目的 比较大鼠骨髓间充质干细胞(BMSC)与脂肪间充质干细胞(ADSC)在调节肝纤维化方面的差异.方法 分别从Sprague-Dawley大鼠骨髓、脂肪分离纯化培养间充质干细胞,流式细胞仪检测干细胞表面标志.采用0.4 μm Transwell小室半透膜建立共培养体系,分别将第3代ADSC、BMSC与肝星状细胞(HSC)共培养,另将大鼠正常肝细胞系(BRL)与HSC共培养作为阴性对照组,单独HSC培养作为空白对照组.培养72 h,采用细胞计数试剂盒(CCK)-8比色法检测HSC增殖情况,Western印迹法检测HSC细胞的α平滑肌肌动蛋白(α-SMA)表达.流式细胞仪检测HSC凋亡情况.分别将BMSC、ADSC和BRL单独培养72 h,ELISA法测定培养液中血管内皮生长因子(VEGF)、IL-10、神经生长因子(NGF)及TGF-β1的浓度.建立大鼠肝纤维化模型,将大鼠分为BMSC治疗组、ADSC治疗组、阴性对照组和空白对照组,每组6只,BMSC治疗组、ADSC治疗组和阴性对照组分别经门静脉输注1.5 mL(5×106个)BMSC、ADSC、BRL细胞悬液,空白对照组输注等量无细胞培养液,2周1次,共4周.检测肝组织病理切片和肝纤维化指标.两样本均数间比较用t检验.多个样本均数间的比较用方差分析.结果 成功分离培养BMSC、ADSC.BMSC与ADSC在细胞表型上类似.ADSC共培养组和BMSC共培养组分别与空白对照组和阴性对照组相比,两者均能抑制HSC增殖、促进凋亡(空白对照组、阴性对照组、ADSC共培养组和BMSC共培养组增殖程度依次为2.43±0.27、2.39±0.33、1.92±0.38、2.18±0.31,FBMSC=25.61,FADSC=38.63,P均＜0.05;凋亡率依次为(5.59±0.40)％、(6.82±0.57)％、(8.31±1.03)％、(9.36±0.54)％,FBMSC=73.69,FADSC=97.41,P均＜0.05),且ADSC比BMSC作用更强(增殖,t=5.76;凋亡,t=5.18,P均＜0.05).ADSC与BMSC分泌的细胞因子水平也有所不同[NGF为(7.46±0.54) pg/mL比(3.95±0.71) pg/mL,t=10.92,P＜0.05; TGF-β1为(8.79±0.93) pg/mL比(6.36±0.85) pg/mL,t=7.58,P＜0.05].大鼠移植实验显示BMSC、ADSC对肝纤维化均具有明显抑制作用.BMSC治疗组、ADSC治疗组的肝脏炎症活动度和纤维化程度分别为9.87±2.07、4.17±0.94和10.13± 1.81、3.98±0.82,与空白对照组(13.78±2.53和5.09±1.15)和阴性对照组(13.34±1.89和4.95±1.22)相比明显降低(FBMSC=51.26和32.29,P＜0.05; FADSC =46.73和40.94,P＜0.05),BMSC治疗组及ADSC治疗组的血清透明质酸水平[(191.5±33.2) μg/L、(178.8±28.2)μg/L]、血清Ⅲ型胶原[(19.9±5.1)μg/L、(21.7±3.3)μg/L]和肝脏羟脯氨酸[(312.6±38.8)μg/g、(325.8±28.2)μg/g]含量均明显低于阴性对照组与空白对照组[透明质酸为(282.3±18.7) μg/L、(287.5±26.7)μg/L,F=73.51;Ⅲ型胶原为(35.3±3.3)μg/L、(32.5±4.3) μg/L,F=76.19;羟脯氨酸为(458.4±38.1)μg/g、(473.9±63.7) μg/g,F=60.37,P均＜0.05],但BMSC治疗组与ADSC治疗组间差异均无统计学意义(P均＞0.05).结论 BMSC与ADSC具有相似的干细胞特征.在抑制HSC活性方面,ADSC与BMSC有所不同,但在大鼠体内抑制肝纤维化方面差异无统计学意义.
