本文關(guān)鍵詞：lncRNA對(duì)SPA作用下人骨髓間充質(zhì)干細(xì)胞成骨分化能力的作用及機(jī)制研究　出處：《第三軍醫(yī)大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 人骨髓間充質(zhì)干細(xì)胞 骨髓炎 葡球菌A蛋白 lncRNA基因芯片 感染性骨不連 成骨分化 長(zhǎng)鏈非編碼RNA NONHSAT009968

【摘要】：研究背景和目的:骨髓炎是一種急性或慢性的骨組織感染,以化膿性炎癥、異常骨重建、難控性骨吸收為特征,常伴有骨缺損、骨不連等骨科疾病的出現(xiàn),導(dǎo)致肢體功能障礙、截肢,甚至危及生命,嚴(yán)重威脅患者身心健康,其治療一直是骨科醫(yī)師面臨的難題之一。骨髓炎并骨不連的發(fā)生發(fā)展機(jī)制并不清楚。金黃色葡萄球菌(Staphylococcus aureus,SA)是最常見的引發(fā)骨髓炎的微生物,葡球菌A蛋白(StaphylococcalproteinA,SPA)是SA的主要毒力成分,研究發(fā)現(xiàn)在骨髓炎發(fā)生發(fā)展過程中,SPA能直接刺激病灶區(qū)成骨細(xì)胞的凋亡,導(dǎo)致病灶區(qū)的骨破壞和骨丟失,說明SPA在骨髓炎的發(fā)生發(fā)展過程起著關(guān)鍵的作用。研究已證實(shí):在骨再生的修復(fù)過程中,骨折部位需要聚集足夠數(shù)量的骨髓間充質(zhì)干細(xì)胞(Bone Mesenchymal Stem Cells,BMSCs),這些干細(xì)胞向成骨細(xì)胞的分化能力直接影響到了骨組織的新骨生成和骨愈合能力。以往研究主要集中于SA的毒力因子引起成骨細(xì)胞的凋亡,而對(duì)SA的菌體成分的研究,如SPA對(duì)成骨細(xì)胞的祖細(xì)胞-BMSCs的分化能力影響尚未見報(bào)道。本課題組前期首次通過SPA對(duì)BMSCs成骨分能力的影響做了相關(guān)研究,發(fā)現(xiàn)SPA確實(shí)能顯著抑制BMSCs的成骨分化能力,提示毒力因子SPA可模擬體內(nèi)的炎癥環(huán)境,同時(shí)對(duì)SPA如何影響B(tài)MSCs成骨分化能力的機(jī)制則不是很清楚。因此,深入研究在SPA作用下hBMSCs成骨分化能力下降的原因,對(duì)進(jìn)一步理解骨髓炎情況下骨生成不足的原因具有重要意義。長(zhǎng)鏈非編碼RNA(long non-coding RNA,lncRNA)是一類在基因組轉(zhuǎn)錄中不參與編碼蛋白、長(zhǎng)度超過200個(gè)核苷酸的轉(zhuǎn)錄產(chǎn)物,涉及一系列的發(fā)育過程和疾病進(jìn)展。研究表明一些lncRNAs如AK141205、ANCR、H19和MEG3在調(diào)節(jié)干細(xì)胞的成骨分化過程中起到重要的作用,可促進(jìn)成骨細(xì)胞分化。以上研究均表明lncRNA對(duì)干細(xì)胞的成骨分化具有正向調(diào)節(jié)作用。而也有研究表明部分lncRNA對(duì)干細(xì)胞的成骨分化存在負(fù)向調(diào)節(jié)作用。研究發(fā)現(xiàn)過表達(dá)AK035085能夠抑制C3H10T1/2細(xì)胞的成骨分化。說明lncRNA對(duì)干細(xì)胞的成骨分化能力有著明顯的調(diào)控作用,但是在骨髓炎狀態(tài)下lncRNA對(duì)hBMSCs的成骨分化能力的作用則未見相關(guān)報(bào)道。因此,我們采用不同濃度的SPA處理hBMSCs,通過檢測(cè)炎癥因子的表達(dá)以及成骨活性變化情況,構(gòu)建模擬骨髓炎細(xì)胞模型;然后,通過基因芯片分析SPA處理hBMSCs成骨分化的過程中l(wèi)ncRNA差異的變化,根據(jù)生物信息學(xué)分析的結(jié)果,確定lncRNA的靶基因;進(jìn)一步分析lncRNA通過調(diào)控其靶基因在SPA處理的hBMSCs成骨誘導(dǎo)中的作用,明確lncRNA在骨髓炎狀態(tài)下調(diào)控hBMSCs成骨分化的機(jī)制。本研究是首次從lncRNA水平去闡述骨髓炎情況下hBMSCs成骨能力不足的原因,為骨髓炎并缺損的研究提供新的理論基礎(chǔ)及研究方向,為下一步的研究提供堅(jiān)實(shí)的基礎(chǔ)。研究方法:1..SPA作用下體外骨髓炎模型的構(gòu)建為了模擬骨髓炎細(xì)胞模型,不同濃度的SPA(0μg/ml、0.1 μg/ml,0.5 μg/ml,1 μg/ml,10μg/mL,100μg/mL)處理hBMSCs細(xì)胞。處理72h后,24孔培養(yǎng)板細(xì)胞上清被收集進(jìn)行ELISA檢測(cè)IL-la、IL-6和TNF-a。添加不同濃度的SPA的成骨分化培養(yǎng)基培養(yǎng)3周,然后收集細(xì)胞進(jìn)行茜素紅染色檢測(cè)鈣結(jié)節(jié)沉積和堿性磷酸酶檢測(cè);2.分析及驗(yàn)證SPA處理下hBMSCs成骨分化過程中IncRNA的變化hBMSCs在含有(SPA組)或者不含有(對(duì)照組)1μg/ml SPA的成骨分化培養(yǎng)基中培養(yǎng)2周后,收集細(xì)胞進(jìn)行l(wèi)ncRNA基因芯片分析。然后分析即表達(dá)差異顯著又和成骨分化密切相關(guān)的lncRNA,將以上差異基因進(jìn)行qRT-PCR驗(yàn)證,篩選出目的基因。3.差異lncRNA對(duì)SPA骨毮炎模型下BMMSC成骨分化功能的影響合成 3 條 NONHSAT009968 干擾序列(siRNA-1,siRNA-2 和 siRNA-3)。培養(yǎng)hBMSCs細(xì)胞,根據(jù)操作說明書,使用Lipofectamine 2000試劑將大約200nmsiRNA轉(zhuǎn)染到hBMSCs。干擾效率采用qRT-PCR檢測(cè)。采用siRNA-2序列進(jìn)行慢病毒包裝。根據(jù)標(biāo)準(zhǔn)操作進(jìn)行慢病毒生產(chǎn)、濃度和滴定測(cè)定、感染條件確認(rèn)以及穩(wěn)定細(xì)胞株篩選。