Construction of human interleukin-26 lentivirus expression plasmid and gene knockout plasmid and their activity verification in HEK293T cells / 中国生物制品学杂志

@#Objective To construct a lentivirus-based expression plasmid and gene knockout plasmid of human interleukin(IL)-26 so as to lay a foundation of studying the function of IL-26 gene in cell signaling pathway and autophagy.Methods IL-26 gene sequence was amplified from human peripheral blood mononuclear cells by RT-PCR and cloned into pCDH-CMVMCS-EF1-copGFP eukaryotic expression vector to construct overexpression plasmid;Four knockout targets,Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sgRNA2,were designed based on the exon sequence of IL-26,and constructed into lentiCRISPRv2 vector by CRISPR/Cas9 technology to construct gene knockout plasmid. The overexpression plasmid and gene knockout plasmid were transiently transfected into HEK293T cells respectively,and the expression of IL-26 was verified by RT-qPCR and Western blot. In addition,amino acid sequence analysis,structure prediction and subcellular localization observation of IL-26 were performed.Results The results of restriction digestion,sequencing and bioinformatics analysis showed that IL-26 was 516 bp in length,encoding 171 amino acids. The IL-26 mRNA level and protein level of HEK293T cells transfected with IL-26 overexpression plasmid increased by 656. 789 times and 1. 978 times respectively with significant differences as compared with the normal control group(t = 17. 976 and 7. 859,P < 0. 000 1 and < 0. 001,respectively). With the transfection of 4 knockout targets Exon1sgRNA1,Exon1sgRNA2,Exon3sgRNA1 and Exon3sg-RNA2 into HEK293T cells,the expression of IL-26 decreased by 0. 930,0. 980,0. 523 3 and 0. 316 9 times,respectively,among which Exon3sgRNA2 significantly down-regulated the expression of IL-26(t = 7. 440,P < 0. 001). IL-26protein showed signal peptide structure and certain transmembrane function in the first 22 amino acids,which existed in cytoplasm.Conclusion IL-26overexpression and gene knockout plasmids were successfully constructed,which laid a foundation of the follow-up study of the function of IL-26.

Establishment and genotype identification of hepatic stellate cell-specific Grk2 gene knockout mouse model / 中国药理学通报

Aim To establish a stable hepatic stellate cell ( HSC ) -specific G protein-coupled receptor kinase 2 ( GRK2 ) knockout mice and provide the important animal model for further studying the biological function of GRK2 in HSC. Methods The loxP-labeled Grk2 gene mouse (Grk2

Effect of intestinal Metrnl gene knockout on intestinal microbiota and ulcerative colitis / 药学实践杂志

Objective To investigate the effect of intestinal Metrnl on dextran sodium sulfate (DSS)-induced ulcerative colitis mouse model and the regulation mechanism of intestinal microbiota. Methods Different concentrations of DSS (3% DSS and 1% DSS) were used to induce ulcerative colitis on C57 mice to determine the experimental conditions. Intestinal epithelial Metrnl specific knockout mice (Metrnl(-/-)) and its control mice (Metrnl(+/+)) were administrated with 3% DSS for 5 d. Then the survival time, body weight, DAI (disease activity index), colon length and pathological changes in colon tissues were observed. 16S ribosomal RNA gene sequencing was used to detect the composition of intestinal microbiota. Results Compared with 1% DSS, 3% DSS could significantly aggravate ulcerative colitis on C57 mice, such as lower survival rate (P<0.05), more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). After administrated to 3% DSS for 5 d, comparing with Metrnl(+/+) mice, Metrnl(-/-) mice showed more weight loss (P<0.05), higher DAI score (P<0.05), shorter colon length (P<0.05) and higher pathology score (P<0.05). The 16S ribosomal RNA results showed that the diversity of intestinal microbiota in Metrnl(-/-) mice significantly decreased. Furthermore, Bacteroidetes and Proteobacteria significantly decreased, while Firmicutes increased. Conclusion Metrnl could protect the DSS-induced ulcerative colitis mouse through regulating intestinal microbiota.

