發(fā)布時間：2018-05-31 06:51

  本文選題：BMSCs + Wnt信號通路　； 參考：《浙江大學(xué)》2014年博士論文

【摘要】：第一部分：純鈦表面Wnt信號通路對BMSCs成骨分化的調(diào)控效應(yīng)研究 實驗一采用了密度梯度離心和貼壁培養(yǎng)相結(jié)合的方法,從SD大鼠股骨內(nèi)獲得高純度的BMSCs.純化后的第三代BMSCs接種至純鈦表面上進行培養(yǎng),加入成骨誘導(dǎo)液定向誘導(dǎo),7天、14天時分別進行成骨分化的檢測,驗證BMSCs具有向成骨分化的能力。 流武細胞儀檢測結(jié)果顯示我們提取的BMSCs純度較高。茜素紅染色以及ALP、OC的檢測結(jié)果均表明BMSCs已經(jīng)分化成為成骨細胞,驗證了BMSCs具有向成骨細胞分化的能力。 實驗二將BMSCs以1×104/cm2密度接種至純鈦表面,設(shè)立誘導(dǎo)組和空白對照組。接種24h后換含體積分數(shù)為10%胎牛血清的DMEM(L)培養(yǎng)基,誘導(dǎo)組加入成骨誘導(dǎo)液,對照組常規(guī)培養(yǎng)。成骨誘導(dǎo)組條件為：DMEM(L)培養(yǎng)液+體積分數(shù)為10%胎牛血清+10mmol/Lβ-甘油磷酸鈉+1*10-7mo1/L地塞米松+50mg/L抗壞血酸C。7天后用Trizol萃取DNA,進行全基因組PCR芯片檢測。通過分析基因芯片獲得BMSCs在成骨誘導(dǎo)條件下向成骨細胞分化的過程中Wnt信號通路調(diào)控基因的變化情況,找出成骨調(diào)控的關(guān)鍵基因。 結(jié)果顯示：Wnt信號通路在調(diào)控BMSCs成骨分化的過程起著重要的作用,但是它并非是單獨作用的,還有MAPK信號通路、TGF-β(BMP)信號通路也參與其中,Wnt信號通路的兩條支路也互相關(guān)聯(lián)。通過基因芯片分析,我們發(fā)現(xiàn)LRP5在BMSCs向成骨分化的過程中上調(diào)倍數(shù)為11.15倍；Wnt5b的下調(diào)倍數(shù)為19.34倍,p-catenin的上調(diào)倍數(shù)沒有顯著性差異,但是p-catenin是Wnt信號通路在胞內(nèi)和胞核之間聯(lián)通的重要因子,也是Wnt信號通路和其他信號通路關(guān)聯(lián)的連接點,因此我們選擇了LRP5、Wnt5b、β-catenin作為目的基因進行后續(xù)的研究。 第二部分：純鈦表面經(jīng)典的Wnt信號通路對BMSCs成骨分化的調(diào)控機制研究 實驗三成功構(gòu)建了含目的基因LRP5的四條慢病毒載體(2723、2724、2725、2726),設(shè)立陰性對照組(NC對照組)和空白對照組BLANK。通過RT-PCR和WB篩選出有效的基因片段(2725、2726)進行后續(xù)檢測。LV3-LRP5(干擾型LRP5慢病毒)侵染BMSCs后,檢測BMSCs向成骨分化過程中LRP5、β-catenin、BMP2基因、蛋白表達量的改變,同時通過ALP、OC和茜素紅染色等成骨指標(biāo)檢測來驗證LV3-LRP5侵染BMSCs后對其成骨分化能力的改變。 根據(jù)每組的差別,我們選擇了侵染72h作為最佳侵染時間。LV3-LPR5侵染BMSCs后可以檢測到LRP5、β-catenin、BMP2基因表達量和蛋白表達量顯著性下降,同時測得7天、14天ALP、OC表達量的顯著性下降,說明作為膜上受體的LRP5在Wnt經(jīng)典信號通路的傳導(dǎo)中起著重要的作用,它通過降低胞內(nèi)β-catenin的集聚,阻斷Wnt經(jīng)典信號通路的傳導(dǎo),阻斷了BMSCs向成骨分化的過程。同時也說明這個調(diào)控過程有TGF-p信號通路的參與。 