本文選題：骨折 + 酸性微環(huán)境��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：現(xiàn)代外科學(xué)的發(fā)展為骨折的治療帶來了新的理念和技術(shù),但術(shù)后骨不連的發(fā)病率仍舊很高,因此骨折愈合的機制及影響因素仍有待于深入研究。成骨細(xì)胞在骨折愈合中扮演重要的角色,其增殖分化與骨折周圍的微環(huán)境息息相關(guān),因此,研究成骨細(xì)胞在骨折局部微環(huán)境中的反應(yīng)有著非常重要的意義。自噬可避免細(xì)胞受外界不利環(huán)境的影響,是用來維持細(xì)胞內(nèi)環(huán)境平衡與穩(wěn)定的重要保護(hù)機制。研究表明,缺血、低氧和氧化應(yīng)激等可導(dǎo)致自噬的發(fā)生。然而,骨折局部同樣存在酸性的微環(huán)境,但是酸性微環(huán)境是否誘導(dǎo)成骨細(xì)胞發(fā)生自噬目前尚不清楚。因此,對成骨細(xì)胞所在酸性微環(huán)境中的自噬反應(yīng)以及相關(guān)機制進(jìn)行研究,可能為促進(jìn)骨愈合以及骨折術(shù)后骨不連的防治提供新的思路。目的:本研究通過構(gòu)建小鼠骨折模型,分析骨折斷端周圍能否發(fā)生自噬反應(yīng)。通過體外細(xì)胞實驗?zāi)M骨折周圍的酸性微環(huán)境,分析酸性環(huán)境對成骨細(xì)胞活力和凋亡的影響,以及酸性微環(huán)境下成骨細(xì)胞是否能發(fā)生保護(hù)性自噬反應(yīng),從而提高成骨細(xì)胞的存活率,為促進(jìn)骨折愈合提供新的策略。方法:1.動物實驗分組及方法實驗選取27只體重在(30±10)g,6周齡雄性KM大鼠,并隨機分為3組:A組(6h),B組(24h),C組(36h),每組9只。小鼠右側(cè)股骨建立骨折損傷,左側(cè)正常側(cè)作為對照。A,B,C三組大鼠分別在骨折6h,24h,36h后處死,取下骨折及正常側(cè)骨組織作為樣本,樣本固定、骨組織脫鈣、石蠟包埋、切片,進(jìn)行組織免疫熒光染色,檢測標(biāo)志蛋白LC3,p62的表達(dá)水平。2.細(xì)胞實驗的分組及方法實驗按pH的不同將培養(yǎng)基隨機分為三組分別為pH 6.4、6.8(實驗組)及7.4組(對照組)。成骨細(xì)胞培養(yǎng)在96孔板內(nèi),經(jīng)過不同pH的培養(yǎng)基處理12h,24h,48h時間后,采用MTT比色法檢測酸性微環(huán)境下成骨細(xì)胞的細(xì)胞活力;細(xì)胞在不同pH的培養(yǎng)基內(nèi)處理24h后,采用AnnexinV-PI染色法檢測不同pH培養(yǎng)基對成骨細(xì)胞凋亡的影響;細(xì)胞免疫熒光用來檢測,經(jīng)不同pH的培養(yǎng)基處理6h后,LC3在細(xì)胞內(nèi)的表達(dá);透射電鏡觀察pH 6.4的培養(yǎng)基處理6h后自噬小體的數(shù)量及其形態(tài)的變化;免疫蛋白印記法檢測自噬標(biāo)志蛋白LC3及p62的表達(dá)以及轉(zhuǎn)化以及監(jiān)測酸性微環(huán)境下成骨細(xì)胞自噬流的產(chǎn)生。通過添加自噬抑制劑CQ抑制成骨細(xì)胞自噬反應(yīng),檢測不同pH培養(yǎng)基培養(yǎng)24h后細(xì)胞凋亡情況,分析酸性微環(huán)境下自噬對凋亡的影響。采用SPSS16.0軟件包進(jìn)行分析,單因素方差分析對數(shù)據(jù)進(jìn)行統(tǒng)計。結(jié)果:1.動物免疫熒光觀察顯示:免疫熒光用來檢測LC3及p62的表達(dá)。骨折組,骨折斷端LC3的表達(dá)相比對照組要強(p0.05),相反p62表達(dá)較對照組弱(p0.05)。即骨折斷端發(fā)生了自噬。2.MTT比色法顯示:實驗組(pH6.4、6.8)與對照組(pH7.4)相比,在各個時間點(12h,24h,48h)細(xì)胞活力均小于對照組(p0.05),即酸性環(huán)境下成骨細(xì)胞的細(xì)胞活力受到抑制,與pH6.8組相比pH6.4組細(xì)胞活力更低,其差異具有顯著性(p0.05)。即酸性pH微環(huán)境對成骨細(xì)胞的細(xì)胞活力是不利的且呈pH及時間依賴性。3.AnnexinV-PI染色法顯示:24小時后,pH6.4和pH6.8組細(xì)胞凋亡數(shù)目較正常組pH(7.4)明顯增多,且pH6.4組細(xì)胞凋亡數(shù)最多。即酸性微環(huán)境促使成骨細(xì)胞凋亡,其差異具有顯著性(p0.05)。4.電鏡觀察顯示:成骨細(xì)胞在酸性為pH6.4的培養(yǎng)液中培養(yǎng)6h后,自噬小體數(shù)目較正常組pH(7.4)明顯增多,成骨細(xì)胞的形態(tài)結(jié)構(gòu)發(fā)生了變化,呈雙層膜包裹著細(xì)胞器或線粒體。即酸性環(huán)境可以誘導(dǎo)成骨細(xì)胞發(fā)生自噬反應(yīng)。5.蛋白質(zhì)免疫印記檢測顯示:pH6.4時,隨著時間的延長(6h,12h,24h),LC3-Ⅱ/Ⅰ比值變低(p0.05),而p62逐漸升高(p0.05);即隨著時間延長自噬減弱;6h時,隨著pH的升高(pH6.4,6.8,7.4)LC3-Ⅱ/Ⅰ比值降低,相反p62逐漸升高(p0.05),即酸性pH越低,自噬越強。在相同條件加入自噬抑制劑氯喹后,因其阻礙了自噬小體與溶酶體的結(jié)合,導(dǎo)致LC3-Ⅱ的表達(dá)明顯增加。即隨著時間的延長自噬減弱。6.細(xì)胞免疫熒光檢測顯示:細(xì)胞在pH6.4的培養(yǎng)液處理6h后,采用免疫熒光檢測LC3,紅色熒光分布在細(xì)胞質(zhì)內(nèi),且隨pH的升高,熒光表達(dá)降低(p0.05)。說明酸性微環(huán)境可以促進(jìn)自噬。7.加入自噬抑制劑后AnnexinV-PI染色法顯:在相同的條件下,加入自噬抑制劑組比不加入抑制劑組細(xì)胞凋亡數(shù)明顯增多(p0.05)。即在酸性微環(huán)境下,抑制自噬,促進(jìn)了成骨細(xì)胞凋亡。結(jié)論:1.體外實驗發(fā)現(xiàn)酸性pH微環(huán)境對成骨細(xì)胞的細(xì)胞活力是不利的,它可以促進(jìn)成骨細(xì)胞凋亡,并且誘導(dǎo)成骨細(xì)胞發(fā)生自噬。2.體內(nèi)實驗發(fā)現(xiàn)成骨細(xì)胞在骨折部位可發(fā)生自噬反應(yīng)。3.成骨細(xì)胞通過自噬抑制細(xì)胞的凋亡,從而提高成骨細(xì)胞在酸性微環(huán)境中的的存活率。