Effect of ACE2 deletion on vasoconstriction reactivity of aortic segments in mice with tourniquet shock / 中国病理生理杂志

AIM:To observe the effect of angiotensin-converting enzyme 2(ACE2)deletion on vasoconstric-tion reactivity of aortic segments in ACE2 knockout(KO)mice with tourniquet shock(TS).METHODS:The 8-month-old male mice with C57BL/6 background were divided into wild-type(WT)control group,WT-TS group,KO group and KO-TS group,with 10 mice in each group,of which five were used for determination of vascular reactivity,and the other five for the other assays.The hindlimbs of the mice in WT-TS group and KO-TS group were ligated with tourniquet for 2 h and loosened for 4 h.The mice in WT group and KO group were subjected to the same treatment except for tourniquet liga-tion.The vasoconstriction reactivity of the aorta was measured on tensiometer.The morphological damage of the aorta was evaluated by vascular histopathology.Western blot was used to detect the expression of AT1,MAS,ACE and ACE2 pro-teins in aorta.The serum levels of angiotensin(Ang)Ⅱ and Ang-(1-7)were determined by enzyme-linked immunosorbent assay.RESULTS:Compared with WT group,the mice in WT-TS group had lower vascular reactivity to norepinephrine(NE)and obvious vascular lesions.The expression of ACE protein increased significantly(P＜0.01),while the expres-sion of ACE2 decreased(P＜0.05).The expression of AT1 protein in aorta decreased significantly,the expression of MAS protein increased significantly,and the AT1/MAS ratio decreased(P＜0.01).Serum Ang II level increased,serum Ang-(1-7)level decreased,and Ang Ⅱ/Ang-(1-7)ratio increased(P＜0.05).Compared with WT group,vascular reactivity in KO group increased at low concentration of NE(＜10-7 mol/L),and decreased at high concentration(＞10-7 mol/L)without vascular lesion.The expression levels of aortic AT1,MAS and ACE were all elevated(P＜0.05).The serum level of Ang Ⅱ increased(P＜0.05),but the level of Ang-(1-7)had no obvious change.Compared with KO and WT-TS groups,the aortic reactivity in KO-TS group subtracted apparently(P＜0.05),representing its curve shifting to the right obviously.The morphological damage aggravated slightly,and the expression of AT1 and ACE increased slightly in KO-TS group com-pared with WT-TS group(P＜0.05).However,the expression of MAS increased significantly in vascular tissue(P＜0.01).The serum levels of Ang Ⅱ and Ang-(1-7)further increased and decreased,respectively,and the Ang Ⅱ/Ang-(1-7)ratio increased(P＜0.01).CONCLUSION:Deficiency of ACE2 induces severe aortic hyporeactivity to NE during TS,which may be related to the increased imbalance of renin-angiotensin system in ACE2 gene knockout mice.

