糖尿病人的成骨细胞对脐带间充质干细胞成骨分化的影响

发布时间：2018-03-13 13:40

本文选题：糖尿病　切入点：成骨细胞　出处：《泰山医学院》2012年硕士论文　论文类型：学位论文

【摘要】：目的观察糖尿人来源的成骨细胞与胰岛素对人脐带间充质干细胞成骨分化的影响，，探讨人脐带间充质干细胞治疗糖尿病人骨折愈合延迟的可行性。方法一、成骨细胞的分离与培养：利用胶原酶消化法分离培养人成骨细胞，利用碱性磷酸酶钙钴法染色、Ⅰ型胶原蛋白免疫细胞化学染色、钙结节茜素红染色鉴定成骨细胞的特性。二、成骨细胞对脐带间充质干细胞成骨分化的影响：利用Transwell细胞培养板，间接共培养成骨细胞与脐带间充质干细胞。实验分三组，A组上室不加细胞作为阴性对照组；B组上室内放置非糖尿病人来源的成骨细胞；C组上室内放置糖尿病人来源的成骨细胞；三组下室内均放置人脐带间充质干细胞。采用CCK-8法于第一周内观察脐带间充质干细胞的生长增殖情况；第10天时行免疫荧光染色，观察Ⅰ型胶原蛋白的表达情况；第7、10、14天时利用细胞碱性磷酸酶活性比色法观察脐带间充质干细胞内的碱性磷酸酶活性表达情况；利用RT-PCR的方法在第21天时，观察脐带间充质干细胞内骨钙素mRNA的相对表达量。三、胰岛素对脐带间充质干细胞分化的影响：实验分为胰岛素处理组与非胰岛素处理组，利用CCK-8法观察脐带间充质干细胞的增殖情况；细胞碱性磷酸酶活性比色法比较脐带间充质干细胞内碱性磷酸酶活性；RT-PCR法比较脐带间充质干细胞内Ⅰ型胶原蛋白mRNA、骨钙素mRNA的相对表达量。结果一、所培养的细胞表达碱性磷酸酶，Ⅰ型胶原蛋白和钙结节，符合成骨细胞的特征，同非糖尿病人来源的成骨细胞相比，糖尿病人来源的成骨细胞表达的碱性磷酸酶、Ⅰ型胶原蛋白、钙结节量少。二、A组的脐带间充质干细胞的增殖较慢；B、C组的脐带间充质干细胞增殖较快，B组的细胞数量多于A、C两组，三组间的差异具有统计学意义（P0.05）。三、脐带间充质干细胞在第10天时行Ⅰ型胶原蛋白免疫荧光染色，A组为阴性，B、C组呈阳性，B、C两组间的差异无统计学意义（P0.05）。四、A组碱性磷酸酶活性一直处于低表达状态，同A组相比，B、C组碱性磷酸酶活性增加，10天时达到高峰，14天时表达降低，B、C组碱性磷酸酶活性表达趋势相同，但C组表达量低，三组间的差异具有统计学意义（P0.05）。五、在第21天时，RT-PCR检测发现B、C组的脐带间充质干细胞内表达骨钙素基因，A组未检测到骨钙素基因的表达，C组表达量低于B组表达量，三组间的差异具有统计学意义（P0.05）。六、同非胰岛素处理组相比，胰岛素处理组脐带间充质干细胞的增殖加快，碱性磷酸酶活性增高，Ⅰ型胶原蛋白基因、骨钙素基因表达量高，两者间的差异具有统计学意义（P0.05）。结论糖尿病人来源的成骨细胞虽然功能受损，但是具有诱导脐带间充质干细胞成骨分化的能力，其诱导分化作用略差于非糖尿病人来源的成骨细胞。胰岛素不仅能够促进受损成骨细胞的功能恢复，并且促进脐带间充质干细胞的增殖和成骨分化。因此推测脐带间充质干细胞联合胰岛素移植在促进糖尿病人的骨折修复领域中具有积极的作用。
[Abstract]:objective
Objective To observe the effect of diabetic human osteoblasts and insulin on the osteogenic differentiation of human umbilical cord mesenchymal stem cells, and to explore the feasibility of human umbilical cord mesenchymal stem cells in the treatment of diabetic patients with fracture healing delay.
Method
First, the isolation and culture of osteoblasts: isolation and culture of human osteoblasts by collagenase digestion. The characteristics of osteoblasts were identified by alkaline phosphatase cobalt cobalt staining, type I collagen immunocytochemical staining and calcium nodule alizarin red staining.
Two, the osteogenic effect of human umbilical cord mesenchymal stem cell into osteogenic differentiation: culture plate using Transwell cells, indirect co cultured osteoblasts and umbilical cord mesenchymal stem cells. The experiment was divided into three groups, A group on the room without cells as a negative control group; Osteoblasts group B was placed in the room without diabetic patient derived osteoblasts; group C was placed in the room of diabetic patients from three groups were placed indoors; human umbilical cord mesenchymal stem cells. In the first weeks of observation of umbilical cord mesenchymal stem cell proliferation by CCK-8 method; tenth days after immunofluorescence staining, we observed the expression of type I collagen the first 7,10,14 days; the alkaline phosphatase activity of cultured human umbilical cord mesenchymal stem cells in the expression of alkaline phosphatase activity assay; using RT-PCR method in twenty-first days, the observation of umbilical cord mesenchymal stem cells in osteocalcin mRN The relative expression of A.
