Background:Liver diseases are a major contributor to both morbidity and mortality.Conditional knockout animals are always produced through crossing floxed animals with a tissue-specific Cre animal.The use of floxed ra...Background:Liver diseases are a major contributor to both morbidity and mortality.Conditional knockout animals are always produced through crossing floxed animals with a tissue-specific Cre animal.The use of floxed rat resource has rapidly increased,but the liver-specific Cre rat lines for studying liver diseases and interested genes are limited,especially in a spatially and temporally restricted manner.Methods:RNA sequencing and real-time polymerase chain reaction(PCR)were used to screen and confirm the presence of liver-specific genes.Apoa4-Cre rats and Cyp2c11-Cre rats were produced by CRISPR/Cas9 knockin.Rosa26-imCherry rats were employed to hybridize with the Cre rats to obtain the Apoa4-Cre/Rosa26-imCherry and Cyp2c11-Cre/Rosa26-imCherry rats.The temporal and spatial patterns of Cre expression were determined by the observation of red fluorescence on tis-sue sections.Hematoxylin-eosin stain was used to evaluate the liver histopathologic changes.The blood biochemical analysis of several liver enzymes and liver lipid profile was performed to evaluate the liver function of Cre rats.Results:Apoa4 and Cyp2c11 were identified as two liver-specific genes.Apoa4-Cre and Cyp2c11-Cre rats were produced and hybridized with Rosa26-imCherry rats.The red fluorescence indicated that the Cre recombinases were specially expressed in the juvenile and adult liver and not in other organs of two hybridized rats.All the blood biochemical parameters except low-density lipoprotein(LDL)did not change signifi-cantly in the Cre rats.No histological alterations were detected in the livers of the Cre rats.Conclusions:Liver-specific Apoa4-Cre and Cyp2c11-Cre rats have been established successfully and could be used to study gene knockout,specifically in juvenile and adult liver.展开更多
Background and Aims:Cholesterol synthesis and gallstone formation are promoted by trimethylamine-N-oxide(TMAO),a derivative of trimethylamine,which is a metabolite of gut microbiota.However,the underlying mechanisms o...Background and Aims:Cholesterol synthesis and gallstone formation are promoted by trimethylamine-N-oxide(TMAO),a derivative of trimethylamine,which is a metabolite of gut microbiota.However,the underlying mechanisms of TMAO-induced lithogenesis remain incompletely understood.This study aimed to explore the specific molecular mechanisms through which TMAO promotes gallstone formation.Methods:Enzyme-linked immunosorbent assays were used to compare serum concentrations of TMAO,apolipoprotein A4(APOA4),and proprotein convertase subtilisin/kexin type 9(PCSK9)between patients with cholelithiasis and normal controls.A murine model of TMAOinduced cholelithiasis was employed,incorporating assays of gallstone weight and bile cholesterol content,along with RNA sequencing of murine hepatic tissue.A TMAO-induced AML12 hepatocyte line was constructed and transfected with targeted small interfering RNAs and overexpression plasmids.In vivo and in vitro experiments were performed to determine the expression and regulation of genes related to cholesterol metabolism.Results:Serum TMAO and PCSK9 levels were elevated,whereas APOA4 levels were reduced in patients with cholelithiasis.Furthermore,our murine model demonstrated that TMAO upregulated hepatic expression of PCSK9,3-hydroxy-3-methylglutaryl-CoA reductase,and ATP-binding cassette sub-family G member 5/8,while reducing APOA4 expression,thereby modulating cholesterol metabolism and promoting lithogenesis.PCSK9 and APOA4 were identified as key regulatory genes in the cholesterol metabolic pathway.PCSK9 knockdown increased APOA4 expression,while APOA4 overexpression led to reduced PCSK9 expression.Conclusions:TMAO upregulated hepatic PCSK9 expression and reduced APOA4 expression,initiating a feedback loop that dysregulated cholesterol metabolism and promoted lithogenesis.展开更多
The incidence of Inactive ovaries of dairy cows in China is relatively high. There is no complete early warning system for the occurrence of ovarian quiescence in clinical cows. This test provides early warning indica...The incidence of Inactive ovaries of dairy cows in China is relatively high. There is no complete early warning system for the occurrence of ovarian quiescence in clinical cows. This test provides early warning indicators for clinical prediction of ovary cessation in dairy cows. This experiment selected blood samples of dairy cows from 60 to 90 days postpartum in the inactive ovaries group and control group. Differential proteins were selected on the basis of proteomics, three energy indexes: AST, Glu, NEFA. Four reproductive hormones: E2, P4, FSH, LH, and four differentially expressed proteins: IGFBP-2, AHSG, APO-A4, and RBP-4. Key enzyme activities: ALDOB, LDHB, ITIH3, GPX3, SPAM1, PKM2. The ELISA test kit was used to detect the content and activity of the above markers in the test bovine serum. Through correlation analysis, binary logistic regression modeling and ROC analysis, a single indicator early warning technique for APOA4 and ITIH3 was established. The early warning values were APOA4 > 28.825 μg/L and ITIH3 > 195.07 ng/L. A multi-index early warning system based on potential biomarkers of APOA4 + ITIH3 and APOA4 + ITIH3 + E2 was established. The former had an early warning value of: APOA4 > 19.55 μg/I;ITIH3 > 191.14 ng/L;the latter has an early warning value: APOA4 > 47.56 μg/L, ITIH4 > 187.80 ng/L, E2 < 69.63 ng/L.展开更多
