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[1]李晉玉,俞興,姜俊杰,等.骨碎補(bǔ)總黃酮對納米羥基磷灰石-膠原復(fù)合材料與成骨細(xì)胞-血管內(nèi)皮細(xì)胞共培養(yǎng)體系中血管內(nèi)皮細(xì)胞增殖的影響[J].中醫(yī)正骨,2019,31(07):1-8.
　LI Jinyu,YU Xing,JIANG Junjie,et al.Effects of osteopractic total flavone on proliferation of vascular endothelial cells of osteoblasts-vascular endothelial cells co-culture system in nano-hydroxyapatite collagen composite[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2019,31(07):1-8.
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骨碎補(bǔ)總黃酮對納米羥基磷灰石-膠原復(fù)合材料與成骨細(xì)胞-血管內(nèi)皮細(xì)胞共培養(yǎng)體系中血管內(nèi)皮細(xì)胞增殖的影響()
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《中醫(yī)正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第31卷
期數(shù):
2019年07期
頁碼:
1-8
欄目:
基礎(chǔ)研究
出版日期:
2019-07-20

文章信息/Info

Title:
Effects of osteopractic total flavone on proliferation of vascular endothelial cells of osteoblasts-vascular endothelial cells co-culture system in nano-hydroxyapatite collagen composite
作者:
李晉玉1; 俞興1; 姜俊杰2; 趙學(xué)千1; 孫旗1; 鄭晨穎1; 白春曉1; 劉楚吟1; 賈育松1
(1.北京中醫(yī)藥大學(xué)東直門醫(yī)院,北京 100700; 2.中國中醫(yī)科學(xué)院中醫(yī)臨床基礎(chǔ)醫(yī)學(xué)研究所,北京 100700)
Author(s):
LI Jinyu1; YU Xing1; JIANG Junjie2; ZHAO Xueqian1; SUN Qi1; ZHENG Chenying1; BAI Chunxiao1; LIU Chuyin1; JIA Yusong1
1.Dongzhimen Hospital of Beijing University of Traditional Chinese Medicine,Beijing 100700,China 2.Institute of Basic Research in Clinical Medicine of China Academy of Chinese Medical Sciences,Beijing 100700,China
關(guān)鍵詞:
骨碎補(bǔ);  黃酮;  細(xì)胞增殖;  血管內(nèi)皮細(xì)胞;  成骨細(xì)胞;  共同培養(yǎng)技術(shù);  羥基磷灰石類;  膠原;  內(nèi)皮生長因子;  成纖維細(xì)胞生長因子2;  組織工程
Keywords:
drynaria fortune;  flavone;  cell proliferation;  vascular endothelial cell;  osteoblasts;  coculture techniques;  hydroxyapatites;  collagen;  endothelial growth factors;  fibroblast growth factor 2;  tissue engineering
摘要:
目的:探討骨碎補(bǔ)總黃酮(osteopractic total flavone,OTF)對納米羥基磷灰石-膠原(nano-hydroxyapatite collagen,nHAC)復(fù)合材料與成骨細(xì)胞-血管內(nèi)皮細(xì)胞共培養(yǎng)體系中血管內(nèi)皮細(xì)胞增殖的影響。方法:分別以濃度為500 μg·mL-1、250 μg·mL-1、100 μg·mL-1、50 μg·mL-1及0 μg·mL-1的OTF溶液干預(yù)生長在nHAC復(fù)合材料上的hFOB1.19人成骨細(xì)胞,干預(yù)24 h后收集5種上清液。再分別以5種上清液和人臍靜脈血管內(nèi)皮細(xì)胞(human umbilical vein endothelial cells,HUVEC)培養(yǎng)基混合培養(yǎng)HUVEC,培養(yǎng)24 h和48 h后進(jìn)行劃痕實驗,計算細(xì)胞遷移率,確定OTF溶液最佳干預(yù)濃度和時間。在4個培養(yǎng)皿中鋪滿nHAC復(fù)合材料,加入DMEM/F12。A培養(yǎng)皿中不添加其他材料; B、D培養(yǎng)皿中接種hFOB1.19人成骨細(xì)胞,預(yù)培養(yǎng)24 h使細(xì)胞貼壁; C、D培養(yǎng)皿中加入OTF溶液,根據(jù)上一步劃痕實驗確定的最佳干預(yù)濃度和時間進(jìn)行干預(yù)。干預(yù)結(jié)束后收集各培養(yǎng)皿中的上清液進(jìn)行后續(xù)實驗。接種HUVEC于HUVEC培養(yǎng)基上,分為空白組和A、B、C、D共5組; 空白組不添加其他材料,A、B、C、D組分別加入A、B、C、D培養(yǎng)皿上清液; 以CCK-8法測定細(xì)胞增殖率,HE染色后觀察HUVEC形態(tài),以ELISA法檢測血管內(nèi)皮細(xì)胞生長因子(vascular endothelial growth factor,VEGF)和成纖維細(xì)胞生長因子-2(fibroblast growth factor-2,FGF-2)含量。結(jié)果:①OTF溶液最佳干預(yù)濃度與時間檢測結(jié)果。5種濃度OTF溶液干預(yù)后,0～24 h細(xì)胞遷移率的差異有統(tǒng)計學(xué)意義[(16.46±4.01)%,(21.71±1.30)%,(24.94±3.47)%,(22.08±2.46)%,(21.34±2.28)%,F=4.564,P=0.013],其中100 μg·mL-1OTF溶液干預(yù)后的細(xì)胞遷移率高于其余4組(P=0.001,P=0.020,P=0.014,P=0.029); 24～48 h細(xì)胞遷移率的差異無統(tǒng)計學(xué)意義[(6.06±1.22)%,(5.37±1.85)%,(5.58±1.88)%,(6.14±1.52)%,(4.99±1.25)%,F=0.374,P=0.823]。提示OTF溶液最佳干預(yù)濃度為100 μg·mL-1,最佳干預(yù)時間為24 h。②HUVEC細(xì)胞活性檢測結(jié)果。