Three, the effect of insulin on differentiation of umbilical cord mesenchymal stem cells: the experiment was divided into insulin group and non insulin treated group, observation of umbilical cord mesenchymal stem cell proliferation by CCK-8 method; alkaline phosphatase activity of the cells colorimetric method of umbilical cord mesenchymal stem cells alkaline phosphatase activity; RT-PCR method of umbilical cord mesenchymal mesenchymal stem cells in collagen type mRNA, the relative expression of osteocalcin mRNA.
Result
First, cultured cells expressed alkaline phosphatase, type I collagen and calcium nodules, which accords with the characteristics of osteoblasts. Compared with osteoblasts derived from non-diabetic patients, alkaline phosphatase, type I collagen and calcium nodules expressed in osteoblasts derived from diabetic patients were less.
Two, the proliferation of umbilical cord mesenchymal stem cells in A group was slower than that in group B; the proliferation of umbilical cord mesenchymal stem cells in group C and C was faster than that in group C, and the number of cells in B group was more than that in A group. The difference between C group was statistically significant (P0.05).
Three, human umbilical cord mesenchymal stem cells staining in tenth days were of type I collagen immunofluorescence, group A was negative, B, C group was positive, B, C was no significant difference between the two groups (P0.05).
Four, the A group has been in the low expression of alkaline phosphatase activity, compared with the A group, B group, C increased alkaline phosphatase activity, reached the peak on day 10, day 14 decreased expression of B, C group expression of alkaline phosphatase activity the same trend, but the C low expression group, and the difference between the three groups with statistical significance (P0.05).
Five, on the twenty-first day, RT-PCR detected the expression of osteocalcin gene in umbilical cord mesenchymal stem cells of B and C group. The expression of osteocalcin gene was not detected in group A, and the expression level in group C was lower than that in B group. The difference between the three groups was statistically significant (P0.05).
Six, compared with the non insulin treatment group, the proliferation of umbilical cord mesenchymal stem cells and the activity of alkaline phosphatase in the insulin treatment group increased. The expression of type I collagen gene and osteocalcin gene was high, and the difference between them was statistically significant (P0.05).
conclusion
Diabetic osteoblasts although the function is impaired, but can induce human umbilical cord mesenchymal stem cells differentiated into bone, bone cells and the differentiation effect is slightly worse than the non diabetes sources. Insulin can not only promote the recovery of bone cells into damaged function, and promote the umbilical cord mesenchymal stem cells the proliferation and osteogenic differentiation. So we speculate that umbilical cord mesenchymal stem cells transplantation combined with insulin plays a positive role in promoting fracture repair in patients with diabetes in the field.

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R587.1;R329

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