慢病毒表達(dá)NONHSAT009968-shRNA或空的慢病毒,感染后3天,收集骨髓間充質(zhì)干細(xì)胞檢測(cè)NONHSAT009968確認(rèn)是否成功沉默。慢病毒表達(dá)NONHSAT009968-shRNA或空的慢病毒感染的hBMSCs細(xì)胞培養(yǎng)14天和21天。采用茜素紅染色檢測(cè)鈣結(jié)節(jié)沉積。收集細(xì)胞進(jìn)行western blot檢測(cè)Runx2、OCN、OPN和COL1A1的表達(dá),ELISA檢測(cè)IL-Ia、IL-6、TNF-a和堿性磷酸酶檢測(cè)。分析干擾NONHSAT009968對(duì)SPA處理下的hBMSCs的成骨分化能力的影響。4差異IncRNA對(duì)SPA骨髓炎模型下BMMSC成骨分化功能影響的作用機(jī)制慢病毒表達(dá)NONHSAT009968-shRNA或空的慢病毒感染的hBMSCs細(xì)胞培養(yǎng)14天和21天。收集細(xì)胞采用qRT-PCR和western blot檢測(cè)Wnt3a、β-catenin和GSK-3[β的表達(dá),分析干擾NONHSAT009968對(duì)Wnt3a、β-catenin和GSK-3β的表達(dá)的影響。研究結(jié)果:1.SPA骨髓炎模型構(gòu)建成功SPA可以顯著增加hBMSCs細(xì)胞成骨分化過程中炎癥因子IL-1a、IL-6和TNF-a的分泌(P0.05),而且炎癥因子的分泌對(duì)SPA呈濃度依賴性(0、0.1、0.5、1、10和100 μg/mL)。另外,茜素紅染色顯示不同濃度的SPA能夠明顯抑制鈣結(jié)節(jié)沉積,而且抑制程度和SPA的濃度有密切聯(lián)系。然后ALP檢測(cè)結(jié)果表明SPA處理能夠抑制ALP的活性(P0.05),并且抑制能力呈濃度依賴性。以上結(jié)果表明在1 μg/ml SPA能夠明顯增加炎癥因子IL-1a、IL-6和TNF-a的分泌,抑制鈣結(jié)節(jié)沉積和ALP的表達(dá)。說明SPA可以促進(jìn)炎癥因子的表達(dá)及抑制hBMSCs成骨能力的下降,這一結(jié)果與體內(nèi)骨髓炎狀態(tài)表現(xiàn)一致。因此,在體外采用1μg/ml SPA處理hBMSCs能夠模擬體內(nèi)骨髓炎狀態(tài),體外可構(gòu)建SPA骨髓炎模型。2.IncRNA基因芯片結(jié)果分析(1)SPA處理的骨髓間充質(zhì)干細(xì)胞和對(duì)照組相比,存在2033個(gè)異常表達(dá)的lncRNAs。其中 641 個(gè) lncRNAs 下調(diào),1392 個(gè) lncRNAs 上調(diào)(倍數(shù)變化2.0,P0.05)。從mRNA表達(dá)分析數(shù)據(jù)中發(fā)現(xiàn)SPA處理的骨髓間充質(zhì)干細(xì)胞和對(duì)照組相比,存在449個(gè)異常表達(dá)的mRNA,其中318個(gè)mRNA顯著下調(diào),131個(gè)mRNA顯著上調(diào)(倍數(shù)變化2.0,P0.05)。(2)基于KEGG途徑分析,表達(dá)上調(diào)的mRNA包含20個(gè)不同的信號(hào)通路,最豐富的通路是唾液分泌,谷氨酸能突觸,嗅覺傳導(dǎo)和胰島素分泌�；贙EGG途徑分析,表達(dá)下調(diào)的mRNA包含20個(gè)不同的信號(hào)通路,最豐富的通路是造血,視黃醇的新陳代謝,藥物代謝,細(xì)胞粘附分子和細(xì)胞色素代謝外源性物質(zhì)。為了理解差異表達(dá)基因的功能,所有的差異表達(dá)基因映射到GO數(shù)據(jù)庫(kù)中并比較其背景�；趯�(duì)生物過程進(jìn)行GO分析,上調(diào)的轉(zhuǎn)錄本主要富集在纖溶酶原激活和水轉(zhuǎn)運(yùn),而下調(diào)的轉(zhuǎn)錄本主要富集在對(duì)糖皮質(zhì)激素刺激的細(xì)胞反應(yīng)和細(xì)胞糖脂化。基于對(duì)分子功能進(jìn)行GO分析,上調(diào)的轉(zhuǎn)錄本主要富集在神經(jīng)遞質(zhì)轉(zhuǎn)運(yùn)體活動(dòng)和鈣調(diào)素結(jié)合,而下調(diào)的轉(zhuǎn)錄本主要富集在對(duì)配體-依賴的核受體轉(zhuǎn)錄共激活和葡萄糖醛酸基轉(zhuǎn)移酶活性。(3)通過cis-lOOk分析,五個(gè)潛在的和成骨分化相關(guān)且表達(dá)差異顯著的lncRNA被鑒定出來,包括 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627和NONHSAT009968。lncRNA的潛在靶向調(diào)控目標(biāo)分別包括骨形態(tài)形成蛋白1、SMAD 1、SMAD 1、膠原蛋白I型αl和Wnt3a,這些蛋白和成骨細(xì)胞分化密切相關(guān)。然后將差異的1ncRNA采用qRT-PCR驗(yàn)證,qRT-PCR結(jié)果表明SPA處理中和對(duì)照組中 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627和NONHSAT009968的表達(dá)顯著上升(P0.05),和基因芯片的結(jié)果一致。其中NONHSAT009968的表達(dá)結(jié)果提高的最為明顯,后續(xù)實(shí)驗(yàn)采用NONHSAT009968以及其靶基因Wnt3a進(jìn)行研究。3.沉默]NONHSAT009968能夠逆轉(zhuǎn)SPA對(duì)hBMSCs細(xì)胞成骨分化的影響在siRNA的檢測(cè)中siRNA-2和siRNA-3轉(zhuǎn)染后能夠顯著降低NONHSAT009968的表達(dá)(P0.05),其中siRNA-2轉(zhuǎn)染后NONHSAT009968的表達(dá)量最低,干擾效率最好,可以用于后續(xù)NONHSAT009968的干擾序列。當(dāng)感染NONHSAT009968-shRNA后采用 qRT-PCR 檢測(cè) NONHSAT009968 的表達(dá),結(jié)果表明 NONHSAT009968-shRNA干擾能夠顯著降低 NONHSAT009968 的表達(dá)(P0.05)。