1.Establishment of a porcine small intestinal epithelial cell line with IRF8 gene knockout based on AAV-SaCas9
Mingliang ZHANG ; Kaiqi LIAN ; Yao WANG ; Bingqian WANG ; Shengming MA ; Yifan ZHANG ; Xinying JI ; Xuekun DOU ; Longfei ZHANG ; Shaoting WENG
Chinese Journal of Veterinary Science 2025;45(6):1169-1177
The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P<0.05)and IFN-λ(P<0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.
2.Establishment of a porcine small intestinal epithelial cell line with IRF8 gene knockout based on AAV-SaCas9
Mingliang ZHANG ; Kaiqi LIAN ; Yao WANG ; Bingqian WANG ; Shengming MA ; Yifan ZHANG ; Xinying JI ; Xuekun DOU ; Longfei ZHANG ; Shaoting WENG
Chinese Journal of Veterinary Science 2025;45(6):1169-1177
The specific mechanisms of interferon regulatory factor 8(IRF8)in porcine intestinal in-nate immunity and resistance to enteric virus infection remain to be elucidated.To investigate the immunoregulatory role of IRF8,establishing an IRF8 gene knockout porcine intestinal epithelial cell(IPEC-J2)monoclonal cell line is of significant importance.This study initially aimed to obtain recombinant adeno-associated virus rAAV-sgIRF8-eGFP capable of knocking out the IRF8 gene through co-transfection of HEK-293T cells with three plasmids.Subsequently,IPEC-J2 cells were infected with the virus,and those expressing eGFP were selected by flow cytometry and cultured to form monoclonal cell lines.These cell lines were then identified by Sanger sequencing and West-ern blot techniques.Lastly,qPCR analysis was used to measure the expression levels of interferon factors IFN-α,IFN-β,IFN-γ and IFN-λ,providing preliminary insights into the impact of IRF8 gene knockout on IPEC-J2 cell immunity.The results demonstrated successful generation of rAAV-sgIRF8-eGFP,which successfully infected IPEC-J2 cells leading to eGFP fluorescence.Flow cytometry followed by cell culture led to the establishment of two monoclonal cell lines,IRF8-KO1 and IRF8-KO3.Sanger sequencing revealed a five-base deletion in IRF8-KO1 and a seven-base dele-tion in IRF8-KO3.Western blot confirmed the absence of IRF8 protein expression in IRF8-KO1,making it an ideal candidate monoclonal cell line.qPCR analysis of interferon factors indicated sig-nificant decrease in IFN-γ(P<0.05)and IFN-λ(P<0.01)transcription level in IRF8-knockout cells,while the transcription levels of IFN-α and IFN-β remained relatively unchanged.This study successfully established an IRF8 gene knockout IPEC-J2 monoclonal cell line,providing a founda-tion for further research on IRF8-related porcine intestinal immune regulation and mechanisms of intestinal virus infection.
3.Artificial intelligence and surgical gesture recognition: a new paradigm for surgical skill asse-ssment in the era of intelligent minimally invasive surgery
Runzhuo MA ; Longfei GOU ; Jiang YU ; Yanfeng HU ; Hao CHEN ; Andrew J. HUANG
Chinese Journal of Digestive Surgery 2025;24(4):480-486
With the advancements in artificial intelligence (AI), computational power, and surgical robotics, the analysis of surgical performance at the granular level of individual surgical gestures has become feasible. Surgical gestures, defined as the smallest independent units of inter-action between surgical instruments and tissues, offer a quantifiable framework for surgical skill assessment. Evidence suggests that the selection and execution of surgical gestures are strongly correlated with the expertise of the surgeon and patient outcomes, underscoring their significance in both surgical training and clinical practice. Moreover, the establishment of a standardized classifi-cation system for surgical gestures and the adoption of uniform terminology have the potential to improve communication efficiency during surgical education and training. The authors synthesize existing classification systems for surgical gestures, with a focus on their applications in diverse tasks such as suturing, exposure and dissection. By examining the latest advancements in AI models applied to surgical gesture, as well as the current research landscape of surgical gesture recognition in digestive surgery, the authors explore the potential applications of such technologies in assisting surgeons during operations in the future.
