本文關鍵詞：糖尿病對口腔種植體骨整合影響的作用機制研究　出處：《山東大學》2015年博士論文　論文類型：學位論文

  更多相關文章： 種植體 骨結合 高糖 糖尿病 PI3K/AKT 整合素 纖維粘連蛋白

【摘要】：背景和目的：口腔種植是治療缺牙患者的最有效途徑。B ranemark在1971提出的“骨結合Osseointegration"概念成為種植體成功的最重要標準。穩(wěn)定的骨結合依賴于成骨細胞在種植體表面的粘附和增殖分化,這一過程受到多種局部和全身因素的影響。糖尿病作為口腔種植治療的相對禁忌癥,一直受到學者們的關注,被公認為是影響種植體骨結合的最重要系統(tǒng)因素之一。本研究擬從體外實驗觀察高糖對MC3T3-E1細胞增殖、成骨分化的影響,并分析PI3K/Akt信號通路在其中的調節(jié)作用機制；從體內實驗,通過糖尿病大鼠模型,檢測不同血糖水平大鼠種植體周圍骨組織變化及整合素α5β1和纖維粘連蛋白(FN)表達水平,綜合評價糖尿病對種植體骨結合影響的作用機制。材料與方法：體外實驗：PI3K/AKT通路對不同高糖狀態(tài)下MC3T3-E1細胞成骨分化的影響根據文獻選取四種培養(yǎng)基糖濃度：5.5mmol/L,15.5mmol/L,25.5mmol/L, 35.5mmol/L,分別模擬人體內正常生理糖濃度及不同程度高血糖濃度,并根據是否添加PI3K/AKT通路抑制劑LY294002來分為八組：1)5.5mM糖濃度組(5.5mM-)；2)15.5mM糖濃度組(15.5mM-);3)25.5mM糖濃度組(25.5mM-);4)35.5mM糖濃度組(35.5mM-);5)5.5mM糖濃度+LY294002組(5.5mM+);6)1 5.5mM糖濃度+LY294002組(15.5mM+);7)25.5mM糖濃度+LY294002組(25.5mM+);8)35.5mM糖濃度+LY294002組(35.5mM+)；其中,5.5mM糖濃度組(5.5mM)為對照組。甲基噻唑基四唑(MTT)法測定1d,3d的細胞增殖；堿性磷酸酶(ALP)活性檢測3d,7d的細胞分化情況；茜素紅染色觀察7d,14d細胞分泌鈣結節(jié)的情況；熒光實時定量PCR測定Run mRNA和OCN mRNA基因分別在3d、7d的表達；蛋白免疫印跡(Western blot)檢測3d、7d磷酸化蛋白激酶(p—Akt)的表達。體內實驗：糖尿病大鼠種植體周圍骨結合的機制研究33只3月齡雄性健康ZDF(Zucker diabetic fatty)大鼠隨機分為3組,每組11只,每只大鼠植入2顆種植體。A組：對照組,ZDF大鼠皮下注射等量生理鹽水,直接植入種植體(11只大鼠,22顆種植體)；B組：ZDF大鼠接受艾塞那肽緩釋納米球治療(皮下注射,0.74%,0.1m]/100g),并同時植入種植體(11只大鼠,22顆種植體)；C組：ZDF大鼠接受艾塞那肽緩釋納米球治療(皮下注射,0.74%,0.1ml/100g),至血清葡萄糖維持在恒定水平(≤16mmol/L),然后植入種植體(11只大鼠,22顆種植體)。種植手術后,在第7d、14d、30d、60d分批次處死大鼠,外科手術取出雙側股骨,去凈肌肉、粘膜等軟組織,隨后使用切割鉆切取種植體近遠中3mm的范圍的骨塊,從而得到一個包含種植體的長方形骨塊標本(第7d,標本數n＝4個/每組,第14d、30d、60d,標本數分別為n=6個/每組)。通過硬組織切片、HE染色觀察種植體周圍骨代謝狀況,并通過免疫組化檢測種植體周圍整合素α5β1和纖維粘連蛋白的表達。結果：MTT實驗結果顯示,在第24h,與對照組相比,15.5mM-和25.5mM-組的吸光度值均沒有顯著變化(PO.05)；然而,35.5mM-組的吸光度值較對照組顯著降低,差異具有統(tǒng)計學意義(P0.05)；在第72h,與對照組相比較,15.5mM-組的吸光度值有顯著升高,差異具有統(tǒng)計學意義(P0.05)；然而,25.5mM-和35.5mM-組的吸光度值較對照組顯著降低,且差異具有統(tǒng)計學意義(P0.05)。此外,在每一時間點,使用LY294002處理過的四組細胞的吸光度值較對照組均顯著下降,差異具有統(tǒng)計學意義(P0.05)ALP實驗結果顯示,在第3d,與對照組相比較,15.5mM-組、25.5mM-組、35.5mM-組的吸光度值均無顯著變化,差異沒有統(tǒng)計學差異(P0.05),然而,添加LY294002處理的四組細胞的吸光度值較對照組均顯著降低,差異具有統(tǒng)計學意義(P0.05)；在第7d,15.5mM-組表現出最高的吸光度值,且與對照組相比具有統(tǒng)計學差異(P0.05),然而,25.5mM-組和35.5 mM-組的吸光度值較對照顯著降低,差異具有統(tǒng)計學意義(P0.05)。此外,經LY294002處理的四組細胞的吸光度值較對照組顯著降低(P0.05),其中,15.5 mM+組的吸光度值高于5.5 mM+組、25.5 mM+組和35.5 mM+組,且差異具有統(tǒng)計學差異(P0.05)。茜素紅染色結果顯示,在第7d,15.5mM-組鈣結節(jié)的積分吸光度值顯著高于對照組及其他實驗組,差異具有統(tǒng)計學意義(P0.05),同時,25.5mM-組和35.5mM-組鈣結節(jié)的積分吸光度值也要高于對照組,且差異具有統(tǒng)計學差異(P0.05),此外,經LY294002處理的四組細胞的鈣結節(jié)積分吸光度值較對照組均顯著降低,差異具有統(tǒng)計學意義(P0.05)；在第14d,與對照組相比,15.5mM-組鈣結節(jié)的積分吸光度值要顯著增高,差異具有統(tǒng)計學意義(P0.05),然而,25.5mM-組和35.5mM-組鈣結節(jié)的積分吸光度值與對照組相比無顯著差異(P0.05),同樣,經LY294002處理的四組細胞的鈣結節(jié)積分吸光度值較對照組均顯著降低,差異具有統(tǒng)計學意義(P0.05)。