NONHSAT009968-shRNA 和NC在1 μg/ml SPA中培養(yǎng)14天和21天。茜素紅染色結(jié)果表明:無論在培養(yǎng)后14天還是21天,NONHSAT009968沉默組和NC組相比,都能夠增加鈣結(jié)節(jié)沉積(P0.05)。堿性磷酸酶活性檢測(cè)結(jié)果表明無論在培養(yǎng)后14天還是21天,NONHSAT009968沉默和NC組相比,都能夠增加堿性磷酸酶活性(P0.05)。另外,沉默NONHSAT009968能夠增加成骨分化相關(guān)的Runx2、OCN、OPN和COL1A1蛋白的表達(dá)(P0.05)。這些結(jié)果表明沉默NONHSAT009968能逆轉(zhuǎn)SPA抑制的hBMSCs成骨分化。4.沉默 NONHSAT009968 促進(jìn) Wnt3a 以及 β-catenin 和 GSK-3β 的表達(dá)SPA處理能夠顯著抑制Wnt3a、β-catenin和GSK-3β的表達(dá)(P0.05),且該抑制結(jié)果還呈時(shí)間依賴性。該結(jié)果還表明干擾NONHSAT009968的表達(dá)能夠增強(qiáng)Wnt3a、β-catenin和GSK-3β的表達(dá)(P0.05)。該結(jié)果表明干擾NONHSAT009968促進(jìn)成骨分化的機(jī)制可能和Wnt3a以及β-catenin和GSK-3β的表達(dá)相關(guān)。研究結(jié)論:(1)本研究1 μg/ml的SPA處理能夠促進(jìn)IL-la、IL-6和TNF-a的表達(dá),降低堿性磷酸酶的表達(dá)和鈣結(jié)節(jié)沉積,抑制Wnt3a、β-catenin和GSK-3β的表達(dá),抑制hBMSCs的成骨分化,成功模擬骨髓炎狀態(tài)。(2)在SPA處理hBMSCs的成骨分化的過程中,通過基因芯片分析發(fā)現(xiàn)存在5個(gè)和成骨分化相關(guān)的 lncRNA,有 NONHSAT125464、ENST00000504555、NONHSAT098635、NONHSAT054627 和 NONHSAT009968。其中 lncRNA NONHSAT009968的表達(dá)上升最為明顯,而且lncRNA NONHSAT009968潛在的cis-regulated mRNA 目標(biāo)為 Wnt3a。(3)進(jìn)一步研究發(fā)現(xiàn)通過干擾lncRNA NONHSAT009968的表達(dá)能夠顯著提高Wnt3a、β-catenin和GSK-3β的表達(dá),促進(jìn)鈣結(jié)節(jié)沉積,提高ALP的表達(dá),促進(jìn)成骨相關(guān)蛋白R(shí)unx2、OCN、OPN和COL1A1的表達(dá),改善SPA抑制的hBMSCs的成骨分化。(4)本研究首次從lncRNA角度闡明骨髓炎狀態(tài)下成骨能力下降的原因及其作用機(jī)制,為骨髓炎狀態(tài)下成骨能力下降的原因提出了新的解釋及研究方向。
[Abstract]:Background and objective: osteomyelitis is a kind of acute or chronic bone infection with purulent inflammation, abnormal bone remodeling, refractory bone absorption, often accompanied by bone defect and nonunion of diseases in Department of orthopedics, leading to limb dysfunction, amputation, and even endanger the life, a serious threat to the physical and mental health of patients and its treatment has been one of the problems in Medical Department of orthopedics. The pathogenesis of osteomyelitis with bone nonunion is not clear. Staphylococcus aureus (Staphylococcus aureus SA) is the most common cause of microorganisms, staphylococcus protein A (StaphylococcalproteinA, SPA) is a main toxic component of SA, the study found that in the process of the occurrence and development of osteomyelitis, SPA lesions can directly stimulate the apoptosis of osteoblasts, resulting in bone destruction and bone lesions. Lost, indicating that SPA plays a key role in the occurrence and development of osteomyelitis. Research has confirmed that in the repairing process of bone regeneration in the fracture site need to gather a sufficient number of bone marrow mesenchymal stem cells (Bone Mesenchymal Stem Cells, BMSCs), these stem cells differentiation into osteogenic cells directly affects the formation of new bone and bone tissue healing ability. Previous studies have focused on the virulence factors of SA, which induce the apoptosis of