甲状旁腺素促进骨质疏松骨折大鼠Ⅰ型胶原代谢的研究

Study on the effects of PTH on type Ⅰcollagen metabolism in rats with osteoporotic fracture

目的监测骨质疏松性骨折愈合过程中甲状旁腺素(parathyroid hormone,PTH)影响下Ⅰ型胶原代谢的变化,及Ⅰ型胶原在骨痂组织的表达状态。方法切除成年雌性大鼠双侧卵巢,构建骨质疏松模型。3个月后将大鼠双侧胫骨锯断,并以克氏针髓内固定,建立大鼠双侧胫骨骨折愈合模型。术后即将大鼠分为两组,A组(PTH组)予以特立帕肽[PTH(1-34)]3 ug/(100 g·d)皮下注射,每天一次;B组(NS组)予以等量生理盐水皮下注射,每天一次。分别于给药后第1、2、4、8周取大鼠胫骨标本,一部分利用荧光定量PCR检测骨痂附近骨组织COL1A1 mRNA的表达水平;另一部分行骨痂附近组织的HE染色及Ⅰ型胶原免疫组化染色。结果 HE染色及Ⅰ型胶原免疫组化染色显示,自第2周起,PTH组大鼠骨痂内Ⅰ型胶原的表达状态即优于NS组,至第4周时表现最为明显;到第8周,PTH组大鼠骨痂中已经观察不到成束的Ⅰ型胶原纤维,仅见大片已修复良好的骨基质;而NS组大鼠骨痂内尚可见部分Ⅰ型胶原表达。荧光定量PCR检测显示,第1周时PTH组大鼠COL1A1mRNA表达量为17.258±1.006,NS组大鼠为10.022±0.308(P<0.001);第2周时PTH组为19.548±0.767,NS组为10.208±0.619(P<0.001);第4周时PTH组为20.892±1.140,NS组为10.182±0.624(P<0.001);第8周时PTH组为20.708±1.249,NS组为10.560±0.453(P<0.001)。结论 PTH可以增加骨折端COL1A1 mRNA的表达,促进Ⅰ型胶原的合成和重塑,有效增加骨痂组织Ⅰ型胶原的含量和结构排列。

Objective To monitor the metabolism of type I collagen and the expression of type I collagen in callus tissue during the process of osteoporotic fracture healing with intermittent PTH treatment. Methods The bilateral ovaries of adult female rats were excised to establish the osteoporosis model of rat. Three months later after OVX,the rats had bilateral tibial osteotomy followed by a Kirschner wire intramedullary fixation. Then the rats were divided into two groups： group A（ PTH group） were treated with teriparatide（ PTH（ 1-34）） 3 ug/100 g/d subcutaneous injections,once daily; Group B（ NS group） were treated with physiological saline（ NS） subcutaneous injection at the same dose. At week 1,2,4,8 after the first treatment,specimens the tibia were collected,with some of them evaluated for the COL1 A1 mRNA expression level of callus tissue,using the fluorescent quantitative PCR detection,and others for HE staining and immunohistochemical staining of type I collagen. Results On the staining,more new typeⅠcollagen expression and more regular arrangement of collagen fibers could be seen in the PTH groups.The difference was most evident at the 4 th week. At the 8 th week,typeⅠcollagen fibers were rare in the PTH groups,instead plenty of repaired bone matrix could be seen. The COL1 A1 mRNA PCR result ： week 1： PTH group 17. 258 ± 1. 006,NS group10. 022 ± 0. 308（ P〈0. 001）; week 2： PTH group 19. 548 ± 0. 767,NS group 10. 208 ± 0. 619（ P〈0. 001）; week 4： PTH group20. 892 ± 1. 140,NS group 10. 182 ± 0. 624（ P〈0. 001）; week 8： PTH group 20. 708 ± 1. 249,NS group 10. 560 ± 0. 453（ P〈0. 001）. Conclusion In the callus,PTH could improve the expression of COL1 A1 mRNA,promote the synthesis and remodeling of typeⅠcollagen,and thus effectively improve the content and arrangement of typeⅠcollagen.