目的 观察没药提取物对人皮肤Fb生物学特性的影响,探讨其促进创面愈合的可能机制. 方法 从人正常包皮组织中分离并培养Fb,取第3～5代细胞用于实验.(1)将Fb接种至96孔板,按随机数字表法分为对照组,1×10-4、1 ×10-3、1×10-2、1 ×10-1、1、10、1×102 g/L没药水提取物组以及上述7种浓度的没药醇提取物组.对照组用含体积分数0.25％小牛血清的DMEM培养液(简称低浓度血清培养液)培养,各浓度没药水及没药醇提取物组分别用含相应终浓度2种没药提取物的低浓度血清培养液培养.培养48 h,用倒置相差显微镜观察细胞形态,噻唑蓝法测定各组Fb增殖活性(以吸光度值表示).(2)将Fb分别接种于培养瓶和培养皿中,均按随机数字表法分为2组:1 g/L没药水提取物组,用含终浓度1 g/L没药水提取物的低浓度血清培养液培养；对照组,采用低浓度血清培养液培养.培养72 h,分别采用流式细胞仪、实时荧光定量PCR法检测Fb周期与Ⅰ、Ⅲ型胶原mRNA的表达.对数据进行LSD-t检验. 结果 (1)各组细胞均呈长梭形生长,1 g/L没药水提取物组细胞融合较对照组更显著.1×10-3、1×10-2、1×10-1、1、10 g/L没药水提取物组Fb吸光度值分别为0.378±0.032、0.402±0.007、0.390±0.038、0.453±0.036、0.390±0.037,均高于对照组的0.332±0.044,t值分别为2.24、2.93、2.69、5.73、2.71,P值均小于0.05.1×10-4、1×102 g/L没药水提取物组吸光度值分别为0.312±0.048、0.154±0.009,前者与对照组比较,差异无统计学意义(t=2.84,P＞0.05)；后者显著低于对照组(t =7.17,P＜0.05).1×10-3、1 ×10-1、1、10、1×102 g/L没药醇提取物组Fb吸光度值显著低于对照组(t值为2.30～24.79,P值均小于0.05).(2)1 g/L没药水提取物组G0/G1期细胞百分比为(74.3±6.3)％,明显少于对照组的(82.2±7.9)％,t=6.77,P＜0.05；S期及G2/M期细胞百分比分别为(16.6±3.4)％、(9.1±1.6)％,明显多于对照组的(13.3±2.3)％、(4.5±0.8)％,t值分别为7.53、6.34,P值均小于0.051 g/L没药水提取物组Fb中Ⅰ型胶原mRNA相对表达量(0.89±0.08)与对照组(1.00±0.06)比较,差异无统计学意义(t =1.17,P＞0.05)；Ⅲ型胶原mRNA相对表达量(1.38±0.12)显著高于对照组(1.00±0.05,t=3.81,P＜0.01). 结论 没药水提取物能显著促进Fb增殖,加快Fb细胞周期进程,上调Fb中Ⅲ型胶原mRNA表达,可能与其促进创面愈合的机制相关.
