【摘要】：miRNA-146a是最早發(fā)現(xiàn)的與骨性關(guān)節(jié)炎相關(guān)的差異性miRNA。我們通過(guò)miRNA芯片技術(shù)研究發(fā)現(xiàn)其在急性壓力損傷后的軟骨細(xì)胞中表達(dá)也會(huì)上調(diào)。然而miRNA-146a在軟骨細(xì)胞壓力損傷中的作用尚不清楚。本研究的目的就是希望找到miRNA-146a的靶基因，并闡明其在軟骨細(xì)胞壓力損傷中的作用機(jī)制。 miRNA芯片技術(shù)研究顯示，相對(duì)于正常軟骨細(xì)胞，壓力損傷后的軟骨細(xì)胞miRNA-146a表達(dá)上調(diào)，采用關(guān)聯(lián)分析的方法，發(fā)現(xiàn)生物信息學(xué)分析預(yù)測(cè)的miRNA-146a的靶基因SMAD4，在聯(lián)合應(yīng)用的全基因組基因芯片結(jié)果中低表達(dá)。因此，我們推測(cè)miRNA-146a通過(guò)SMAD4介導(dǎo)的方式來(lái)調(diào)控軟骨細(xì)胞的壓力損傷。研究目的： 在這一假設(shè)的基礎(chǔ)上，本實(shí)驗(yàn)擬通過(guò)研究闡明miRNA-146a在軟骨細(xì)胞壓力損傷中的調(diào)控作用及其分子機(jī)制；證明SMAD4是miRNA-146a的直接靶基因，并參與介導(dǎo)miRNA-146a對(duì)軟骨細(xì)胞壓力損傷的調(diào)控。我們希望此研究可以為臨床上急性關(guān)節(jié)損傷的治療帶來(lái)新的研究思路和切入點(diǎn)。 研究方法： 1.原代分離及培養(yǎng)人軟骨細(xì)胞和骨性關(guān)節(jié)炎細(xì)胞，使用光鏡下觀察其生長(zhǎng)情況，MTT法繪制生長(zhǎng)曲線，采用甲苯胺藍(lán)染色、II型膠原免疫組化染色等方法對(duì)比兩種細(xì)胞的差別。 2.研制一套具有自主知識(shí)產(chǎn)權(quán)的實(shí)用多功能細(xì)胞壓力加載系統(tǒng)，為進(jìn)行細(xì)胞力學(xué)相關(guān)實(shí)驗(yàn)奠定基礎(chǔ)。使用該系統(tǒng)構(gòu)建軟骨細(xì)胞壓力損傷模型，力求盡量模擬高能損傷的力學(xué)特點(diǎn)。 3.聯(lián)合應(yīng)用miRNA芯片和全基因組基因芯片篩選正常及壓力損傷后軟骨細(xì)胞的差異miRNA和基因。采用Realtime RT-PCR分析的方法，，驗(yàn)證壓力損傷組特異性高表達(dá)的miRNA-146a表達(dá)情況是否與芯片結(jié)果一致，采用Realtime RT-PCR的方法及Western Blot的方法，驗(yàn)證芯片結(jié)果中SMAD4和VEGF的差異表達(dá)。 4.通過(guò)向細(xì)胞內(nèi)轉(zhuǎn)染miRNA-146a的模擬物、抑制物、無(wú)關(guān)序列和空白對(duì)照組的方式，調(diào)控miRNA-146a的表達(dá)，進(jìn)一步研究其功能。 5.觀察調(diào)控miRNA-146a的表達(dá)后，對(duì)軟骨細(xì)胞生物學(xué)特性的影響，并使用實(shí)時(shí)定量RT-PCR和Western-blot檢測(cè)各組中SMAD4和VEGF的表達(dá)情況。 6.觀察miRNA-146a的上調(diào)和下調(diào)后對(duì)軟骨細(xì)胞壓力損傷作用的影響 7.采用雙熒光素酶報(bào)告基因系統(tǒng)證明SMAD4的mRNA3’UTR和miRNA-146a之間可以直接結(jié)合，驗(yàn)證了SMAD4是miRNA-146a的直接靶基因。 研究結(jié)果： 1.通過(guò)細(xì)胞表型和生物學(xué)活性的檢測(cè)，保證原代培養(yǎng)所得細(xì)胞為狀態(tài)良好的軟骨細(xì)胞，可以進(jìn)行下一步實(shí)驗(yàn)。 2.根據(jù)對(duì)比MTT等試驗(yàn)結(jié)果，選擇對(duì)細(xì)胞生物學(xué)特性影響加大，且大部分存活的參數(shù)，10Mpa60min作為成模參數(shù)。 3. miRNA-146a是芯片結(jié)果中差異最顯著的一個(gè)，聯(lián)合基因芯片結(jié)果并根據(jù)生物信息學(xué)方法預(yù)測(cè)其靶基因，找出了在芯片結(jié)果中與預(yù)測(cè)結(jié)果相一致的潛在靶基因SMAD4，并成功驗(yàn)證了芯片結(jié)果。 4.轉(zhuǎn)染外源性miRNA-146a mimics后，人正常軟骨細(xì)胞中miRNA-146a的表達(dá)水平顯著上升。轉(zhuǎn)染外源性miRNA-146a inhibitor后，人正常軟骨細(xì)胞中miRNA-146a的表達(dá)水平顯著下調(diào)。 5.在正常人軟骨細(xì)胞中上調(diào)miRNA-146a的表達(dá)，可以使SMAD4的表達(dá)下調(diào)并使VEGF的表達(dá)上調(diào)，miRNA-146a的表達(dá)下調(diào)時(shí)可引起相反的作用。 6.實(shí)驗(yàn)結(jié)果顯示miRNA-146a具有抑制細(xì)胞增殖，促進(jìn)壓力損傷軟骨細(xì)胞的凋亡的能力。 7. SMAD4是miRNA-146a的直接靶基因。 研究結(jié)論： 1.軟骨細(xì)胞壓力損傷后，miRNA-146a的表達(dá)升高，SMAD4表達(dá)下調(diào)而VEGF的表達(dá)升高。 2. SMAD4是miRNA-146a的直接靶基因。 3. miRNA-146a可以通過(guò)SMAD4介導(dǎo)的方式調(diào)控VEGF的表達(dá)，具有促進(jìn)軟骨細(xì)胞凋亡的作用。 4. miRNA-146a可能通過(guò)下調(diào)SMAD4的方式阻斷TGF-β通路，上調(diào)VEGF的表達(dá)水平，促進(jìn)骨性關(guān)節(jié)炎的發(fā)生。
[Abstract]:MiRNA-146a is the first identified differential miRNA. associated with osteoarthritis. We found that the expression in the cartilage cells after acute stress injury is also up regulated by miRNA chip technology. However, the role of miRNA-146a in cartilage pressure damage is not clear. The purpose of this study is to find the target base of miRNA-146a. To elucidate its mechanism in the stress injury of chondrocytes.