成骨相關轉錄因子(Runt-related transcription factor 2, RunX2)表達結果顯示,在第3d,5.5mM+組的基因相對表達量顯著低于對照組,且差異具有統(tǒng)計學意義(P0.05),其余各組的基因相對表達量與對照組相比沒有明顯差異(P0.05)；在第7d,15.5mM-組的基因相對表達量較對照組明顯增高,差異具有統(tǒng)計學意義(P0.05),同時,15.5mM+組的基因相對表達量與對照組相比無顯著差異(P0.05),其余各組的基因相對表達量均較對照組顯著降低(P0.05)。鋅指結構轉錄因子(Osterix)表達結果顯示,在第3d,5.5mM+組的基因相對表達量顯著低于對照組,且差異具有統(tǒng)計學意義(P0.05),15.5mM-組的基因相對表達量顯著高于對照組,且差異具有統(tǒng)計學意義(P0.05),其余各組的基因相對表達量與對照組相比沒有明顯差異(P0.05)在第7d,15.5mM-組及15.5mM+組的基因相對表達量較對照組均明顯增高,差異具有統(tǒng)計學意義(P0.05),同時,5.5mM+組的基因相對表達量與對照組相比明顯降低,且有統(tǒng)計學意義(P0.05),其余各組的基因相對表達量與對照組相比沒有明顯差異(P0.05)。骨橋蛋白(Osteoprotegerin, OPN)結果顯示,在第3d與7d兩個時間點各組的變化趨勢一致,15.5mM-組、25.5mM-組及15.5mM+組的基因相對表達量顯著高于照組,且差異具有統(tǒng)計學意義(P0.05),35.5mM+組的基因相對表達量與對照組相比顯著降低,且差異具有統(tǒng)計學意義(P0.05),其余各組的基因相對表達量與對照組相比沒有明顯差異(P0.05)。骨鈣蛋白(osteocalcin, OCN)結果顯示,在第3d,15.5mM-組的基因相對表達量顯著高于對照組,且差異具有統(tǒng)計學意義(P0.05),5.5mM+組的基因相對表達量顯著低于對照組,且差異具有統(tǒng)計學意義(P0.05),其余各組的基因相對表達量與對照組相比沒有明顯差異(P0.05)；在第7d,15.5mM-組、25.5mM-組及25.5mM-組的基因相對表達量較對照組明顯增高,差異具有統(tǒng)計學意義(P0.05),同時,5.5mM+組、25.5mM+組及35.5mM+組的的基因相對表達量顯著低于對照組,且差異具有統(tǒng)計學意義(P0.05),15.5mM+組的基因相對表達量與對照組相比無顯著差異(P0.05)。蛋白免疫印跡(Western blot)檢測P-Akt的表達結果顯示,在第3d,15.5mM-組的P-AKT表達量顯著高于對照組,且差異具有統(tǒng)計學意義(P0.05),25.5mM-組的P-AKT的表達量較對照組無明顯變化(P0.05),其余各組的P-AKT表達量均較對照組顯著降低(P0.05)；在第7d,15.5mM-組和25.5mM-組的P-AKT表達量較對照組無顯著差異(P0.05),其余各組的P-AKT表達量均較對照組顯著降低(P0.05)。硬組織切片觀察結果顯示,術后30天,A組：骨沒有填滿種植體與骨組織之間的空腔；B組：在種植體與骨組織之間的空腔中,填滿了新形成的編織骨,且骨與種植直接接觸,沒有纖維組織細胞的干擾；C組：和B組相比較,新形成的編織骨更加緊致規(guī)則,種植體表面的骨開始重建并出現類骨質沉淀。術后60天,C組的種植體周圍包繞著非常致密的骨組織,可見骨細胞沉積在板層骨上,新舊骨組織之間可觀察到結合線；A組和B組的骨-種植體結合狀況與C組類似。HE染色結果顯示：術后7天：三組均未見明顯的新生骨,并且有大量的炎性細胞浸潤；術后14天,對照組和實驗組均未見明顯的新生骨,可見較多活躍的破骨細胞,C組的炎性細胞明顯少于A組和B組,且C組的松質骨較其他兩組更為致密。A組和B組的破骨細胞數目在14天后達到峰值,C組在第7天達到峰值,并隨時間逐漸下降；但C組的平均破骨細胞數目較A組、B組沒有顯著差異,p0.05。免疫組化染色觀察種植體周圍整合素α5β1及纖維連接蛋白的表達結果顯示,纖維連接蛋白及其受體主要在種植體周圍骨組織中及破骨細胞表面表達,而整合素α5β1則主要在成骨細胞表面表達。纖維連接蛋白及整合素α5β1在C組中的表達要高于其他兩組。整合素α5β1的光密度值表達顯示,在整個實驗過程中,C組種植體周圍骨組織中整合素α5β1的表達要顯著高于A組和B組(p=0.003)。在第7天,A組和B組的整合素α5β1表達量相同(p0.05),但是,在第14天,B組的整合素α5β1表達量要顯著高于A組(p=0.027),第30天以后,A組和B組的差異沒有統(tǒng)計學意義(p0.05)。纖維粘連蛋白的光密度值表達顯示,在整個實驗過程中,C組種植體周圍骨組織中纖維粘連蛋白的光密度值表達顯著高于A組和B組(p=0.012)。在第7天、14天、30天,A組和B組的纖維粘連蛋白沒有統(tǒng)計學差異(p0.05),但是,在第60天開始,B組的纖維粘連蛋白表達量要顯著高于A組(p=0.001)。結論：1.適當的高糖(如15.5mM)能促進MC3T3-E1細胞的增殖及成骨分化,過高的糖濃度(25.5 mM和35.5 mM)則起抑制作用；2.P13K/Akt信號通路在成骨細胞的增殖、成骨分化等生理過程中起到重要作用；3.血糖濃度得到穩(wěn)定控制的糖尿病大鼠,較對照組更早的開始成骨代謝,其種植體周圍能形成良好的骨結合；4.整合素α5 β1及纖維粘連蛋白在種植體-骨結合過程中起到重要調節(jié)作用,糖尿病得到良好控制的大鼠表現出更高的整合素α5β1及纖維粘連蛋白的表達。
