碱热处理对钛片表面成骨细胞整合素影响的实验研究

发布时间：2018-04-23 17:58

本文选题：碱热处理 + 钛片表面　；参考：《昆明医科大学》2014年硕士论文

【摘要】：[目的]研究碱热处理的钛片表面对成骨细胞整合素α2β1,整合素α5β1的影响。 [材料和方法]本实验用SD胎鼠颅顶骨组织块培养法进行成骨细胞原代培养。用倒置显微镜观察原代成骨细胞的生长情况,并利用HE染色、碱性磷酸酶染色及钙化结节茜素红染色进行成骨细胞鉴定。将直径为15mm,厚lmm的医用纯钛钛片分组进行表面处理：①以240目、360目、600目砂纸逐级打磨得到机械打磨组(grooved,G组)；②采用356-411μmTiO2喷砂处理,得到喷砂组(sandblasted,SB组)；③以63.6%的H2S04和10.6%的HCL混合液在60。C时酸蚀SB组的钛片10分钟得到酸蚀组(sandblasted and acid-etched,SLA组)；④由G组、SB组、SLA组,经5moL/L氢氧化钠60。C水浴14小时,600。C处理1小时,得到光滑碱热组(grooved alkali-heated,AH1组)、喷砂碱热组(sandblasted alkali-heated,AH2组)、喷砂酸蚀碱热组(acid-etched and alkali-heated,AH3组)。用扫描电镜、轮廓测量仪检测6组钛片表面理化性质。将成骨细胞接种于六组钛片上,在1、3、5天收集所培养的成骨细胞。利用RT-PCR定量免疫荧光法检测成骨细胞整合素亚基α2、整合素亚基a5、整合素亚基β1mRNA的表达水平。数据采用单因素方差分析及配对t检验(a=0.05)。 [结果]本实验采用的体外组织块法操作简便,骨量消耗小,细胞产量大、纯度高,能满足实验需求。培养的成骨细胞经鉴定符合成骨细胞的生物学特性,并具有良好的活性。体外培养的大鼠成骨细胞膜上均能持续表达整合素亚基α2、整合素亚基α5、整合素亚基β1。整合素亚基p1表达水平最高,其次为整合素亚基α2,整合素亚基α5相对最低。本实验中,SB组钛片表面处理后成骨细胞整合素表达水平最高,AH1组较G组、AH3组较SLA组的表达水平要高,AH1组、AH1组和AH3组表达水平接近。在本实验观察时段中,随着时间的延长,整合素亚基α2、整合素亚基α5、整合素亚基β1mRNA的表达水平有一定程度的增长。 [结论]1.实验采用组织块法培养成骨细胞,操作简便,骨量消耗小,细胞产量大、纯度高,能满足实验需求。获得的成骨细胞经鉴定符合成骨细胞的生物学特性,并具有良好的活性。2.实验引物特异性佳,扩增效率高,成功检测到3种目标整合素亚基,扩增出相应目的片段,合乎实验要求。3.SB组钛片成骨细胞整合素亚基a2、整合素亚基a5、整合素亚基p1表达量最大,SB组有利于成骨细胞粘附。4.碱热处理表面对成骨细胞整合素亚基a2、整合素亚基α5、整合素亚基β1表达有一定促进作用,有利于成骨细胞粘附。
[Abstract]:[objective] to study the effect of alkaline heat treated titanium sheet on integrin 伪 2 尾 1 and integrin 伪 5 尾 1 in osteoblasts. Materials and methods the primary culture of osteoblasts was carried out by the method of SD fetal rat skullcap tissue mass culture. The growth of primary osteoblasts was observed by inverted microscope. Osteoblasts were identified by HE staining, alkaline phosphatase staining and alizarin red staining of calcified nodules. The titanium sheet with a diameter of 15 mm and thickness of lmm was divided into two groups. The surface treatment was carried out with 240 mesh / 360 mesh / 600 mesh sandpaper, and the mechanical grinding group was treated with 356-411 渭 mTiO2 sandblasting, and the mechanical grinding group was treated with 356-411 渭 mTiO2. The sand-blast group and blasted SB group were treated with 63.6% H2S04 and 10.6% HCL mixture solution for 10 minutes at 60.C, and then treated with 5moL/L sodium hydroxide 60.C for 14 hours and 600.C, respectively, and then treated with 5moL/L sodium hydroxide 60.C for 14 hours or 600.C, then treated with 5moL/L sodium hydroxide for 14 hours or 600.C. The smooth alkali-hested AH1 group, sandblasted alkali-hested AH2 group and acid-etched and alkali-heaved AH3 group were obtained in the smooth alkali fever group, sandblasted alkali-hested AH _ 2 group and sandblasted alkali-heated AH _ 2 group. The surface physicochemical properties of 6 groups of titanium sheets were examined by scanning electron microscope (SEM) and profilometer. Osteoblasts were inoculated on six groups of titanium slices and cultured osteoblasts were collected for 5 days. The expression levels of integrin subunit 伪 2, integrin subunit a5 and integrin subunit 尾 1mRNA in osteoblasts were detected by RT-PCR quantitative immunofluorescence assay. The data were analyzed by single factor ANOVA and paired t test. [results] the method of tissue mass in vitro was simple, the bone mass consumption was small, the cell yield was large, the purity was high, and it could meet the needs of the experiment. The cultured osteoblasts were confirmed to be in accordance with the biological characteristics of osteoblasts and had good activity. In vitro cultured rat osteoblasts, integrin subunit 伪 2, integrin subunit 伪 5 and integrin subunit 尾 1 were continuously expressed. The expression level of integrin subunit p1 was the highest, followed by integrin subunit 伪 2, and integrin subunit 伪 5 was the lowest. In this experiment, the expression level of integrin in osteoblasts of group B was higher than that of group G, AH3 and SLA. The expression level of integrin in osteoblasts of group B was higher than that of group SLA. The expression level of integrin in group AH1 was higher than that in group SLA. The expression level of integrin in group AH1 was similar to that in group AH3. During the observation period, the expression of integrin subunit 伪 2, integrin subunit 伪 5 and integrin subunit 尾 1mRNA increased to some extent. [conclusion] 1. Osteoblasts were cultured by tissue mass method, which was easy to operate, low bone consumption, high cell yield and high purity. The obtained osteoblasts were confirmed to be in accordance with the biological characteristics of osteoblasts and had good activity. 2. 2. Three target integrin subunits were successfully detected and the corresponding target fragments were amplified. In accordance with the experimental requirements, the osteoblasts in group SB had the highest expression of integrin subunit a2, integrin subunit a5 and integrin subunit p1, and the SB group was in favor of osteoblast adhesion of .4. The surface of alkali heat treatment can promote the expression of integrin subunit a2, integrin subunit 伪 5 and integrin subunit 尾 1 in osteoblasts, which is beneficial to osteoblast adhesion.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R783.6

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