時間因素與分組因素存在交互效應(yīng)(F=14.039,P=0.000); 5組HUVEC細(xì)胞增殖率總體比較,差異有統(tǒng)計學(xué)意義,即存在分組效應(yīng)(F=83.285,P=0.000); 干預(yù)開始后不同時點之間HUVEC細(xì)胞增殖率的差異有統(tǒng)計學(xué)意義,即存在時間效應(yīng)(F=1 968.467,P=0.000); 5組HUVEC細(xì)胞增殖率隨時間變化均呈升高趨勢,各組的變化趨勢不完全相同(1.05±0.06,1.81±0.09,2.53±0.05,6.10±0.17,11.29±1.21,F=527.347,P=0.000; 0.99±0.11,2.37±0.40,2.92±0.19,7.35±0.19,14.41±1.35,F=913.030,P=0.000; 0.98±0.20,3.50±0.18,4.35±0.16,8.53±0.99,15.42±0.73,F=1 637.304,P=0.000; 1.02±0.10,2.10±0.13,2.59±0.15,4.70±0.25,11.41±1.21,F=494.876,P=0.000; 1.01±0.06,2.81±0.37,3.31±0.08,5.95±0.24,12.74±2.03,F=878.982,P=0.000); 干預(yù)開始后0 d時,各組細(xì)胞增殖率比較,差異無統(tǒng)計學(xué)意義; 干預(yù)開始后1 d、2 d時,B組HUVEC細(xì)胞增殖率最高、D組次之,其余3組增殖率較為接近; 干預(yù)開始后3 d、4 d時,B組HUVEC細(xì)胞增殖率最高、A組次之,其余3組增殖率較為接近。③HUVEC形態(tài)觀察結(jié)果。細(xì)胞形態(tài)觀察結(jié)果顯示,干預(yù)開始后0 d、1 d、2 d、3 d、4 d時各組HUVEC的形態(tài)均無明顯差異。④VEGF和FGF-2含量檢測。A、B、C、D 4組VEGF含量比較,差異無統(tǒng)計學(xué)意義(0.059±0.012,0.057±0.016,0.059±0.018,0.057±0.014,F=0.060,P=0.980)。4組FGF-2含量比較,差異有統(tǒng)計學(xué)意義[(215.83±19.56)pg·mL-1,(565.83±47.14)pg·mL-1,(228.33±17.67)pg·mL-1,(445.00±17.69)pg·mL-1,F=317.377,P=0.000]; B組的FGF-2含量高于其余3組(P=0.009,P=0.010,P=0.025),D組的FGF-2含量高于A組和C組(P=0.013,P=0.017),A組和C組FGF-2含量的差異無統(tǒng)計學(xué)意義(P=0.050)。結(jié)論:人成骨細(xì)胞和HUVEC在nHAC復(fù)合材料上共培養(yǎng),均可以良好貼附、生長和增殖; OTF不能促進(jìn)nHAC復(fù)合材料與人成骨細(xì)胞-HUVEC共培養(yǎng)體系中HUVEC增殖; HUVEC的增殖可能與成骨細(xì)胞分泌FGF-2有關(guān)。
Abstract:
Objective:To explore the effects of osteopractic total flavone(OTF)on proliferation of vascular endothelial cells(VECs)of osteoblasts(OBs)-VECs co-culture system in nano-hydroxyapatite collagen(nHAC)composite.Methods:The hFOB1.19 human OBs were cultured in nHAC composite and were intervened by OTF solution with concentration of 500,250,100,50 and 0 μg/mL respectively for 24 hrs,and then 5 kinds of supernatants were collected.The HUVECs were cultured in human umbilical vein endothelial cells(HUVEC)medium supplemented with the 5 kinds of supernatants respectively.The scratch tests were performed after 24- and 48-hour cell cultivation to calculate the cell migration rate and determine the optimal intervention concentration and duration of OTF solution.Four petri dishes were overspread with nHAC composites and were added with DMEM/F12 medium respectively.No other mediums was added into petri dish A,while petri dish B and D were inoculated with hFOB1.19 human OBs and the OBs were precultured for 24 hrs till cell adhesion.The petri dish C and D were supplemented with OTF solution and the optimal intervention concentration and duration were determined by above scratch tests.The supernatant in each petri dish was collected for the following experiments after the end of intervention.HUVEC were inoculated into HUVEC medium and were divided into blank group and group A,B,C and D.The blank group were not added with other mediums,and group A,B,C and D were added with supernatants that collected from petri dish A,B,C and D respectively.The HUVEC proliferation rate was detected by using CCK-8 method,and the morphology of HUVEC was observed through HE staining.The contents of vascular endothelial growth factor(VEGF)and fibroblast growth factor-2(FGF-2)were detected by using ELISA method.Results:After intervention by OTF solution with concentrations of 500,250,100,50 and 0 μg/mL,there was statistical difference in 0-24 hour migration rate of HUVEC between the 5 groups(16.46+/-4.01,21.71+/-1.30,24.94+/-3.47,22.08+/-2.46,21.34+/-2.28%,F=4.564,P=0.013),and the migration rate was