The incidence of breast cancer is still increasing, and with improved cancer treatment, more women live longer with the side effects of their treatment. The response of normal tissue to radiation continues for years a...The incidence of breast cancer is still increasing, and with improved cancer treatment, more women live longer with the side effects of their treatment. The response of normal tissue to radiation continues for years after the treatment is completed. The influence of radiotherapy on the outcome of breast reconstructtive surgery remains unpredictable. The combination of two surgical sites of which one is previously irradiated, is rarely encountered in humans and thus compiles a unique opportunity to study the implications of irradiation followed by surgery. The aim of this study was to examine the long-term effect of radiation therapy on the proteins expressed in the wound tissue after a breast reconstruction. Ten patients were included in the study, all treated with radiotherapy after a mastectomy and breast reconstruction with a contralateral pedicled TRAM flap. Expanded poly-tetrafluoretylene polymer tubes were implanted for 10 days, subcutaneously, below the inframammary fold and below the donor site. The protein from the newly synthesized granulation tissue in the tubes was extracted and analyzed for differences in protein expression with 2D gel electrophoresis and mass spectrometry. A total of 676 proteins were detected;of these, 4 proteins changed significantly and were successfully identified. TPM4 and APOA4 from the radiation treated tissue were shown to be significantly decreased, whereas IGKC and VDAC1 were found to be significantly increased. The proteomic technique combined with the ePTFE tube wound model can elucidate some of the molecular alterations in the wound healing induced by radiation therapy. The protein modifications of TPM4, APOA4, IGKC and VDAC1 may influence the cell proliferation, apoptosis and the inflammation of the tissue repair process.展开更多
基金CAMS Innovation Fund for Medical Sciences(CIFMS),Grant/Award Number:2021-I2M-1-035National Natural Science Foundation of China,Grant/Award Number:31970508。
文摘Background:Liver diseases are a major contributor to both morbidity and mortality.Conditional knockout animals are always produced through crossing floxed animals with a tissue-specific Cre animal.The use of floxed rat resource has rapidly increased,but the liver-specific Cre rat lines for studying liver diseases and interested genes are limited,especially in a spatially and temporally restricted manner.Methods:RNA sequencing and real-time polymerase chain reaction(PCR)were used to screen and confirm the presence of liver-specific genes.Apoa4-Cre rats and Cyp2c11-Cre rats were produced by CRISPR/Cas9 knockin.Rosa26-imCherry rats were employed to hybridize with the Cre rats to obtain the Apoa4-Cre/Rosa26-imCherry and Cyp2c11-Cre/Rosa26-imCherry rats.The temporal and spatial patterns of Cre expression were determined by the observation of red fluorescence on tis-sue sections.Hematoxylin-eosin stain was used to evaluate the liver histopathologic changes.The blood biochemical analysis of several liver enzymes and liver lipid profile was performed to evaluate the liver function of Cre rats.Results:Apoa4 and Cyp2c11 were identified as two liver-specific genes.Apoa4-Cre and Cyp2c11-Cre rats were produced and hybridized with Rosa26-imCherry rats.The red fluorescence indicated that the Cre recombinases were specially expressed in the juvenile and adult liver and not in other organs of two hybridized rats.All the blood biochemical parameters except low-density lipoprotein(LDL)did not change signifi-cantly in the Cre rats.No histological alterations were detected in the livers of the Cre rats.Conclusions:Liver-specific Apoa4-Cre and Cyp2c11-Cre rats have been established successfully and could be used to study gene knockout,specifically in juvenile and adult liver.
基金supported by the National Natural Science Foundation of China(No.82270598)the National Natural Science Foundation of China(No.82100675)the Open Fund of the Key Laboratory of Hepatosplenic Surgery,Ministry of Education,Harbin,China(No.GPKF202404).
