發(fā)布時(shí)間：2018-06-20 11:44

  本文選題：脂肪來(lái)源干細(xì)胞 + 軟骨細(xì)胞��； 參考：《中國(guó)修復(fù)重建外科雜志》2017年05期

【摘要】：目的探討大鼠脂肪來(lái)源干細(xì)胞(adipose-derived stem cells,ADSCs)對(duì)紫外線照射造成的軟骨細(xì)胞DNA損傷的修復(fù)作用。方法取3~4周齡SD大鼠(體質(zhì)量100~120 g)腹股溝脂肪,利用Ⅰ型膠原酶消化法體外分離培養(yǎng)ADSCs并傳代;取第3代細(xì)胞采用流式細(xì)胞儀檢測(cè)其表面相關(guān)標(biāo)記,行成骨及成脂誘導(dǎo)鑒定其多向分化潛能。另取SD大鼠關(guān)節(jié)軟骨,利用酶消化法分離培養(yǎng)軟骨細(xì)胞并傳代,并行甲苯胺藍(lán)染色鑒定。取第3代軟骨細(xì)胞,采用40 J/m~2劑量紫外線照射;照射后細(xì)胞分別以正常培養(yǎng)基培養(yǎng)(照射組)、以含ADSCs培養(yǎng)上清的培養(yǎng)基培養(yǎng)(ADSCs上清組)或與ADSCs共培養(yǎng)(ADSCs組)24h。以不進(jìn)行紫外線照射的正常第3代軟骨細(xì)胞作為對(duì)照(對(duì)照組)。采用MTS法檢測(cè)細(xì)胞增殖情況,免疫熒光染色、Western blot法檢測(cè)軟骨細(xì)胞DNA損傷標(biāo)志蛋白磷酸化組蛋白2A變異體(phosphorylatedhistone family 2A variant,γH2AX)的表達(dá)情況。結(jié)果經(jīng)流式細(xì)胞儀檢測(cè)及成骨、成脂誘導(dǎo)鑒定,所培養(yǎng)細(xì)胞為ADSCs。對(duì)照組、照射組及ADSCs上清組吸光度(A)值分別為2.20±0.10、1.34±0.04、1.57±0.06,照射組及ADSCs上清組顯著低于對(duì)照組,照射組顯著低于ADSCs上清組,比較差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。免疫熒光染色示,照射組、ADSCs上清組及ADSCs組γH2AX蛋白熒光強(qiáng)度明顯高于對(duì)照組,但ADSCs上清組及ADSCs組γH2AX蛋白熒光強(qiáng)度明顯弱于照射組,ADSCs組和ADSCs上清組則無(wú)明顯區(qū)別。Western blot法檢測(cè)示,照射組、ADSCs上清組及ADSCs組γH2AX蛋白相對(duì)表達(dá)量均明顯高于對(duì)照組,但ADSCs上清組及ADSCs組顯著低于照射組,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);ADSCs組和ADSCs上清組比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論大鼠ADSCs有助于恢復(fù)紫外線照射后的軟骨細(xì)胞的增殖,降低DNA損傷標(biāo)志蛋白γH2AX的表達(dá),對(duì)紫外線照射所致軟骨細(xì)胞損傷具有修復(fù)作用。
[Abstract]:Objective to investigate the repair effect of adipose-derived stem cells (ADSCs) on chondrocyte DNA damage induced by ultraviolet radiation in rats. Methods the inguinal fat of SD rats aged 3 ~ 4 weeks (body mass 100 ~ 120 g) was isolated and cultured by type I collagenase digestion in vitro, and the surface related markers of ADSCs were detected by flow cytometry (FCM) in the third passage of SD rats. The multidirectional differentiation potential was identified by osteogenesis and adipogenic induction. The articular cartilage of SD rats was isolated and cultured by enzyme digestion, and identified by toluidine blue staining. The third passage of chondrocytes were irradiated with 40 J / m ~ (2) dose of ultraviolet radiation. After irradiation, the cells were cultured in normal medium (irradiation group, culture medium containing ADSCs supernatant) or co-cultured with ADSCs for 24 hours. The normal third passage chondrocytes without ultraviolet radiation were used as control group (control group). The expression of phosphorylated histone family 2A variant (phosphorylatedhistone family 2A variant, 緯 H 2AX) in chondrocytes was detected by MTS and immunofluorescence staining. Results the cultured cells were identified as ADSCs by flow cytometry and osteogenesis. The absorbance (A) values of the control group, the irradiation group and the ADSCs supernatant group were 2.20 鹵0.10 鹵1.34 鹵0.04 鹵1.57 鹵0.06, respectively, which were significantly lower in the irradiation group and the ADSCs supernatant group than in the ADSCs supernatant group, and the difference was statistically significant (P 0.05). Immunofluorescence staining showed that the fluorescence intensity of 緯 -H2AX protein in ADSCs supernatant group and ADSCs group was significantly higher than that in control group, but the fluorescence intensity of 緯 -H2AX protein in ADSCs supernatant group and ADSCs group was significantly lower than that in ADSCs supernatant group and ADSCs supernatant group. The relative expression of 緯 H2AX protein in ADSCs supernatant group and ADSCs group was significantly higher than that in control group, but the expression of 緯 H2AX protein in ADSCs supernatant group and ADSCs group was significantly lower than that in irradiation group. There was no significant difference between ADSCs supernatant group and ADSCs supernatant group. Conclusion ADSCs can restore the proliferation of chondrocytes after ultraviolet irradiation, decrease the expression of DNA damage marker 緯 H _ 2AX, and repair the damage of chondrocytes induced by ultraviolet radiation.
【作者單位】： 廣州市紅十字會(huì)醫(yī)院暨南大學(xué)醫(yī)學(xué)院附屬?gòu)V州紅十字會(huì)醫(yī)院創(chuàng)傷外科研究所;廣州市紅十字會(huì)醫(yī)院暨南大學(xué)醫(yī)學(xué)院附屬?gòu)V州紅十字會(huì)醫(yī)院燒傷整形科;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(81272222) 廣州市衛(wèi)生局一般引導(dǎo)項(xiàng)目(20131A011038) 廣州市科技計(jì)劃項(xiàng)目(201508020262)~~
【分類號(hào)】：R68

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