Mechanism of Renshentang in Treatment of Atherosclerosis Based on Autophagic Effect of TRPV1 on Arterial Smooth Muscle / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of Renshentang， recorded in Synopsis of Golden Chamber， in the treatment of atherosclerosis （AS） based on the autophagic effect of transient receptor potential vanilloid subtype 1 （TRPV1） on arterial smooth muscle. MethodFourteen SPF-grade 8-week-old male C57BL/6J mice were assigned to the normal group and 70 8-week-old apolipoprotein E knockout （ApoE-/-） mice were assigned to the experimental group. The AS model was induced by a high-fat diet in the mice in the experimental group for eight weeks. The model mice were then randomly divided into model group， low-， medium-， and high-dose Renshentang groups （2.715， 5.43， and 10.68 g·kg-1·d-1）， and simvastatin group （0.02 g·kg-1·d-1）. Drug treatment lasted eight weeks. Serum was taken and serum total cholesterol （CHO）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， and high-density lipoprotein cholesterol （HDL-C） levels were measured by assay kits to observe the changes in lipid levels in mice. The aorta was stained with hematoxylin-eosin （HE） to observe the overall pathology of the aortic root and oil red O staining was used to detect the lipid deposition in the aortic plaque and calculate the percentage of the aortic root area to the lumen area. The protein expression of TRPV1， adenylate-activated protein kinase （AMPK）， phosphorylated AMPK （p-AMPK）， autophagy effector-1 （Beclin-1）， and microtubule-associated protein 1 light chain 3 （LC3Ⅱ） in mouse aortic tissues was determined by Western blot. ResultCompared with the normal group， the model group showed increased serum CHO， TG， and LDL-C levels， decreased HDL-C， and increased aortic root plaque area （P<0.01）. Compared with the model group， the Renshentang groups showed decreased levels of CHO， TG， and LDL-C in serum （P<0.05， P<0.01）， especially in the low- and medium-dose Renshentang groups （P<0.01）. Compared with the normal group， the simvastatin group and the Renshentang groups showed reduced aortic root plaque area （P<0.05）， especially in the high-dose Renshentang group （P<0.01）. Compared with the normal group， the model group showed decreased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05， P<0.01）. Compared with the model group， the medium- and high-dose Renshentang groups showed increased relative expression levels of TRPV1， p-AMPK/AMPK， Beclin-1， and LC3Ⅱ/LC3Ⅰ（P<0.05，P<0.01）. ConclusionThe anti-AS effect of Renshentang recorded in Synopsis of Golden Chamber may be achieved by up-regulating TRPV1 expression to restore the level of autophagy mediated by AMPK.

Macrophage MED1 deficiency promotes the development of atherosclerosis in female ApoE and LDLR knockout mice / 西安交通大学学报(医学版)

【Objective】 To study the effect of macrophage mediator 1 (MED1) deficiency on atherosclerosis in female mice. 【Methods】 ApoE knockout (ApoE-/-), LDLR knockout (LDLR-/-), MED1fl/fl, and macrophage MED1 knockout (MED1△Mac) mice were recruited in the study. Two types of mouse model were constructed:ApoE and macrophage MED1 double knockout (MED1△Mac/ApoE-/-) mice and their littermate controls (MED1fl/fl/ApoE-/-). ② LDLR knockout (LDLR-/-) mice receiving bone marrow from MED1△Mac (MED1△Mac→LDLR-/-) or MED1fl/fl (MED1fl/fl→LDLR-/-) mice. Female mice from these two models were fed a Western diet (21% fat and 0.15% cholesterol) for 12 weeks to promote the development of atherosclerosis. Body weight, total cholesterol (TC), and total triglyceride (TG) content in plasma were measured dynamically. After Western diet feeding for 12 weeks, aortic tree and aortic root were collected and hematoxylin-eosin (H&E) and oil red O staining were performed. 【Results】 Plasma TC and TG did not significantly differ between MED1fl/fl/ApoE-/- control group and MED1△Mac/ApoE-/-experimental group. However, the plaque area in aortic tree and aortic root was significantly increased in MED1△Mac/ApoE-/-mice. Moreover, compared with that in MED1fl/fl→LDLR-/- control group, the plaque area of aortic tree and aortic root had an increasing trend in MED1△Mac→LDLR-/- mice group. 【Conclusion】 MED1 deficiency in macrophages promotes the development of atherosclerosis in female ApoE or LDLR knockout mice.

