| Objective:Congenital cataract is defined as existing before or after birth,or in childhood illness.Congenital cataracts can lead to infants amblyopia or even blindness.It is a serious eye disease cause blindness,affecting children's visual acuity seriously,congenital cataract has became the second cause of blindness in children.Congenital cataracts can not only separate the onset but also accompanied by other systemic dysplasia.The current treatment of congenital cataract is mainly through surgery.Muscle Segment Homeobox 2?Msx2?is used to regulate gene transcription.Previous studies on gene Msx2 mainly focused on the development of tissues such as teeth,bones,mammary glands and so on.In recent years,it has been found that abnormal expression of gene Msx2 can affect the development of lens resulting in small eyeball deformity.Studies have shown that lens dysplasia can associated with other tissues dysplasia,indicating that the development of lens is crucial in eye development process.Calcium signaling pathway is mainly involved in maintenance the homeostasis and the development of lens cells.Elevated calcium-dependent proteases are accompanied by an increase in lens intracellular,resulting in irreversible degradation of important intracellular structural proteins,depolymerization of proteins and chelates and an increase in non-soluble proteins.All of these changes will lead to cells apoptosis.Previous studies have shown that the maintenance of intracellular homeostasis depends mainly on calcium-dependent proteases,calcium pump,calcium channel to remove extra from lens cells in maintaining the low concentration of Ca2+.Only in this way,lens cells can hold the normal physiology functions.Calcium signaling pathway can affect lens formation and transparency.At present,there is no report on the mechanism that Msx2 affects lens development in calcium signaling pathway.In this study,based on the foundation that Msx2 knockout can lead to the deformity of small eyeball,we explore the mechanism of Msx2 in surface ectoderm impacting eye development on calcium signaling pathway.The results of this study in revealing the molecular basis of congenital cataract and elucidating its pathophysiological mechanism is of great significance.At the same time,this study has important clinical significance and provides some basis and therapeutic targets for the diagnosis and treatment of congenital cataracts.Methods:1.Generation of Conditional Deletion of Msx2 Mice:Cre-loxP approach was employed to specifically inactivate Msx2 on the eye surface ectoderm.PCR was performed to detect the genotype of mice with DNA samples extracted from the mice tails or embryo yolk sac.Le-Cre;Msx2flox/flox?Msx2-CKO?mice were used as experimental group.Littermate mice carrying Msx2flox/flox were used as controls.PCR and immunofluorescence techniques were employed to certify gene Msx2 was knocked out in the lens.2.General Observation of Experimental Group and Control Group:Observation mice eyes detail changes in slit lamp after droping on mydriatic medicine.After mice were sacrificed,eyeballs were dissected to observe the differences under the dissecting microscope.Then,eyeballs were dissected to get mice lens in observing the detail differences.The ocular paraffin sections of 2 months mice in experimental and control groups were prepared for observation.Hematoxylin and Eosin?HE?staining were used to observe the morphological differences.3.Observe the Development of Eye in Conditional Deletion of Msx2 Mice:Mice embryos and postnatal mice were used to compare by dissecting microscope.Preparation of Msx2 gene knockout mice embryos and postnatal mouse eye ball paraffin sections,and sections were stained with Hematoxylin and Eosin to observe the development of lens.4.BrdU immunohistochemical analysis:BrdU?100 mg/g body weight?was injected intraperitonealy in pregnant mice for 1 hour before sacrifice.Immunostaining was performed in observing the labeled BrdU+cells subsequently.5.TUNEL Assay of Lens Cell Apoptosis:TUNEL apoptosis kit labeled Msx2 CKO and Msx2 WT mice lens apoptosis cells.Paraffin section underwent dewaxing,rehydration;digestion by protease K incubate at 37?for 20 minutes;using 5ul TdT enzyme+45ul fluorescent labeling solution incubated in dark for 1 hour;DAPI counterstain for 1 minute;slides were mounted with anti-fade medium and photography.6.RNA-seq Analysis of Mice Lens:2 month old mice eyeballs of experimental group and control group were put in DEPC-PBS buffer,removal lens extract surrounding tissues completely and placed in RNA-free EP tubes,marking the genotypes,sending samples to Beijing Novogene Biotechnology Company underwent mRNA sequencing analysis of mice lens.Analysis of the results after results were obtained.7.Quantitative PCR of lens Tissue:Trizol reagent was used to extract the total RNA from the lens of experimental group and control group according to the instructions,then RNA quality were detected by micro spectrophotometer?NanoDrop ND-5000?.After that,two sets of RNA were reverse transcribed into cDNA.The expression level of Capn1,Tgm2,Camk2B,Gja8,crystalline???Cryab?,crystalline??1?Cryba1?,crystalline?b1?Crybb1?and crystalline?b3?Crybb3?were detected by