纯钛表面WNT信号通路调控BMSCs成骨分化的机制研究

发布时间：2018-05-31 06:51

  本文选题：BMSCs + Wnt信号通路　； 参考：《浙江大学》2014年博士论文

【摘要】：第一部分：纯钛表面Wnt信号通路对BMSCs成骨分化的调控效应研究 实验一采用了密度梯度离心和贴壁培养相结合的方法,从SD大鼠股骨内获得高纯度的BMSCs.纯化后的第三代BMSCs接种至纯钛表面上进行培养,加入成骨诱导液定向诱导,7天、14天时分别进行成骨分化的检测,验证BMSCs具有向成骨分化的能力。 流武细胞仪检测结果显示我们提取的BMSCs纯度较高。茜素红染色以及ALP、OC的检测结果均表明BMSCs已经分化成为成骨细胞,验证了BMSCs具有向成骨细胞分化的能力。 实验二将BMSCs以1×104/cm2密度接种至纯钛表面,设立诱导组和空白对照组。接种24h后换含体积分数为10%胎牛血清的DMEM(L)培养基,诱导组加入成骨诱导液,对照组常规培养。成骨诱导组条件为：DMEM(L)培养液+体积分数为10%胎牛血清+10mmol/Lβ-甘油磷酸钠+1*10-7mo1/L地塞米松+50mg/L抗坏血酸C。7天后用Trizol萃取DNA,进行全基因组PCR芯片检测。通过分析基因芯片获得BMSCs在成骨诱导条件下向成骨细胞分化的过程中Wnt信号通路调控基因的变化情况,找出成骨调控的关键基因。 结果显示：Wnt信号通路在调控BMSCs成骨分化的过程起着重要的作用,但是它并非是单独作用的,还有MAPK信号通路、TGF-β(BMP)信号通路也参与其中,Wnt信号通路的两条支路也互相关联。通过基因芯片分析,我们发现LRP5在BMSCs向成骨分化的过程中上调倍数为11.15倍；Wnt5b的下调倍数为19.34倍,p-catenin的上调倍数没有显著性差异,但是p-catenin是Wnt信号通路在胞内和胞核之间联通的重要因子,也是Wnt信号通路和其他信号通路关联的连接点,因此我们选择了LRP5、Wnt5b、β-catenin作为目的基因进行后续的研究。 第二部分：纯钛表面经典的Wnt信号通路对BMSCs成骨分化的调控机制研究 实验三成功构建了含目的基因LRP5的四条慢病毒载体(2723、2724、2725、2726),设立阴性对照组(NC对照组)和空白对照组BLANK。通过RT-PCR和WB筛选出有效的基因片段(2725、2726)进行后续检测。LV3-LRP5(干扰型LRP5慢病毒)侵染BMSCs后,检测BMSCs向成骨分化过程中LRP5、β-catenin、BMP2基因、蛋白表达量的改变,同时通过ALP、OC和茜素红染色等成骨指标检测来验证LV3-LRP5侵染BMSCs后对其成骨分化能力的改变。 根据每组的差别,我们选择了侵染72h作为最佳侵染时间。LV3-LPR5侵染BMSCs后可以检测到LRP5、β-catenin、BMP2基因表达量和蛋白表达量显著性下降,同时测得7天、14天ALP、OC表达量的显著性下降,说明作为膜上受体的LRP5在Wnt经典信号通路的传导中起着重要的作用,它通过降低胞内β-catenin的集聚,阻断Wnt经典信号通路的传导,阻断了BMSCs向成骨分化的过程。同时也说明这个调控过程有TGF-p信号通路的参与。 实验四成功构建了含目的基因β-catenin的四条慢病毒载体(3081、3082、3083、3084),设立NC对照组和空白对照组BLANK。通过RT-PCR和WB筛选出有效的基因片段(3083)。LV3-β-catenin慢病毒侵染BMSCs后,检测BMSCs向成骨分化过程中的β-catenin、LRP5、BMP2基因、蛋白表达量的改变,同时通过ALP、OC和茜素红染色等成骨指标检测来验证LV3-β-catenin侵染BMSCs后对其成骨分化能力的改变。 根据每组的差别,我们选择了侵染72h作为最佳侵染时间。LV3-β-catenin侵染BMSCs后可以检测到p-catenin、LRP5、BMP2基因表达量和蛋白表达量的显著性下降,同时测得7天、14天ALP、OC表达量的下降,说明p-catenin被干扰后阻断了BMSCs向成骨分化的过程,这个调控过程有BMP信号通路的参与。 第三部分：纯钛表面非经典的Wnt信号通路对BMSCs向成骨分化的调控机制研究 实验五成功构建了含目的基因Wnt5b的四条慢病毒干扰载体(484、918、975、1579)和过表达慢病毒载体(v5053),设立NC对照组和空白对照组BLANK.通过RT-PCR和WB筛选出有效的基因片段(1579, v5053). LV3-Wnt5b和LV5-Wnt5b(过表达型慢病毒)分别侵染BMSCs后,检测BMSCs向成骨分化过程中Wnt5b、LRP5、BMP2、β-catenin基因、蛋白表达量的改变,同时通过ALP、OC和茜素红染色等成骨指标检测来验证BMSCs向成骨分化的能力。 我们选择了侵染72h作为最佳侵染时间。当BMSCs有效转染LV3-Wnt5b后,Wnt5b被干扰,从RT-PCR结果和WB结果来看,Wnt5b、LRP5、 BMP2, β-catenin的表达量均下调,结合ALP、OC、茜素红的结果,发现BMSCs成骨分化的进程被阻断；当BMSCs转染LV5-Wnt5b,即Wnt5b过表达,可发现Wnt5b、LRP5、BMP2、β-catenin的表达量均上调,进一步通过ALP、OC、茜素红的结果验证了LV5-Wnt5b在促进BMSCs的成骨分化过程中发挥作用,可以认为Wnt5b参与的Wnt非经典信号通路也参与调节BMSCs向成骨分化的过程,它和Wnt经典通路共同作用,对Wnt经典通路起一个正性调节的作用。总结： 1,密度梯度离心法和传代贴壁筛选法结合提纯的BMSCs纯度高,可以满足做为体外成骨细胞诱导模型的实验要求。 2,纯钛表面上Wnt经典信号通路对BMSCs成骨分化具有促进作用 3,LRP5的干扰,阻断了Wnt/β-catenin信号的转导,阻断了BMSCs向成骨细胞分化的进程。 4,在BMSCs成骨分化的过程中,BMP2与β-catenin在时间上和空间上产生了一定的协同作用。 5, β-catenin的干扰,阻碍了BMSCs向成骨细胞分化的过程。 6,Wnt非经典通路中的Wnt5b参与调节BMSCs成骨分化的过程。