Development and application of a rapid gene manipulating toolbox for Pseudomonas aeruginosa / 生物工程学报

Manipulation of genes, including knock-out or knock-in, replacement of gene elements (such as promoters), fusion with a fluorescent protein gene, and construction of in situ gene reporter, is required in most of the biotechnological laboratories. The widely used gene manipulating methods based on two-step allelic exchange are cumbersome in terms of constructing plasmids, transforming and screening. In addition, the efficiency of using this method for long fragment knockout is low. To simplify the process of gene manipulation, we constructed a minimized integrative vector pln2. When a gene needs to be inactivated, an internal fragment of the target gene (non-frameshift) is cloned into the pln2 plasmid. Once the single-crossover recombination between genome and the constructed plasmid occurs, the endogenous gene is segmented by the plasmid backbone and thus inactivated. We developed a toolbox based on pln2 that can be used for different genomic operation mentioned above. With the help of this toolbox, we successfully knocked out large fragments of 20-270 kb.

Generation of Mlk3 KO mice by CRISPR/Cas9 and its effect on blood pressure / 生物工程学报

To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.

Berberine Inhibits Atherosclerosis by Regulating Lipophagy Via Targeting Wnt5a/NPC1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the regulatory effect and molecular mechanism of berberine （BBR） on lipophagy in the prevention and treatment of atherosclerotic （AS） lesions in mice. MethodFifty apolipoprotein E-knockout （ApoE-/-） mice were randomly divided into an AS model group， an atorvastatin group （5 mg·kg-1）， and low-， medium-， and high-dose BBR groups （2.5， 5， 10 mg·kg-1）. Ten C57BL/6J mice were assigned to the control group. After 12 weeks， hematoxylin-eosin （HE） and oil red O staining were performed to assess the histopathological changes of AS plaques in the aorta. Biochemical analysis was used to measure serum lipid levels， and enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory cytokines interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α）， oxidative stress marker reactive oxygen species （ROS）， and serum lipophagy marker Beclin1 and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）. The xanthine oxidase method was used to measure serum superoxide dismutase （SOD） activity. Immunohistochemistry （IHC） was used to detect the distribution of wingless-type MMTV integration site family member 5a （Wnt5a） and Nieman Pick type C1 （NPC1） in the aorta， and Western blot was used to determine the protein expression of Wnt5a and NPC1 in the aorta. ResultCompared with the control group， the AS model group showed significant AS plaque formation， significantly elevated levels of serum total cholesterol （TC）， triglycerides （TG）， low-density lipoprotein cholesterol （LDL-C）， IL-6， TNF-α， and ROS， aortic Wnt5a distribution and protein expression （P<0.01）， and significantly reduced levels of serum high-density lipoprotein cholesterol （HDL-C）， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.01）. Compared with the AS model group， the atorvastatin group， and high- and medium-dose BBR groups showed a significant reduction in AS plaque area （P<0.05， P<0.01）， significantly decreased levels of serum TC， TG， LDL-C， IL-6， TNF-α， ROS， and aortic Wnt5a distribution and protein expression （P<0.05， P<0.01）， and significantly increased levels of serum HDL-C， SOD， Beclin1， LC3Ⅱ， and aortic NPC1 distribution and protein expression （P<0.05， P<0.01）. There was no statistically significant difference in the above indicators between the atorvastatin group and the medium-dose BBR group. ConclusionBBR can competitively bind to Wnt5a to activate NPC1 expression， upregulate lipophagy levels， reduce blood lipids， and inhibit the release of inflammatory mediators and oxidative stress damage， thereby exerting a preventive and therapeutic effect on AS.

Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout in vivo

The lysosome is responsible for protein and organelle degradation and homeostasis and the cathepsins play a key role in maintaining protein quality control. Cathepsin D (CTSD), is one such lysosomal protease, which when deficient in humans lead to neurolipofuscinosis (NCL) and is important in removing toxic protein aggregates. Prior studies demonstrated that CTSD germ-line knockout-CtsdKO (CDKO) resulted in accumulation of protein aggregates, decreased proteasomal activities, and postnatal lethality on Day 26 ± 1. Overexpression of wildtype CTSD, but not cathepsin B, L or mutant CTSD, decreased α-synuclein toxicity in worms and mammalian cells. In this study we generated a mouse line expressing human CTSD with a floxed STOP cassette between the ubiquitous CAG promoter and the cDNA. After crossing with Nestin-cre, the STOP cassette is deleted in NESTIN + cells to allow CTSD overexpression-CTSDtg (CDtg). The CDtg mice exhibited normal behavior and similar sensitivity to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration. By breeding CDtg mice with CDKO mice, we found that over-expression of CTSD extended the lifespan of the CDKO mice, partially rescued proteasomal deficits and the accumulation of Aβ42 in the CDKO. This new transgenic mouse provides supports for the key role of CTSD in protecting against proteotoxicity and offers a new model to study the role of CTSD enhancement in vivo.