4.Artificial intelligence and surgical gesture recognition: a new paradigm for surgical skill asse-ssment in the era of intelligent minimally invasive surgery
Runzhuo MA ; Longfei GOU ; Jiang YU ; Yanfeng HU ; Hao CHEN ; Andrew J. HUANG
Chinese Journal of Digestive Surgery 2025;24(4):480-486
With the advancements in artificial intelligence (AI), computational power, and surgical robotics, the analysis of surgical performance at the granular level of individual surgical gestures has become feasible. Surgical gestures, defined as the smallest independent units of inter-action between surgical instruments and tissues, offer a quantifiable framework for surgical skill assessment. Evidence suggests that the selection and execution of surgical gestures are strongly correlated with the expertise of the surgeon and patient outcomes, underscoring their significance in both surgical training and clinical practice. Moreover, the establishment of a standardized classifi-cation system for surgical gestures and the adoption of uniform terminology have the potential to improve communication efficiency during surgical education and training. The authors synthesize existing classification systems for surgical gestures, with a focus on their applications in diverse tasks such as suturing, exposure and dissection. By examining the latest advancements in AI models applied to surgical gesture, as well as the current research landscape of surgical gesture recognition in digestive surgery, the authors explore the potential applications of such technologies in assisting surgeons during operations in the future.
5.A case report of recurrent posterior circulation infarction caused by bilateral bow hunter's syndrome
Jun ZHANG ; Huan ZHANG ; Pingping WANG ; Yuan WANG ; Ting YANG ; Qingfeng MA ; Longfei WU
Chinese Journal of Cerebrovascular Diseases 2025;22(9):631-635
Bow hunter's syndrome,also referred to as rotational vertebral artery occlusion syndrome,is a rare etiological factor of posterior circulation infarction.This article reported a case of a young male patient who experienced recurrent posterior circulation infarctions caused by bilateral bow hunter's syndrome.Carotid ultrasonography confirmed a marked reduction in blood flow velocity in both vertebral arteries during neck rotation.High-resolution MR angiography and CT angiography of the head and neck revealed dissection involving the V3 segment of the left vertebral artery.The findings suggested that bow hunter's syndrome may be associated with thrombus formation secondary to repetitive mechanical compression of the vertebral artery intima,which could potentially lead to arterial embolism and subsequent cerebral infarction.This paper presents the patient's diagnostic and therapeutic course and includes a review of relevant literature aimed to enhance clinical awareness and understanding of this uncommon condition.
6.A case report of recurrent posterior circulation infarction caused by bilateral bow hunter's syndrome
Jun ZHANG ; Huan ZHANG ; Pingping WANG ; Yuan WANG ; Ting YANG ; Qingfeng MA ; Longfei WU
Chinese Journal of Cerebrovascular Diseases 2025;22(9):631-635
Bow hunter's syndrome,also referred to as rotational vertebral artery occlusion syndrome,is a rare etiological factor of posterior circulation infarction.This article reported a case of a young male patient who experienced recurrent posterior circulation infarctions caused by bilateral bow hunter's syndrome.Carotid ultrasonography confirmed a marked reduction in blood flow velocity in both vertebral arteries during neck rotation.High-resolution MR angiography and CT angiography of the head and neck revealed dissection involving the V3 segment of the left vertebral artery.The findings suggested that bow hunter's syndrome may be associated with thrombus formation secondary to repetitive mechanical compression of the vertebral artery intima,which could potentially lead to arterial embolism and subsequent cerebral infarction.This paper presents the patient's diagnostic and therapeutic course and includes a review of relevant literature aimed to enhance clinical awareness and understanding of this uncommon condition.
