發(fā)布時間：2018-04-29 00:00

  本文選題：機械牽伸 + 力生長因子�。� 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：前言肌腱病(Tendinopathy)是一種常見的多種因素引起的肌腱功能障礙的綜合征,其治療方式多樣,但總體治療效果有限,究其原因在于肌腱病的發(fā)病機制尚未完全闡明[1-3]。肌腱干細胞(tendon stem cells,TSCs)作為肌腱來源的特殊干細胞在維持肌腱正常生理功能及肌腱病病理生理過程中都扮演重要角色[4,5]。如何調控TSCs正常分化為肌腱細胞修復肌腱微損傷,對于提高肌腱病的防治水平具有重要意義。目前已研究證實TSCs可受多種因素調控,如機械應力[6-11]、炎癥因子[11-15]、皮質醇激素[16]、細胞因子[14,17-21]等。在眾多調控因素中,機械應力是肌腱病發(fā)病始動因素,對于TSCs調控作用重要性不言而喻。力生長因子(Mechano growth factor,MGF),也稱為機械生長因子,廣泛存在于骨、骨骼肌、肌腱、腦等組織[22-25],是在受到機械應力、電刺激或者組織損傷時產生的一種胰島素樣生長因子-1的選擇性剪接異構體[24-27]。自從MGF被發(fā)現(xiàn)以來,其獨特的E肽結構及其特有的功能吸引著眾多學者的關注,現(xiàn)已證實MGF對多種細胞的增殖、遷移及分化有重要作用[28-36]。另外有研究發(fā)現(xiàn)肌腱組織經機械應力刺激后,MGF表達會顯著升高,且反應時間明顯早于IGF-1[37-41]。然而,TSCs作為肌腱病發(fā)病過程中的“明星細胞”,在受到機械牽伸應力作用后MGF表達水平如何變化以及MGF對其增殖分化及遷移能力是否起到一定的調節(jié)作用卻未見相關報道。同時,機械牽伸和MGF對于TSCs的分化調控是單獨作用還是共同作用?是相互協(xié)同作用還是拮抗作用呢?這一系列問題均亟待解決。因此,本課題研究目的主要是:1.驗證機械牽伸對TSCs分化的調控及對MGF表達的影響;2.明確MGF對TSCs增殖、分化、遷移的影響及調控機制;3.闡明MGF在機械牽伸條件下對TSCs分化調控的影響及其機制。方法第一部分:進行大鼠TSCs分離培養(yǎng)鑒定;通過Real-time PCR、Western Blot分別從轉錄和翻譯水平檢測不同機械牽伸條件處理后TSCs中成骨、成脂及成肌腱分化的相關基因mRNA及蛋白表達水平,同時檢測TSCs中MGF mRNA表達水平。第二部分:通過CCK8及實時無標記動態(tài)細胞分析技術檢測TSCs經不同濃度MGF處理后,細胞增殖變化情況;通過Transwell、劃痕試驗及激光共聚焦顯微鏡檢測不同濃度MGF處理對TSCs遷移能力及細胞骨架變化的影響;通過Western Blot檢測TSCs經不同濃度MGF及MAPK/ERK通路抑制劑PD98059處理后,成腱分化相關基因蛋白表達變化。第三部分:通過Real-time PCR和Western Blot分別檢測機械牽伸與MGF共同處理TSCs后,成肌腱、成骨及成脂三系分化相關基因mRNA及蛋白相對表達變化。結果第一部分:1.4%1Hz組牽伸48h成腱分化指標TNC和成骨分化指標RUNX2相對表達量較靜態(tài)培養(yǎng)組均顯著升高(P0.05);4%2Hz組各牽伸時間點TNC相對表達量較靜態(tài)培養(yǎng)組均明顯升高(P0.05),同時牽拉24h時CEBPα表達受到顯著抑制(P0.05),RUNX2相對表達量較對照組無明顯差異(P0.05);8%1Hz組牽拉24h RUNX2表達水平顯著高于靜態(tài)培養(yǎng)組(P0.05),同時成肌腱分化及成脂肪分化相關基因表達較靜態(tài)培養(yǎng)組無顯著變化(P0.05);8%2Hz組牽拉48h時CEBPα表達水平顯著高于靜態(tài)培養(yǎng)組(P0.05),同時RUNX2相對表達量較靜態(tài)培養(yǎng)組無顯著差異(P0.05),TNC相對表達量較靜態(tài)培養(yǎng)組顯著降低(P0.05)。2.不同條件機械牽伸會引起TSCs中MGF mRNA相對表達量出現(xiàn)不同水平的增加。4%2Hz及8%1Hz組牽伸12小時即可見MGF mRNA水平顯著升高(P0.05),牽伸24h時MGF mRNA表達量達到頂峰,差異有統(tǒng)計學意義(P0.05)。第二部分:1.CCK-8結果顯示:TSCs經5ng/ml MGF處理后,24小時開始OD值即出現(xiàn)顯著升高,與其余各組相比差異有統(tǒng)計學意義(P0.05),但其余各組間吸光度無明顯差異(P0.05)。RTCA結果顯示:經1ng/ml及5ng/ml MGF處理后,TSCs細胞指數較其余各組均升高,差異有統(tǒng)計學意義(P0.05),但兩組間無顯著性差異(P0.05);經10ng/ml、25ng/ml和50ng/ml MGF及5ng/ml MGF+10μg/ml PQ401處理后,細胞指數較對照組顯著降低(P0.05);5ng/mlMGF+50μM PD98059組較其余各組,細胞指數明顯降低,差異有統(tǒng)計學意義(P0.05)。2.劃痕實驗結果顯示:經1ng/ml、5ng/ml和10ng/ml MGF處理后,TSCs遷移能力較對照組均明顯加快,其中5ng/ml MGF處理組TSCs的遷移速度最快。Transwell結果顯示:TSCs經過不同濃度MGF處理后,TSCs遷移數量均有顯著增加,OD值顯著升高(P0.05),其中以5ng/ml和7.5ng/ml組最為明顯;另外5ng/ml MGF+50μM PD98059組與5ng/ml MGF組相比,TSCs遷移數量明顯減少,OD值顯著降低,差異有統(tǒng)計學意義(P0.05);而5ng/ml MGF+10μg/ml PQ401組細胞形態(tài)發(fā)生明顯改變,細胞遷移數量最少,OD值最低。激光共聚焦顯微鏡觀察可見:經0、1.0、2.5、5.0、7.5及10.0ng/ml MGF處理的TSCs,細胞骨架連續(xù)性良好,熒光強,細胞成典型長梭形,肌動蛋白纖維排列規(guī)則;經高濃度(25ng/ml及50ng/ml)MGF處理后,TSCs形態(tài)成不規(guī)則形,細胞骨架斷裂,排列紊亂,熒光強度變弱;經10μg/ml PQ401處理后,TSCs細胞形態(tài)出現(xiàn)明顯改變,細胞骨架斷裂,呈顆粒樣,熒光強度極弱。3.Western Blot結果顯示TSCs經5ng/ml及10ng/mlMGF處理72h后,TNC、SCX蛋白表達量均較對照組顯著升高,差異有統(tǒng)計學意義(P0.05)。而經1ng/ml及25ng/ml MGF處理后,蛋白表達較對照組無明顯差異(P0.05)。