| [Background] Casein kinase 2 interacting protein 1(The casein kinase 2 interacting protein 1,CKIP-1)gene is highly conservative,which were expressed in the vast majority of adult and embryonic tissues.Its translation protein is a key factor regulating the subcellular localization and the activity of various proteins,and plays a key role in the occurrence and development of various diseases by participating in the regulation of cell morphology,proliferation,differentiation,apoptosis and migration.In 2008,Lu K et al.found that CKIP-1 is a negative regulator of bone formation,which can regulate bone mass change and osteoblastic activity after birth.Based on this characteristic,Zhang G,et al.invented a bone tissue targeted RNA interference technology in 2012,specifically silencing CKIP-1 gene in bone tissue of rats.This method significantly promoted bone formation,enhanced bone microstructure and increased bone mass in healthy rats and osteoporosis rats.Subsequently,a number of laboratories also reported the application of CKIP-1 in distraction osteogenesis and osseointegration of titanium implants,and achieved promising results.This suggests that CKIP-1 is a potential target molecule for the treatment of osteoporosis and other bone-related diseases.As a potential target molecule for clinical application,we need to consider not only its efficacy,but also its application complication.A large number of literatures show that CKIP-1 has obvious effects on many other tissues and organs besides bone tissue.Studies have confirmed that CKIP-1 may have a certain correlation with cardiac hypertrophy,cerebral hemorrhage,lung cancer,gastric cancer,colon cancer,diabetic nephropathy,arteriosclerosis,fatty liver and other diseases.Therefore,when treating osteoporosis with CKIP-1,it may have serious adverse effects on other important tissues and organs.However,the current research on the effects of CKIP-1 on these tissues and organs has some limitations: Firstly,studies on craniomaxillofacial tissues and organs besides the craniomaxillofacial bone by CKIP-1 are lacking.Preliminary experiments in this project found that CKIP-1 gene knockout mice also had changes in craniomaxillofacial morphology,such as round blunt face shape,microear deformity,which suggested that CKIP-1 may have an impact on craniomaxillofacial tissues and organs.A large number of studies have confirmed that,because of the embryonic development sources of craniomaxillofacial and whole body tissues are different,the regulation of the same molecule on similar tissues in these two parts are different.Therefore,it is also instructive to conduct a comparative study of craniomaxillofacial and whole body tissues to further discuss the influence of key genes on tissues in different parts.Secondly,most of these studies focus on the cellular and molecular level,lacking histological evaluation of the animal as a whole.Histological analysis is the gold standard of tissue structure analysis,pathological examination and disease diagnosis.Thirdly,previous reports mainly build disease models,and there are few studies on the effects of CKIP-1 on various tissues and organs in healthy state.[Purpose] In this study,the key gene of CKIP-1 was studied by comparing the CKIP-1 gene knockout mice with wild-type mice,and 11 craniomaxillofacial and 15 important tissues and organs of the whole body were preliminarily screened through general observation.The differences of hard tissues(bone and teeth)in mice were analyzed by imaging.And above tissues and organs were evaluated by histological methods.It is expected to provide experimental basis for the application of this molecule to target a single tissue(such as bone tissue)for treatment,to prevent its adverse effects on other tissues and organs,and to provide reference for the research on craniomaxillofacial cartilage,fat,skin and other tissues concerned by this discipline.[Method] 1.The gross phenotypic observation method was used to compare CKIP-1-knockout mice with wild-type mice,focusing on the comparison of body shape,appearance,body length,weight,fertility rate,serum biochemical detection,diet and behavior of mice.2.Micro-CT technique was used to analyze the differences among the parietal bone,nasal bone,vertebra,incisor and molar of mice,to determine whether the change trend of craniomaxillofacial hard tissues of mice was consistent with that of the whole body.3.Histological staining method was used to assess the changes of tissues and organs.Toluidine blue staining,alizarin red staining and safranin fast green staining were used to analyze chondrocyte differentiation,matrix secretion and mineralization in ear,nose,intervertebral disc and the knee joint.HE staining,Masson staining and immunohistochemical staining were used to analyze the change of collagen fiber and important molecular expression in skins and mucosae.HE staining was used to analyze the change of muscle fibers,adipocyte size,fat accumulation condition of liver and the histological change of testis,epididymis,ovary,brain,eye,parotid gland,spleen,lung and kidney.The histological differences of tissues and organs between CKIP-1-knockout mice and wild-type mice were systematically analyzed.