SYBR green method.GAPDH was used as an internal control.At the end of the cycle,the dissolution profile was used to ensure the specificity of the products.Data were obtained from three independent experiments and analyzed based on equation RQ=2-??CT.8.Immunofluorescence:The expression of Capn1,Tgm2,Camk2B,Gja8 were observed in experimental group and control group.Mice embryos paraffin sections underwent dewaxed,rehydrated,antigen-repaired and incubated corresponding antibodies overnight at 4?,secondary antibodies were incubated for two hours at room temperature.DAPI counterstain for 1 minute,slides were mounted with anti-fade medium,merge pictures and take pictures.9.Immunohistochemistry:The expression of Cryab,Cryba1,Crybb1 and Crybb3were observed in experimental group and the control group.Mice embryos paraffin sections underwent dewaxed,rehydrated,antigen-repaired and incubated corresponding antibodies overnight at 4?,secondary antibodies were incubated for two hours at room temperature.DAB staining,dehydration,xylene,slides were mounted with resin,observing slides under microscope and take pictures.10.Plasmids preparation:In this experiment,mice lens epithelial cells??-TN4?cells were selected as transfection vector and pegfp-c1 as empty vector.Vector NTI was used to analyze the monoclonal enzyme cutting site.Sal?and BmaH?were used as the suitable restriction sites.The 804bp Msx2 fragment was synthesized and added with the same restriction enzyme sites in two sections.Then,the empty vector and fragments were double-digested to obtain complementary sticky ends.After that,start linking reactions.Transformation,shaking bacteria,coating,picking monoclonal bacteria,bacterial PCR,sending sequences,transfecting cells after plasmids extracted.11.Cells Transfection:Transfection was started when cells grew to 70%-80%.DMEM-/-and lipofectamin 2000 were mixed in 3:1,experimental and the control plasmid were mixed with lipofectamin 2000 in the same proportion.The plasmid needs to be added dropwise to the transfection reagent,allowing the surface to be fully charged.Both incubated at room temperature for 5min and then two mixtures were mixed at room temperature for 15-20min after adding cells for culture.Because of the strong toxicity of transfection reagent,fresh medium were changed after transfection in 6 hours,and after24 hours transfection results were observed.12.Western Blot:Discarded cell culture and washed cells three times with PBS and then dropped cell lysate on the cells.After scraping off cells,centrifuging and freezing on the ice to collect the lysate,centrifuging again,absorbing the supernatant and quantifying the protein.After protein was quantified,protein electrophoresis,transplanting the s,cut the membranes and blocked.Primary antibodies of Capn1,Tgm2,Camk2B and Gja8were incubated overnight at 4?,washed twice with TBST and incubated second antibody at room temperature for 2 hours.EL star developed after washed the membranes with TBST.13.Transmission Electron Microscopy:We utilized Msx2 CKO and Msx2 WT of P60 for transmission electron microscopy observation.From sacrificing mice to extracting lens,mice eyeballs were carried out in the fixative.Fixed overnightat at 4?,the organizations were sent to the Department of Cell Biology,China Medical University,electron microscope room for observation.Then,the samples were washed in PBS and dehydrated in gradient ethanol series and were embedded in Epon resin.Embedded samples were cut into ultrathin sections and were counterstained with uranyl acetate and lead citrate.All samples were observed using a HITAC H IH-7650 electron microscope.14.Statistical Analysis:GraphPad statistical analysis software for statistical analysis.Data are presented as meanąstandard deviation and analyzed using independent samples T-test.p<0.05 was considered statistically significant.Result:1.In this study,Cre-LoxP was used to perform conditional targeting gene knockout technology.Pax6 promoter-driven Cre recombinase was used to knock out gene Msx2 on the surface ectoderm in mice embryos.The genotypes were identified by PCR.Immunofluorescence showed that expression of Msx2 was absent in the lens of Msx2 CKO mice.2.Compared with the control group,the experimental mice were linear hair defects around the its eyes,reduced eyelid and palpebral fissure,irregular eyelashes and some of them were missing.Slit lamp showed experimental mice pupil can not be dispersed.The experimental group eyeball showed that the volume of eyes were decreased,iris attached to the corneal,lens volume decreased with varying degrees of internal opacity.HE showed that the whole eyeball volume decreased in experimental group compared with the control group.The normal angle structure disappeared,the volume of lens were reduced and the shape was irregular,not round,cloudy and accompanied by many cavities in the middle.Retina were irregular thickening and easy to detachment.The weight of lens in experimental group were less than that of control group,and it was statistically significant.3.From embryonic observation period of 12.5 days to 8 days after birth,compared with the control group Msx2 knockout mice showed varying degrees of small eyeball deformity,congenital