[Abstract]:Part one: regulation of Wnt signaling pathway on osteogenic differentiation of BMSCs on pure titanium surface
In Experiment 1, a combination of density gradient centrifugation and adherent culture was used to inoculate third generation BMSCs of high purity BMSCs. from the femur of SD rats to be inoculated on the surface of pure titanium to be cultured on the surface of pure titanium. The osteogenic induction was added to the osteogenic induction solution, and the osteogenesis was detected at 7 days and 14 days respectively to verify the ability of BMSCs to differentiate into osteogenic differentiation.
The test results of the FCM showed that the purity of the extracted BMSCs was higher. Alizarin red staining and the results of ALP and OC showed that BMSCs had been differentiated into osteoblasts, which proved that BMSCs had the ability to differentiate into osteoblasts.
In experiment two, BMSCs was inoculated to the surface of pure titanium with 1 x 104/cm2 density, and the induction group and blank control group were set up. After 24h inoculation, the DMEM (L) medium containing the volume fraction of 10% fetal bovine serum was changed. The induction group was added into the osteogenic induction solution and the control group was routinely cultured. The condition of the osteogenic induction group was DMEM (L) culture and the volume fraction of 10% fetal bovine serum +10mmol/L beta. Sodium glycerphosphate +1*10-7mo1/L, dexamethasone +50mg/L ascorbic acid, C.7 days after C.7, was used to extract DNA for whole genome PCR chip detection. By analyzing gene chip, the changes of the regulation gene of Wnt signaling pathway during the osteoblast differentiation of BMSCs under osteogenic induction were obtained, and the key genes of osteogenesis regulation were found.
The results show that Wnt signaling pathway plays an important role in the regulation of BMSCs osteogenesis, but it is not alone, and the MAPK signaling pathway, and the TGF- beta (BMP) signaling pathway is also involved, and the two branches of the Wnt signaling pathway are related to each other. By gene chip analysis, we found that LRP5 has been differentiated from BMSCs to osteogenesis in BMSCs. The up-regulation multiplier in the process is 11.15 times, the downregulation multiplier of Wnt5b is 19.34 times, and there is no significant difference between the up regulation of P-catenin, but P-catenin is an important factor connecting the Wnt signaling pathway between the intracellular and the nucleus, and is also the connection point associated with the Wnt signaling pathway and other signaling pathways, so we chose LRP5, Wnt5b, and beta -catenin as a choice. A follow-up study of the target gene.