Identification of a new C-23 metabolite in sterol degradation of Mycobacterium neoaurum HGMS2 and analysis of its metabolic pathways / 生物工程学报

Mycobacterium neoaurum has the ability to produce steroidal intermediates known as 22-hydroxy-23, 24-bisnorchol-4-en-3-one (BA) upon the knockout of the genes for either the hydroxyacyl-CoA dehydrogenase (Hsd4A) or acyl-CoA thiolase (FadA5). In a previous study, we discovered a novel metabolite in the fermentation products when the fadA5 gene was deleted. This research aims to elucidate the metabolic pathway of this metabolite through structural identification, homologous sequence analysis of the fadA5 gene, phylogenetic tree analysis of M. neoaurum HGMS2, and gene knockout. Our findings revealed that the metabolite is a C23 metabolic intermediate, named 24-norchol-4-ene-3, 22-dione (designated as 3-OPD). It is formed when a thioesterase (TE) catalyzes the formation of a β-ketonic acid by removing CoA from the side chain of 3, 22-dioxo-25, 26-bisnorchol-4-ene-24-oyl CoA (22-O-BNC-CoA), followed by spontaneously undergoing decarboxylation. These results have the potential to contribute to the development of novel steroid intermediates.

Síndrome de Baller-Gerold. A propósito de un caso clínico y revisión de la literatura

El síndrome de Baller-Gerold es secundario a mutaciones en el gen RECQL4 (8q24.3). Este gen pertenece a la familia de las RecQhelicasas y está implicado en otras enfermedades de predisposición al cáncer. El diagnóstico se basa en criterios clínicos y debido al elevado número de diagnósticos diferenciales, encontrar una mutación en el gen puede ayudar a precisar el espectro diagnóstico, el consejo genético y su tratamiento. Se han descrito en la literatura alrededor de 30 casos, aunque se sabe que se ha presentado en menos de 200 000 personas en el mundo, por lo que se considera, una condición clínica rara. Se presenta el caso de una paciente que desde su nacimiento se constataron múltiples malformaciones músculo-esqueléticas: aplasia radial, pulgares aplásicos, malformaciones de la parrilla costal, clinodactilia de todos los dedos de miembros superiores, antebrazos hipoplásicos, clinodactilia del miembro inferior izquierdo. Se le realizó alimentación parenteral por varios días, fue imposible establecer vía oral adecuada por ausencia y/o incoordinación de los reflejos de succión-deglución como expresión de sus malformaciones a nivel de sistema nervioso central. Se presenta el caso por su baja incidencia y prevalencia por lo que es considerada una enfermedad rara.

Baller-Gerold syndrome is secondary to mutations in the RECQL4 gene (8q24.3). This gene belongs to the RecQhelicase family and is implicated in other diseases predisposing to cancer. Diagnosis is based on clinical criteria and due to the high number of differential diagnoses, finding a mutation in the gene can help to specify the diagnostic spectrum, genetic counseling and treatment. Around 30 cases have been described in the literature, although it is known that it has occurred in less than 200,000 people in the world, being considered a rare clinical condition. We present the case of a newborn, who at birth was found to have multiple musculoskeletal malformations: radial aplasia, aplastic thumbs, malformations of the rib cage, clinodactyly of all the fingers of the upper limbs, hypoplastic forearms, clinodactyly of the left lower limb. She was given parenteral feeding for several days, it was impossible to establish an adequate oral route due to the absence and/or incoordination of the sucking-swallowing reflexes as an expression of her malformations at the central nervous system level. The case is presented due to its low incidence and prevalence, which is why it is considered a rare disease.