[Result] 1.Gross phenotypic observation showed that CKIP-1-knockout mice had increased body length,body size,auricle deformity,stiff spine,loose,white and dull hair with harsh handle,normal diet,but some mice were slightly slow and sluggish.The body weight of CKIP-1-knockout mice was statistically significant.Statistical analysis of fertility rate showed that the average number of litter size of CKIP-1-knockout mice was lower than that of wild-type mice.No significant difference was found in serum biochemical examination of cholesterol,riglyceride,HDL and LDL.2.Through the clue of " increasing body length",we focused on the detection of parietal bone,nasal bone,vertebra,incisor tooth and molar of mice.Cancellous bone thickness of CKIP-1-1-knockout mice were increased,suggesting that the craniomaxillofacial bone tissue and the whole body bone change trend were consistent after CKIP-1 knockout.The analysis of incisors and molars in mice showed that after the knockout of CKIP-1 gene,there was no abnormality in the thickness of enamel in mice,but the pulp cavity became smaller and the thickness of dentin increased,showing a statistical difference.3.Through the clue of "loose and white hair with harsh hanfeel",we focused on the detection of skin and mucosa.It was found that the skin of mice with CKIP-1 gene knockout showed obvious fibrosis changes,that is,the skin keratosis increased,the epidermis and dermis thickened,the collagen fibers twisted and thickened,and the arrangement was more dense.The content of collagen in KO mice was significantly increased by Masson staining,with statistical significance.Immunohistochemical staining showed that TGF-β1 and Collagen-1 expression in KO mice dermis increased significantly,especially it is important to note that the TGF-β1 in the dermis and epidermis layer junction expression increased significantly.Labial mucous also appears irregular thickening phenomenon of cutinized layer.4.Through the clue of "auricle deformity and spinal rigidity",we focused on the detection of the ear,nose,trachea and ribs,intervertebral disc and knee joint,which represent three different types of cartilage: elastic cartilage,hyaline cartilage and fibrous cartilage.Abnormal differentiation of chondrocytes in auricular and nasal alar cartilage was found after CKIP-1 gene knockout,and cartilage matrix changes and abnormal mineralization were found in trachea,ribs,intervertebral disc and knee joint.5.Through the clue of "enlarged body size",we focused on Tongue muscle,myocardial muscle and vascular smooth muscle.It was found that the weight and size of the heart of KO mice were significantly increased compared with that of WT mice,and all the three muscle fibers were more robust,compact and less spaced.6.Through the clue of "weight gain",we focused on the detection of adipose tissue and the liver which is prone to fat accumulation.Mice aged 2 months,6 months and 9 months were selected to represent human adolescents,middle-aged and elderly,respectively.It was found that the adipocyte size of KO mice aged 2 months and 6 months increased,while the adipocyte size of KO mice aged 9 months was not significantly different from that of WT mice.However,in the healthy physiological state,no fat accumulation was found in the liver of KO mice,only the widening of the hepatic cord.7.Through the clue of "low fertility",we focused on the reproductive organs,namely the testes,epididymis and ovaries.However,no obvious abnormality was found in the initial morphological and histological analysis.Brain,eye,parotid gland,spleen,lung and kidney were selected for preliminary screening,and the number of glial cells was increased.The lymphatic follicles of the spleen are fused and irregular.Pulmonary alveoli capillary network is dense.There were no significant differences among eyes,parotid gland and kidney.[Conclusion] As a star molecule,the study of CKIP-1 on bone has entered the application stage,so it is important to study of its application complications,especially its adverse effects on other tissues and organs other than bone needs to be focused on.Therefore,the systematic screening of tissues and organs is of great significance.At the same time,as the first step of the follow-up extended project,it is also a key starting point.The project provides clues and experimental basis for the next research direction from phenotypic screening,imaging observation,histological analysis and preliminary mechanism discussion.Based on the above experimental results,suggesting the molecular not only has the potential of osteoporosis treatment,it can also has a wide prospect of clinical application in these fileds including cranial and maxillofacial soft tissue repair,treatment of articular cartilage,scar tissue,fat fill,oral implantology and so on,and the related subject in the field of research also has certain guiding significance.The specific conclusions are as follows: 1.CKIP-1 gene can negatively regulate the changes in bone mass of cancellous bone of craniomaxillofacial bone and the whole body in mice,and negatively regulate the dentin mineralization of teeth in mice.2.The knockout of CKIP-1 gene can lead to "abnormal differentiation" and "abnormal mineralization" of cartilage.3.Knockout of CKIP-1 gene can cause skin fibrosis of mice.4.CKIP-1 regulates the morphology and size of different muscle fibers.5.CKIP-1 knockout may accelerate the maturation and differentiation of adipocytes,but it does not affect the pathological changes of liver and fat metabolism. |
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