[Abstract]:Background and purpose: oral implant is the most effective way to treat the patients with tooth deficiency. The "bone binding Osseointegration" concept proposed by B ranemark in 1971 has become the most important criterion for the success of the implant. A stable bone binding depends on the adhesion and proliferation of osteoblasts on the surface of the implant. This process is affected by a variety of local and systemic factors. Diabetes as a relative contraindication of dental implant therapy has attracted the attention of scholars. It is generally recognized as one of the most important factors affecting implant bone union. This study intends to observe in vitro high glucose on MC3T3-E1 cell proliferation, osteogenic differentiation, and analysis of PI3K/Akt signal pathway in the regulation mechanism; from in vivo experiments, through the model of diabetic rats, detect the blood glucose levels of rats bone around the implant and changes of integrin alpha 5 beta 1 and fibronectin (the expression level of FN), comprehensive evaluation of the impact of the mechanisms of diabetes mellitus combined with bone implant. Materials and methods: in vitro: the PI3K/AKT pathway in osteogenic differentiation of MC3T3-E1 cells under high glucose condition according to the different literature selected four kinds of radical concentrations of glucose: 5.5mmol/L, 15.5mmol/L, 25.5mmol/L, 35.5mmol/L, to simulate the normal physiological concentration of glucose in the body and in different degrees of high blood glucose concentration, and according to whether adding PI3K/AKT to LY294002 pathway inhibitors into eight groups: 1) 5.5mM glucose concentration group (5.5mM-); 2) 15.5mM sugar concentration group (15.5mM-); 3) 25.5mM sugar concentration group (25.5mM-); 4) 35.5mM sugar concentration group (35.5mM-); 5) 5.5mM sugar concentration +LY294002 group (5.5mM+); 6) +LY294002 group (1 5.5mM glucose concentration 15.5mM+); 7) 25.5mM sugar concentration +LY294002 group (25.5mM+); 8) 35.5mM sugar concentration +LY294002 group (35.5mM+); the 5.5mM sugar concentration group (5.5mM) as the control group. Methyl thiazolyl four triazole (MTT) method for the determination of 1D, 3D cell proliferation; alkaline phosphatase (ALP) detection of 3D activity, cell differentiation of 7D; alizarin red staining 7d, 14d cells secrete calcium nodules; Determination of fluorescence real-time quantitative PCR gene Run mRNA and OCN mRNA were expressed in 3D and 7d Western blot (Western; blot) for detection of 3D and phosphorylation of 7D protein kinase (P Akt) expression. In vivo experiment: the mechanism of osseointegration around implants in diabetic rats. A total of 33 healthy male ZDF (Zucker diabetic fatty) rats were randomly divided into 3 groups, 11 rats in each group, and 2 implants in each of the 3 month old rats. A group: control group, ZDF rats were injected with normal saline, direct implants (11 rats, 22 implants); group B: ZDF rats received exenatide release nanospheres treatment (subcutaneous injection, 0.74%, 0.1m]/100g), and at the same time implants (11 rats rats, 22 implants); group C: ZDF rats received exenatide release nanospheres treatment (subcutaneous injection, 0.74%, 0.1ml/100g), and serum glucose maintained at a constant level (less than 16mmol/L), and then implants (11 rats, 22 implants). Implant after surgery, in 7d, 14d, 30d, 60d rats were killed in batches, surgical removal of bilateral femur to net muscle, mucosa and soft tissue, then use bone implant drill cutting range cut near 3mm, resulting in a rectangular bone implant containing specimens (section 7d, the number of specimens of n = 4 / n, 14d, 30d, 60d, the number of specimens were n=6 / N). The bone metabolism status around the implants was observed by hard tissue slices and HE staining. The expression of integrin alpha 5 beta 1 and fibronectin around the implants were detected by immunohistochemistry. Results: MTT results showed that in the 24h, compared with the control group, 15.5mM- group and 25.5mM- absorbance values showed no significant change (PO.05); however, the absorbance of 35.5mM- group was significantly lower than the control group, the difference was statistically significant (P0.05); in the 72h, compared with the control group, the absorbance of 15.5mM- the value of the group increased significantly, the difference was statistically significant (P0.05); however, the absorbance of 25.5mM- and 35.5mM- group was significantly lower than the control group, and the difference was statistically significant (P0.05). In addition, at each time point, the absorbance using four groups of cells