目的 比較大鼠骨髓間充質榦細胞(BMSC)與脂肪間充質榦細胞(ADSC)在調節肝纖維化方麵的差異.方法 分彆從Sprague-Dawley大鼠骨髓、脂肪分離純化培養間充質榦細胞,流式細胞儀檢測榦細胞錶麵標誌.採用0.4 μm Transwell小室半透膜建立共培養體繫,分彆將第3代ADSC、BMSC與肝星狀細胞(HSC)共培養,另將大鼠正常肝細胞繫(BRL)與HSC共培養作為陰性對照組,單獨HSC培養作為空白對照組.培養72 h,採用細胞計數試劑盒(CCK)-8比色法檢測HSC增殖情況,Western印跡法檢測HSC細胞的α平滑肌肌動蛋白(α-SMA)錶達.流式細胞儀檢測HSC凋亡情況.分彆將BMSC、ADSC和BRL單獨培養72 h,ELISA法測定培養液中血管內皮生長因子(VEGF)、IL-10、神經生長因子(NGF)及TGF-β1的濃度.建立大鼠肝纖維化模型,將大鼠分為BMSC治療組、ADSC治療組、陰性對照組和空白對照組,每組6隻,BMSC治療組、ADSC治療組和陰性對照組分彆經門靜脈輸註1.5 mL(5×106箇)BMSC、ADSC、BRL細胞懸液,空白對照組輸註等量無細胞培養液,2週1次,共4週.檢測肝組織病理切片和肝纖維化指標.兩樣本均數間比較用t檢驗.多箇樣本均數間的比較用方差分析.結果 成功分離培養BMSC、ADSC.BMSC與ADSC在細胞錶型上類似.ADSC共培養組和BMSC共培養組分彆與空白對照組和陰性對照組相比,兩者均能抑製HSC增殖、促進凋亡(空白對照組、陰性對照組、ADSC共培養組和BMSC共培養組增殖程度依次為2.43±0.27、2.39±0.33、1.92±0.38、2.18±0.31,FBMSC=25.61,FADSC=38.63,P均＜0.05;凋亡率依次為(5.59±0.40)％、(6.82±0.57)％、(8.31±1.03)％、(9.36±0.54)％,FBMSC=73.69,FADSC=97.41,P均＜0.05),且ADSC比BMSC作用更彊(增殖,t=5.76;凋亡,t=5.18,P均＜0.05).ADSC與BMSC分泌的細胞因子水平也有所不同[NGF為(7.46±0.54) pg/mL比(3.95±0.71) pg/mL,t=10.92,P＜0.05; TGF-β1為(8.79±0.93) pg/mL比(6.36±0.85) pg/mL,t=7.58,P＜0.05].大鼠移植實驗顯示BMSC、ADSC對肝纖維化均具有明顯抑製作用.BMSC治療組、ADSC治療組的肝髒炎癥活動度和纖維化程度分彆為9.87±2.07、4.17±0.94和10.13± 1.81、3.98±0.82,與空白對照組(13.78±2.53和5.09±1.15)和陰性對照組(13.34±1.89和4.95±1.22)相比明顯降低(FBMSC=51.26和32.29,P＜0.05; FADSC =46.73和40.94,P＜0.05),BMSC治療組及ADSC治療組的血清透明質痠水平[(191.5±33.2) μg/L、(178.8±28.2)μg/L]、血清Ⅲ型膠原[(19.9±5.1)μg/L、(21.7±3.3)μg/L]和肝髒羥脯氨痠[(312.6±38.8)μg/g、(325.8±28.2)μg/g]含量均明顯低于陰性對照組與空白對照組[透明質痠為(282.3±18.7) μg/L、(287.5±26.7)μg/L,F=73.51;Ⅲ型膠原為(35.3±3.3)μg/L、(32.5±4.3) μg/L,F=76.19;羥脯氨痠為(458.4±38.1)μg/g、(473.9±63.7) μg/g,F=60.37,P均＜0.05],但BMSC治療組與ADSC治療組間差異均無統計學意義(P均＞0.05).結論 BMSC與ADSC具有相似的榦細胞特徵.在抑製HSC活性方麵,ADSC與BMSC有所不同,但在大鼠體內抑製肝纖維化方麵差異無統計學意義.
