条件性敲除Indian Hedgehog对出生后小鼠生长板骨化早期的影响

发布时间：2018-07-07 07:30

本文选题：生长板 + 骨化　；参考：《山西医科大学》2016年硕士论文

【摘要】：目的:1、观察条件性敲除Ihh对小鼠股骨、胫骨长度的影响;2、观察Ihh基因条件性敲除小鼠生长板开始骨化前后一天生长板组织形态学变化,以期观察到四个区的演变过程;3、初步探讨Ihh对生长板骨化早期的影响机制;4、为Ihh异常导致的相关疾病如:BDA1、骨关节炎、骨质疏松等疾病的防治提供理论依据。方法:1、取基因型为Col2α1-Cre ERT2;Ihhfl/fl的刚出生小鼠28只,随机分为实验组Ta组(6天组)、对照组Ca组(6天组),每组14只。再取Col2α1-Cre ERT2;Ihhfl/fl的小鼠12只,随机分为实验组Tb组(8天组)、对照组Cb组(8天组),每组6只。实验组于出生后第一天开始腹腔注射玉米油溶解的TM(Tamoxifen)连续注射3天。对照组腹腔注射等量的玉米油作为对照。2、RT-PCR检测实验组Ta组、对照组Ca组,胫骨近端骨骺中Ihh基因m RNA的相对表达量,明确其敲除率(n=6)。3、测量Ta、Ca、Tb、Cb组左股骨、左胫骨长度,观察对股骨、胫骨长度的影响(n=6)。4、小鼠左胫骨近端X照射及组织切片Von kossa染色,明确实验组胫骨近端生长板开始骨化的前一天。5、小鼠左胫骨近端生长板组织切片番红O染色,观察胫骨近端生长板形态学变化。6、RT-PCR检测实验组小鼠胫骨近端生长板开始骨化前一天,胫骨近端骨骺中RUNX2基因m RNA表达水平的变化(n=6)。结果:1、实验组Ta组骨骺中Ihh基因表达量明显低于对照组Cb组(P0.05),基因敲除率为65.17%。2、出生后第6天及出生后第8天,实验组股骨长度、胫骨长度较对照组股骨长度、胫骨长度明显缩短(P0.05)。3、胫骨近端X线及生长板组织切片Von Kossa染色均发现小鼠在出生后第6天,生长板均未发生骨化,而实验组出生后第8天,生长板骨化明显。确认条件性敲除Ihh生长板开始骨化前一天为出生后第6天。4、实验组番红O染色显示:生长板组织结构紊乱,增殖区消失,未发现典型的排列成柱状的增殖区细胞,生长板被肥大软骨细胞所占据,蛋白聚糖分泌减少,分布紊乱。对照组胫骨近端生长板组织结构正常。5、实验组开始骨化前一天胫骨近端骨骺组织RUNX2基因表达量明显高于对照组(P0.05)。结论:条件性敲除生长板来源的Ihh导致小鼠胫骨、股骨长度缩短,胫骨近端生长板组织结构紊乱、生长板骨化提前,生长板中RUNX2表达提高。敲除生长板来源的Ihh,生长板骨化提前的机制可能与RUNX2的表达相关,为Ihh异常导致的相关疾病如:BDA1、骨关节炎、骨质疏松等疾病的防治提供了理论依据。
[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 伪 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 伪 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.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R68

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