treated with LY294002 values were significantly decreased compared with the control group, the difference was statistically significant (P0.05) ALP experimental results show that, in the 3D, compared with the control group, 15.5mM- group, 25.5mM- group and 35.5mM- group absorbance values were not significant change, no statistically significant difference between the absorbance (P0.05), however, adding four groups of cells treated with LY294002 were significantly lower than the control group, the difference was statistically significant (P0.05); in the 7d, the 15.5mM- group showed the highest absorbance value, and compared with the control group with significant difference (P0.05), however. 25.5mM- group and mM- group of 35.5 absorbance values significantly decreased, the difference was statistically significant (P0.05). In addition, the absorbance values of the four groups treated by LY294002 were significantly lower than those of the control group (P0.05), and the absorbance values of the 15.5 mM+ group were higher than those of the 5.5 mM+ group, the 25.5 mM+ group and the 35.5 mM+ group, and the difference was statistically significant (P0.05). Alizarin red staining showed that in the 7d group, 15.5mM- integral absorbance value of calcium nodules was significantly higher than the control group and the experimental group, the difference was statistically significant (P0.05), at the same time, the integral absorbance of 25.5mM- group and 35.5mM- group of calcium nodules value is higher than that of the control group, and the difference had statistical differences (P0.05) in addition. The integral absorbance of calcium nodules, four groups of cells treated with LY294002 were significantly lower than the control group, the difference was statistically significant (P0.05); in the 14d, compared with the control group, 15.5mM- group of integral absorbance value to calcium nodules increased significantly, the difference was statistically significant (P0.05), however, the integral absorbance of 25.5mM- group 35.5mM- group and calcium nodules were compared with the control group had no significant difference (P0.05), also, the calcium nodules integral absorbance of four groups of cells treated with LY294002 were significantly lower than the control group, the difference has statistical significance Yi (P
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R587.2;R783