목적 비교대서골수간충질간세포(BMSC)여지방간충질간세포(ADSC)재조절간섬유화방면적차이.방법 분별종Sprague-Dawley대서골수、지방분리순화배양간충질간세포,류식세포의검측간세포표면표지.채용0.4 μm Transwell소실반투막건립공배양체계,분별장제3대ADSC、BMSC여간성상세포(HSC)공배양,령장대서정상간세포계(BRL)여HSC공배양작위음성대조조,단독HSC배양작위공백대조조.배양72 h,채용세포계수시제합(CCK)-8비색법검측HSC증식정황,Western인적법검측HSC세포적α평활기기동단백(α-SMA)표체.류식세포의검측HSC조망정황.분별장BMSC、ADSC화BRL단독배양72 h,ELISA법측정배양액중혈관내피생장인자(VEGF)、IL-10、신경생장인자(NGF)급TGF-β1적농도.건립대서간섬유화모형,장대서분위BMSC치료조、ADSC치료조、음성대조조화공백대조조,매조6지,BMSC치료조、ADSC치료조화음성대조조분별경문정맥수주1.5 mL(5×106개)BMSC、ADSC、BRL세포현액,공백대조조수주등량무세포배양액,2주1차,공4주.검측간조직병리절편화간섬유화지표.량양본균수간비교용t검험.다개양본균수간적비교용방차분석.결과 성공분리배양BMSC、ADSC.BMSC여ADSC재세포표형상유사.ADSC공배양조화BMSC공배양조분별여공백대조조화음성대조조상비,량자균능억제HSC증식、촉진조망(공백대조조、음성대조조、ADSC공배양조화BMSC공배양조증식정도의차위2.43±0.27、2.39±0.33、1.92±0.38、2.18±0.31,FBMSC=25.61,FADSC=38.63,P균＜0.05;조망솔의차위(5.59±0.40)％、(6.82±0.57)％、(8.31±1.03)％、(9.36±0.54)％,FBMSC=73.69,FADSC=97.41,P균＜0.05),차ADSC비BMSC작용경강(증식,t=5.76;조망,t=5.18,P균＜0.05).ADSC여BMSC분비적세포인자수평야유소불동[NGF위(7.46±0.54) pg/mL비(3.95±0.71) pg/mL,t=10.92,P＜0.05; TGF-β1위(8.79±0.93) pg/mL비(6.36±0.85) pg/mL,t=7.58,P＜0.05].대서이식실험현시BMSC、ADSC대간섬유화균구유명현억제작용.BMSC치료조、ADSC치료조적간장염증활동도화섬유화정도분별위9.87±2.07、4.17±0.94화10.13± 1.81、3.98±0.82,여공백대조조(13.78±2.53화5.09±1.15)화음성대조조(13.34±1.89화4.95±1.22)상비명현강저(FBMSC=51.26화32.29,P＜0.05; FADSC =46.73화40.94,P＜0.05),BMSC치료조급ADSC치료조적혈청투명질산수평[(191.5±33.2) μg/L、(178.8±28.2)μg/L]、혈청Ⅲ형효원[(19.9±5.1)μg/L、(21.7±3.3)μg/L]화간장간포안산[(312.6±38.8)μg/g、(325.8±28.2)μg/g]함량균명현저우음성대조조여공백대조조[투명질산위(282.3±18.7) μg/L、(287.5±26.7)μg/L,F=73.51;Ⅲ형효원위(35.3±3.3)μg/L、(32.5±4.3) μg/L,F=76.19;간포안산위(458.4±38.1)μg/g、(473.9±63.7) μg/g,F=60.37,P균＜0.05],단BMSC치료조여ADSC치료조간차이균무통계학의의(P균＞0.05).결론 BMSC여ADSC구유상사적간세포특정.재억제HSC활성방면,ADSC여BMSC유소불동,단재대서체내억제간섬유화방면차이무통계학의의.
