本文選題：生長板 + 骨化��； 參考：《山西醫(yī)科大學(xué)》2016年碩士論文

【摘要】：目的:1、觀察條件性敲除Ihh對小鼠股骨、脛骨長度的影響;2、觀察Ihh基因條件性敲除小鼠生長板開始骨化前后一天生長板組織形態(tài)學(xué)變化,以期觀察到四個區(qū)的演變過程;3、初步探討Ihh對生長板骨化早期的影響機(jī)制;4、為Ihh異常導(dǎo)致的相關(guān)疾病如:BDA1、骨關(guān)節(jié)炎、骨質(zhì)疏松等疾病的防治提供理論依據(jù)。方法:1、取基因型為Col2α1-Cre ERT2;Ihhfl/fl的剛出生小鼠28只,隨機(jī)分為實驗組Ta組(6天組)、對照組Ca組(6天組),每組14只。再取Col2α1-Cre ERT2;Ihhfl/fl的小鼠12只,隨機(jī)分為實驗組Tb組(8天組)、對照組Cb組(8天組),每組6只。實驗組于出生后第一天開始腹腔注射玉米油溶解的TM(Tamoxifen)連續(xù)注射3天。對照組腹腔注射等量的玉米油作為對照。2、RT-PCR檢測實驗組Ta組、對照組Ca組,脛骨近端骨骺中Ihh基因m RNA的相對表達(dá)量,明確其敲除率(n=6)。3、測量Ta、Ca、Tb、Cb組左股骨、左脛骨長度,觀察對股骨、脛骨長度的影響(n=6)。4、小鼠左脛骨近端X照射及組織切片Von kossa染色,明確實驗組脛骨近端生長板開始骨化的前一天。5、小鼠左脛骨近端生長板組織切片番紅O染色,觀察脛骨近端生長板形態(tài)學(xué)變化。6、RT-PCR檢測實驗組小鼠脛骨近端生長板開始骨化前一天,脛骨近端骨骺中RUNX2基因m RNA表達(dá)水平的變化(n=6)。結(jié)果:1、實驗組Ta組骨骺中Ihh基因表達(dá)量明顯低于對照組Cb組(P0.05),基因敲除率為65.17%。2、出生后第6天及出生后第8天,實驗組股骨長度、脛骨長度較對照組股骨長度、脛骨長度明顯縮短(P0.05)。3、脛骨近端X線及生長板組織切片Von Kossa染色均發(fā)現(xiàn)小鼠在出生后第6天,生長板均未發(fā)生骨化,而實驗組出生后第8天,生長板骨化明顯。確認(rèn)條件性敲除Ihh生長板開始骨化前一天為出生后第6天。4、實驗組番紅O染色顯示:生長板組織結(jié)構(gòu)紊亂,增殖區(qū)消失,未發(fā)現(xiàn)典型的排列成柱狀的增殖區(qū)細(xì)胞,生長板被肥大軟骨細(xì)胞所占據(jù),蛋白聚糖分泌減少,分布紊亂。對照組脛骨近端生長板組織結(jié)構(gòu)正常。5、實驗組開始骨化前一天脛骨近端骨骺組織RUNX2基因表達(dá)量明顯高于對照組(P0.05)。結(jié)論:條件性敲除生長板來源的Ihh導(dǎo)致小鼠脛骨、股骨長度縮短,脛骨近端生長板組織結(jié)構(gòu)紊亂、生長板骨化提前,生長板中RUNX2表達(dá)提高。敲除生長板來源的Ihh,生長板骨化提前的機(jī)制可能與RUNX2的表達(dá)相關(guān),為Ihh異常導(dǎo)致的相關(guān)疾病如:BDA1、骨關(guān)節(jié)炎、骨質(zhì)疏松等疾病的防治提供了理論依據(jù)。
[Abstract]:Objective to observe the effect of conditioned knockout Ihh on the length of femur and tibia in mice and observe the histomorphologic changes of growth plate of Ihh gene conditioned knockout mice before and after ossification. In order to observe the evolution process of the four regions, the influence mechanism of Ihh on the early ossification of the growth plate was discussed preliminarily, which provided the theoretical basis for the prevention and treatment of diseases related to the abnormal Ihh such as: BDA1, osteoarthritis, osteoporosis and so on. Methods Twenty eight newly born mice with the genotype Col2 偽 1-CRE ERT _ 2 Ihhfl-fl were randomly divided into experimental group Ta (6-day group) and control group Ca (6-day group) with 14 mice in each group. Twelve mice of Col2 偽 1-CRE ERT2Ihfl-fl were randomly divided into experimental group (TB group) (8 days group) and control group (CB group (8 days group) with 6 mice in each group). The experimental group was injected intraperitoneally with TM (Tamoxifen) on the first day after birth. The relative expression of Ihh mRNA in the proximal tibia epiphysis of the control group (Ca group) and the control group (Ta group) were detected by intraperitoneal injection of the same amount of corn oil as control group. The knockout rate of Ihh gene was determined. The left femur and length of the left tibia were measured in the TahCaTbTbCb group, and the relative expression of Ihh mRNA in the proximal tibial epiphysis of the control group was determined. The effect on the length of femur and tibia was observed (nong6) .4. the proximal tibia of mice was irradiated by X ray and by Von kossa staining. It was determined that the proximal tibia growth plate of experimental group was ossified on the day of ossification, and that the tissue section of mouse left tibia proximal tibia was stained with fenflon O, the other day before the ossification of the proximal tibia. Morphological changes of proximal tibia growth plate were observed by RT-PCR. The expression of RUNX2 gene mRNA in proximal tibial epiphysis was detected one day before ossification of proximal tibia growth plate in experimental group (nong6). Results the expression of Ihh gene in epiphysis of Ta group in experimental group was significantly lower than that in control group (P0.05). The knockout rate was 65.17.2.The length of femur and tibia in experimental group was higher than that in control group on the 6th day after birth and the 8th day after birth. The length of tibia was significantly shortened (P0.05). The proximal tibia and Von Kossa staining showed that there was no ossification of the growth plate in the mice on the 6th day after birth, but in the experimental group, the ossification of the growth plate was obvious on the 8th day after birth. It was confirmed that the first day before the initiation of ossification of the Ihh growth plate was on the 6th day after birth. In the experimental group, the tissue structure of the growth plate was disordered, the proliferative zone disappeared, and no typical columnar proliferating zone cells were found. The growth plate is occupied by mast chondrocytes, the secretion of proteoglycan is decreased and the distribution is disordered. The expression of RUNX2 gene in the proximal tibia epiphysis of the control group was significantly higher than that in the control group one day before ossification (P0.05). Conclusion: conditional knockout of Ihh from growth plate can shorten the length of femur, disorder the tissue structure of proximal tibia, advance the ossification of growth plate, and increase the expression of RUNX2 in the growth plate. The mechanism of early ossification of growth plate may be related to the expression of RUNX2, which may provide a theoretical basis for the prevention and treatment of diseases such as: BDA1, osteoarthritis, osteoporosis and so on.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R68

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