MiRNA chip technology studies show that the expression of miRNA-146a in cartilage cells after stress damage is up regulation relative to normal cartilage cells. Using correlation analysis, the target gene SMAD4 of miRNA-146a, predicted by bioinformatics analysis, is low expressed in the results of the combined whole genome gene chip. Therefore, we speculate that miRNA-146a passes through the results. SMAD4 mediated the regulation of stress damage in chondrocytes.
On the basis of this hypothesis, this study intends to elucidate the regulatory role of miRNA-146a in cartilage cell stress damage and its molecular mechanism. It is proved that SMAD4 is the direct target gene of miRNA-146a and is involved in the regulation of miRNA-146a's regulation of cartilage cell stress damage. We hope this study can be a clinical acute joint injury. Treatment brings new research ideas and breakthrough points.
Research methods:
1. the human chondrocytes and osteoarthritis cells were isolated and cultured in the original generation. The growth of the cells was observed under the light microscope. The growth curve was drawn by MTT method. The difference of the two cells was compared with the methods of toluidine blue staining and the immunohistochemical staining of type II collagen.
2. a practical multifunction cell pressure loading system with independent intellectual property rights is developed to lay the foundation for the experiment of cell mechanics. The system is used to construct the model of cartilage cell stress damage, and try to simulate the mechanical characteristics of high energy damage.
3. miRNA chip and whole genome microarray were used to screen the difference miRNA and gene of cartilage cells after normal and stress damage. The method of Realtime RT-PCR analysis was used to verify whether the specific high expression of miRNA-146a expression in the stress group was in accordance with the results of the chip. The method of using Realtime RT-PCR and the prescription of Western Blot were used. Methods to verify the differential expression of SMAD4 and VEGF in the chip results.
4. to regulate the expression of miRNA-146a by transfecting miRNA-146a mimic, inhibitor, irrelevant sequence and blank control group to further study its function.
5. the effects of the expression of miRNA-146a on the biological characteristics of chondrocytes were observed, and the expression of SMAD4 and VEGF in each group was detected by real-time quantitative RT-PCR and Western-blot.
6. to observe the effect of miRNA-146a up regulation and down regulation on the stress injury of chondrocytes.
7. dual luciferase reporter gene system is used to prove that mRNA3 'UTR and miRNA-146a can directly bind to SMAD4, which proves that SMAD4 is the direct target gene of miRNA-146a.
The results of the study:
1. through the detection of cell phenotype and biological activity, we can ensure that primary cultured cells are well preserved chondrocytes, and we can carry out the next experiment.
2. based on the comparison of MTT and other experimental results, 10Mpa60min was chosen as the model parameter to increase the influence of cell biological characteristics and most of the survival parameters.
3. miRNA-146a is the most significant difference in the result of the chip. Combining the results of the gene chip and predicting the target gene according to the bioinformatics method, the potential target gene SMAD4 which is consistent with the prediction results in the result of the chip is found, and the result of the chip is verified successfully.
After transfection of exogenous miRNA-146a mimics, the expression level of miRNA-146a in normal human cartilage cells increased significantly. After transfection of exogenous miRNA-146a inhibitor, the expression level of miRNA-146a in normal human cartilage cells decreased significantly.
5. up regulation of the expression of miRNA-146a in normal human chondrocytes can reduce the expression of SMAD4 and increase the expression of VEGF. The down regulation of miRNA-146a can cause the opposite effect.
6. the experimental results showed that miRNA-146a could inhibit cell proliferation and promote the ability of stress to damage chondrocyte apoptosis.
7. SMAD4 is the direct target gene of miRNA-146a.
The conclusions are as follows:
1. the expression of miRNA-146a increased and the expression of SMAD4 was downregulated and the expression of VEGF increased after the stress injury of chondrocytes.
2. SMAD4 is the direct target gene of miRNA-146a.
3. miRNA-146a can regulate the expression of VEGF through SMAD4 mediated manner, and has the effect of promoting chondrocyte apoptosis.
4. miRNA-146a may block the TGF- beta pathway by up regulating SMAD4, up regulate the expression level of VEGF, and promote the occurrence of osteoarthritis.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R684.3

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