人脐带间充质干细胞通过调控OPG/RANKL比值对骨质疏松大鼠的影响及机制研究

Effects and mechanism of human umbilical cord mesenchymal stem cells on osteoporosis rats by regulating the ratio of osteoprotegerin/receptor activator of nuclear factor-κB ligand

摘要:

目的： 探讨人脐带间充质干细胞（human umbilical cord mesenchymal stem cells，HUC-MSCs）对骨质疏松大鼠的影响及机制。方法： 将30只SPF级6月龄雌性SD大鼠随机分为3组：假手术组（Sham组）、卵巢切除组（OVX组）、卵巢切除+HUC-MSCs干预组（4×10⁶/3 d，HUC-MSCs干预组），每组各10只，Sham组切除大鼠腹腔少量脂肪，其余各组均去除双侧卵巢建立骨质疏松大鼠模型。建模成功后，HUC-MSCs组用干细胞干预12周，其余2组用等剂量生理盐水干预。干预结束后，通过骨代谢血清指标血钙（calcium，Ca）、血磷（phosphorus，P）、碱性磷酸酶（alkaline phosphatase，ALP）、Ⅰ型胶原C端肽（c-terminal peptide of typeⅠcollagen，CTX）、骨组织形态计量学参数测量、骨密度检测、成骨细胞表面积/骨表面积比（osteoblast surface/bone surface，ObS/BS）和破骨细胞表面积/骨表面积比（osteoclast surface area/bone surface，OcS/BS），评估大鼠骨质疏松改善情况，同时通过Western blot探究各组大鼠骨保护素（osteoprotegerin，OPG）/核因子-κB受体活化因子配体（receptor activator of nuclear factor-κB ligand，RANKL）、β-连环蛋白（β-catenin）、Runt相关转录因子2（Runt-related transcription factor 2，Runx2）的表达情况。结果： HUC-MSCs组大鼠血清Ca、ALP含量明显高于OVX组（2.320±0.188 vs.1.822±0.139；79.584±8.349 vs.43.644±5.369；均P=0.000），P、CTX含量明显低于OVX组（1.651±0.068 vs.1.864±0.076，P=0.000；119.832±18.180 vs.144.272±15.629，P=0.039）；HUC-MSCs组骨小梁结构得到明显改善，骨密度、骨小梁数目、骨小梁面积比、ObS/BS均明显高于OVX组（161.114±16.726 vs.126.558±7.100，P=0.009；1.702±0.220 vs.1.051±0.323，P=0.020；33.111±6.101 vs.19.575±5.629，P=0.022；45.279±3.947 vs.33.948±7.339，P=0.041），骨小梁分离度、OcS/BS明显低于OVX组（0.592±0.066 vs.0.845±0.165，P=0.018；37.532±3.910 vs.44.891±3.615，P=0.026）；Western blot结果显示HUC-MSCs组OPG/RANKL比值、β-catenin、Runx2的蛋白表达量明显高于OVX组（0.776±0.045 vs.0.097±0.063，P=0.000；0.590±0.057 vs.0.202±0.088，P=0.000；0.595±0.078 vs.0.239±0.071，P=0.001）。结论： HUC-MSCs可以通过Wnt/β-catenin/Runx2通路调节OPG/RANKL比值，促进骨形成，抑制骨吸收，改善骨质疏松。

Abstract:

Objective: To investigate the effect of human umbilical cord mesenchymal stem cells (HUC-MSCs) on osteoporosis rats and its mechanism. Methods: Thirty SPF grade 6-month-old female rats were randomly divided into three groups: sham operation group (Sham group), ovariectomy group (OVX group) and HUC-MSCs intervention group (HUC-MSCs group) (4×10⁶/3 day), with 10 rats in each group. In Sham group, a small amount of abdominal fat was removed, and the other groups were removed bilateral ovaries to establish osteoporosis rat model. After successful modeling, HUC-MSCs group was intervened with stem cells for 12 w, while the other two groups were intervened with equal dose of normal saline. After the intervention, calcium (Ca), phosphorus (P), alkaline phosphatase (ALP), c-terminal peptide of type I collagen (CTX), histomorphometric parameters of femur, bone mineral density (BMD), osteoblast surface/bone surface ratio (ObS/BS) and osteoclast surface area/bone surface (OcS/BS) were used to evaluate the improvement of osteoporosis in rats. Meanwhile, the expression of osteoprotegerin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), β-catenin and Runt-related transcription factor 2 (Runx2) were detected by Western blot. Results: The contents of serum Ca and ALP in HUC-MSCs group were significantly higher than those in OVX group (2.320±0.188 vs.1.822±0.139; 79.584±8.349 vs.43.644±5.369; all P=0.000) ; P and CTX contents were significantly lower than those in OVX group (1.651±0.068 vs.1.864±0.076, P=0.000; 119.832±18.180 vs.144.272±15.629, P=0.039) ; BMD, the number of trabecular bone, the ratio of trabecular area and ObS/BS in HUC-MSCs group were significantly higher than those in OVX group (161.114±16.726 vs.126.558±7.100, P=0.009; 1.702±0.220 vs.1.051±0.323, P=0.020; 33.111±6.101 vs.19.575±5.629, P=0.022; 45.279±3.947 vs.33.948±7.339, P=0.041) ; the bone trabecular separation and OcS/BS in HUC-MSCs group were significantly lower than those in OVX group (0.592±0.066 vs.0.845±0.165, P=0.018; 37.532±3.910 vs.44.891±3.615, P=0.026) ; the results of Western blot showed that the ratio of OPG/RANKL and the protein expression of β-catenin and Runx2 in HUC-MSCs group were significantly higher than those in OVX group (0.776±0.045 vs.0.097±0.063, P=0.000; 0.590±0.057 vs.0.202±0.088, P=0.000; 0.595±0.078 vs.0.239±0.071, P=0.001) . Conclusion: HUC-MSCs can regulate OPG/RANKL ratio through Wnt/β-catenin/Runx2 pathway, thus promoting bone formation, inhibiting bone resorption and improving osteoporosis.