第三部分:1.Real-time PCR結果顯示:TSCs經0ng/ml、2.5ng/ml以及5.0ng/ml MGF處理后,在4%2Hz機械牽伸條件下牽伸24h,SCX和TNC mRNA相對表達較靜態(tài)培養(yǎng)組明顯升高,差異有統(tǒng)計學意義(P0.05)。RUNX2、DLX5以及AP2、PPARγmRNA相對表達量較靜態(tài)培養(yǎng)組明顯降低,差異有統(tǒng)計學意義(P0.05)。50μM PD98059+5ng/ml MGF組TSCs經4%2Hz機械牽伸24h后,全部基因的mRNA相對表達量較未添加抑制劑的5ng/ml MGF組變化倍數顯著降低,差異有統(tǒng)計學意義(P0.05)。2.Western blot結果顯示:TSCs經0、2、5ng/ml MGF處理后,在4%2Hz機械牽伸下牽拉24h,TNC蛋白相對表達量較靜態(tài)培養(yǎng)組升高,差異有統(tǒng)計學意義(P0.05)。RUNX2以及CEBPα蛋白相對表達量較靜態(tài)培養(yǎng)組顯著降低,差異有統(tǒng)計學意義(P0.05)。50μM PD98059+5ng/ml MGF組TSCs經4%2Hz機械牽伸24h后,全部分化相關基因的蛋白相對表達量較未添加抑制劑的5.0ng/mlMGF組變化倍數顯著降低,差異有統(tǒng)計學意義(P0.05)。結論第一部分:不同條件的機械牽伸會引起TSCs向不同方向分化。4%2Hz 24h為誘導成肌腱分化的最佳牽伸條件,8%1Hz 24h為誘導成骨分化的最佳牽伸條件,8%2Hz 48h為誘導成脂肪分化的最佳牽伸條件。機械牽伸可上調TSCs中MGF表達水平,且與成肌腱分化相關。第二部分:MGF可增強TSCs遷移及增殖能力,并且可誘導TSCs向肌腱細胞方向分化,其中對增殖和遷移促進作用與MAPK/ERK通路激活相關。第三部分:機械牽伸與MGF協(xié)同促進TSCs向肌腱方向分化,抑制成脂及成骨分化。機械牽伸條件下MGF對TSCs分化的誘導調控是通過激活MAPK/ERK通路發(fā)揮作用。
[Abstract]:Preface tendons disease (Tendinopathy) is a common cause of a variety of factors caused by tendon dysfunction syndrome, its treatment is diverse, but the overall treatment effect is limited, the reason is that the pathogenesis of tendon disease is not fully elucidated by the [1-3]. tendon stem cell (tendon stem cells, TSCs) as a tendon source of special stem cells in maintenance The normal physiological function of tendon and the pathophysiological process of tendon disease all play an important role [4,5]. how to regulate the normal differentiation of TSCs into tendon cells to repair the tendon micro injury, which is of great significance for the improvement of the prevention and treatment of tendon disease. At present, it has been proved that TSCs can be regulated by many factors, such as mechanical stress [6-11], inflammatory factor [11-15], skin [16], cytokine [14,17-21] and so on. In many regulatory factors, mechanical stress is the starting factor of the pathogenesis of tendon disease, and it is self-evident for the importance of TSCs regulation. Mechano growth factor (MGF), also known as mechanical growth factor, is widely found in bone, skeletal muscle, tendon, brain and other tissues, which are in the machine. The selective splicing isomer of an insulin-like growth factor -1, produced by mechanical stress, electrical stimulation or tissue damage, has attracted many scholars' attention since the discovery of MGF, and its unique E peptide structure and its unique functions have been shown to play an important role in the proliferation, migration and differentiation of various cells, [28-36]., and [28-36].. It was found that the expression of MGF increased significantly after the mechanical stress stimulation, and the reaction time was significantly earlier than that of IGF-1[37-41].. TSCs was the "star cell" in the pathogenesis of tendon disease, and how the expression of MGF was changed after the mechanical drafting stress was applied and whether MGF played a role in the proliferation and