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相關期刊論文 前8條

1 趙晶蕾;江凌勇;房兵;;叉頭框蛋白O1在骨改建中的作用[J];國際口腔醫(yī)學雜志;2014年02期

2 楊璐;魏守海;;鵝FoxO1基因cDNA序列的克隆及組織表達[J];中國畜牧獸醫(yī);2014年08期

3 彭娟;鄧夏青;敖鈺舒;袁建平;;巖藻黃素抗肥胖和抗糖尿病活性研究進展[J];現代食品科技;2015年09期

4 朱允和;周海寧;楊波;張永恒;;FoxO1基因研究進展[J];中華實用診斷與治療雜志;2014年05期

5 王玉霞;趙陽;鄧霖;遲希明;索琳娜;紀紅梅;;遼寧地區(qū)漢族人群中FoxO1基因rs17446614多態(tài)性與2型糖尿病的相關性[J];中國生物制品學雜志;2014年08期

6 辛雪;蘇琳;靳燁;;肌纖維及其相關基因對肉品質的影響[J];食品工業(yè);2014年09期

7 徐碧林;申甜;喻明;汪紅平;章志建;朱近悅;;白藜蘆醇對游離脂肪酸誘導的胰島β細胞凋亡的影響[J];山東醫(yī)藥;2015年08期

8 鄒磊;王玉華;蔡春友;魏鳳江;楊付花;焦紅肖;凌超;時文濤;李衛(wèi)東;;PCAF通過調控FOXO1活性參與3T3-L1前脂肪細胞分化[J];天津醫(yī)科大學學報;2015年03期

相關博士學位論文 前10條

1 楊曉紅;miR-705對絕經后骨質疏松小鼠的骨髓間充質干細胞分化功能異常的調控作用[D];第四軍醫(yī)大學;2013年

2 宋丹丹;蛋白酪氨酸磷酸酶1B抑制成脂并介導TNFα在肥胖中的作用[D];第二軍醫(yī)大學;2013年

3 沈寧;早期應激蛋白Egr-1及其相關信號通路調控機體血糖穩(wěn)態(tài)的研究[D];南京大學;2013年

4 孫文星;二花臉豬皮下與肌內脂肪組織基因表達譜比較及脂肪差異沉積調控機制初探[D];南京農業(yè)大學;2013年

5 江書忠;豬脂肪沉積關鍵基因的篩選及鋅指蛋白KLF13的功能研究[D];華中農業(yè)大學;2014年

6 楊莘;PI3K/Akt信號通路與天然免疫限制性因子SAMHD1在PRRSV感染過程中的作用研究[D];中國農業(yè)科學院;2014年

7 曾轉萍;HDAC、SIRT1基因多態(tài)性及環(huán)境因素與2型糖尿病關系的病例對照研究[D];南方醫(yī)科大學;2014年

8 楊曉紅;miR-705對絕經后骨質疏松小鼠的骨髓間充質干細胞分化功能異常的調控作用[D];第四軍醫(yī)大學;2013年

9 朱淑斌;姜曲海豬肉質特性及其分子遺傳學基礎的研究[D];揚州大學;2013年

10 臧坤;Brd2在3T3-L1前脂肪細胞向脂肪細胞分化過程中的作用及機制研究[D];復旦大學;2012年

相關碩士學位論文 前10條

1 宋佳巍;營養(yǎng)水平對不同品種鵝生長發(fā)育及MyoG、Foxo1基因表達的影響[D];吉林農業(yè)大學;2013年

2 陳小龍;衰老小鼠視網膜中相關基因的表達及山茱萸多糖的干預作用[D];佳木斯大學;2013年

3 羅瑋;n-6/n-3多不飽和脂肪酸構成對3T3-L1脂肪細胞脂聯素和PPARγ表達的調節(jié)研究[D];南方醫(yī)科大學;2013年

4 楊威;白藜蘆醇對糖尿病大鼠骨折愈合過程的影響[D];遼寧醫(yī)學院;2013年

5 熊燕;C/EBPβ和FoxO1通過反饋環(huán)和蛋白互作調控豬前體脂肪細胞分化[D];西北農林科技大學;2013年

6 孫雨佳;秦川牛FoxO1基因遺傳變異及組織表達譜分析[D];西北農林科技大學;2013年

7 滕云;小鼠排卵前卵泡閉鎖過程中FSH對FOXO1基因的表達調控[D];南京農業(yè)大學;2013年

8 李凡;磷酸化FoxO1在高糖誘導人腎小管上皮細胞脂質沉積中的作用[D];河北醫(yī)科大學;2014年

9 楊文靜;小鼠DsbA-L基因的轉錄調控機制研究[D];蘇州大學;2014年

10 劉文秀;FOXC2、APOE、eNOS基因多態(tài)性與云南省納西族2型糖尿病的相關性研究[D];昆明醫(yī)科大學;2014年

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