血小板源性生长因子BB对大鼠肌腱愈合和肌腱粘连的影响

目的 了解血小板源性生长因子BB(PDGF-BB)基因转染大鼠肌腱细胞对肌腱愈合及肌腱粘连的影响. 方法 将90只SD大鼠制成跟腱损伤模型,按随机数字表法分为3组,每组30只:实验组,肌腱断端注射20μL转染PDGF-BB基因的大鼠肌腱细胞(1×108个/mL);对照组,肌腱断端注射20μL未行转染的大鼠肌腱细胞(1×108个/mL);空白对照组,不做任何处理.6-0丝线行改良Kessler法缝合跟腱,管型石膏固定1周.通过基因测序及RT-PCR鉴定转染PDGF-BB基因的大鼠肌腱细胞.分别于术后3 d和1、2、4、8周取各组大鼠肌腱组织样本,行大体、组织学观察以及生物力学检测,对比各组肌腱粘连度、组织中Fb数量与胶原纤维含量、肌腱最大抗拉力及最大滑动距离、组织中PDGF-BB的浓度.对数据行t检验. 结果(1)转染的肌腱细胞经RT-PCR以及基因测序证实在体外稳定表达PDGF-BB mRNA.(2)各组大鼠术后3 d肌腱均出现较明显肿胀及炎性细胞浸润,实验组改变较其他组明显轻微;随后各组情况均逐渐好转.术后4、8周肌腱粘连度分级组间比较未见明显差异.(3)实验组Fb数在术后2、4、8周显著低于对照组和空白对照组(t值分别为2.94、4.26、5.76和4.00、3.83、6.12,P＜0.05或P＜0.01).(4)实验组术后4周胶原纤维含量为(43±6)%,较对照组(55±8)%]与空白对照组(61±8)%]显著下降(t值分别为2.94和4.41,P＜0.05或P＜0.01).(5)术后4、8周实验组肌腱最大滑动距离为(3.25±0.33)、(3.65±0.21)mm,显著高于对照组的(2.29±0.40)、(2.21±0.37)mm和空白对照组的(2.01±0.23)、(1.89±0.24)mm(t值分别为4.53、8.29和7.55、13.52,P值均小于0.01),但其肌腱最大抗拉力与另2组比较差异无统计学意义(t值分别为0.41、0.41和0.77、0.72,P值均大于0.05).(6)术后3 d和2、4周,实验组肌腱组织中PDGF-BB浓度为(12.95±1.36)、(8.32±0.94)、(9.10±1.06)ng/mL,均显著高于对照组的(1.13±0.21)、(2.07±0.48)、(3.85±0.39)ng/mL(t值分别为21.04、14.50、11.39,P值均小于0.01). 结论 转染PDGF-BB基因肌腱细胞有促进肌腱内源性愈合、减轻肌腱粘连的作用.

Effect of platelet-derived growth factor-BB on the healing and adhesion of rat tendon

Objective To study the effect of platelet-derived growth factor-BB (PDGF-BB) gene transfected rat tendon cells on the healing and adhesion of rat tendon. Methods A model of heel tendon injury was reproduced in 90 rats. They were randomly divided into three groups: experiment group with injection of 20 μL rat tendon cells(1 × 108 cell/mL) transfected with PDGF-BB gene into the injured tendon ends], control group with injection of 20 μL non-transfected rat tendon cells (1 × 108 cell/mL) into the injured tendon ends] , and blank control group(without treatment) , with 30 rats in each group. Heel tendon ends were sutured with 6-0 thread by modified Kessler method and immobilized with tube-type plaster of Paris cast for one week. Rat tendon cells transfected with PDGF-BB gene were identified with gene sequencing and RT-PCR. Tendon tissue sample was harvested 3 days or 1, 2, 4, 8 week(s) after operation (POD or POW) for morphology and histology observation, and bio-mechanical test. The degree of tendon adhesion,the number of Fb and collagen fiber content in tissue, maximum tensile strength and sliding distance of tendon, and concentration of PDGF-BB in tendon tissue among groups were compared. Data were processed with t test. Results (1) PDGF-BB mRNA expressed stably in PDGF-BB gene transfected tendon cells as testified by RT-PCR and gene sequencing. (2) Obvious edema and inflammatory cells infiltration were observed in each group on POD 3, but they were less pronounced in experiment group. And the changes in all groups were ameliorated gradually. The difference in grading of tendon adhesion was not obvious among groups in POW 4 and 8. (3) Fb number in experiment group in POW 2, 4, 8 was respectively fewer than that of control group and blank control group(with t value respectively 2.94, 4. 26, 5.76 and 4.00, 3.83,6.12, P ＜0.05 orP ＜0.01). (4) Collagen fiber content in rat tendon of experimental group in POW 4was (43 ± 6) %, which was significantly lower as compared with that of control group (55 ± 8) %] and blank control group (61 ± 8) %](with t value respectively 2.94 and 4.41, P ＜ 0. 05 or P ＜ 0. 01). (5)The largest sliding distance of tendon in experiment group in POW 4 and 8 were (3.25 ±0.33) and (3.65 ±0.21) mm, which were significantly longer than those in control group (2.29 ±0.40), (2.21 ±0.37)mm] and blank control group (2.01 ± 0.23),(1.89 ± 0. 24) mm](with t value respectively 4.53, 8.29and 7.55, 13.52, P values all below 0.01). There was no statistical significant difference among the three groups in the maximum tensile strength of tendon (with t value respectively 0. 41,0.41,0.77, 0.72, P values all above 0. 05). (6) Content of PDGF-BB in tendon tissue of experimental group on POD 3 and in POW 2, 4 were(12.95 ± 1.36),(8.32 ± 0. 94), (9.10 ± 1.06) ng/mL, all significantly higher than those in control group (1.13 ±0.21),(2.07 ± 0.48),(3.85 ± 0.39) ng/mL](with t value respectively 21.04,14. 50, 11.39, P values all below 0. 01). Conclusions PDGF-BB gene transfected rat tendon cells can promote endogenous healing of tendon and prevent tendon adhesion.