目的 觀察沒藥提取物對人皮膚Fb生物學特性的影響,探討其促進創麵愈閤的可能機製. 方法 從人正常包皮組織中分離併培養Fb,取第3～5代細胞用于實驗.(1)將Fb接種至96孔闆,按隨機數字錶法分為對照組,1×10-4、1 ×10-3、1×10-2、1 ×10-1、1、10、1×102 g/L沒藥水提取物組以及上述7種濃度的沒藥醇提取物組.對照組用含體積分數0.25％小牛血清的DMEM培養液(簡稱低濃度血清培養液)培養,各濃度沒藥水及沒藥醇提取物組分彆用含相應終濃度2種沒藥提取物的低濃度血清培養液培養.培養48 h,用倒置相差顯微鏡觀察細胞形態,噻唑藍法測定各組Fb增殖活性(以吸光度值錶示).(2)將Fb分彆接種于培養瓶和培養皿中,均按隨機數字錶法分為2組:1 g/L沒藥水提取物組,用含終濃度1 g/L沒藥水提取物的低濃度血清培養液培養；對照組,採用低濃度血清培養液培養.培養72 h,分彆採用流式細胞儀、實時熒光定量PCR法檢測Fb週期與Ⅰ、Ⅲ型膠原mRNA的錶達.對數據進行LSD-t檢驗. 結果 (1)各組細胞均呈長梭形生長,1 g/L沒藥水提取物組細胞融閤較對照組更顯著.1×10-3、1×10-2、1×10-1、1、10 g/L沒藥水提取物組Fb吸光度值分彆為0.378±0.032、0.402±0.007、0.390±0.038、0.453±0.036、0.390±0.037,均高于對照組的0.332±0.044,t值分彆為2.24、2.93、2.69、5.73、2.71,P值均小于0.05.1×10-4、1×102 g/L沒藥水提取物組吸光度值分彆為0.312±0.048、0.154±0.009,前者與對照組比較,差異無統計學意義(t=2.84,P＞0.05)；後者顯著低于對照組(t =7.17,P＜0.05).1×10-3、1 ×10-1、1、10、1×102 g/L沒藥醇提取物組Fb吸光度值顯著低于對照組(t值為2.30～24.79,P值均小于0.05).(2)1 g/L沒藥水提取物組G0/G1期細胞百分比為(74.3±6.3)％,明顯少于對照組的(82.2±7.9)％,t=6.77,P＜0.05；S期及G2/M期細胞百分比分彆為(16.6±3.4)％、(9.1±1.6)％,明顯多于對照組的(13.3±2.3)％、(4.5±0.8)％,t值分彆為7.53、6.34,P值均小于0.051 g/L沒藥水提取物組Fb中Ⅰ型膠原mRNA相對錶達量(0.89±0.08)與對照組(1.00±0.06)比較,差異無統計學意義(t =1.17,P＞0.05)；Ⅲ型膠原mRNA相對錶達量(1.38±0.12)顯著高于對照組(1.00±0.05,t=3.81,P＜0.01). 結論 沒藥水提取物能顯著促進Fb增殖,加快Fb細胞週期進程,上調Fb中Ⅲ型膠原mRNA錶達,可能與其促進創麵愈閤的機製相關.
목적 관찰몰약제취물대인피부Fb생물학특성적영향,탐토기촉진창면유합적가능궤제. 방법 종인정상포피조직중분리병배양Fb,취제3～5대세포용우실험.(1)장Fb접충지96공판,안수궤수자표법분위대조조,1×10-4、1 ×10-3、1×10-2、1 ×10-1、1、10、1×102 g/L몰약수제취물조이급상술7충농도적몰약순제취물조.대조조용함체적분수0.25％소우혈청적DMEM배양액(간칭저농도혈청배양액)배양,각농도몰약수급몰약순제취물조분별용함상응종농도2충몰약제취물적저농도혈청배양액배양.배양48 h,용도치상차현미경관찰세포형태,새서람법측정각조Fb증식활성(이흡광도치표시).(2)장Fb분별접충우배양병화배양명중,균안수궤수자표법분위2조:1 g/L몰약수제취물조,용함종농도1 g/L몰약수제취물적저농도혈청배양액배양；대조조,채용저농도혈청배양액배양.배양72 h,분별채용류식세포의、실시형광정량PCR법검측Fb주기여Ⅰ、Ⅲ형효원mRNA적표체.대수거진행LSD-t검험. 결과 (1)각조세포균정장사형생장,1 g/L몰약수제취물조세포융합교대조조경현저.1×10-3、1×10-2、1×10-1、1、10 g/L몰약수제취물조Fb흡광도치분별위0.378±0.032、0.402±0.007、0.390±0.038、0.453±0.036、0.390±0.037,균고우대조조적0.332±0.044,t치분별위2.24、2.93、2.69、5.73、2.71,P치균소우0.05.1×10-4、1×102 g/L몰약수제취물조흡광도치분별위0.312±0.048、0.154±0.009,전자여대조조비교,차이무통계학의의(t=2.84,P＞0.05)；후자현저저우대조조(t =7.17,P＜0.05).1×10-3、1 ×10-1、1、10、1×102 g/L몰약순제취물조Fb흡광도치현저저우대조조(t치위2.30～24.79,P치균소우0.05).(2)1 g/L몰약수제취물조G0/G1기세포백분비위(74.3±6.3)％,명현소우대조조적(82.2±7.9)％,t=6.77,P＜0.05；S기급G2/M기세포백분비분별위(16.6±3.4)％、(9.1±1.6)％,명현다우대조조적(13.3±2.3)％、(4.5±0.8)％,t치분별위7.53、6.34,P치균소우0.051 g/L몰약수제취물조Fb중Ⅰ형효원mRNA상대표체량(0.89±0.08)여대조조(1.00±0.06)비교,차이무통계학의의(t =1.17,P＞0.05)；Ⅲ형효원mRNA상대표체량(1.38±0.12)현저고우대조조(1.00±0.05,t=3.81,P＜0.01). 결론 몰약수제취물능현저촉진Fb증식,가쾌Fb세포주기진정,상조Fb중Ⅲ형효원mRNA표체,가능여기촉진창면유합적궤제상관.
