音猬因子修饰的骨髓间充质干细胞治疗大鼠骨质疏松的实验研究

doi:10.3877/cma.j.issn.2096-0263.2019.04.006

目的

探讨转染音猬因子的骨髓间充值干细胞移植对卵巢切除骨质疏松大鼠的治疗作用。

方法

选取40只雌性SD大鼠，分为假手术组、模型组、骨髓间充值干细胞（BMSCs）移植组和音猬因子修饰的骨髓间充值干细胞（Shh-BMSCs）移植组，每组10只动物。假手术组手术中仅摘除卵巢附近的脂肪组织，但不摘除卵巢。其余的3组均进行双侧的卵巢切除手术，建立骨质疏松模型，在骨质疏松造模后的2个月，BMSCs移植组和Shh-BMSCs移植组分别通过大鼠的尾静脉注射BMSCs与Shh-BMSCs（第四代，约2*10⁶个干细胞），模型组动物注射等量PBS液体。在干细胞移植2 w后，检测尾静脉中血清钙、磷、碱性磷酸酶水平，同时采用双能X射线骨密度仪测量大鼠的股骨、腰椎及全身骨密度，micro-CT扫描进行胫骨的形态计量学指标分析。

结果

（1）与假手术组相比，模型组的大鼠骨小梁厚度、骨小梁数量和骨体积分数均明显减少，大鼠的骨密度、血清钙与碱性磷酸酶明显降低，而大鼠的血清磷水平明显升高，差异有统计学意义（P<0.05），以上表明骨质疏松大鼠模型成功制备。（2）与模型组相比，BMSCs组的大鼠血清钙和和碱性磷酸酶明显升高，而血清磷水平明显降低，差异有统计学意义（P<0.05）。与模型组、BMSCs组相比，Shh-BMSCs组大鼠血清钙、碱性磷酸酶水平明显升高，而血清磷水平明显降低，差异有统计学意义（P<0.05）。（3）同模型组的骨密度相比，BMSCs组大鼠的骨密度明显升高，差异有统计学意义（P<0.05）；与模型组、BMSCs组相比，Shh-BMSCs组大鼠的骨密度明显升高，差异有统计学意义（P<0.05）。（4）同模型组相比，BMSCs组的大鼠骨小梁厚度、骨小梁数量和骨体积分数均明显增加，差异有统计学意义（P<0.05），与模型组和BMSCs组相比，Shh-BMSCs组的大鼠骨小梁厚度、骨小梁数量和骨体积分数均明显增加，差异有统计学意义（P<0.05）。

结论

Shh-BMSCs细胞移植后能够治疗大鼠卵巢切除后的骨质疏松。

关键词: 间充质干细胞, 音猬因子蛋白，大鼠, 骨质疏松, 细胞移植

Objective

To research the effect of bone marrow stromal cells transferred with sonic hedgehog gene (Shh-BMSCs) transplantation about osteoporosis in ovariectomized rats.

Methods

Forty female SD rats were divided into control group, model group, bone marrow stem cells (BMSCs) transplantation group and bone marrow stromal cells transferred with sonic hedgehog gene (Shh-MSCs) transplantation group, each group has ten animals. Rats in control group were only removed adipose tissue around the ovaries, not their ovaries. The rest of the three groups were performed bilateral ovariectomy, and osteoporosis model was established. Two months after modeling, rats in the BMSCs or Shh-BMSCs transplantation group were respectively given BMSCs or Shh-BM SCs via the rat tail intravenous (fourth generation, about 2*10⁶ stem cells), and the same amount of PBS was injected in the model group. Two weeks after treatment, levels of serum calcium, phosphorus, alkaline phosphatase were detected in rats, and bone mineral density was measured using dual energy X-ray, at the same timetibial form metrology was analyzed by micro -CT scan.