Effects of Bcl3 gene knockout on composition of spleen immune cells and antitumor ability of mice / 中华微生物学和免疫学杂志

Objective:To investigate the effects of Bcl3 gene knockout on the composition of spleen immune cells and antitumor ability of mice.Methods:Bcl3 gene knockout mice (Bcl3 -/-) were established by CRISPR/Cas9 genome editing technology. Blood routine test and flow cytometry were used to detect the immune cell composition in Bcl3 -/- mice. Lung metastasis models were established by injecting mice with B16F10 melanoma cells. The number of tumor nodules in lung and the survival time of mice were used to assess the antitumor ability of wild-type (WT) and Bcl3 -/- mice. Results:Bcl3 -/- mice were successfully bred to a strain with normal growth rate and normal breeding performance. Furthermore, no embryonic death occurred. Compared with WT mice, Bcl3 -/- mice showed splenomegaly and a significant increase in the number of spleen immune cells ( P<0.05). The counts and percentages of platelets and neutrophils in Bcl3 -/- mice were significantly lower than those in WT mice. The proportion of CD19 + B cells showed no significant change, while the proportions of CD3 + T cells and T cell subsets (CD4 + , CD8 + , Treg) increased significantly ( P<0.05). The proportions of NK cells (NK1.1 + ) and neutrophils (Gr1 + ) decreased ( P<0.05), while no significant change in the proportion of DC (CD11b + ) was observed. There were a large number of tumor nodules formed by melanoma cells in the lung of Bcl3 -/- tumor bearing mice, and their survival time was shortened dramatically. Conclusions:Knockout of Bcl3 gene affected the development, differentiation and function of immune cells, thereby reducing the antitumor ability of mice.

Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury / 中南大学学报(医学版)

OBJECTIVES@#Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation.@*METHODS@#Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL.@*RESULTS@#Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (P<0.01 or P<0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both P<0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (P<0.05 or P<0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both P<0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05).@*CONCLUSIONS@#Knockout of FXR gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.

Effects of CACNA1H gene knockout on autistic-like behaviors and the morphology of hippocampal neurons in mice / 北京大学学报(医学版)

OBJECTIVE@#To investigate the effects of CACNA1H gene knockout (KO) on autistic-like behaviors and the morphology of hippocampal neurons in mice.@*METHODS@#In the study, 25 CACNA1H KO mice of 3-4 weeks old and C57BL/6 background were recruited as the experimental group, and 26 wild type (WT) mice of the same age and background were recruited as the control group. Three-chamber test and open field test were used to observe the social interaction, anxiety, and repetitive behaviors in mice. After that, their brain weight and size were measured, and the number of hippocampal neurons were observed by Nissl staining. Furthermore, the CACNA1H heterozygote mice were interbred with Thy1-GFP-O mice to generate CACNA1H-/--Thy1+(KO-GFP) and CACNA1H+/+-Thy1+ (WT-GFP) mice. The density and maturity of dendritic spines of hippocampal neurons were observed.@*RESULTS@#In the sociability test session of the three-chamber test, the KO mice spent more time in the chamber of the stranger mice than in the object one (F1, 14=95.086, P < 0.05; Post-Hoc: P < 0.05), without any significant difference for the explored preference index between the two groups (t=1.044, P>0.05). However, in the social novelty recognition test session, no difference was observed between the time of the KO mice spend in the chamber of new stranger mice and the stranger one (F1, 14=18.062, P < 0.05; Post-Hoc: P>0.05), and the explored preference index of the KO mice was less than that of the control group (t=2.390, P < 0.05). In the open field test, the KO mice spent less time in the center of the open field apparatus than the control group (t=2.503, P < 0.05), but the self-grooming time was significantly increased compared with the control group (t=-2.299, P < 0.05). Morphological results showed that the brain weight/body weight ratio (t=0.356, P>0.05) and brain size (t=-0.660, P>0.05) of the KO mice were not significantly different from those of the control group, but the number of neurons were significantly reduced in hippocampal dentate gyrus compared with the control group (t=2.323, P < 0.05). Moreover, the density of dendritic spine of dentate gyrus neurons in the KO-GFP mice was significantly increased compared with the control group (t=-2.374, P < 0.05), without any significant difference in spine maturity (t=-1.935, P>0.05).@*CONCLUSION@#CACNA1H KO mice represent autistic-like behavior, which may be related to the decrease in the number of neurons and the increase in the density of dendritic spine in the dentate gyrus.