differentiation and migration ability of MGF. No related reports have been reported. At the same time, mechanical drafting and MGF play a separate role or a common effect on TSCs differentiation, are they synergistic or antagonistic? This series of problems need to be solved. Therefore, the main purpose of this study is to verify the regulation of TSCs differentiation by mechanical drafting and the expression of MGF. The influence of MGF on the proliferation, differentiation, migration and regulation mechanism of TSCs, and 3. to elucidate the effect and mechanism of MGF on the regulation of TSCs differentiation under the condition of mechanical drafting. Method first part: TSCs isolation and culture identification of rats, and Real-time PCR, Western Blot respectively from the transcriptional and Translation levels to detect different mechanical drafting conditions. Bone formation, mRNA and protein expression levels of lipid and tendon differentiation in TSCs, and the level of MGF mRNA expression in TSCs. The second part: CCK8 and real-time unmarked dynamic cell analysis were used to detect the proliferation of TSCs after different concentrations of MGF, and through Transwell, scratch test and confocal laser confocal The effects of different concentrations of MGF on the mobility and cytoskeleton changes of TSCs were detected by the microscope. The expression of gene protein expression related to the differentiation of the tendon was detected by Western Blot after the treatment of TSCs with different concentrations of MGF and MAPK/ERK pathway inhibitor PD98059. The third part: the mechanical drafting was detected by Real-time PCR and Western Blot, respectively. After the same treatment with TSCs, the relative expression of mRNA and protein in the three lineage differentiation related genes of the tendon, osteogenesis and fat formation. Results the first part: the relative expression of TNC and the osteogenic differentiation index RUNX2 in the 1.4%1Hz group was significantly higher than that in the static culture group (P0.05), and the relative expression of TNC in the 4% 2Hz group was more than that in the static culture. The expression of CEBP alpha was significantly inhibited (P0.05) and the relative expression of RUNX2 was significantly higher than that in the control group (P0.05), while the RUNX2 expression level of 24h in 8%1Hz group was significantly higher than that in the static culture group (P0.05), and the expression of CEBP in 8%1Hz group was significantly higher than that in the static culture group (P0.05). There was no significant change in the expression of the adult tendon differentiation and the expression of the adipose differentiation related genes in the 8%1Hz group than that in the static culture group (P). 