兔骨髓间质干细胞在PRF膜上增殖和分化的实验研究

发布时间：2018-11-15 10:29

【摘要】：目的 通过兔骨髓间质干细胞在PRF膜上增殖和分化的实验，研究PRF膜在诱导骨髓间质干细胞向成骨细胞转化的作用。为PRF膜应用于口腔种植学领域提供分子生物学的依据。 方法 取7天生新西兰大白兔股骨和胫骨，分离骨髓间质干细胞进行体外培养，在各自的培养条件下分为三组：空白对照组（MSCs）、实验组（PRF膜+MSCS)和阳性对照组（BMP-2+MSCs）。其中，实验组根据PRF膜剂量的不同，分为五个实验亚组，分别是PRF膜1ml组，2ml组,3ml组,4ml组和5ml组。三组细胞分别在24h，48h，72h采用MTT法测定细胞增殖率、在72h用PNPP法测定碱性磷酸酶(ALP)的表达、免疫组化检测Ⅰ型胶原蛋白，骨钙素的表达。通过以上方法检测细胞的成骨特性。 结果 细胞形态学观察，MSCS分化后，细胞形态从长梭形变成三角形，多角形，立方形；MTT测定细胞增殖显示，实验组五个亚组呈剂量反应关系（即随着PRF膜剂量的增加，细胞增殖数量增加）与空白对照组比较，各实验亚组具有统计学意义（F=89.548，P=0.000）；与阳性对照组比较，PRF膜剂量为1ml，2ml和3ml时，细胞增殖数量随着PRF膜剂量的增大而增加，但均小于阳性对照组；PRF膜剂量为4ml和5ml时，，与阳性对照组差别不大，无统计学意义（F=3.364，P=0.142）；ALP检测结果显示，MSCS分化后，实验组五个亚组呈剂量反应关系（即随着PRF膜剂量的增加，ALP活性增强）与空白对照组比较，各实验亚组具有统计学意义。与阳性对照组比较，PRF膜剂量为1ml，2ml和3ml时，ALP活性随着PRF膜剂量的增大而增强，但均小于阳性对照组。PRF膜剂量为4ml和5ml时，与阳性对照组差别不大，无统计学意义。免疫组化标记空白对照组，PRF膜4ml组，阳性对照组的Ⅰ型胶原蛋白和骨钙素，结果均显示阳性。即棕褐色颗粒沉着，阳性颗粒出现在细胞浆，细胞膜和细胞质基质。提示，细胞处于Ⅰ型胶原；骨钙素合成分泌的不同时期。推测，PRF膜5ml组细胞也会具有相同的阳性反应。 结论 PRF膜可以诱导骨髓间质干细胞分化为成骨细胞，分化的细胞具有成骨细胞的特性，可以作为自体材料应用于口腔种植学领域里骨缺损的修复。
[Abstract]:Objective to investigate the effect of PRF membrane on the proliferation and differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) into osteoblasts. It provides molecular biology basis for the application of PRF membrane in the field of stomatology. Methods Bone marrow mesenchymal stem cells (BMSCs) were isolated from femur and tibia of 7 born New Zealand white rabbits and cultured in vitro. They were divided into three groups: blank control group (MSCs),). Experimental group (PRF membrane MSCS) and positive control group (BMP-2 MSCs).) According to the dosage of PRF membrane, the experimental group was divided into five subgroups: PRF membrane 1ml group, 2ml group, 3ml group, 4ml group and 5ml group. The proliferation rate of the three groups was measured by MTT assay at 24 h and 48 h at 72 h, the expression of alkaline phosphatase (ALP) by PNPP method at 72 h, and the expression of type I collagen and osteocalcin by immunohistochemistry. The osteogenic characteristics of the cells were detected by the above methods. Results after MSCS differentiation, cell morphology changed from long spindle to triangle, polygonal and square. MTT assay showed that there was a dose-response relationship among the five subgroups in the experimental group (that is, with the increase of the PRF membrane dose, the number of cell proliferation increased), which was significantly different from that of the control group (F _ (89.548) P _ (0.000). Compared with the positive control group, the number of cell proliferation increased with the increase of PRF membrane dose when the PRF membrane dose was 1ml and 3ml, but both were smaller than those of the positive control group. When the dosage of PRF membrane was 4ml and 5ml, there was no significant difference between the positive control group and the 4ml membrane dose (FN 3.364 P 0.142). The results of ALP showed that after MSCS differentiation, the five subgroups of the experimental group showed dose-response relationship (that is, with the increase of PRF membrane dose, the activity of ALP increased), compared with the control group, the experimental subgroups had statistical significance. Compared with the positive control group, the activity of ALP increased with the increase of PRF membrane dose when the PRF membrane dose was 1ml / L and 3ml, but they were smaller than those of the positive control group. When the PRF membrane dosage was 4ml and 5ml, the difference was not significant between the positive control group and the positive control group, and there was no significant difference between the two groups. Immunohistochemical staining showed positive expression of type I collagen and osteocalcin in the blank control group, PRF membrane 4ml group and positive control group. That is, brown granules, positive granules appear in the cytoplasm, cell membrane and cytoplasmic matrix. These results suggest that the cells are at different stages of osteocalcin synthesis and secretion. It is speculated that the cells in PRF membrane 5ml group also have the same positive reaction. Conclusion PRF membrane can induce bone marrow mesenchymal stem cells to differentiate into osteoblasts. The differentiated cells have the characteristics of osteoblasts and can be used as autogenous materials to repair bone defects in the field of dental implants.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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