Effects of dexmedetomidine on ADAMTS5 mediated matrix degradation in chondrocytes and expressions of related factors / 西安交通大学学报(医学版)

【Objective】 To investigate the effects of dexmedetomidine (DEX) intervention on the expressions of chondrocytes and related factors in vitro and its possible molecular mechanisms. 【Methods】 C28/I2 normal human chondrocyte lines were cultured in vitro, and dexmedetomidine at the concentration of 1 μmol/L was selected to intervene for 24 h and 48 h, respectively. The morphology and cell density of chondrocytes were observed after DEX culture at different time points. Immunofluorescence technique was used to detect the expression levels of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) in chondrocytes in each group. The expression levels of Adamts5, aggrecan (Acan), versican (Vcan), Furin, proprotein convertase subtilisin/kexin type 6 (Pcsk6), collagen type Ⅰ alpha 1 (Col1a1), collagen type Ⅱ alpha 1 (Col2a1), collagen type X alpha 1 (Col10a1), and SRY2 related high mobility group box gene9 (Sox9) were detected by RT-PCR. Adamts5 gene knockout chondrocytes were constructed by lentivirus transfection technology and treated with DEX; RT-PCR was used to detect the effects of DEX on the expression levels of Acan, Vcan, Furin, Pcsk6 and Sox9 after Adamts5 gene knockout. 【Results】 After 24 and 48 h of intervention with 1 μmol/L DEX, the morphology and size of chondrocytes did not change significantly, but the cell density increased slightly. Immunofluorescence assay showed that the expression of ADAMTS5 increased at first and then decreased after DEX treatment for 24 and 48h, respectively (P=0.032). RT-PCR results showed that with the extension of intervention time, the expression of Adamts5 first increased and then decreased. The expression difference between 48 and 24 h after culture was statistically significant (P=0.032). The change trend of Pcsk6 was the same as that of Adamts5, while the change trend of Acan expression was opposite that of Adamts5. Chondrocytes knocked out Adamts5 gene and intervened with DEX for 24 and 48 h. The results of RT-PCR showed that the expression of Pcsk6 decreased while that of Acan increased and the changes were significant. 【Conclusion】 Dexmedetomidine may activate ADAMTS5 zymogen through Pcsk6, thereby promoting proteoglycan degradation in chondrocytes.

Establishment and identification of a human megakaryocytic cell line with low Ley expression / 中国输血杂志

【Objective】 To establish a stable human megakaryocytic cell line with low expression of Ley antigen to further study the role of Ley on activation of platelets. 【Methods】 The expression level of the Ley antigen in a human megakaryocytic cell line, DAMI, was determined using Western Blot and flow cytometry. The expression level of genes that encode fucosyltransferase (FUTs), which was involved in the biosynthesis of Ley antigen, was also determined to identify the candidate genes to be knocked out. The candidate FUT gene was knocked out via a CRISPR/Cas 9 gene knockout system and cells with low Ley antigen expression were sorted by flow cytometry. The sorted cell line was cultured and characterized. 【Results】 The Ley was expressed intensively on DAMI cell. FUT1 and FUT4 mRNA was expressed relatively higher, both may be key enzymes for the biosynthesis of the Ley antigen. In the DAMI cell line with the knockout of FUT1 gene, the expression of the Ley adntigen was remarkedly reduced, while cell proliferation was not affected compared to the wildtype control cells. 【Conclusions】 Since various FUTs contributes to the biosynthesis of the Ley antigen, the knockout of the primary one of them cannot totally block its biosynthesis, but only reduce its expression. In this study, a stable FUT gene knockout human megakaryocyticcell line is established using CRISPR/Cas 9 technology, which provides basis for the study of the impact of the Ley antigen on platelet functions.