實驗四成功構(gòu)建了含目的基因β-catenin的四條慢病毒載體(3081、3082、3083、3084),設(shè)立NC對照組和空白對照組BLANK。通過RT-PCR和WB篩選出有效的基因片段(3083)。LV3-β-catenin慢病毒侵染BMSCs后,檢測BMSCs向成骨分化過程中的β-catenin、LRP5、BMP2基因、蛋白表達量的改變,同時通過ALP、OC和茜素紅染色等成骨指標(biāo)檢測來驗證LV3-β-catenin侵染BMSCs后對其成骨分化能力的改變。 根據(jù)每組的差別,我們選擇了侵染72h作為最佳侵染時間。LV3-β-catenin侵染BMSCs后可以檢測到p-catenin、LRP5、BMP2基因表達量和蛋白表達量的顯著性下降,同時測得7天、14天ALP、OC表達量的下降,說明p-catenin被干擾后阻斷了BMSCs向成骨分化的過程,這個調(diào)控過程有BMP信號通路的參與。 第三部分：純鈦表面非經(jīng)典的Wnt信號通路對BMSCs向成骨分化的調(diào)控機制研究 實驗五成功構(gòu)建了含目的基因Wnt5b的四條慢病毒干擾載體(484、918、975、1579)和過表達慢病毒載體(v5053),設(shè)立NC對照組和空白對照組BLANK.通過RT-PCR和WB篩選出有效的基因片段(1579, v5053). LV3-Wnt5b和LV5-Wnt5b(過表達型慢病毒)分別侵染BMSCs后,檢測BMSCs向成骨分化過程中Wnt5b、LRP5、BMP2、β-catenin基因、蛋白表達量的改變,同時通過ALP、OC和茜素紅染色等成骨指標(biāo)檢測來驗證BMSCs向成骨分化的能力。 我們選擇了侵染72h作為最佳侵染時間。當(dāng)BMSCs有效轉(zhuǎn)染LV3-Wnt5b后,Wnt5b被干擾,從RT-PCR結(jié)果和WB結(jié)果來看,Wnt5b、LRP5、 BMP2, β-catenin的表達量均下調(diào),結(jié)合ALP、OC、茜素紅的結(jié)果,發(fā)現(xiàn)BMSCs成骨分化的進程被阻斷；當(dāng)BMSCs轉(zhuǎn)染LV5-Wnt5b,即Wnt5b過表達,可發(fā)現(xiàn)Wnt5b、LRP5、BMP2、β-catenin的表達量均上調(diào),進一步通過ALP、OC、茜素紅的結(jié)果驗證了LV5-Wnt5b在促進BMSCs的成骨分化過程中發(fā)揮作用,可以認為Wnt5b參與的Wnt非經(jīng)典信號通路也參與調(diào)節(jié)BMSCs向成骨分化的過程,它和Wnt經(jīng)典通路共同作用,對Wnt經(jīng)典通路起一個正性調(diào)節(jié)的作用。總結(jié)： 1,密度梯度離心法和傳代貼壁篩選法結(jié)合提純的BMSCs純度高,可以滿足做為體外成骨細胞誘導(dǎo)模型的實驗要求。 2,純鈦表面上Wnt經(jīng)典信號通路對BMSCs成骨分化具有促進作用 3,LRP5的干擾,阻斷了Wnt/β-catenin信號的轉(zhuǎn)導(dǎo),阻斷了BMSCs向成骨細胞分化的進程。 4,在BMSCs成骨分化的過程中,BMP2與β-catenin在時間上和空間上產(chǎn)生了一定的協(xié)同作用。 5, β-catenin的干擾,阻礙了BMSCs向成骨細胞分化的過程。 6,Wnt非經(jīng)典通路中的Wnt5b參與調(diào)節(jié)BMSCs成骨分化的過程。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R783

【參考文獻】

相關(guān)期刊論文 前1條

1 劉峰,韓驊;鼠骨髓基質(zhì)干細胞的特性及向成骨細胞的分化[J];醫(yī)學(xué)分子生物學(xué)雜志;2004年02期

，

本文編號：1958712