[Abstract]:The development of modern surgery has brought new ideas and techniques for the treatment of fracture, but the incidence of postoperative bone nonunion is still very high. Therefore, the mechanism and influencing factors of fracture healing still need to be studied. Osteoblasts play an important role in fracture healing, and their proliferation and differentiation are closely related to the microenvironment around fracture. It is of great significance to study the response of osteoblasts in the local microenvironment of fracture. Autophagy can avoid the effect of the adverse environment on the external environment, and it is an important protective mechanism for maintaining the balance and stability of the cell environment. In acidic microenvironment, it is not clear whether acid microenvironment induces autophagy in osteoblasts. Therefore, the study of autophagy and related mechanisms in the acidic microenvironment of osteoblasts may provide new ideas for the promotion of bone healing and the prevention and treatment of bone nonunion after fracture. A mouse model of fracture was used to analyze the autophagic reaction around the broken end of the fracture. The effects of acid environment on the activity and apoptosis of osteoblasts and the protective autophagy in the acidic microenvironment were analyzed by simulating the acid microenvironment around the fracture. To provide a new strategy to promote fracture healing. Methods: 1. group and method experiment of 1. animals were divided into 3 groups (30 + 10) and 6 weeks old male KM rats, and were randomly divided into 3 groups: A group (6h), B group (24h), C group (36h), 9 rats in each group. The left normal side was used as control.A, B, C three rats respectively in the fracture 6h, 3, 3. 3 After 6h, the fracture and the normal lateral bone tissue were taken as samples, the samples were fixed, bone tissue decalcified, paraffin embedded, sliced, tissue immunofluorescence staining, and tissue immunofluorescence staining, LC3, p62 expression level.2. cell experiments were divided into three groups randomly divided into pH 6.4,6.8 (experimental group) and 7.4 respectively according to the difference of pH. Group (control group). Osteoblasts were cultured in 96 orifice plates. After 12h, 24h and 48h were treated with different pH medium, MTT colorimetric assay was used to detect the cell viability of osteoblasts in acidic microenvironment. After the cells were treated with 24h in the culture base of different pH, the effect of different pH medium on the apoptosis of osteoblasts was detected by AnnexinV-PI staining. Cell immunofluorescence was used to detect the expression of LC3 in the cell after treating 6h with different pH medium. The number and morphological changes of autophagic corpuscles after 6h was treated with pH 6.4 medium; the expression of autophagic marker protein LC3 and p62 was detected by the immunoglobulin imprint method and the autophagy was transformed and the osteoblast under the acid microenvironment was monitored from the acid microenvironment. The effect of autophagy by adding autophagic inhibitor CQ to autophagy in osteoblasts was detected and apoptosis in different pH cultures was detected and the effect of autophagy on apoptosis in acid microenvironment was analyzed. SPSS16.0 software package was used to analyze the cell apoptosis. The data were analyzed by single factor analysis of variance. Results: 1. animal immunofluorescence observation showed that: 1. The expression of LC3 and p62 was detected by immunofluorescence. The expression of LC3 in fracture fracture group was stronger than that of the control group (P0.05), but the expression of p62 was weaker than that of the control group (P0.05). That is, the autophagic.2.MTT colorimetric method of the fracture end showed that the experimental group (pH6.4,6.8) was less than the control