0.05); in group 8%2Hz, the expression level of CEBP a was significantly higher than that in the static culture group (P0.05), while the relative expression of RUNX2 was not significantly different than that in the static culture group (P0.05). The relative expression of TNC was significantly lower than that in the static culture group (P0.05). (P0.05) the different conditions of the mechanical drafting would lead to the increase of the relative expression of MGF mRNA in TSCs. The level of MGF mRNA increased significantly (P0.05) for 12 hours in group Z and 8%1Hz, and the expression of MGF mRNA reached the peak at the time of drawing 24h. The difference was statistically significant (P0.05). The second part: 1.CCK-8 results showed that TSCs after 24 hours, the OD value began to rise significantly, and the difference was statistically significant compared with the rest of the other groups. However, there was no significant difference in absorbency between the other groups (P0.05).RTCA results showed that after 1ng/ml and 5ng/ml MGF treatment, the TSCs cell index was higher than the rest of the other groups, and the difference was statistically significant (P0.05), but there was no significant difference between the two groups (P0.05). Compared with the other groups, the cell index of 5ng/mlMGF+50 M PD98059 group decreased significantly, and the difference was statistically significant (P0.05).2. scratch test results showed that after 1ng/ml, 5ng/ml and 10ng/ml MGF treatment, TSCs migration ability was significantly faster than that of the control group. The results showed that after the treatment of TSCs with different concentrations of MGF, the number of TSCs migration increased significantly, and the OD value increased significantly (P0.05), among which the 5ng/ml and 7.5ng/ml groups were the most obvious. In addition, the number of migration in 5ng/ml MGF+50 mu M PD98059 group was significantly reduced and the OD value decreased significantly. The cell morphology of the g/ml PQ401 group changed obviously, the number of cell migration was the least, and the OD value was the lowest. The confocal laser scanning microscope showed that the cytoskeleton was good, the cytoskeleton was good, the fluorescence was strong, the cell became the typical long shuttle shape, the myocutaneous fibrin was arranged regularly, and the high concentration (25ng/ml and 50ng/ml) MGF (25ng/ml and 50ng/ml) MGF were observed by laser confocal microscopy. After treatment, the morphology of TSCs was irregular, the cytoskeleton was broken, the arrangement was disorderly and the fluorescence intensity was weak. After 10 u g/ml PQ401 treatment, the morphology of TSCs cells changed obviously, the cytoskeleton broke, the particle like, the fluorescence intensity was very weak.3.Western Blot results showed that TSCs was treated with 5ng/ ml and 10ng/mlMGF 72h, TNC, the expression of TNC and protein expression were both compared to the control. The difference was statistically significant (P0.05). The protein expression was not significantly different from the control