cataract and anterior segment dysplasia,the performance of the lens volume was significantly smaller than the control group,the lens cells arranged disorder.The anterior and posterior diameters of lens in the experimental group were all less than those in the control group.The corneal thickness of experimental group became thinner.Retina folding,thickening and detachment were observed in the experimental group.4.TUNEL results showed that the apoptotic rate of Msx2 gene knockout mice was higher than that of the control group from E12.5 and the difference was statistically significant.BrdU labeling immunofluorescence showed that since the observation day E12.5 the number of proliferating cells in control group was higher than the experimental group,the difference between the two groups were gradually reduced and the differences were statistically significant.5.In order to further investigate the molecular mechanism of Msx2 in epidermal ectoderm affecting lens development,we use mRNA sequencing analysis to explore the lens of 2 months old experimental mice and control littermates.We found that there were1911 differentially expressed genes?DEGs?,including 1586 up-regulated genes and 325down-regulated genes.Enrichment analysis results showed as follows:?1?GO?Gene Ontology,GO?enrichment analysis includes three biological networks:biological processes?BP?,cellular components?CC?and molecular functions?MF?.In BP,it was found that the ocular developmental abnormalities ranked Number 5,the complex ion channel ranked the first one was found in CC,the second was the ion channel in MF,the 14th in the calcium ion binding,the lens structural component ranked17th.?2?KEGG?Kyoto Encyclopedia of Genes and Genomes,KEGG?enrichment analysis with eye closely related to the development of calcium signaling pathway,ranked No.4,as the focus of the study.?3?Reactome enrichment analysis showed visual phototransduction ranked eighth.6.Through the analysis of mRNA sequencing,it was found that calcium signaling pathway was closely related to eye development,and Real-Time PCR was used to verify differentially expressed genes at the gene level.Compared with the control group,the expression of Gja8 in the experimental group was down-regulated,the expressions of Capn1,Tgm2 and Camk2B were up-regulated,and the expressions of the crystalline??,crystalline??1,crystalline?b1 and crystalline?b3 genes were down-regulated.The difference was statistically significant.7.Transfecting the prepared Pegfp-c1 and Pegfp-c1-Msx2 plasmids into mice lens epithelial cells??-TN4?.After successful transfected,extracting protein and conducting Western Blot analysis.The results showed that the expression of Gja8 was down-regulated in the transfected Pegfp-c1 plasmid and the expression of Capn1,Tgm2and Camk2B was up-regulated in the transfected Pegfp-c1 plasmid compared with the control group.8.Immunofluorescence results showed that Gja8 was down-regulated in experimental mice at E14.5,and Capn1,Tgm2 and Camk2B were up-regulated in experimental mice.9.The results of immunohistochemistry showed that the expression of crystalline??,crystalline??1,crystalline?b1,crystalline?b3 in the experimental group was down-regulated in the experimental group at E14.5.10.Transmission electron microscopy showed that compared with the experimental group mice lens cells gap junction is not uniform and broaden in experimental group,fat deposition was found in experimental mice lens cells gap junction and cells gap junctional structure was damaged.The experimental group lens cells shape were irregular,disordered,and a wide range of fusion.In contrast,the mice lens cells in the control group had normal morphology and arrangement and normal connections.11.Alternative splicing?AS?includes retained intron?RI?,variable 3'end of alternative 5'splice site?A5SS?Alternative 3'splice site?A3SS?,skipped exon?SE?and mutually exclusive exons?MXE?.RNA-seq showed that among them,A3SS occurred in Capn1,RI occurred in Camk2B,11 species of SE,1 specie of A3SS and 5 species of MXE occurred in Cryba1,2 species of A5SS in occurred Crybb1.RNA-seq results also showed that the biological types of Capn1,Camk2B,UPS,Gja3,Cryaa,Cryab,Cryba1,Cryba4,Crybb1,Crybb2,Crybb3,BF-SP2 have already changed.Conclusion:1.Gene Msx2 was conditional knockout on the surface ectoderm in early stage of mice embryos by conditional gene targeting using Cre-Loxp system,while the expression of Msx2 in other tissues was not affected.2.Msx2 knockout on surface ectoderm lead to lens dysplasia,varying degrees of small-eyeball deformity and severe congenital cataracts.3.Msx2 knockout on surface ectoderm showed lens cells apoptosis increased in experimental group while cells proliferation decreased compared with the control group.4.RNA-seq analysis showed gene Msx2 conditional knockout on surface ectoderm lead to congenital cataract closely related to the signaling pathway.5.Expression of Gja8,Capn1,Tgm2 and Camk2B in calcium signaling pathway were changed in mice lens of conditional knockout gene Msx2 on epidermal ectoderm.The expression of the downstream crystallin and lens-specific protein were changed as well. |
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