Cui G S, Zeng J Y, Zhang J, Lu R
Department of Prosthodontics, Capital Medical University School of Stomatology, Beijing 100050, China(Present address: Hexi Clinic, Tianjin Stomatological Hospital, Tianjin 300061, China).
Department of Prosthodontics, Capital Medical University School of Stomatology, Beijing 100050, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2018 Feb 9;53(2):97-102. doi: 10.3760/cma.j.issn.1002-0098.2018.02.005.
To study the effects of nerve growth factor (NGF) on the proliferation, osteogenic differentiation and mineralization of type 2 diabetic mice bone marrow stromal cell (BMSC), providing basis for clinical application of NGF. Three 8-week-old male db/db mice and two 8-week-old male C57BL/6J mice were used in the study. BMSC derived from femur were cultured though adherence method. BMSC of C57BL/6J mice and db/db mice was divided into normal group and diabetic group to conduct the osteogenic potential experiment, named experiment one. In experiment two, diabetic BMSC was divided into 3 groups: diabetic control group, NGF group, and K252a+NGF group [K252a was the inhibitor of tyrosine kinase A (TrkA), which was the high affinity receptor of NGF], to investigate effect of NGF on osteogenic potential of diabetic mice BMSC. After seeding BMSC, K252a was added into K252a+NGF group, then NGF was added 30 min later. NGF was added into NGF group and K252a+NGF group, but not diabetic control group. The proliferation of BMSC at 1, 3, 5 and 7 d in experiment one and the proliferation of BMSC at 1, 2 and 3 d in experiment two were evaluated through methyl thiazolyl tetrazolium, and the level of alkaline phosphatase (ALP) at 3, 5 and 7 d in both experiments were measured. After being osteogenic induced for 14 d, mineralized nodules in both experiments were quantitated by alizarin red calcium stain. Five holes were set in every group, and all experiments were repeated 3 times. The BMSC proliferation of diabetic group was significantly higher than that of the normal group at 3, 5 and 7 d (0.05). After being osteogenic inducted for 3, 5 and 7 d, ALP level of diabetic group were significantly lower than that of normal group (0.05). After being osteogenic inducted for 14 d, calcium nodule count of diabetic group [(23.1±6.4) nodule/field] were significantly lower than that of normal group (36.9±7.9) nodule/field. At 1, 2 and 3 d, BMSC proliferations of diabetic control group, NGF group and K252a+NGF group were not statistically different (0.05). After being osteogenic inducted for 3 and 5 d, ALP level of NGF group was significantly higher than that of diabetic control group (0.05). After being osteogenic inducted for 3, 5, and 7 d, ALP level of K252a+NGF group was significantly lower than that of NGF group (0.05) and diabetic control group (0.05). After being osteogenic induced for 14 d, calcium nodule count of NGF group [(45.2±6.8) nodule/field] was significantly more than that of diabetic control group (23.1±6.4) nodule/field; while calcium nodule count of K252a+NGF group [(18.0±4.5) nodule/field] was significantly less than that of NGF group (0.05) and diabetic control group (0.05). The differentiation and mineralization of type 2 diabetic mice BMSC was significantly reduced. NGF promoted the osteoblastic differentiation and mineralization of diabetic mice BMSC though combining with TrkA.
研究神经生长因子（NGF）对2型糖尿病小鼠骨髓间充质干细胞（BMSC）增殖、成骨分化及矿化的影响，为NGF的临床应用提供依据。本研究选用3只8周龄雄性db/db小鼠和2只8周龄雄性C57BL/6J小鼠。采用贴壁法培养从股骨分离的BMSC。将C57BL/6J小鼠和db/db小鼠的BMSC分为正常组和糖尿病组进行成骨潜能实验，即实验一。在实验二中，将糖尿病BMSC分为3组：糖尿病对照组、NGF组和K252a + NGF组[K252a为酪氨酸激酶A（TrkA）的抑制剂，TrkA是NGF的高亲和力受体]，以研究NGF对糖尿病小鼠BMSC成骨潜能的影响。接种BMSC后，向K252a + NGF组加入K252a，30分钟后再加入NGF。向NGF组和K252a + NGF组加入NGF，糖尿病对照组不加入。通过甲基噻唑基四氮唑法评估实验一中第1、3、5和7天BMSC的增殖情况以及实验二中第1、2和3天BMSC的增殖情况，并检测两个实验中第3、5和7天的碱性磷酸酶（ALP）水平。成骨诱导14天后，通过茜素红钙染色对两个实验中的矿化结节进行定量。每组设置5个孔，所有实验重复3次。糖尿病组在第3、5和7天的BMSC增殖明显高于正常组（P<0.05）。成骨诱导3、5和7天后，糖尿病组的ALP水平明显低于正常组（P<0.05）。成骨诱导14天后，糖尿病组的钙结节计数[（23.1±6.4）个结节/视野]明显低于正常组[（36.9±7.9）个结节/视野]（P<0.05）。在第1、2和3天，糖尿病对照组、NGF组和K252a + NGF组的BMSC增殖无统计学差异（P>0.05）。成骨诱导3和5天后，NGF组的ALP水平明显高于糖尿病对照组（P<0.05）。成骨诱导3、5和7天后，K252a + NGF组的ALP水平明显低于NGF组（P<0.05）和糖尿病对照组（P<0.05）。成骨诱导14天后，NGF组的钙结节计数[（45.2±6.8）个结节/视野]明显多于糖尿病对照组[（23.1±6.4）个结节/视野]（P<0.05）；而K252a + NGF组的钙结节计数[（18.0±4.5）个结节/视野]明显少于NGF组（P<0.05）和糖尿病对照组（P<0.05）。2型糖尿病小鼠BMSC的分化和矿化明显降低。NGF通过与TrkA结合促进糖尿病小鼠BMSC的成骨细胞分化和矿化。