The second part: the regulatory mechanism of classic Wnt signaling pathway on osteogenic differentiation of BMSCs on pure titanium surface.
In experiment three, four lentivirus carriers (2723272427252726) containing the target gene LRP5 were successfully constructed. The negative control group (NC control group) and the blank control group were selected by RT-PCR and WB screening effective gene fragment (27252726) for subsequent detection of.LV3-LRP5 (interfering LRP5 lentivirus) infection of BMSCs, and detection of BMSCs to osteogenic differentiation. The changes in LRP5, beta -catenin, BMP2 gene and protein expression during the process of ALP, OC and alizarin red staining were used to verify the changes in the osteogenic differentiation of LV3-LRP5 after BMSCs infection.
According to the difference of each group, we selected the infection 72h as the best infection time,.LV3-LPR5 infection BMSCs, can detect the significant decrease in LRP5, beta -catenin, BMP2 gene expression and protein expression, at the same time, 7 days, 14 days ALP, OC expression significantly decreased, indicating that as the membrane receptor LRP5 in Wnt classic signaling pathway conduction It plays an important role in blocking the transmission of Wnt classical signaling pathway by reducing the concentration of intracellular beta -catenin, blocking the process of BMSCs differentiation into osteogenesis, and also indicating that the regulatory process is involved in the TGF-p signaling pathway.
In Experiment four, four lentivirus carriers (3081308230833084) containing the target gene beta -catenin were successfully constructed. The NC control group and the blank control group were set up by RT-PCR and WB screening effective gene fragments (3083).LV3- beta -catenin lentivirus infected BMSCs, and the beta -catenin, LRP5, BMP2 gene, protein in the process of BMSCs to osteogenesis were detected. The change of expression level was detected by ALP, OC and alizarin red staining. The osteogenic differentiation ability of LV3- beta -catenin after infection with BMSCs was verified.
According to the difference of each group, we selected the infection 72h as the best infection time.LV3- beta -catenin infection BMSCs to detect the significant decrease in P-catenin, LRP5, BMP2 gene expression and protein expression, at the same time, 7 days, 14 days ALP, the decrease of OC expression, indicating that P-catenin was interfered to block the process of BMSCs osteogenesis. This regulation process is involved in the BMP signaling pathway.
The third part: the regulation mechanism of non classical Wnt signaling pathway on osteogenic differentiation of BMSCs on pure titanium surface.
In experiment five, four lentivirus interference carriers (4849189751579) and overexpressed lentivirus carrier (v5053) were successfully constructed with target gene Wnt5b, and NC control group and blank control group were established by RT-PCR and WB screening effective gene fragments (1579, v5053). LV3-Wnt5b and LV5-Wnt5b (overexpressed lentivirus) infected BMSCs respectively. The changes in Wnt5b, LRP5, BMP2, beta -catenin gene and protein expression during the osteogenic differentiation of BMSCs were measured, and the bone differentiation ability of BMSCs to osteogenesis was tested by ALP, OC and alizarin red staining.
We chose the infection 72h as the best infection time. When BMSCs transfected LV3-Wnt5b, Wnt5b was disturbed. From the results of RT-PCR and WB, the expression of Wnt5b, LRP5, BMP2, and beta -catenin were all down, and the results of ALP, OC, and alizarin red were blocked. It was found that the expression of Wnt5b, LRP5, BMP2, and beta -catenin were all up-regulated. The results of ALP, OC and alizarin red showed that LV5-Wnt5b played a role in promoting the osteogenesis of BMSCs, and that the Wnt non classical signaling pathways involved in Wnt5b participated in the process of regulating BMSCs osteogenesis. NT classical pathway plays a positive regulatory role.
1, the purity of BMSCs purified by density gradient centrifugation and passage adherence screening method is high, which can be used as an experimental requirement for osteoblast induction models in vitro.
2, Wnt signaling pathway on pure titanium promotes BMSCs osteogenic differentiation.
3, the interference of LRP5 blocked the transduction of Wnt/ beta -catenin signal and blocked the differentiation process of BMSCs into osteoblasts.
4, in the process of osteogenic differentiation of BMSCs, BMP2 and beta -catenin play a synergistic role in time and space.
5, the interference of beta -catenin hindered the differentiation of BMSCs into osteoblasts.
6, Wnt5b in non classical pathway of Wnt participates in the process of BMSCs osteogenic differentiation.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R783

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