group after 1ng/ml and 25ng/ml MGF treatment (P0.05). The third part: 1.Real-time PCR results showed that TSCs was drafted under the condition of 0ng/ml, 2.5ng/ml and 5.0ng/ml MGF. The difference was statistically significant (P0.05).RUNX2, DLX5 and AP2, and the relative expression of PPAR gamma mRNA was significantly lower than that in the static culture group (P0.05), the difference was statistically significant (P0.05).50 u M PD98059+5ng/ml MGF group after TSCs via mechanical draft, the relative expression of all genes was more than that of the non inhibitor. The difference was statistically significant (P0.05).2.Western blot results showed that after 0,2,5ng/ml MGF treatment, TSCs was pulled 24h under 4%2Hz mechanical drafting, and the relative expression of TNC protein was higher than that in the static culture group, and the difference was statistically significant (P0.05).RUNX2 and the relative expression of CEBP alpha protein was significantly lower than that in the static culture group, and the difference was found. Statistical significance (P0.05).50 mu M PD98059+5ng/ml MGF group TSCs after 4%2Hz mechanically drafted 24h, the relative expression of all the related genes was significantly lower than that of the 5.0ng/mlMGF group without the inhibitor, and the difference was statistically significant (P0.05). Conclusion the first part: the mechanical drafting of different conditions will cause TSCs to different directions. Differentiation.4%2Hz 24h is the best drawing condition for inducing tendon differentiation, 8%1Hz 24h is the best drawing condition for inducing osteogenic differentiation, 8%2Hz 48h is the best drawing condition for inducing adipose differentiation. Mechanical drawing can increase the level of MGF expression in TSCs, and is related to the differentiation of tendon. The second part: MGF can enhance the ability of TSCs migration and proliferation, and It can induce the differentiation of TSCs to tendon cells, and the promotion of proliferation and migration is related to the activation of MAPK/ERK pathway. The third part: the synergistic effect of mechanical drafting and MGF to promote the direction differentiation of TSCs to the tendons and inhibit the formation of lipid and osteogenic differentiation. The induced regulation of MGF to TSCs differentiation under the condition of mechanical drawing is to play the role by activating the MAPK/ERK pathway.

【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R686.1

【參考文獻】

相關期刊論文 前5條

1 李元靖;劉歡;樊欣;周躍;;MGF對人軟骨終板干細胞增殖和遷移的影響[J];中國細胞生物學學報;2015年03期

2 楊廣華;唐康來;馬林;周梅;陳萬;穆米多;袁成松;胡超;李賽英;;體外細胞單軸循環(huán)牽伸載荷模型建立的實驗研究[J];第三軍醫(yī)大學學報;2014年11期

3 胡超;唐康來;陳萬;馬林;楊廣華;;大鼠跟腱來源肌腱干細胞的分離培養(yǎng)及鑒定[J];第三軍醫(yī)大學學報;2013年11期

4 潘同斌;王曉雪;唐芳;左偉;劉躍兵;溫慧霞;;大負荷運動及其恢復期間大鼠骨骼肌超微結構及MGF的變化[J];中國運動醫(yī)學雜志;2012年10期

5 曹洪輝;唐康來;陳磊;常廷杰;董世武;鄭紅;陳前博;陶旭;James H-C Wang;;新型肌腱細胞循環(huán)牽伸載荷系統(tǒng)的研制及運行效果觀察[J];第三軍醫(yī)大學學報;2010年06期

，

本文編號：1817457