7.Acupotomy ameliorates knee osteoarthritis-related collagen deposition and fibrosis in rabbit skeletal muscle through the TGF-β/Smad pathway
Tingyao Hu ; Einar Khavaza ; Chuxi Liang ; Longfei Xing ; Xilin Chen ; Yue Xu ; Weiwei Ma ; Farid Mokhtari ; Juan Lu ; Changqing Guo
Journal of Traditional Chinese Medical Sciences 2024;11(3):376-385
Objective:
To investigate the effects of acupotomy on skeletal muscle fibrosis and collagen deposition in a rabbit knee osteoarthritis (KOA) model.
Methods:
Rabbits (n = 18) were randomly divided into control, KOA, and KOA + acupotomy (Apo) groups (n = 6). The rabbits in the KOA and Apo groups were modeled using the modified Videman's method for 6 weeks. After modeling, the Apo group was subjected to acupotomy once a week for 3 weeks on the vastus medialis, vastus lateralis, rectus femoris, biceps femoris, and anserine bursa tendons around the knee. The behavior of all animals was recorded, rectus femoris tissue was obtained, and histomorphological changes were observed using Masson staining and transmission electron microscopy. The expression of transforming growth factor-β1 (TGF-β1), Smad 3, Smad 7, fibrillar collagen types I (Col-I) and III (Col-III) was detected using Western blot and real-time polymerase chain reaction (RT-PCR).
Results:
Histological analysis revealed that acupotomy improved the microstructure and reduced the collagen volume fraction of rectus femoris, compared with the KOA group (P = .034). Acupotomy inhibited abnormal collagen deposition by modulating the expression of fibrosis-related proteins and mRNA, thus preventing skeletal muscle fibrosis. Western blot and RT-PCR analysis revealed that in the Apo group, Col-I, and Col-III protein levels were significantly lower than those in the KOA group (both P < .01), same as Col-I and Col-III mRNA levels (P = .0031; P = .0046). Compared with the KOA group, the protein levels of TGF-β1 and Smad 3 were significantly reduced (both P < .01), as were the mRNA levels of TGF-β1 and Smad 3 (P = .0007; P = .0011). Conversely, the levels of protein and mRNA of Smad 7 were significantly higher than that in the KOA group (P < .01; P = .0271).
Conclusion
Acupotomy could alleviate skeletal muscle fibrosis and delay KOA progress by inhibiting collagen deposition through the TGF-β/Smad pathway in the skeletal muscle of KOA rabbits.
8.Research Progress of Microneedle Delivery System in the Treatment of Skin Tumors
Yiting LUO ; Ting YU ; Yue QIU ; Longfei LIAO ; Xue MA ; Hongtao XIAO ; Tingting QI
Herald of Medicine 2024;43(8):1235-1244
As an emerging drug delivery technology,microneedles can puncture the skin's stratum corneum to create micron-sized conduits,painlessly,minimally invasive,and efficiently deliver drugs into viable epidermis or dermis for local or systemic therapeutic effects.This paper reviews the current clinical trials of microneedles used in the treatment of various diseases,elaborates on the characteristics of various types of microneedles,and summarizes the latest research progress of microneedles used to treat skin tumors,including chemotherapy,photothermal and photodynamic therapy,immunotherapy,gene therapy,and combination therapy.This review provides ideas and directions for further research on microneedles in treating skin tumors.