Objective To compare the difference between bone marrow stomal cell (BMSC) and adipose-derived stem cell (ADSC) of liver fibrosis in rats.Methods BMSC and ADSC of Sprague-Dawley (SD) rats were isolated and purified.The stem cell markers were detected with flow cytometry.The coculture system was set up with 0.4 μm Transwell insert semipermeable membrane.ADSC or BMSC were co-cultured with hepatic stellate cells (HSC).Normal hepatocyte cell line of rat (BRL) was co-cultured with HSC as negative control group and HSC cultured alone was blank control group.After cultured for 72 hours,the proliferation of HSC was determined by cell counting kit-8 (CCK-8) method.The expression of α-smooth muscle actin (α-SMA) of HSC was detected by Western blotting.The apoptosis of HSC was examined by flow cytometry.After BMSC,ADSC and BRL cultured alone for 72 hours,expression level vascular endothelial growth factor (VEGF),interleukin-10 (IL-10),nerve growth factor (NGF) and transforming growth factor-β1 (TGF-β1) in the culture medium were detected by enzymelinked immunosorbent assay (ELISA) method.The rats model of liver fibrosis were established.The rats were divided into BMSC treatment group,ADSC treatment group,BRL group and culture medium group,six rats in each group,which were injected with 1.5 mL BMSC,ADSC and BRL cells suspension (5 × 106) through portal vein,respectively,and same volume of culture medium was injected to the rate of culture medium group,once every two weeks for four weeks.The pathological changes of liver tissue sections were observed and liver fibrosis markers were tested.T test was performed for comparison between two samples and analysis of variance was used for comparison among multiple groups.Results BMSC and ADSC were successfully isolated and cultured.The phenotype of BMSC and ADSC was similar.Compared with blank control group and negative control group,both ADSC and BMSC could inhibit the proliferation of HSC and promote apoptosis (proliferation,2.43±0.27,2.39±0.33,1.92±0.38 and 2.18±0.31,FBMSC =25.61,FADSC =38.63,both P＜0.05 ;apoptosis rate,(5.59 ± 0.40)％,(6.82±0.57)％,(8.31± 1.03) ％ and (9.36 ± 0.54) ％,FBMSC =73.69,FADSC =97.41,both P＜ 0.05).The effects of ADSC were more significant than those of BMSC (t=5.76 and 5.18,both P＜0.05).There was difference in the cytokine levels secreted by ADSC and BMSC (NGF,(7.46 ± 0.54) pg/mL vs (3.95 ± 0.71) pg/mL,t =10.92,P＜0.05; TGF-β1,(8.79 ±0.93) pg/mL vs (6.36±0.85) pg/mL,t=7.58,P＜0.05).The cell transplantation experiment indicated that both BMSC and ADSC had significant inhibitory effect on liver fibrosis.The activity index of inflammation and degree of fibrosis in BMSC treatment group and ADSCs treatment group were 9.87±2.07,4.17 ± 0.94 and 10.13 ± 1.81,3.98 ± 0.82,which were significantly lower than those in blank control group (13.78±2.53 and 5.09±1.15)and negative control group (13.34± 1.89 and 4.95± 1.22,FBMSC=51.26 and 32.29,P＜0.05; FADSC =46.73 and 40.94,P＜0.05).The level of hyaluronic acid ((191.5±33.2) μg/L and (178.8±28.2) μg/L),type Ⅲ collagen ((19.9±5.1) μg/L and (21.7± 3.3) μg/L) and hydroxyproline ((312.6±38.8) μg/g and (325.8±28.2) μg/g) of BMSC treatment group and ADSC treatment group were significantly lower than those of negative control group and blank control group (hyaluronic acid,(282.3 ± 18.7) μg/L and (287.5 ± 26.7) μg/L),F =73.51 ; type Ⅲ collagen,(35.3± 3.3) μg/L and (32.5±4.3) μg/L,F=76.19; hydroxyproline,(458.4 ± 38.1) μg/g and (473.9 ± 63.7) μg/g,F=60.37,all P＜0.05).However,there was no difference between BMSC treatment group and ADSC treatment (all P＜0.05).Conclusions ADSC and BMSC had similar stem cell characteristics.There was difference in inhibiting the activation of HSC between ADSC and BMSC.But there was no significant difference in inhibiting liver fibrosis of rats in vivo.