Objective To observe the effects of myrrh extract on biological characteristics of human dermal fibroblasts (Fb),and to explore its possible mechanisms in promoting wound healing. Methods Normal Fb was isolated from human foreskin tissue and cultured in vitro.The third to fifth passages of Fb were used in the experiment. ( 1 ) Fb were planted onto 96-well plate and divided into control group,and 1 × 10-4,1 × 10-3,1 × 10-2,1 × 10-1,1,10,1 × 102 g/L myrrh water extract groups and myrrh ethanol extract groups according to the random number table.Fb in control group were cultured with DMEM medium containing 0.25％ calf serum (briefly called low-concentration serum medium),and those in various concentrations of myrrh water extract and myrrh ethanol extract groups respectively with low-concentration serum medium containing corresponding concentration of 2 kinds of myrrh extract. After being cultured for 48 h,cell morphology was observed with inverted-phase contrast microscope,and Fb proliferation activity ( denoted as absorbance value) was determined with MTT method. (2) Fb were respectively planted into flasks and dishes and divided into two groups according to the random number table.Fb in control group were cultured with low-concentration serum medium,and that in 1 g/L myrrh water extract group with low-concentration serum medium containing 1 g/L myrrh water extract. After being cultured for 72 h.Fb cell cycle and the type Ⅰ and Ⅲ collagen mRNA expression were respectively determined by flow cytometry and real-time fluorescent quantitative PCR.Data were processed with LSD- t test. Results ( 1 ) Fb in all groups grew in long-spindle shape,but the cell fusion was much obvious in 1 g/L myrrh water extract group than in control group.Fb absorbance value in 1 ×10-3,1 ×10-2,1 ×10-1,1,10 g/L myrrh water extract groups was respectively 0.378 ± 0.032,0.402 ± 0.007,0.390 ± 0.038,0.453 ± 0.036,0.390 ± 0.037,all higher than that in control group (0.332 ±0.044,with t value respectively 2.24,2.93,2.69,5.73,2.71,P values all below 0.05 ).Compared with that in control group,Fb absorbance value in 1 × 10 -4 g/L myrrh water extract group was not statistically different (0.312 ± 0.048,t =2.84,P ＞ 0.05 ),while that in 1 × 102 g/Lmyrrh water extract group was significantly lower (0.154 ± 0.009,t =7.17,P ＜ 0.05).Fb absorbance values in 1 × 10-3,1 × 10 -1,1,10,1 × 102 g/L myrrh ethanol extract groups were significantly lower than that in control group ( with t values from 2.30 to 24.79,P values all below 0.05 ).(2) Compared with those in control group [(82.2 ±7.9)％ and (13.3 ±2.3)％,(4.5 ±0.8)％],the percentage of cells in G0/G1 phase in 1 g/L myrrh water extract group was obviously decreased [ (74.3 ± 6.3 ) ％,t =6.77,P ＜0.05],while those in S and G2/M phases increased [(16.6±3.4)％,(9.1 ±1.6)％,witht value respectively 7.53,6.34,P values below 0.05 ].Compared with those in control group ( 1.00 ± 0.05,1.00 ±0.06),the mRNA level of collagen Ⅲ in 1 g/L myrrh water extract group was significantly up-regulated ( 1.38 ± 0.12,t =3.81,P ＜ 0.01 ),while that of collagen Ⅰ was not statistically different (0.89 ± 0.08,t =1.17,P ＞ 0.05). Conclusions Myrrh water extract can notably promote the proliferation of Fb,accelerate the cell cycle of Fb,and up-regulate the mRNA expression of type Ⅲ collagen in Fb,which may be related to its mechanisms in promoting wound healing.