group (pH7.4) at all time points (12h, 24h, 48h) cells were less than those of the control group. The cell viability of osteoblasts in the acidic environment was inhibited, and the cell viability of the pH6.4 group was lower than that in the pH6.8 group, and the difference was significant (P0.05). That is, the acid pH microenvironment was negative to the cell viability of the osteoblasts and was shown by pH and time dependent.3.AnnexinV-PI staining: 24 hours later, pH6.4 and pH6.8 groups finely. The number of apoptotic cells increased significantly compared with the normal group pH (7.4), and the number of apoptotic cells in the pH6.4 group was the most. That is, the acid microenvironment resulted in the apoptosis of osteoblasts. The difference was significant (P0.05).4. electron microscope observation showed that the number of autophagic corpuscles in the acidic pH6.4 culture medium, the number of autophagic corpuscles increased significantly than the normal group pH (7.4), and osteoblasts The morphological structure changed, and the cell organelle or mitochondria were wrapped in the double layer membrane. That is, the acid environment can induce autophagy to induce autophagic reaction.5. protein immuno imprint detection show: pH6.4, with the prolongation of time (6h, 12h, 24h), LC3- II / I ratio decreases (P0.05), and p62 gradually increases (P0.05); that is, the autophagy decreases with time; At 6h, with the decrease of the ratio of pH (pH6.4,6.8,7.4) LC3- II / I, the opposite p62 gradually increased (P0.05), that is, the lower the acid pH, the stronger the autophagy. After adding the autophagic inhibitor chloroquine, the expression of the autophagosome and the lysosome was hindered by the combination of the autophagic inhibitor, which resulted in a significant increase in the expression of LC3- II. That is, the autophagy attenuated the.6. cell with the time of autophagy. Immunofluorescence detection showed that after the cells were treated with 6h in pH6.4 culture, LC3 was detected by immunofluorescence, the red fluorescence was distributed in the cytoplasm, and the fluorescence expression decreased with the increase of pH (P0.05). It indicated that the acid microenvironment could promote autophagic.7. to add autophagic inhibitor after AnnexinV-PI staining method: under the same condition, the autophagy inhibition was added. The number of apoptotic cells in the agent group was significantly increased (P0.05). That is, in the acidic microenvironment, inhibiting autophagy and promoting the apoptosis of osteoblasts. Conclusion: 1. in vitro, the acid pH microenvironment was found to be unfavorable to the cell viability of osteoblasts. It could promote osteoblast apoptosis and induce autophagy in.2. body. It was found that autophagy could induce autophagy.3. osteoblasts to inhibit the apoptosis of cells by autophagy, thus improving the survival rate of osteoblasts in the acid microenvironment.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R683

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張志超;李雅南;來慶國;袁奎封;;酸性pH環(huán)境對成骨細(xì)胞增殖和凋亡的影響[J];中國口腔頜面外科雜志;2017年01期

2 云帆;王瑞;趙建寧;;破骨細(xì)胞的骨吸收機制[J];中國骨傷;2014年06期

3 李曉峰;趙勁民;蘇偉;范鍥;羅世興;馬愛國;;大鼠成骨細(xì)胞的原代培養(yǎng)和鑒定[J];中國組織工程研究與臨床康復(fù);2011年06期

4 李楠,王和鳴;骨折愈合基礎(chǔ)研究新進(jìn)展[J];中醫(yī)正骨;1999年04期

相關(guān)博士學(xué)位論文 前1條

1 李強;低氧誘導(dǎo)成骨細(xì)胞增殖分化兩種機制的研究[D];南方醫(yī)科大學(xué);2016年

，

本文編號：1880268