osteoblasts. However, the effect of SPA on the differentiation of osteoblasts from progenitor cells, such as -BMSCs, has not been reported in SA. The research group for the first time through the early effects of SPA on osteogenic ability of BMSCs to do related research, found that SPA can significantly inhibit BMSCs osteogenic differentiation, suggesting that virulence factor SPA can mimic the in vivo inflammatory environment, at the same time the effect of SPA on osteogenic differentiation of the BMSCs mechanism is not very clear. Therefore, in-depth study of the causes of the decline of hBMSCs osteogenic differentiation under the action of SPA is of great significance for further understanding the cause of osteogenesis. Long chain non coding RNA (long non-coding RNA, lncRNA) is a class of transcription products that do not participate in encoding protein and over 200 nucleotides in genome transcription, and involves a series of development processes and disease progression. Studies have shown that some lncRNAs, such as AK141205, ANCR, H19 and MEG3, play an important role in regulating the osteogenic differentiation of stem cells, and promote osteoblast differentiation. All of these studies suggest that lncRNA has a positive regulatory effect on the osteogenic differentiation of stem cells. However, some studies have shown that partial lncRNA has a negative regulatory effect on the osteogenic differentiation of stem cells. It was found that the expression of AK035085 could inhibit the osteogenic differentiation of C3H10T1/2 cells. It indicates that lncRNA plays a significant role in regulating the osteogenic differentiation ability of stem cells. However, in the condition of osteomyelitis, the effect of lncRNA on the osteogenic differentiation of hBMSCs has not been reported. Therefore, we use the SPA treatment of different concentrations of hBMSCs, by detecting the expression of inflammatory factors and changes of bone activity, construction simulation of osteomyelitis cell model; then, changes of lncRNA SPA hBMSCs during osteogenic differentiation by gene chip analysis of the difference, according to the results of a bioinformatics analysis, to identify the target gene of lncRNA lncRNA; further analysis through the regulation of its target gene into bone in the induction of SPA treatment of hBMSCs, lncRNA in the clear state of osteomyelitis regulation of hBMSCs differentiation mechanism. This study is the first time to expound the causes of insufficient hBMSCs osteogenesis ability from lncRNA level, and provide a new theoretical basis and research direction for the study of osteomyelitis and defect, and provide a solid foundation for further research. Research method: 1..SPA was used to construct the model of extracorporeal