higher in group that was intervented by OTF solution with concentration of 100 μg/mL compared to the other 4 groups(P=0.001,P=0.020,P=0.014,P=0.029).There was no statistical difference in 24-48 hour migration rate of HUVEC between the 5 groups(6.06+/-1.22,5.37+/-1.85,5.58+/-1.88,6.14+/-1.52,4.99+/-1.25%,F=0.374,P=0.823).Above results suggested that the optimal intervention concentration of OTF solution was 100 g/mL and the optimal intervention duration was 24 hrs.There was interaction between time factor and group factor in HUVEC activity(F=14.039,P=0.000).There was statistical difference in HUVEC proliferation rate between the 5 groups in general,in other words,there was group effect(F=83.285,P=0.000).There was statistical difference in HUVEC proliferation rate between different timepoints after the beginning of intervention,in other words,there was time effect(F=1 968.467,P=0.000).The HUVEC proliferation rate presented a time-dependent increasing trend in the 5 groups,while the 5 groups were inconsistent with each other in the variation tendency(1.05+/-0.06,1.81+/-0.09,2.53+/-0.05,6.10+/-0.17,11.29+/-1.21,F=527.347,P=0.000; 0.99+/-0.11,2.37+/-0.40,2.92+/-0.19,7.35+/-0.19,14.41+/-1.35,F=913.030,P=0.000; 0.98+/-0.20,3.50+/-0.18,4.35+/-0.16,8.53+/-0.99,15.42+/-0.73,F=1 637.304,P=0.000; 1.02+/-0.10,2.10+/-0.13,2.59+/-0.15,4.70+/-0.25,11.41+/-1.21,F=494.876,P=0.000; 1.01+/-0.06,2.81+/-0.37,3.31+/-0.08,5.95+/-0.24,12.74+/-2.03,F=878.982,P=0.000).There was no statistical difference in HUVEC proliferation rate between the 5 groups at the beginning of intervention.The HUVEC proliferation rate was highest in group B and second highest in group D,and was similar to each other in the other 3 groups at 1 and 2 days after the beginning of intervention.The HUVEC proliferation rate was highest in group B and second highest in group A,and was similar to each other in the other 3 groups at 3 and 4 days after the beginning of intervention.The results of observation on HUVEC morphology showed that there was no obvious difference in HUVEC morphology at the beginning of intervention and at 1,2,3 and 4 days after the beginning of intervention.There was no statistical difference in content of VEGF between group A,B, C and D(0.059+/-0.012,0.057+/-0.016,0.059+/-0.018,0.057+/-0.014,F=0.060,P=0.980).There was statistical difference in content of FGF-2 between group A,B,C and D(215.83+/-19.56,565.83+/-47.14,228.33+/-17.67,445.00+/-17.69 pg/mL,F=317.377,P=0.000).The content of FGF-2 was higher in group B compared to the other 3 groups and was higher in group D compared to group A and group C(P=0.009,P=0.010,P=0.025; P=0.013,P=0.017).There was no statistical difference in content of FGF-2 between group A and group C(P=0.050).Conclusion:Both human OBs and HUVEC,co-cultured in nHAC composite,can adhere,grow and proliferate well.OTF can not promote the proliferation of HUVEC of OBs-HUVECs co-culture system in nHAC composite.The proliferation of HUVEC may be related to FGF-2 secreted by OBs.

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備注/Memo

備注/Memo:
基金項目:國家自然科學(xué)基金項目(81503601; 81603517); 北京中醫(yī)藥大學(xué)東直門醫(yī)院“青苗人才培養(yǎng)計劃”項目(DZMYS-201802); 北京中醫(yī)藥大學(xué)2018年度基本科研業(yè)務(wù)項目(2018-JYBZZ-JS096); 中華中藥學(xué)會青年人才托舉工程項目(CACM-2018-QNRC-C08) 通訊作者:賈育松 E-mail:[email protected](收稿日期:2018-12-08 本文編輯:李曉樂)
更新日期/Last Update: 2019-07-20