Results

(1) Compared with the control group, the bone trabecular thickness, trabecular bone quantity and bone volume fraction were significantly decreased in the model group (P<0.05). There was a significant reduction in the bone mineral density of the rat femur, lumbar spine and whole body as well as the levels of serum calcium and alkaline phosphatase, however the phosphorus was increased significantly (P<0.05). These findings indicate the successful preparation of the ostesporosis rat model. (2) Compared with the ovariedomized group, the levels of serum calcium and alkaline phosphatase were significantly higher in the BMSCs group, however the phosphorus was decreased significantly (P<0.05). Compared with the ovariedomized and BMSCs groups, the levels of serum calcium and alkaline phosphatase were significantly higher in the Shh-BMSCs group, however the phosphorus was decreased significantly (P<0.05). (3) Compared with the ovariedomized group, the bone mineral density of the rat femur, lumbar spine or whole body was significantly higher in the BMSCs group(P<0.05). Compared with the ovariedomized and BMSCs groups, the bone mineral density of the rat femur, lumbar spine or whole body was significantly higher in the Shh-BMSCs group(P<0.05). (4) Compared with the ovariedomized group, the number and thickness of trabeculae and bone volume fraclio was significantly higher in the BMSCs group(P<0.05). Compared with the ovariedomized and BMSCs groups, the number and thickness of trabeculae and bone volume fraclio was significantly higher in the Shh-BMSCs group(P<0.05).

Conclusion

Shh-BMSCs exerts therapeutic effects on osteoporosis in ovariectomized rats.

Key words: Marrow stromal cells, Sonic hedgehog protein, rat, Osteoporosis, Cell transplantaion

图2 流式细胞仪检测结果

图5 Shh-BMSCs中Shh蛋白的表达情况，其中A组为BMSCs，B组为Shh-BMSCs

表1 SD大鼠的血清生化指标和密度值的比较（±s，n=10）

组别	钙（mmol/L）	磷（mmol/L）	碱性磷酸酶（U/L）	腰椎	股骨	全身
假手术组	3.59±0.63	3.69±0.23	201.31±29.23^abc	187.21±12.32	257.29±19.73	137.29±8.82
模型组	2.52±0.75^a	5.26±0.86^a	225±31.54	97.32±10.35^a	198.29±23.96^a	100.29±9.75^a
BMSCs组	3.08±0.64^ab	4.30±0.42^ab	132.29±19.25^a	122.19±10.84^ab	211.18±16.75^ab	112.02±11.45^ab
Shh-BMSCs组	3.31±0.68^abc	3.98±0.29^abc	170.52±25.06^ab	143.89±10.53^abc	230.62±22.51^abc	121.31±10.34^abc

表1 SD大鼠的血清生化指标和密度值的比较（±s，n=10）

组别	钙（mmol/L）	磷（mmol/L）	碱性磷酸酶（U/L）	腰椎	股骨	全身
假手术组	3.59±0.63	3.69±0.23	201.31±29.23^abc	187.21±12.32	257.29±19.73	137.29±8.82
模型组	2.52±0.75^a	5.26±0.86^a	225±31.54	97.32±10.35^a	198.29±23.96^a	100.29±9.75^a
BMSCs组	3.08±0.64^ab	4.30±0.42^ab	132.29±19.25^a	122.19±10.84^ab	211.18±16.75^ab	112.02±11.45^ab
Shh-BMSCs组	3.31±0.68^abc	3.98±0.29^abc	170.52±25.06^ab	143.89±10.53^abc	230.62±22.51^abc	121.31±10.34^abc

表2 各组大鼠骨组织微结构形态计量学指标比较（±s）

组别	例数（只）	骨小梁厚度（mm）	骨小梁数量（mm^-1）	骨小梁分离度（mm）	骨体积分数
假手术组	10	0.047±0.008	4.6729±0.131	0.208±0.036	0.196±0.021
模型组	10	0.025±0.009^a	3.127±0.263^a	0.369±0.531^a	0.098±0.009^a
BMSCs组	10	0.036±0.012^ab	3.988±0.284^ab	0.273±0.352^ab	0.136±0.021^ab
Shh-BMSCs组	10	0.042±0.010^abc	4.239±0.327^abc	0.239±0.326^abc	0.158±0.019^abc

表2 各组大鼠骨组织微结构形态计量学指标比较（±s）

组别	例数（只）	骨小梁厚度（mm）	骨小梁数量（mm^-1）	骨小梁分离度（mm）	骨体积分数
假手术组	10	0.047±0.008	4.6729±0.131	0.208±0.036	0.196±0.021
模型组	10	0.025±0.009^a	3.127±0.263^a	0.369±0.531^a	0.098±0.009^a
BMSCs组	10	0.036±0.012^ab	3.988±0.284^ab	0.273±0.352^ab	0.136±0.021^ab
Shh-BMSCs组	10	0.042±0.010^abc	4.239±0.327^abc	0.239±0.326^abc	0.158±0.019^abc

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Effect of bone marrow stromal cells transferred with sonic hedgehog gene on osteoporosis in ovariectomized rats

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Effect of bone marrow stromal cells transferred with sonic hedgehog gene on osteoporosis in ovariectomized rats