33.97±0.38;空質(zhì)粒組b FGF陽性率分別為:19.23±1.02、25.16±1.11、30.27±0.93、27.22±1.17。結(jié)果見自第1d、第3d、第7d,VEGF及b FGF陽性率逐漸增加,第14d陽性表達(dá)較第7d稍降低,IL-10-h AMSCs組、h AMSCs組及空質(zhì)粒組陽性率在各時間點(diǎn)均高于PBS組,其中IL-10-h AMSCs組陽性表達(dá)最高,差異具有統(tǒng)計學(xué)意義(P0.05)。結(jié)論:(1)采用慢病毒載體搭載IL-10基因修飾后的h AMSCs除細(xì)胞增殖能力受到一定程度的抑制外,不影響h AMSCs的表型特征和多向分化能力的基本生物學(xué)特性;(2)IL-10可增強(qiáng)h AMSCs的遷移能力,移植IL-10-h AMSCs能夠上調(diào)創(chuàng)面組織VEGF以及b FGF的表達(dá),促進(jìn)血管再生,并促進(jìn)創(chuàng)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.
【學(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R622

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本文編號：2041027