摘要
目的探讨与机械牵张损伤后的大鼠子宫韧带成纤维细胞间接共培养,对大鼠骨髓间充质干细胞(BMSC)定向分化的影响。方法利用细胞牵张损伤模型,对大鼠子宫韧带成纤维细胞施以10%牵张变形(1Hz,连续12h单向机械牵张)后与大鼠BMSC间接共培养(牵张损伤组)3、6、12d,采用SP法检测间接共培养后BMSC中Ⅰ、Ⅲ型胶原蛋白表达水平(以平均灰度值表示);实时定量PCR技术检测间接共培养后BMSC中Ⅰ、Ⅲ型胶原mRNA表达水平(以其与磷酸甘油醛脱氢酶的比值表示),以BMSC自身共培养为对照组。结果(1)蛋白表达情况:牵张损伤组细胞间接共培养3d后,BMSC的Ⅰ、Ⅲ型胶原蛋白表达水平分别为82.4±3.4和76.8±2.5,与对照组(分别为80.2±2.6和74.6±1.1)比较,差异无统计学意义(P〉0.05);细胞间接共培养6d后,牵张损伤组BMSC的Ⅰ、Ⅲ型胶原蛋白表达水平分别为126.6±2.2和118.6±1.4,与对照组(分别为82.7±3.0和76.2±1.3)比较,差异有统计学意义(P〈0.05);细胞间接共培养12d后,牵张损伤组BMSC的Ⅰ、Ⅲ型胶原蛋白表达水平分别为135.3±3.4和128.7±2.6,与对照组(分别为86.6±1.3和81.8±1.4)比较,差异也有统计学意义(P〈0.05)。(2)mRNA表达情况:牵张损伤组细胞间接共培养3d后,BMSC的Ⅰ、Ⅲ型胶原mRNA表达水平分别为2.10±0.20和1.20±0.30,与对照组(分别为2.01±0.12和1.13±0.21)比较,差异无统计学意义(P〉0.05);细胞间接共培养6d后,牵张损伤组BMSC的Ⅰ、Ⅲ型胶原mRNA表达水平分别为5.60±0.21和2.61±0.20,与对照组(分别为3.704-0.33和1.82±0.14)比较,差异有统计学意义(P〈0.05);细胞间接共培养12d后,牵张损伤组BMSC的Ⅰ、Ⅲ型胶原mRNA表达水平分别为5.91±0.31和2.92±0.23,与对照组(分别为4.0d±0.21和2.04±0.13)比较,差异也有统计学意义(P〈0.05)。结论机械牵张损伤后的大鼠子宫韧带成纤维细胞与BMSC间接共培养,可以促进BMSC的Ⅰ、Ⅲ型胶原蛋白的合成,并诱导其分化为成纤维细胞。
Objective To investigate the effect on the differentiation of bone marrow mesenchymal stem cells (BMSC) with non-contact co-culture with mechanical stimulated ligament fibroblasts. Methods A cyclic 10% uniaxia strain at 1 Hz was applied on rat pelvic ligament fibroblasts, then were co-cultured with BMSC for 3, 6 and 12 days in non-contact condition. The protein expression of collagen Ⅰ ,Ⅲ in BMSC were detected by SP method and revealed by the mean gray value. The mRNA expressions of collagens type Ⅰ and type Ⅲ in the BMSCs were measured with real-time(RT)-PCR ,and the results were indicated by the ratio between the mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Results ( 1 ) Protein expression: after 3 days co-culture with pelvic ligament fibroblasts, expression of collagen Ⅰ and Ⅲ in BMSC are 82. 4 ± 3.4 and 76. 8 ± 2. 5. When compared with 80. 2 ± 2. 6 and 74. 6 ± 1.1 in BMSC withoutco-culture, there was no significant difference ( P 〉 0.05 ). After 6 days co-culture with pelvic ligament fibroblasts, the expression of collagen I and m of 126. 6 ±2. 2 and 118.6 ± 1.4 in BMSC were significantly higher than 82. 7 ± 3.0 and 76. 2 ± 1.3 in BMSC without co-culture (P 〈 0. 05 ). Similarly, after 12 days co-culture with pelvic ligament fibroblasts, the expression of collagen Ⅰ and Ⅲ of 135 .3 ±3. 4 and 128.7 ± 2. 6 in BMSC were significantly higher than 86. 6 ± 1.3 and 81.8 ± 1.4 in BMSC without co-culture ( P 〈 0. 05 ). (2) mRNA expression : after 3 days co-culture with pelvic ligament fibroblasts , the mRNA expression of type Ⅰ and type Ⅲ collagens in BMSC are 2. 10 ±0. 20 and 1.20 ±0. 30. When compared with mRNA expression of 2. 01 ± 0. 12 and 1.13 ± 0. 21 in BMSC without co-culture, no significant difference were observed ( P 〉 0. 05 ). After 6 days co-culture with pelvic ligament fibroblasts , the mRNA expressions of type Ⅰ and type Ⅲ collagens mRNA were 5.60 -± 0. 21 and 2. 61 ± 0. 20, which were significantly higher than 3.70 ±0. 33 and 1.82 ± 0. 14 in BMSC without eoculture ( P 〈 0.05 ). After 12 days co-culture with pelvic ligament fibroblasts, the mRNA expressions of type Ⅰ and type Ⅲ collagens of 5.91 ± 0. 31 and 2. 92 ± 0. 23 were significantly higher than 4. 04 ± 0. 21 and 2.04 ± 0. 13 in BMSC without co-culture ( P 〈 0. 05 ). Conclusion Non-contact co-culture with mechanical stretch stimulated ligament fibroblasts, it might promote synthesis of types Ⅰ and Ⅲ collagen in rat BMSCs and induced BMSC differentiated into pelvic ligament fibroblasts.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2011年第7期527-532,共6页
Chinese Journal of Obstetrics and Gynecology
关键词
子宫
韧带
成纤维细胞
间质干细胞
细胞分化
胶原Ⅰ型
胶原Ⅲ型
Uterus
Ligaments
Fibroblasts
Masenchymal stem cells
Cell differentiation
Collagen type Ⅰ
Collagen type Ⅲ