Practical Application Strategies of Cre-LoxP Conditional Gene Knockout / 中国生物化学与分子生物学报

Cre-loxP is an efficient recombination system originated from P1 phage. Its specific recombination patterns based on the locus of X-over P1 make it one of the most commonly used tools for gene editing in recent years. This paper focuses on the practical application of Cre-loxP system. Firstly, the functions and advantages of CRISPR/Cas9 system in Cre sequence insertion and loxP sequence insertion are analyzed. Then, a series of practical application problems of Cre-loxP system are described. For example, in this paper, the selection of Cre recombinase sequence in situ and safe site, the strategy of loxP sequence insertion, the identification of Cre recombinase tag protein, fluorescence identification of "ectopic" expression, primer design of PCR identification and reproductive strategy of mouse were described. At the same time, the optimization of Cre-loxP system in conditional gene knockout is introduced, such as ligand-induced Cre, promoter activated Cre, photo-induced Cre and activity modification of Cre. Through these optimized applications, we can obtain time-controlled conditional gene knockout, regulate the activity of Cre recombinase, and even avoid the toxicity of Cre recombinase itself. Finally, this paper discusses the defects and challenges of Cre-loxP system, and looks into the future development direction of Cre-loxP system. In summary, this paper reviews the practical application of gene knockout based on Cre-loxP system, summarizes the latest research progress and optimization strategies of Cre-loxP system, and prospects the future gene editing based on Cre-loxP system. This paper aims to provide theoretical guidance for solving practical operation problems based on Cre-loxP system, and to provide new research ideas for more accurate, more controllable and more adaptive gene editing in the future.

Construction of aquaporin 9 gene knockout mice using CRISPR/Cas9 gene editing system / 解剖学报

Objective To construct homozygous aquaporin 9(AQP-9)

Effect of Smad7 deficiency on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen I secretion in vitro / 解剖学报

Objective To investigate the effects of Smad7 knock down by lentivirus on rat cardiac fibroblasts proliferation, migration, cell differentiation and collagen secretion in vitro. Methods The primary cardiac fibroblasts were separated from the hearts of ten SD rats and identified by immunohistochemical method. The lentivirus transfection knocked down the expresson of Smad7 in cardiac fibroblasts, Western blotting was used to detect the efficiency of Smad7 knock down by lentivirus. The proliferation of cardiac fibroblasts was quantified by real-time unlabeled cell analyzer. Cell migration was evaluted by cell wound scratch assay. Western blotting was used to detect expression of α-smooth muscle actin（α-SMA） and collagen Ⅰ（Col Ⅰ）. Results Myocardial fibroblasts were successfully cultured and identified by immunocytochemical methods. The multiplicity of infection（MOI） that lentivirus transduction of myocardial fibroblasts was 100. After lentivirus transduction, 88.33% myocardial fibroblasts expressed green fluorescent protein, showed that the lentivirus could significantly reduce the protein expression of Smad7. Smad7 deficiency decreased the proliferation and migration of cardiac fibroblasts, increased the protein expression of α-SMA and decreased collagen secretion. The results indicated that Smad7 deficiency significantly down-regulated the proliferation and migration of cardiac fibroblasts, increased α-SMA protein expression and reduced ColⅠ protein expression. Conclusion Smad7 deficiency can significantly change the cardiac fibroblasts function, that is related to the pathological mechanism that lead to myocardial fibrosis