机械牵张损伤后的大鼠子宫韧带成纤维细胞对大鼠骨髓间充质干细胞定向分化的影响

Study on oriented differentiation of bone marrow mesenchymal stem cells by fibroblast in rat uterine ligament with mechanical stretch

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.