文摘Background and Aims:Cholesterol synthesis and gallstone formation are promoted by trimethylamine-N-oxide(TMAO),a derivative of trimethylamine,which is a metabolite of gut microbiota.However,the underlying mechanisms of TMAO-induced lithogenesis remain incompletely understood.This study aimed to explore the specific molecular mechanisms through which TMAO promotes gallstone formation.Methods:Enzyme-linked immunosorbent assays were used to compare serum concentrations of TMAO,apolipoprotein A4(APOA4),and proprotein convertase subtilisin/kexin type 9(PCSK9)between patients with cholelithiasis and normal controls.A murine model of TMAOinduced cholelithiasis was employed,incorporating assays of gallstone weight and bile cholesterol content,along with RNA sequencing of murine hepatic tissue.A TMAO-induced AML12 hepatocyte line was constructed and transfected with targeted small interfering RNAs and overexpression plasmids.In vivo and in vitro experiments were performed to determine the expression and regulation of genes related to cholesterol metabolism.Results:Serum TMAO and PCSK9 levels were elevated,whereas APOA4 levels were reduced in patients with cholelithiasis.Furthermore,our murine model demonstrated that TMAO upregulated hepatic expression of PCSK9,3-hydroxy-3-methylglutaryl-CoA reductase,and ATP-binding cassette sub-family G member 5/8,while reducing APOA4 expression,thereby modulating cholesterol metabolism and promoting lithogenesis.PCSK9 and APOA4 were identified as key regulatory genes in the cholesterol metabolic pathway.PCSK9 knockdown increased APOA4 expression,while APOA4 overexpression led to reduced PCSK9 expression.Conclusions:TMAO upregulated hepatic PCSK9 expression and reduced APOA4 expression,initiating a feedback loop that dysregulated cholesterol metabolism and promoted lithogenesis.
文摘The incidence of Inactive ovaries of dairy cows in China is relatively high. There is no complete early warning system for the occurrence of ovarian quiescence in clinical cows. This test provides early warning indicators for clinical prediction of ovary cessation in dairy cows. This experiment selected blood samples of dairy cows from 60 to 90 days postpartum in the inactive ovaries group and control group. Differential proteins were selected on the basis of proteomics, three energy indexes: AST, Glu, NEFA. Four reproductive hormones: E2, P4, FSH, LH, and four differentially expressed proteins: IGFBP-2, AHSG, APO-A4, and RBP-4. Key enzyme activities: ALDOB, LDHB, ITIH3, GPX3, SPAM1, PKM2. The ELISA test kit was used to detect the content and activity of the above markers in the test bovine serum. Through correlation analysis, binary logistic regression modeling and ROC analysis, a single indicator early warning technique for APOA4 and ITIH3 was established. The early warning values were APOA4 > 28.825 μg/L and ITIH3 > 195.07 ng/L. A multi-index early warning system based on potential biomarkers of APOA4 + ITIH3 and APOA4 + ITIH3 + E2 was established. The former had an early warning value of: APOA4 > 19.55 μg/I;ITIH3 > 191.14 ng/L;the latter has an early warning value: APOA4 > 47.56 μg/L, ITIH4 > 187.80 ng/L, E2 < 69.63 ng/L.
文摘The incidence of breast cancer is still increasing, and with improved cancer treatment, more women live longer with the side effects of their treatment. The response of normal tissue to radiation continues for years after the treatment is completed. The influence of radiotherapy on the outcome of breast reconstructtive surgery remains unpredictable. The combination of two surgical sites of which one is previously irradiated, is rarely encountered in humans and thus compiles a unique opportunity to study the implications of irradiation followed by surgery. The aim of this study was to examine the long-term effect of radiation therapy on the proteins expressed in the wound tissue after a breast reconstruction. Ten patients were included in the study, all treated with radiotherapy after a mastectomy and breast reconstruction with a contralateral pedicled TRAM flap. Expanded poly-tetrafluoretylene polymer tubes were implanted for 10 days, subcutaneously, below the inframammary fold and below the donor site. The protein from the newly synthesized granulation tissue in the tubes was extracted and analyzed for differences in protein expression with 2D gel electrophoresis and mass spectrometry. A total of 676 proteins were detected;of these, 4 proteins changed significantly and were successfully identified. TPM4 and APOA4 from the radiation treated tissue were shown to be significantly decreased, whereas IGKC and VDAC1 were found to be significantly increased. The proteomic technique combined with the ePTFE tube wound model can elucidate some of the molecular alterations in the wound healing induced by radiation therapy. The protein modifications of TPM4, APOA4, IGKC and VDAC1 may influence the cell proliferation, apoptosis and the inflammation of the tissue repair process.