9.Short-term efficacy and safety analysis of PD-1 inhibitor combined with SOX neoadjuvant therapy for locally advanced gastric adenocarcinoma
Tao WANG ; Lulu ZHANG ; Shuxiang DU ; Han ZHANG ; Longfei MA ; Gang WU ; Peichun SUN
Tumor 2023;43(8):635-645
Objective:To explore the short-term efficacy and safety for the new adjuvant therapy of programmed cell protein-1(PD-1)inhibitor combined with Oxaliplatin+Tegafur(SOX)in locally advanced gastric adenocarcinoma. Methods:Retrospective collection of clinical data from 72 locally advanced(stage Ⅲ)gastric adenocarcinoma patients admitted to our gastrointestinal surgery department from January 2020 to July 2020,according to different treatment plans,patients were divided into a control group(n=36)and an observation group(n=36).The control group received SOX chemotherapy regimen,while the observation group received PD-1 inhibitor combined with SOX regimen.Both groups of patients underwent chemotherapy for 3 cycles,and were re-evaluated after resting for 3-4 weeks.Patients with no disease progression and tolerable surgery underwent D2 gastric cancer radical surgery.The overall survival(OS),pathological complete response(pCR)rate,progression free survival period(PFS),RO resection rate,objective response rate(ORR),disease control rate(DCR)and adverse drug reaction were compared between the two groups. Results:The pCR rate,ORR and DCR of the observation group were significantly higher than those of the control group[pCR rate:25%(9/36)vs 5.6%(2/36),x2=5.258,P=0.022;ORR:58.33%(21/36)vs 25.00%(9/36),x2=8.229,P=0.004;DCR:88.89%(32/36)vs 69.44%(25/36),x2=4.1 26,P=0.042].The incidence of adverse reactions such as elevated ALT,elevated AST,and hand foot syndrome during neoadjuvant chemotherapy in the observation group was significantly higher than those in the control group(P<0.05).The incidence of other adverse reactions was not significantly difference between the two groups of patients(P>0.05),and there were no neoadjuvant chemotherapy related death events in both groups.The R0 resection rates of the two groups were 97.22%and 91.67%,respectively(x2=1.059,P=0.303).The median OS and median PFS of 72 patients were 36 and 1 3 months,respectively.The 2-year OS rate of the two groups were 82.2%and 71.5%,respectively.Kaplan Meier survival curve analysis showed that the OS rate of the observation group was significantly higher than that of the control group(x2=4.240,P=0.039);the median PFS of the observation group was higher than that of the control group at 14 and 1 1 months,respectively(x2=4.173,P=0.041);COX univariate regression analysis did not identify any factors affecting survival of locally advanced gastric adenocarcinoma. Conclusion:The neoadjuvant treatment of locally advanced gastric adenocarcinoma with PD-1 inhibitor combined with SOX regimen is safe and effective,and the adverse reactions are controllable.
10.Single-cell profiling reveals Müller glia coordinate retinal intercellular communication during light/dark adaptation via thyroid hormone signaling.
Min WEI ; Yanping SUN ; Shouzhen LI ; Yunuo CHEN ; Longfei LI ; Minghao FANG ; Ronghua SHI ; Dali TONG ; Jutao CHEN ; Yuqian MA ; Kun QU ; Mei ZHANG ; Tian XUE
Protein & Cell 2023;14(8):603-617
Light adaptation enables the vertebrate visual system to operate over a wide range of ambient illumination. Regulation of phototransduction in photoreceptors is considered a major mechanism underlying light adaptation. However, various types of neurons and glial cells exist in the retina, and whether and how all retinal cells interact to adapt to light/dark conditions at the cellular and molecular levels requires systematic investigation. Therefore, we utilized single-cell RNA sequencing to dissect retinal cell-type-specific transcriptomes during light/dark adaptation in mice. The results demonstrated that, in addition to photoreceptors, other retinal cell types also showed dynamic molecular changes and specifically enriched signaling pathways under light/dark adaptation. Importantly, Müller glial cells (MGs) were identified as hub cells for intercellular interactions, displaying complex cell‒cell communication with other retinal cells. Furthermore, light increased the transcription of the deiodinase Dio2 in MGs, which converted thyroxine (T4) to active triiodothyronine (T3). Subsequently, light increased T3 levels and regulated mitochondrial respiration in retinal cells in response to light conditions. As cones specifically express the thyroid hormone receptor Thrb, they responded to the increase in T3 by adjusting light responsiveness. Loss of the expression of Dio2 specifically in MGs decreased the light responsive ability of cones. These results suggest that retinal cells display global transcriptional changes under light/dark adaptation and that MGs coordinate intercellular communication during light/dark adaptation via thyroid hormone signaling.
Animals
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Mice
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Dark Adaptation
;
Light
;
Retina
;
Retinal Cone Photoreceptor Cells/metabolism*
;
Adaptation, Ocular
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Neuroglia/physiology*
;
Cell Communication
;
Thyroid Hormones


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