osteomyelitis. In order to simulate the osteomyelitis cell model, hBMSCs cells were treated with different concentrations of SPA (0 g/ml, 0.1 g/ml, 0.5 g/ml, 1 g/ml, 10 g/mL, 100 g/mL). After the treatment of 72h, the 24 hole culture plate cell supernatant was collected for ELISA detection of IL-la, IL-6 and TNF-a. The effects of different concentrations of SPA osteogenic differentiation medium for 3 weeks, then the cells were collected for detection of alizarin red staining calcium nodules and alkaline phosphatase deposition; 2. analysis and verification of SPA under the treatment of hBMSCs hBMSCs IncRNA changes in the differentiation of bone containing (SPA group) or without (control group) after 2 weeks 1 g/ml SPA osteogenic differentiation medium, cells were collected for analysis of lncRNA gene chip. Then the analysis is to express the lncRNA which is closely related to the differentiation of osteogenesis, and the above differentiating genes are verified by qRT-PCR and the target genes are screened. 3. different effects of lncRNA on osteogenic differentiation of BMMSC SPA bone Sha inflammation model of the synthesis of 3 NONHSAT009968 interference sequence (siRNA-1, siRNA-2 and siRNA-3). HBMSCs cells were cultured and Lipofectamine 2000 reagents were used to transfect about 200nmsiRNA to hBMSCs according to the operation instructions. The interference efficiency was detected by qRT-PCR. The siRNA-2 sequence is used to pack the lentivirus. Lentivirus production, concentration and titration, infection condition confirmation and stable cell line screening were carried out according to the standard operation. The lentivirus expressed NONHSAT009968-shRNA or empty lentivirus, and 3 days after infection, bone marrow mesenchymal stem cells were collected to determine whether NONHSAT009968 was successfully silent. The lentivirus expressed NONHSAT009968-shRNA or empty lentivirus infection of hBMSCs cells for 14 days and 21 days. Alizarin red staining was used to detect the deposition of calcium nodules. The cells were collected for Western blot to detect the expression of Runx2, OCN, OPN and COL1A1, and ELISA was used to detect IL-Ia, IL-6, TNF-a and alkaline phosphatase. The effects of interfering NONHSAT009968 on the osteogenic differentiation of hBMSCs treated with SPA were analyzed. 4, the mechanism of the effect of differential IncRNA on the osteogenic differentiation function of BMMSC in SPA osteomyelitis model. Lentivirus expressing NONHSAT009968-shRNA or empty lentivirus infected hBMSCs cells were cultured for 14 days and 21 days. Collecting cells using qRT-PCR and Western blot to detect Wnt3a, beta -catenin and GSK-3
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R681.2

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