本文選題：糖尿病　切入點：成骨細胞　出處：《泰山醫(yī)學院》2012年碩士論文　論文類型：學位論文

【摘要】：目的 觀察糖尿人來源的成骨細胞與胰島素對人臍帶間充質(zhì)干細胞成骨分化的影響，，探討人臍帶間充質(zhì)干細胞治療糖尿病人骨折愈合延遲的可行性。 方法 一、成骨細胞的分離與培養(yǎng)：利用膠原酶消化法分離培養(yǎng)人成骨細胞，利用堿性磷酸酶鈣鈷法染色、Ⅰ型膠原蛋白免疫細胞化學染色、鈣結(jié)節(jié)茜素紅染色鑒定成骨細胞的特性。 二、成骨細胞對臍帶間充質(zhì)干細胞成骨分化的影響：利用Transwell細胞培養(yǎng)板，間接共培養(yǎng)成骨細胞與臍帶間充質(zhì)干細胞。實驗分三組，A組上室不加細胞作為陰性對照組；B組上室內(nèi)放置非糖尿病人來源的成骨細胞；C組上室內(nèi)放置糖尿病人來源的成骨細胞；三組下室內(nèi)均放置人臍帶間充質(zhì)干細胞。采用CCK-8法于第一周內(nèi)觀察臍帶間充質(zhì)干細胞的生長增殖情況；第10天時行免疫熒光染色，觀察Ⅰ型膠原蛋白的表達情況；第7、10、14天時利用細胞堿性磷酸酶活性比色法觀察臍帶間充質(zhì)干細胞內(nèi)的堿性磷酸酶活性表達情況；利用RT-PCR的方法在第21天時，觀察臍帶間充質(zhì)干細胞內(nèi)骨鈣素mRNA的相對表達量。 三、胰島素對臍帶間充質(zhì)干細胞分化的影響：實驗分為胰島素處理組與非胰島素處理組，利用CCK-8法觀察臍帶間充質(zhì)干細胞的增殖情況；細胞堿性磷酸酶活性比色法比較臍帶間充質(zhì)干細胞內(nèi)堿性磷酸酶活性；RT-PCR法比較臍帶間充質(zhì)干細胞內(nèi)Ⅰ型膠原蛋白mRNA、骨鈣素mRNA的相對表達量。 結(jié)果 一、所培養(yǎng)的細胞表達堿性磷酸酶，Ⅰ型膠原蛋白和鈣結(jié)節(jié)，符合成骨細胞的特征，同非糖尿病人來源的成骨細胞相比，糖尿病人來源的成骨細胞表達的堿性磷酸酶、Ⅰ型膠原蛋白、鈣結(jié)節(jié)量少。 二、A組的臍帶間充質(zhì)干細胞的增殖較慢；B、C組的臍帶間充質(zhì)干細胞增殖較快，B組的細胞數(shù)量多于A、C兩組，三組間的差異具有統(tǒng)計學意義（P0.05）。 三、臍帶間充質(zhì)干細胞在第10天時行Ⅰ型膠原蛋白免疫熒光染色，A組為陰性，B、C組呈陽性，B、C兩組間的差異無統(tǒng)計學意義（P0.05）。 四、A組堿性磷酸酶活性一直處于低表達狀態(tài)，同A組相比，B、C組堿性磷酸酶活性增加，10天時達到高峰，14天時表達降低，B、C組堿性磷酸酶活性表達趨勢相同，但C組表達量低，三組間的差異具有統(tǒng)計學意義（P0.05）。 五、在第21天時，RT-PCR檢測發(fā)現(xiàn)B、C組的臍帶間充質(zhì)干細胞內(nèi)表達骨鈣素基因，A組未檢測到骨鈣素基因的表達，C組表達量低于B組表達量，三組間的差異具有統(tǒng)計學意義（P0.05）。 六、同非胰島素處理組相比，胰島素處理組臍帶間充質(zhì)干細胞的增殖加快，堿性磷酸酶活性增高，Ⅰ型膠原蛋白基因、骨鈣素基因表達量高，兩者間的差異具有統(tǒng)計學意義（P0.05）。 結(jié)論 糖尿病人來源的成骨細胞雖然功能受損，但是具有誘導臍帶間充質(zhì)干細胞成骨分化的能力，其誘導分化作用略差于非糖尿病人來源的成骨細胞。胰島素不僅能夠促進受損成骨細胞的功能恢復，并且促進臍帶間充質(zhì)干細胞的增殖和成骨分化。因此推測臍帶間充質(zhì)干細胞聯(lián)合胰島素移植在促進糖尿病人的骨折修復領域中具有積極的作用。
[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.

【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R587.1;R329

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